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1 DOPAMINERGIC CYSTATIN C: A KEY PLAYER IN NEURON MICROGLIA INTERPLAY By GARIMA DUTTA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Garima Dutta
3 To my family
4 ACKNOWLEDGMENTS Words will never be enough to thank my advisor Dr Bin Liu who not only gave me the liberty to think independently as a scientist but also pr ovided me with strength, conviction and guidance at all those innumerable times when things went awry. I would be forever indebted to him for being extremely understanding and patient with me throughout my PhD career especially the last two years. I m dee ply grateful to my committee members; Dr Maureen Keller Wood, Dr David Barber, Dr Mike King and Dr Michael Katovich for their unwavering faith in my capabilities and constant suggestions towards successful completion of my research projects. I would like to thank Dr Keller Wood for making me a part of one of her projects while I was rotating in her lab and including my name in the manuscript. She always extended a helping hand whenever I needed support and was always willing to go an extra mile to help me better as a scientist. My project could never have been completed without the help of Dr Barber for with my research and infact went out of his way to accommodate me. I sincerely appreciate all his help. research were thought provoking. He helped me broaden my experience by introducing me to a group of engineers who were working on shock and made every effort to ensure that I learnt something new. It was truly an enriching experience and I sincerely thank him for that. Dr Katovich was always the person who brought me back to reality when I got overambitious or too lax. He would always make sure that I was thinking rationally and
5 clearly enough to achieve my goal s in a practical setting. His continuous support and guidance throughout my PhD is highly appreciated. My colleagues and friends Dr Heera Sharma, Yue Liu and Debapriya Dutta need a s pecial mention as they really helped me stay sane through the ups and downs of my professional and personal life. Their support and care helped me overcome setbacks and face life with a fresh outlook. I greatly value their friendship and deeply appreciate the ir belief in me. A special heartfelt thanks also goes to Dr Ping Zhang for lending an ear everytime I wanted to vent and giving me advice that forwarded my scientific and personal pursuits. I also appreciate all the help I got from Dr Haoyu Mao and Raj iv T i kamdas in furthering my research. Even though my name is on the cover of this dissertation, my family is the actual entity and niece are my pillars of strength and without en dared to dream of coming to s plete without thanking my grand dad for introducing me to the field of p harmacy. He was the one who showed me this career and I owe it all to him. Each and ev ery person mentioned in my acknowledgment contributed significantly in making this dissertation a possibility and in making my journey here an experience to be cherished forever.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 15 ................................ ................................ ....................... 15 History ................................ ................................ ................................ ..................... 17 Etiology and Risk Factors ................................ ................................ ....................... 18 Genetics and PD ................................ ................................ .............................. 18 Alpha Synuclein ................................ ................................ ............................... 18 LRRK2 ................................ ................................ ................................ .............. 21 Parkin ................................ ................................ ................................ ............... 22 UCHL 1 ................................ ................................ ................................ ............ 23 PINK1 ................................ ................................ ................................ ............... 24 DJ 1 ................................ ................................ ................................ .................. 25 Environmental Factors and PD ................................ ................................ ......... 25 Beneficial Environmental Agents ................................ ................................ ...... 26 Toxic Environmental Agents ................................ ................................ ............. 27 Metals ................................ ................................ ................................ ........ 27 Rotenone ................................ ................................ ................................ ... 28 Paraquat ................................ ................................ ................................ .... 29 Maneb ................................ ................................ ................................ ........ 30 Organochlorine pesticides ................................ ................................ .......... 30 Dieldrin ................................ ................................ ................................ ....... 31 Experimental DA neurotoxins ................................ ................................ ..... 32 MPTP ................................ ................................ ................................ ......... 32 6 OHDA ................................ ................................ ................................ ..... 34 Interaction Between Genes and Environmental Factors ................................ .. 35 Aging ................................ ................................ ................................ ................ 36 Pathogenesis ................................ ................................ ................................ .......... 40 Mitochondrial Dysfunction and Oxidative Stress ................................ .............. 42 Neuromelanin ................................ ................................ ................................ ... 44 Impairment of Ubiquitin Proteasome System ................................ ................... 46 Neuroinflammation ................................ ................................ ................................ .. 47 Glia and Neurodegeneration ................................ ................................ ............ 47 Microglia ................................ ................................ ................................ ........... 49
7 Microglial Activation and PD ................................ ................................ ............. 54 LPS model ................................ ................................ ................................ 54 MPTP model ................................ ................................ .............................. 56 Alpha synuclein model ................................ ................................ ............... 57 Proinflammatory Factors and PD ................................ ................................ ..... 57 Cytokines ................................ ................................ ................................ ... 58 NADPH oxidase ................................ ................................ ......................... 59 Nitric oxide synthase (NOS) ................................ ................................ ....... 60 Nitric oxide (NO) and PD ................................ ................................ ............ 6 1 Cyclooxygenase ................................ ................................ ......................... 63 ................................ ................................ ...... 63 Treatment ................................ ................................ ................................ ............... 64 Levodopa ................................ ................................ ................................ .......... 64 Dopamine Agonists ................................ ................................ .......................... 65 MAO B and COMT Inhibitors ................................ ................................ ............ 65 Anticholinergics ................................ ................................ ................................ 66 Alpha 2 Adrenergic Anatgonists ................................ ................................ ....... 66 Adenosine A2A Receptor Antagonists ................................ ............................. 66 Surgical Targets: A blation and Deep Brain Stimulation ................................ .... 67 Gene Therapy ................................ ................................ ................................ .. 68 Gene transfer approach ................................ ................................ ............. 68 RNA modification ................................ ................................ ....................... 69 Stem cell therapy ................................ ................................ ....................... 69 Gene Therapy for PD ................................ ................................ ....................... 69 Fetal Tissue Transplantation ................................ ................................ ............ 71 Stem Cells ................................ ................................ ................................ ........ 72 2 EXPERIMENTAL PROTOCOLS ................................ ................................ ............. 73 Cell Culture ................................ ................................ ................................ ............. 73 Cell Viability Assay ................................ ................................ ................................ 74 Preparation of N27 Neuronal Conditioned Media (CM) ................................ ........... 74 Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) ............................... 75 Microglial Activation ................................ ................................ ................................ 76 Nitrite Assay ................................ ................................ ................................ ............ 76 DCF Assay for ROS Detection ................................ ................................ ................ 77 Protein Assay ................................ ................................ ................................ .......... 77 Immunoblotting ................................ ................................ ................................ ....... 78 Immunodepletion ................................ ................................ ................................ .... 79 Immun ofluorescence ................................ ................................ ............................... 79 ELISA ................................ ................................ ................................ ...................... 80 Deglycosylation ................................ ................................ ................................ ....... 81 Statistical Analysis ................................ ................................ ................................ .. 82 3 DOPAMINERGIC NEURONS RELEASE SIGNALS THAT ACTIVATE MICROGLIA ................................ ................................ ................................ ............ 85
8 Introduction ................................ ................................ ................................ ............. 85 Results ................................ ................................ ................................ .................... 90 Toxicity of Dieldrin and MPP + on N27 DA Neuro ns ................................ .......... 90 Soluble Factors Released From Neurotoxicant Injured Dopamine Neurons Induce Microglial Activation ................................ ................................ .......... 91 Lack of a Direct Effect of Neurotoxicants on Microglial Activation .................... 91 Proteomic Profiling of Soluble Factors Released From Neurotoxicant Injured DA Neurons ................................ ................................ ....................... 92 Cystatin C Levels Increase in Toxicant Treated DA Neurons ........................... 92 Discussion ................................ ................................ ................................ .............. 93 4 ROLE OF DOPAMINERGIC CYSTATIN C IN MICROGLIAL ACTIVATION AND DOWNSTREAM NEUROTOXICITY ................................ ................................ ..... 103 Introduction ................................ ................................ ................................ ........... 103 Results ................................ ................................ ................................ .................. 104 Absence of Cystatin C From Neuronal CM D Atten uates CM D Induced Microglial Activation ................................ ................................ .................... 104 Cystatin C Depletion Decreases Microglial ROS Production and Cytokine Release ................................ ................................ ................................ ....... 104 Cystatin C is an Important Contributor to Microglial Activation Caused by CM M ................................ ................................ ................................ ............ 104 Neuronal Cystatin C Activates Microglia and Amplifies Downstream Neurotoxicity ................................ ................................ ............................... 105 Recombinant Cystatin C Does Not Activate Microglia ................................ .... 105 Discussion ................................ ................................ ................................ ............ 106 5 GLCOSYLATION STATUS OF CYSTATIN C IS IMPORTANT FOR ITS MICROGLIA ACTIVATING POTENTIAL ................................ .............................. 117 Introduction ................................ ................................ ................................ ........... 117 Results ................................ ................................ ................................ .................. 120 Neuronal Cystatin C is Unique ................................ ................................ ....... 120 O Glycosylation and Sialylation are Important for Cystatin C Mediated Microglial iNOS Upregulation ................................ ................................ ...... 120 Removal of O Glycosyl and Sialic Acid Linkages Reduce Cystatin C Induced Microglial Cytokine Production ................................ ...................... 121 Differential Levels o f Cystatin C in Neuronal and Glial Cells Under Normal Growth Conditions and Serum Deprivation ................................ ................. 121 Cystatin C from challenged microg lia does not activate microglia .................. 122 Neuronal and Microglial Cystatin C are Distinct from Each Other .................. 122 CM from B35 Cortical Neuronal Cells Cannot Upregulate Microglial iNOS .... 123 B35 Cortical Neurons Release Significantly Less Cystatin C Compared to N27 DA Neurons ................................ ................................ ......................... 123 Discussion ................................ ................................ ................................ ............ 123 6 OVERALL SUMMARY AND CONCLUSIONS ................................ ...................... 135
9 LIST OF REFERENCES ................................ ................................ ............................. 142 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 174
10 LIST OF TABLES Table page 3 1 Pathway analysis of identified proteins ................................ ............................. 101 3 2 Proteins with increased abundance following dieldrin treatment ...................... 102
11 LIST OF FIGURES Figure page 2 1 Experimental Design. ................................ ................................ ......................... 83 2 2 Deglycosylation by enzymatic treatment ................................ .......................... 84 3 1 Effect of neurotoxicants MPP + and dieldrin on viabi lity of N27 DA neuronal cells. ................................ ................................ ................................ .................. 97 3 2 CM from toxicant injured DA neurons induce microglial iNOS upregulation ...... 98 3 3 Effect of direct treatment with dieldrin or MPP + on microglial iNOS upregulation ................................ ................................ ................................ ....... 99 3 4 Neurotoxic assault promotes DA neuronal cystatin C release. ......................... 100 4 1 Immuno depletion of cystatin C reduces the ability of CM from dieldrin treated DA neurons to induce microglial iNOS upregulation ............................. 110 4 2 Immunoblot to confirm successful removal of cystatin C from CM D ................. 111 4 3 Effect of cystatin C immuno depletion on CM D induced microglial ROS pr oduction and cytokine release. ................................ ................................ ..... 112 4 4 Effect of cystatin C immuno depletion on the ability of CM from MPP + treated DA neurons to induce microglial iNOS upregulation ................................ ......... 113 4 5 Immuno depletion of cystatin C reduces the ability of DA neuronal CM to cause mic roglia mediated neurotoxicity ................................ ........................... 114 4 6 Qua ntification of DA neurotoxicity ................................ ................................ .... 115 4 7 Rat recombinant or human urine cystatin C fails to induce microglial iNOS upregulation. ................................ ................................ ................................ .... 116 5 1 Cystatin C released from toxin inj ured DA ne urons is glycosylated. ................ 127 5 2 Glycosylation status influences microglial activation potential of se creted DA neuronal cystatin C ................................ ................................ ........................... 128 5 3 O linked glycosylation and sialylation of DA neuronal cystatin C are important for microglial cytokine production ................................ ................................ ..... 129 5 4 Comparison of the abundance of cystatin C in norm al growth media and serum free media. ................................ ................................ ........................... 130
12 5 5 Lack of an effect on microglial activation by cystatin C r eleased from microglial cells. ................................ ................................ ................................ 131 5 6 Microglial cystatin C has a different composition from neuronal cystatin C. ..... 132 5 7 C ystatin C from toxicant injured B35 cortical neuronal cells fails to induce microglial iNO S upregulation ................................ ................................ ............ 133 5 8 Effect of dieldrin treatment on cystatin C release in B35 cortical neurons ........ 134 6 1 Neuron microglia in ter play ................................ ................................ ........... 141
13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DOPAMINERGIC CYSTATIN C: A KEY PLAYER IN NEURON MICROGLIA INTERPLAY By Garima Dutta May 2012 Chair: Bin Liu Major: Pharmaceutical Sciences Activation of microglia, the resident immune cells in the brain, contribute s to dopaminergic (DA) neurodegeneration in the pathogenesis of Factors released from DA neurons under toxic insult may trigger the activation of microglia and set in motion a vicious cycle of neuronal injury and inflammation that drive s progressive neurodegeneration. In this study, we have detected increased release of cystatin C as well as several other proteins in to conditioned media (CM) from mildly injured rat N27 DA neuronal cells following exposure to the neurotoxicants 1 methy 4 phenylpyridinium and dieldrin Immunodepletion of cyst atin C significantly reduced the ability of DA neuronal CM to induce the activation of rat HAPI microglial cells as determined by upregulation of inducible nitric oxide synthase (iNOS), production of free radicals and release of proinflammatory cytokines a s well as activated microglia mediated DA neurotoxicity in vitro. In contrast, CM from toxin injured cortical neurons and serum starved microglia were ineffective in inducing microglial activation probably due to the low abundance or differential glycosyl ation profile of cystatin C respectively. DA neuronal cystatin C existed as multiple glyco forms and treatment of CM with enzymes that remove O and sialic acid but not N linked carbohydrate modifications
14 markedly lowered the ability of CM to induce micr oglial activation. Taken together, our results demonstrate that cystatin C released from injured DA neurons is an important mediator of microglial activation related DA neurodegeneration.
15 CHAPTER 1 LITERATURE REVIEW isease (PD) Seldom occur ring before the age of fifty, and frequently yielding but little inconvenience for several months, it is generally considered as the irremediable diminution of the nervous influence, naturally resulting from declining life; and remedies therefore are seldo m sought for. James Parkinson, An Essay on t he Shaking Palsy Parkinson's disease (PD) is a chronic and progressive disorder of the nervous system that affects more than 1 million people in the United Stat es. It is more prevalent in the geriatric population affecting 1000 of every 100, 000 people over the age of 65 (N. J. Diederich et al., 2003) It often starts with non motor symptoms like depression, olfactory dysfunction, orthostatic hypot ension and urinary incontine nce which last for years before the appearance of motor symptomatology (R. L. Doty, 2011) The three cardinal motor abnormalities seen in PD are bradykinesia, cogwheel rigidity and r esting tremors. The fourth cardinal sign is postural reflex compromise or gait instability which occurs at a later stage of the disease (K. L. Double, 2011) PD is mainly caused by loss of dopaminergic (DA) neurons in s u bstantia nigra pars c ompacta (SN pc) which leads to a decrease in striatal dopamine levels. Non dopaminergic neurons including cholinergic neurons, norepinephrinergic neurons in locus coeruleus, neurons in brainstem and basal forebrain and serotonergic neurons in raphae nuclei are also affected albeit to a lesser extent. Abnormal accumulatio synuclein in the brain seen in the form of lewy bodies is another pathological hallmark of the disease (S. Phani et al., 2011) Lewy bodies are eosinophilic structures locate d within the cytoplasm of neurons. Lewy bodies may be the result of altered neurofilament transport or metabolism due to neuronal damage. Classical L ewy bodies (s een in PD)
16 are circular and have a dense protein core surrounded by a peripheral halo (H. Braak et al., 2002) On the other hand, cortical L ewy bodies have a random arrangement of intermediate filaments and do not ha ve a dense protein core. They also lack a halo. Recently, accumulation of unfolde d or misfolded proteins in the endoplasmic r eticulum termed as ER s tress or unfolded protein response (UPR) stress has been shown to be involved in the etiology of the idi opathic form of PD (H. Q. Wang and R. Takahashi, 2007) along with the well established risk factors like age, oxidative stress, mitochondrial dysfunction, genetic predisposition, environmental chemicals like MPTP, insecticides like DDT, infectious agents and degeneration of dopamine by monoamine oxidase (MAO B ) and catechol O methyl transferase ( COMT ) (L. Soreq et al., 2011) Familial PD accounts for less than 5% of total cases. An autosomal recessive mutation in Parkin which under normal conditions functions as ubiquitin ligase to target misfolded or da maged proteins is the most common form of familial PD (N. Abbas et al., 1999) Autosomal dominant m utations are commonly seen in leucine rich repeat kinase synuclein genes (A. Brice, 2005) Unfortuna tely, to date there are no early stage diagnostic biomarkers for the disease and the sympto ms start emerging only after > 6 0% loss of DA neurons in the SN and 80% decrease of DA levels in t he striatum Apparently, afferents to the dendrites of DA neurons in SN have been reported to have compensatory responses which can endure deficits of DA neurons without symptomatic presentation (P. Anglade et al., 1995) An increased metabolic turnover and thus heightened activity of the remaining DA neurons is one type of compensatory mechanism. Secondly, increased post synaptic DA receptor density and/or sensitivity can be plausible (R. Deumens et
17 al., 2002) Post mortem studies show significant D1 and D2 receptor binding in the putamen of PD patients. As a result of these compensatory mechanisms some of the PD cases remain undiagnosed and the symptoms start appearing really late and are sometimes correlate d with old age. Hence an early stage diagnostic marker for the dis ease is imperative to facilitate proper therape utic intervention. Also, no current animal model of PD encompasses all the prevailing symptoms of the disease making the formulation of treatment strategies a difficult task. History PD was described by physician Galen as shaking palsy in AD 175 (C.G. Goe tz, 2011) In 1817, James Parkinson, a London based doctor publish ed a detailed report entitled an essay on shaking palsy' which recognised PD as a medical condition. It w as not until 1861 or 1862 that F rench N eurologist Jean Martin Charcot added greatly to the understanding of the disease and renamed it as PD after Ja mes Parkinson (E. D. Louis, 1997) In 1912, German P athologist Frederick Lewy reported neuronal cytoplasmic inclusions in a variety of brain regions. Tertiakoff obs erved that the pigmented neurons of SNp c were most commonly affected in PD. In 1950s, Arvid Carlsson demonstrated that DA was more than just a precursor for norepinephrine. He found that dopamine was a neurotransmitter in the brain and its l evels were particularly high in basal ganglia an area of the brain involved in motor coordination. He further demonstrated that re serpine administration decrease d DA levels and resulted in lo ss of movement control similar to PD (V. K. Yeragani et al., 2010) Paul Greengard contributed significantly in deciphering the cellular signaling pathways of DA (J. W. Kebabian et al., 1972) Both Carlsson and Greengard received Nobel Prize in medicine for their discoveries. In 1961 Birkmayer and Hornykiewicz administered levodopa, a
18 precursor for dopamine, intravenously to Parkinsonian patients (C.G. Goetz, 2011). Later, George C Cotzias discovered that gr adually administering C arbidopa (a peripheral dopa deca rboxylase inhibitor ) with levodopa could alleviate the symptoms of PD without toxic side effects. This eventually led to the development of Sinemet by Dupont in 1970s which became one of the most popular drugs for treatment of PD. Etiology and Risk Factors The e tiology of P D is multifactorial. This makes pinpointing one particular factor as the most probable causative agent an improbable task. Most relevant risk factors with their proposed hypothesis for involvement in PD are discussed below. Genetics and PD The m ost commo n genes associated w synuclein, LRRK2, parkin, UCHL 1, PINK1 and DJ1 (E. Maries et a l., 2003) These genes show a c lassical recessive or dominant Mendelian inheritance pattern. Alpha S ynuclein The r ole of synuclein in PD was discovered in 1997 when a missense mutation in the gene caused familial PD in a small group of people (M. H. Polymeropoulos et al., 1997) A53T and A30P are the t w o most common mutations in synuclein observed in pa tients with autosomal dominant early onset form of PD. E46K mutation in synuclein is also observed in som e patients. synuclein is a 140 amino acid protein expressed predominantly in presynaptic terminals of SN neurons an d lewy bodies Other areas of the brain harboring the protein include hippocampus, cerebellum, neocorte x and thalamus. Structurally, synuc lein has an amphipathic N terminal region, a central hydrophobic region involved in protein aggregation, and a highly acidic and proline rich region (V. N. Uversky et al., 2001a)
19 The hydrophobic region is a 35 amino acid non amyloid protein component of isease amyloid (NAC) w hich self aggregates to form amyloid and is an efficient seed for A fibril formation (E. A. Waxman et al., 2009) The p hysiological role o synuclein is unclear but it is implicated in synaptic plasticity and regulation of DA vesicle release. It is proposed to be a negative regulator synuclein K/O mice released more DA than normal after paired stimulus depression (PSD). Th synuclein gene that cause PD are caused by a gain of function and that it maybe a presynaptic regulator of DA release (A. Abeliovich et al., 2000 ) synuclein si gnificantly reduces the levels of tyrosine hydroxylase (TH) and DA both in v itro and in vivo (Zhou et al., 2000 ) (D. Kirik et al., 2002b) synuclein significantly interacts with tubulin and is involved in functioning of neuronal golgi apparatus (K. Beyer, 2007) and formation of SNARE complexes (J. Burre et al., 2010) synuclein belongs to a larger family of molecules including synuclein, gamma synuclein and synoretin. A study showed that transgenic mice expressing wild type human synuclein develop non fibrillar neuronal inclusions. Cross breeding these mice with a second transgenic line expressing synuclein produced mice with reduced nonfibrillar inclusions and reduced motor deficits suggesting that synuclein the non amyloidogenic homolog of synuclein might be an anti aggregation factor (M. Hashimoto et al., 2001) The synuclein molecule is a natively unstructured soluble protein which can aggregate in pathologi cal conditions to form oligomers and fibrillar polymers Increase in the population of pleated form of th e protein is seen in lewy bodies. This
20 conformation binds other proteins such as ubiquitin, neurofilaments, microtubule associated proteins, synphilin 1, parkin and anti apoptotic protein 14 3 3. One hypothesis for formation of lewy bodies is that synu clein monomers become oligomers (protofibrils) which later form fibrils constituting lewy bodies. While the monomers and oligomers are soluble, the fibrils and lewy bodies are insoluble structures. Conflicting views exist in literature regarding the role o f different physical conformations of the protein Some researchers believe it is the protofibrillar form of the protein that is detrimental to neuronal health and not the fibrils themselves. In an i n vitro study, A30P mutation of synuclein gene seen in PD cases acted by slowing the rate of fibril formation from profibrillar oligomers. However, A53T mutation was shown to expedite fibril formation (K. A. Conway et al., 2000) It is hypothesi zed that protofibrils form elliptical or circular amyloid pores that are similar to bacterial toxins. These pores can rupture cell membranes resulting in cell death (M. J. Volles and P. T. Lansbury, Jr., 2002) Interestingly, cytoplasmic DA was reported to be actively involved in stabilizin g synuclein protofibrils possibly through its metabolite DA orthoquinone (K. A. Conway et al., 2001) This explains in part the selective degeneration of DA neurons in PD and the presence of lewy bodies mainly in S synuclein induced apoptosis in human DA neuronal cell line while being neuroprotective for non DA cell line. Of all the neurotransmitters DA has a higher potential of being toxic. This toxicity is normally prevented within the neurons by its sequestration in vesicles for its transport from synuclein can cause a defect in this ER trafficking and thus a buildup of DA in the neurons (P. Jiang et al., 2010)
21 LRRK2 An a utosomal domina nt inheritance pattern is also seen with mutations in leucine rich repeat kinase 2 (LRRK2) gene linked to PARK8 locus that codes for protein dardarin (W. C. Nichols et al., 2005) Gly2019Ser and R1441C mutations are common in PD patients with LRRK2 gene defect and lead to an increase in kinase activity. These patients however have late onset of the disease which is different from other PD cases that are genetically inherited and are mostly early onset. LRRK2 gene enc odes a large protein of 286 KDa and contains multiple independent domains. Since mutations in this gene cause PD that is almost indistiguishable from sporadic PD this protein has emerged as a very important player in PD pathogenesis (J. P. Taylor et al., 2006) LRRK2 is a member of the ROCO protein family. It has two distinct enzymatic domains kinase domain involved in phospho rylation and the Roc COR GTPase domain involved in GTP GDP hydrolysis. LRRK2 might serve as a scaffold for assembling multiprotein signaling complexes. It is also implicated in the regulation of neurogenesis, vesicular trafficking and cytoskeletal motility (H. L. Melrose et al., 2007) .This protein is present throughout the nigrostriatal DA pathway with highest levels in striatum and low levels in SN. LRRK2 is also a component of the lewy bodies in PD brains (E. Greggio et al., 2008) (E. Greggio et al., 2011) Hsp90 is seen to interact with LRRK2 through its kinase domain to assist in proper folding and activation of the protein (C. J. Gloeckner et al., 2006) Inhibition of Hsp90 blocked this interaction and caused proteasomal degradation of LRRK2 (A. Hurtado Lorenzo and V. S. Anand, 2008) Hence it is suggested that pharmacological inhibitors of Hsp90 could help in reducing levels of mutated LRRK2 in PD patients. PD linked mutations also increase the interaction between LRRK2 and FA DD (Fas associated
22 protein with death domain). Inhibition of FADD reduced LRRK2 mediated neurodegeneration (C. C. Ho et al., 2009) However, more conclusive studies are required to delineate the mechanism of LRRK2 in PD pathogenesis as it is a relatively new direction towards familial PD. Parkin Almost 50% of hereditary PD cases are due to Parkin mutations. Parkin is the causative gene for the autosomal recessive form of early onset juvenile PD (AR JP). AR JP is cha racterised by selective DA neuronal death without formation of lewy bodies (T. Kitada et al., 1998) Park in is mainly found in the cytoplasm but also localizes in synaptic veiscles, ER, golgi complexes and mitochond rial outer membrane. It belongs to the really interesting new gene (RING) finger class of E3 ubiquitin ligase (H. Shimura et al., 2000) Ubiquitin is a small regulatory protein tha t attaches to other proteins and labels them for destruction by proteasome. Polyubiquitinated proteins are recognized by the 20S proteasome subunit and targeted for degradation. Recessive mutations in parkin could eliminate DA neurons by accumulation of ne urotoxic protein substrates when there is insufficient parkin for its proteasomal degradation (P. J. Kahle et al., 2000) To date, several candidates have been identified as substrates for parkin and shown to be present in lewy bo synuclein, synphilin, p38 and synaptotagmin. Parkin associated endothelin receptor like receptor (Pael R) is a novel susbtrate of parkin that accumulates in the core of lewy bodies in PD patients. Overexpression of Pael R initiated ER stress respo nse which prompted upregulation of parkin to mark the protein for ubiquitination and proteasomal degradation (Y. Imai et al., 2001) It is unclear as to how the misfolding of Pael R contributes to selective loss of DA neurons in parkin
23 mediated autosomal recessive juvenile parkinsonism (AR JP) (P. J. Kahle and C. Haass, 2004) Parkin has also been implicated in the elimination of polyglutamine polypeptides, ataxin 3 and sythetic GFP. In addition to the synuclein, a 22 KDa glycosylated for synuclein (alphaSp22) is also regulated by parkin (H. Shimura et al., 2001) Mutated parkin failed to b ind to alphaSp22 and alphaSp22 accumulated in a non ubiquitinated form in parkin deficient PD brains. Ove rall, it would be safe to say that functional parkin contributes to the formation of lewy bodies in human brains. In the conventional Lewy body associated PD wild type parkin polyubiquitinates substrates including alphaSp22. However, they are not properly processed by the proteasomal machinery due to other genetic or environmental causes and thus accumulate as lewy body inclusions. Knockdown of parkin combined with unfolded pr otein stress was neurotox ic to mammalian neuronal cells (L. Petrucelli et al., 2002) Parkin deficient mice showed increased levels of extracellular DA. Also DA metabolism was enhanced as evidenced by high levels of 3, 4 dihydroxyph enylacetic acid (J. Itie r et al., 2003) This conferred oxidative stress and decreased mitochondrial respiratory cap acity in parkin deficient mice (J. Palacino et al., 2004) Thus, parkin may also pl ay a role in the regulation of mitochondrial function in addition to lewy body formation. UCHL 1 Ubiqui tin carboxy terminal hydrolase 1 (UCHL 1) is one of the most abundant prot eins in the brain comprising about 2% of total brain proteins. It cleaves polymeric ubiquitin to monomers and hydrolyses bonds between ubiquitin molecules. It is a member of the deubiquitinating family of enzymes and is a component of lewy bodies in P D bra ins (E. Leroy et al., 1998) Involvement of this protein supports the ubiquitin
24 proteasome hypothesis in PD pathogenesis. Inverse association of S18Y mutation in UCHL 1 with PD was seen in a large cohort of Caucasian population (A. Carmine Belin et al., 2007) This sparked a lot of interest in the protein. However, studies carried out later have not been able to confirm this finding and it remains doubtful to date. PINK1 Parkinson disease 6/phosphate and tensin homolog (PTEN) induced putative kinase 1 (PINK1) is a mitochondrial serine/threonine protein kinase encoded by the PINK1 gene. This kinase protein has a high degree of homology to the Ca + /calmodulin kinase family. Mutations in PINK1 gene (G309D mainly) cause autosomal recessive PD, PARK6 (V. Bonifati, 2011) PARK6 is an early onset PD which has clinical features similar to the classical PD. Two specialized regions of PINK1 are essential for the protein to function properly. Since the protein is pro duced outside the mitochondria, mitochondrial targeting motif delivers it to the mitochondria and the kinase domain is responsible for protecting it from cellular stresses and proteasome mediated apoptosis (L. Pallanck and J. T. Greenamyre, 2006) Genetic and environmental models show that PINK1 acts upstream of Parkin to maintain mitochondrial inte grity and preserve DA neurons. Loss of function mutations in the gene resulted in profound mitochondrial morphology (J. C. Greene et al., 2003) PINK1 and parkin mutants suffered from impaired mitochondrial fission, increased free radical stress and ultimately DA neurodegeneration (A. C. Poole et al., 2008) Wild type PINK1 could provide DA neuroprotection against MPTP both in vitro and in vivo (M. E. Haque et al., 2008) Interestingly, a PINK1 mutant lacking the putative mitochondrial targeting motif also protected neurons from MPP + toxicity indicating that
25 targeting of the protein to mitochondria is not imperative to its neuroprotectiv e function (M. E. Haque et al., 2008) DJ 1 DJ 1 is a protein encoded by the PARK7 gene. Defects in this gene cause autosomal recessive early onset PD (PARK7). Only about 1% of familial PD cases report mutations i n this gene. Structurally, the protein is a homodimer with a highly conserved cysteine residue which is extremely sensitive to radiation damage and other forms of oxidative stress. Loss of function mutation L166P found i n PARK7 could be due to the destabil ization of this dimer interface (M. A. Wilson et al., 2003) DJ 1 is a cytoplasmic protein but can translocate to the mitochondria. It is highly expressed in both neuronal and glial cells. It functions as a redox se nsitive chaperone, protecting neurons from oxidative stress and cell death. It prevented the synuclein in a cellular model of oxidative stress (S. Batelli et al., 2008) It plays an important role in antioxidant stress reaction though induction of self oxidation (T. Taira et al., 2004) DJ 1 knockout mice w ere highly sensitive to MPTP mediated DA neurodegeneration and oxidative stress (R. H. Kim et al., 2005) Expression of DJ 1 increased after oxidative stress induced by paraquat (H. J. Kwon et al ., 2011) .This antioxidant capacity of DJ 1 could be important in protecting DA neurons which are highly susceptible to oxidative stress (L. M. Bekris et al., 2010) Like PINK1, DJ 1 also interacts with parkin, wher eby parkin acts as ubiquitin ligase to remove mutated DJ 1. Environmental Factors and PD Interest in the hypothesis of environment and PD was fuelled immensely by Tanner and colleagues who studied a large population of monozygotic and dizygotic
26 twins to sh ow that heredity is not a major contributing factor in the etiology of PD and non genetic factors need to be revisited (C. M. Tanner et al., 1999) Beneficial Environmental A gents A couple of epidemiological studies have shown neuroprotective effects of caffeine consumption in re levance to PD (R. D. Altman et al., 2011) (N. Amin et al., 2011) (S. Yadav et al., 2011) Another interesting finding is the inverse correlation between cigarette smoking and PD. Smoking has been estimated to reduce risk of PD by 50% (C. M. Tanner et al., 2002) (B. Ritz et al., 2007) Nicotine, one of the major components of tobacco is believed to hav e a modulatory effect on nigrostriatal DA pathway as well as microglial activation (F. M. Zhou et al., 2001) Treatment of rat primary microglial cells with varying concentrations of nicotine (0.1 100m) 30 minutes before stimulation with LPS (100ng/ml) decreased microglial activation significantly (H. J. Park et al., 2007) Extension of this study to in vivo LPS PD model confirmed the aforementioned results with both acute an d chronic nicotine pretreatment (H. J. Park et al., 2007) However, the neuroprotective effects of nicotine could only be seen at lower concentrations which was in accordance with earlier studies reporting a desens itization of nAch receptors at higher doses (F. M. Zhou et al., 2001) Moreover, considering the harmful effects associated with cigarette smoking or rather with the the use of any type of tobacco, a drug that targ ets a selective subtype of nicotinic acetylcholine receptor wou ld be more suitable than using nicotine in general which would interact with multiple nAch receptor types and cause numerous side effects.
27 Toxic Environmental A gents Metals Increased levels of nigral iron and copper content is seen in PD brains (D. T. Dexter et al., 1989) Iron is involved in the generation of hydrogen peroxide during oxidation of DA and generation of hydroxyl radicals via fenton reaction. In vitro studies s how involvement of metals in lewy body formation by p synuclein (V. N. Uversky et al., 2001b) In occupational settings long term exposure to a combination of metals increases the risk of PD In vitro and in vivo PD models have shown that overexposure to low levels of certain metals such as manganese can induce gli al activation and subsequent DA neurodegeneration (P. Zhang et al., 20007) (P. Zhang et al., 2009). Pesticides: Rotenone, Paraquat and Maneb. Evidence for the role of pesticides as DA neurotoxins is provided by clinical and experimental research. Pestici de exposure was related to a high risk of PD as reported by a number of epidemiological studies (K. Rugbjerg et al., 2011) (F. D. Dick et al., 2007) The odds ratio varied from 2 7. The cases showed a dose dependent effect, with agricultural workers and miners having longer time exposu re being more prone to the disease. Specifically the insecticide rotenone, herbicide paraquat and the fungicide maneb have been associated with PD. Farming and movement evaluation (FAME), a case control study showed that exposure to pesticides rotenone or paraquat or both predisposed farmers to develop ing PD 2.5 times more than non users (C. M. Tanner et al., 2011) An important aspect of human pesticide exposure is that it is to a number of agents, rather than a sin gle one, and it occurs over a long time span. Hence, exposure to multiple pesticides is suggested to
28 (H. Sharma et al., 2009) Rote none Rotenone belongs to the family of isoflavonoids and is naturally found in the roots and stems of several plants. It is a broad spectrum pesticide. Being highly lipophilic, it can easily cross the blood brain barrier and get accumulated in mitochondria l complex I. It does not depend on the dopamine transporter ( DAT ) for its transport into neurons unlike other neurotoxins (R. Betarbet et al., 2002) It is still unclear as to how it is selectively toxic to DA neurons. It causes mitochondrial dysfunction by inhibiting the transfer of electrons from iron sulfur centers to ubiquinone (A. Panov et al., 2005) This generates ROS which damages DNA of neuronal cells. Recently, it was also shown to inhibit proteasomal activity (A. P. Chou et al., 2010) In vitr o, rotenone causes synuclein, oxidative damage and caspase dependent death (T. B. Sherer et al., 2003) Rotenone and lipopolysaccharide (LPS), a bacterial endotoxin, exhibited synergistic toxic eff ect s on DA neurons in midbrain cultures (H. M. Gao et al., 2003b) Rotenone model of PD depicts clinical features reminiscent of the disease. Rotenone infusion via osmotic mini pumps produced hypokinesia, rigidity, postural imbalance, and even resting tremors. These symptoms correlated with the irreversible degeneration of DA neurons. Further, motor deficits were ameliorated on administration of apomorphine, a mixed D1/D2 agonist confi rming involvement of DA neuron s Loss of TH immunoreactivity was seen at the striatal level in a dose dependent manner. Lewy synuclein were also discerned in DA neurons (R. Betarbet et al., 2000) More recently, intra oral (M. Inden et al., 2007) and intra nasal (A. I. Rojo et al.,
29 2007) route of administration of the insecticide are being explored to mimic human exposure to rotenone. However, so me reports suggest that the rotenone model does not reproduce PD pathology but rather pathological changes akin to atypical parkinsonism (G. U. Hoglin ger et al., 2006) Paraquat Paraquat is one of the most widely used herbicide in the world and bears close structural resemblance to MPTP. In the US paraquat is classified under restricted use whereas in Europe its use was banned in 2007. It enters the br ain by neutral amino acid transporters in a sodium dependent manner and induces mitochondrial dysfunction indirectly in the cytosol by altering redox cycling (P. R. Castello et al., 2007) It exhibits weak inhibitory activity for mitochondrial complex I. In Taiwan, where paraquat was sprayed in rice fields, a strong association to PD was reported (H. H. Liou et al., 1997) Time of exposure was directly linear to the risk of PD development. In vitro studies indicate that paraquat exerts its toxicity through DAT and decreasing levels of antioxidants (W. Yang et al., 2010) (P. M. Rappold et al., 2011) Combined exposure to paraquat and iron resulted in accelerated degeneration of the nigral DA neurons which was rescued by superoxide dismutase (J. Peng et al., 2007) Similar to metals, paraquat and rotenone also promoted the fibrillization of synuclein (A. B. Manning Bog et al., 2002) (T. B. Sherer et al., 2003) synuclein pathology can be triggered or exacerbated by exposure to pesticides. Mouse models of paraquat show selective nigrostriatal degeneration and formation of intraneu ronal aggregates (A. B. Manning Bog et al., 2002) However, no change s in DA levels or behavioral abnormalities were seen in this model. Recent reports suggest that paraquat induced apoptosis may involve Bak protein, a Bcl2 family member (Q. Fei et al., 2008)
30 Maneb Maneb is an irritant to the respiratory tract and can cross the blood brain barrier to inhibit mitochondrial complex III. Exposure to Maneb in animals causes loss of DA fibers, decrease in TH protein expression and increase in DOPAC (3, 4 dihydroxyphenylacetic acid, a metabolite of dopamine) levels coupled with behavioral abnormal ities (M. Thiruchelvam et al., 2000a) Paraquat and maneb together, synergistically result in greater DA neurodegeneration and locomotor abnormalities in rats (M. Thiruchelvam et al., 2000b) Administration of paraquat/maneb postnatally to mice caused a 38% loss of DA neurons in adulthood Interestingly, a re exposure in adulthood accelerated the loss to 70% and showed motor defects in these animals ( F. Garcia Garcia et al., 2005) Prenatal maneb exposure followed by adult paraquat administration showed similar effects of nigral cell loss, decrease in striatal DA and 95% decrease in locomotor activities (B. K. B arlow et al., 2003) Paraquat/Maneb model has successfully demonstrated the synergistic effect of various environmental agents in producing PD features in animals. It has also provided support for the multiple hit hypothesis of PD (F. Cicchetti et al., 2009) Organochlorine pesticides Organochlorine Pesticides (OCP); DDE (a long lasting metabolite of DDT) and dieldrin have been detected in PD postmortem brains (A. Seidler et al., 1996) (W. Koller et a l., 1990) Levels of dieldrin were significantly higher in the SN compared to age matched controls (L. Fleming et al., 1994) (F. M. Corrigan et al., 1996) OCPs are extremely persistent in the environment due to their high lipophilicity and tend to bioaccumulate as we go higher up in the food chain. Agricultural application of OCPs
31 was banned in periods between 1970 1990. However, human exposure to OCPs continues to be a health concern (A. G. Kanthasamy et al., 2005) Dieldrin Dieldrin is a chlorinated cyclodiene compound with chemical formula C 12 H 10 C l6 O. It is synthesized through Diers Alder reaction. In addition to being highly lipophilic, die ldrin has a low vapor pressure which does not allow ready migration making it a highly persistent environmental chemical. It is largely stored in adipose tissue and can cross the blood brain barrier easily to gain access to neurons. It is metabolized throu gh hydrolysis or by enzymatic oxidation. Typically, half life of dieldrin in the environment is upto 25 years (S. N. Meijer et al., 2001) Infact it is one of the 12 most persistent and toxic chemicals classified by US EPA. It is also one of the top 20 hazardous chemicals to humans accordi ng to the agency for toxic substances and drug r egistry (ATSDR). US EPA banned the use of dieldrin in 1987. Despite the ban, some developing countries continue to use it as an insecticide (M. Suwalsky et al., 2002) Dieldrin continues to be detected in the environment including rivers, agricultural products, vegetables, meat and seafood (R. A. Doong et al., 2002) (K. Kannan et al., 1997) Daily acceptable intake limit of dieldrin is established to be around 100 ng/kg. However, population st udies report much greater levels (1 1.3g), indicating a continued threat from this toxic insecticide (J. W. Brock et al., 1998) Several in vitro and in vivo studies have been conducted to decipher the mechanism of dieldrin toxicity in relation to P D. Animals exposed to low doses of dieldrin chronically, showed a significant reduction in the levels of both DA and norepinephrine although DA levels were more severely affected (G. H. Heinz et al., 1980) (R. P. Sharma et al., 1976) (S. R. Wagner and F. E. Greene, 197 8) Another
32 study showed increased DAT binding activity in the striatum of rats exposed to dieldrin indicating its role in DA reuptake (S. Purkerson Parker et al., 2001) Dieldrin increased synuclein fibril formation in vitro suggesting its role in lewy body formation (V. N. Uversky et al., 2002) (V. N. Uversky, 2003) Short term exposure to dieldrin increased the levels of other ubiquitinated pr oteins as well, in addition to synuclein, and upon chronic exposure it resulted in the inhibition of ubiquitin proteasome system in the rat synuclein (F. Sun et al., 2005) Mitochondria are speculated to be one of the major targets of dieldrin. In vitro, it produces both dose and time dependent alterations in mitochondrial activity (M. Kitazawa et al., 2001) Both mitochondrial membrane potential and cytochrome c release were affected. It has shown to induce oxidative stress and ultimately apoptosis of DA neurons through activation of caspases (M. Kitazawa et al., 2003) (K. Kannan et al., 2000) Pretreatment with SOD significantly attenuated dieldrin mediated ROS production (M. Kitazawa et al., 2001) Dieldrin treatment ind uced PARP (DNA cleaving enzyme), a substrate for caspase 3 in PC12 cell s (M. Kitazawa et al., 2004) Sanchez Ramos et al showed selective toxicity of dieldrin to DA neurons over GABA neurons in the midbrain cultures (J. Sanchez Ramos et al., 1998 ) Another study proposed that dieldrin can inhibit GABA(A) receptors leading to massive calcium influx and ultimately glutamate excitotoxicity (X. Zhao et al., 2003) Experimental DA n eurotoxins MPTP F irst incidenc es of PD reported in the 20 th century were believed to be due to the influenza pandemic of encephalitis lethargica in 1917 1928. However, since the disease incidences continued to rise years following the epidemic, this theory did not hold
33 ground for long. A parkinsonism syndrome strongly resembling PD was seen in 1970s after intravenous injection of MPTP, a toxic meperidine derivative (G. C. Davis et al., 1979) Later in 1983, Langston and colleagues reported development of PD in the narcotic addicts who injected themselves with an illicit recreation drug having MPTP as an impurity (J. W. Langston et al., 1983) This lead researchers to investigate further into the role of MPTP and other environmental toxicants like insecticides, herbicides and metals that might be etiologically related to PD (D. A. Di Monte, 2003) (A. Kanthasamy et al. 2009 ) (F. Tsang and T. W. Soong, 2003) MPTP is highly lipophilic and can easily cross the blood b rain barrier (BBB) but it is not toxic by itself. It is converted to MPP + by MAO B present mainly in the astrocytes and serotonergic neurons. Inside the cell it acts by inhibiting complex I of the electron transport chain. Dysfunctional mitochondrial comp lex I is seen in SN of autopsied PD brains (C. Huerta et al., 2005) (A. H. Schapira et al., 1990) MPTP can produce specific cell loss of SN in many vertebrate species from man to mouse. Today, MPTP represents the gold standard for parkinsonian toxin s applied in animal models (M. F. Beal, 2001) (S. Przedborski et al., 2001) It has an advantage over other animal models of PD due to the following reasons: It is taken up into the neurons b y the dopamine transporter (DAT) which could be one of the reasons for its preferential toxicity to DA neurons. Interestingly, MPP + is also a substrate for VMAT (vesicular monoamine transporter) which sequesters it into synaptic vesicles containing DA (M. Del Zompo et al., 1993) Furthermore, MPTP can be administered by various routes like gavage, stereotaxy, or systemic administration (subcutaneous, intramuscular, intravenous, intraperitoneal). Different schedu les of MPTP administration can be used to capture
34 distinct stages of the disease. Characterization of MPTP i nduced parkinsonism confirmed its similarity to PD in humans but pointed a major difference the lack of lewy body pathology (M. Shimoji et al., 2005) This corroborates the multifactorial etiology of PD where one to xin cannot completely recapitulate the disease. Age related effects, toxins and genes are all acting in cohort to produce PD. Intraneuronal lewy bodies were seen in old monkeys treated with MPTP (L. S. Forno et al., 1986) Combination of environmental risk factors exerts a more severe loss of DA neurons as evidenced in a number of experimental studies. Subtoxic doses of MPTP caused striatal DA depletion and nigral cell degeneration when administered alo ng with fungicide diethyldithiocarbamate (T. L. Walters et al., 1999) This is in line with the multiple hit hypothesis of PD which suggests that while the brain may be able to compensate for the effect of a single toxin, multiple toxins acting at multiple targets would compromise the homeostatic ability of the brain thus leading to a more robust effect even at lower doses. 6 OHDA 6 Hydroxydopamine (6 OHDA) is a catecholamine neurotoxin. It is a hydroxylated analog of the natural neurotransmitter DA (D. Blum et al., 2001) It is easily oxidisable and can take part i n the free radical forming reactions (A. Schober, 2004) It is transported into the cell bodies and fibers of both DA and non DA neurons (A. Schober, 2004) Its toxicity is based on the inhi bition of mitochondrial complexes I and IV (R. Betarbet et al., 2002) and formation of the superoxide radicals (S. Singh et al., 2010) Inside the neuron, it accumulates in the cytosol and induces cell death without apoptotic characteristics (B. S. Jeon et al., 1995) 6 OHDA treatment reduces striatal glutathione
35 and SOD enzyme activity (A. S. Perumal et al., 198 9) Endogenous 6 OHDA has been found to accumulate in the brains of patients suffering from PD (R. Andrew et al., 1993) Reasonable selectivity of 6 OHDA for DA is achieved by pretreating the subjects with desimipramine, a noradrenaline transport blocker Further, the toxin can be directly injected stereotactically into distinct parts of the ascending nigros triatal pathway for high selectivity. Systemically, it cannot cross the BBB. Injection of 6 OHDA into the rat medial forebrain bundle or SN significantly decreased striatal DA levels along with loss of SN DA neurons. DA neurons began to degenerate within 1 2 h and striatal DA levels were depleted 2 3 days later. Locomotor abnormalities were also evident in these rats (R. L. Faull and R. Laverty, 1969) Howe ver, extensive loss of cells in VTA was also seen. 6 OHDA mediated ventrolateral lesion of rat caudate putamen (since putamen is mainly affected in PD and vent rolateral section of rat caudate putamen is more close to human putamen) closely depicted PD pathology. Striatal DA levels reduced by 75% and both motor and cognitive deficits were seen. It induced a more progressive and retrograde neuronal death (S. Przedborski et al., 1995) However, 6 OHDA model does not form lewy body inclusions and produces more acute effects which differs significantly from the slowly progressive pathology of human PD (R. Betarbet et al., 2002) Interaction Between Genes and Envir onmental Factors Pathogenesis of PD is multifactorial and involves gene environment interaction. synuclein has a major role in sporadic PD as it is an essential component of lewy bodies. The process of synuclein fibrilization and formation of lewy bodies can be accelerated by nucleation iron and copper and ROS generation. Also, toxins like MPTP generate free radicals in the neu ronal surroundings which further promote
36 oligomerization of synuclein and ultimately death of DA neurons (K. Steece Collier et al., 2002) This highlights the importance of interactions between endogenous (constitutive proteins) and exogenous factors (environmental agents) in the process of neurodegeneration. Aging There is a long standing debate ove r the connection between pathological processes of aging and PD. If we live long enough are we more liable to develop ing PD or is PD independent of aging? Indeed, prevalence of PD increases with aging with a mean age of onset being mid 70s. Patients with a late onset of disease have a faster rate of motor progression due to a significantly shorter duration for settling in PD compared to those who have an early onset of the disease. Also, gait and postural instability, two of the cardinal features of PD are non responsive to Levodopa therapy with increasing age. This shows the involvement of non DA neurons and thus aging process as a contributing factor (G. Levy et al., 2000) Neuropsychological studies have shown that there is severe dementia associated with the onset of PD in older patients compared to early onset individuals. PD related dementia was approximately 1 2.5% in the patients between 50 59 years of age whereas it was close to 70% in individuals close to 80 years or more (R. Mayeux et al., 1992) Aging is thought to be a stochastic process leading to cellular damage and weakening of cellular repair mechanisms. It leads to mitochondrial dysfunction, free radical production and oxidative stress. De cline in the synuclein is also seen in aged brains although it is much less compared to PD brains. There is a reduction in the striatal tyrosine hydroxylase and DA neurons. However the remaining pigmented neurons are believe d to undergo hypertrophy as a compensatory mechanism in normal
37 aging brain (G. Levy et al., 2005) (W. R. Gibb and A. J. Lees, 1 987) (P. L. McGeer et al., 1988) Despite age being an important factor in PD progression, involvement of genetic and environmental factors cannot be ignored as they determine whether an individual develops a neurodegenerative disorder durin g aging. These factors can either exacerbate or counteract the molecular and cellular mechanisms of aging. Lifetsyle changes like low calorie diet and intermittent fasting can reduce the oxidative stress generated during glucose metabolism. This reduced ca lorie intake which is experienced during periods of increased metabolism as in exercise and studying render a mild partial stress on neurons and prepare them for fighting successive larger stresses which might result from environmental toxins or other caus es. This process is known as hormesis. It also helps in the release of neurotrophins like NGF and BDNF, sirtuins (histone deacetylases which play an important role in stress regulation) and also produces mitochondrial uncoupling proteins that promote a pro ton leak across the mitochondrial membrane thereby decreasing oxidative phosphorylation and ROS production (L. Fontana et al., 2004) (C. W. Cotman and N. C. Berchtold, 2002) Aging is one of th e largest risk factor for PD but a couple of things need to be looked at in order to view its relevance to PD. Age relates to the clinical progression of the disease rather than the clinical onset of disease initiation and involves its actions on non DA ne urons rather than DA neurons (G. Levy, 2007) (J. V. Hindle, 2010) Also topographic location of lewy bodies in PD is distinct from that seen in normal aged brain.
38 Braak classified PD into stages according to the progression of lewy bodies in as cending order. In Braak stage 1, changes are confined to the dorsal motor nucleus and olfactory bulb, leading to loss of smell which is a preclinical feature of PD and not associated with aging. PD patients have a greater loss of smell compared to the age matched controls, long before the appearance of motor symptoms (C. H. Hawkes, 2008) Braak stage 2 involves the formation of lewy bodies in pons and medulla, stage s 3 and 4 produce clinical motor symptoms and stage s 5 and 6 involve neo cortex, cognitive problems and dementia. Post mortem analysis of brains of patients with no dementia or PD showed that 50% of patients with diffuse cortical lewy bodies had no history of dementia in life and the pattern of lewy body distribution only parti ally (L. Parkkinen et al., 2008) In PD, cell loss occurs mainly in the ventral tier of SNpc whereas in normal aging the dorsal tier is mainly affected at a ratio of 3:1 (G. Rudow et al., 2008) The cause of selective vulnerability of cells affected in PD (SNpc, medium spiny neurons in striatum and lower motor neurons in spinal cord) is unclear but a number of possibilities have been proposed. These neurons are relatively large and span over long distances in the CNS. Large projection neurons in addition to having hi gh energy requirements, rely on a xonal transport for sustained function and trophic support, and endorse a large cell surface area that increases exposure of cells to toxic environmental conditions. Also, the ir cytoskeleton is more susceptible to dysfunction by aggregation and displacemen t of axonal neurofilaments (M. P. Mattson and T. Magnus, 2006) DA itself might contribute to the demise of neurons producing it by inducing oxidative stres s in the presynaptic terminals (S. Jana et al., 2007) Density of microglia is the highest in
39 midbrain and also midbrain DA neurons have low antioxidant capacity due to reduced ynuclein can interact with and increase the activity of DAT thereby increasing levels of DA in cytoplasm (F. J. Lee et al., 2001) and it also downregulates VMAT leading to increased DA levels and oxidative stress (J. T. Guo et al., 2008) Levels of neuromelanin are another contributing factor. Monkeys and humans have high levels of neuromelanin in the SN. Neuromelanin is a pigment produced by the non enzymatic oxidation of cytosolic (non vesicular ) DA, a process that generates ROS. Neuromelanin has the capacity to store and release large quantities of iron and serves as a catalyst for free hydroxyl ion generation through fenton reaction (T. Shima et al., 1997) Neuromelanin in the SN cell bodies also serves as a depot which gradually releases MPP + (W. Dauer and S. Przedborski, 2003) Furthermore, DA has been shown to sensitize the neurons to excitotoxic cell death (G. S. Schierle and P. Brundin, 1999) This is due to the high num bers of NMDA and AMPA receptors and low levels of protective calcium binding proteins, calbindin and parvalbumin in SN. The v entral tegmental area (VTA) has high levels of VMAT2 which promotes vesicular sequestration of DA and low levels of DAT that pump DA into the cytoplasm. DA neurons in the VTA have a lower sens itivity to Katp channels due to an intrinsic capacity to buffer ROS. Brain uncoupling protein 2 (UCP 2) are more abundant in VTA than SN DA neurons. The se VTA neurons also possess an inherently better antioxidant system. Glia l cells having glutathione peroxidase and catalase activities are higher in VTA. Overall, the mechanism of PD and aging is complex and interrelated to a certain extent. As already pointed out aging accelerates the progressio n of the disease but
40 might not be a contributive factor to its initiation. Normal aging is associated with very mild parkinsonian signs but PD has a distinct clinical picture (J. V. Hindle, 2010) There is a failure of the normal compensatory mechanisms in PD which is increased by genetic and environmental factors and most importantly age. Pathogenesis Molecular mechanisms underlying PD are not well understood. A variety of mechanisms have been pro posed. Mitochondrial dysfunction is one of the most common pathogenetic mechanisms Before talking about mitochondrial defects associated with PD lets take a look into normal physiological role of mitochondria in generation of ROS and the involvement of m itochondria nucleus signaling in regulating free radical levels. Mitochondria are the major source of reactive oxygen generation. Reactive oxygen (O2 ), a non diffusible oxygen radical, is generated by the mitochondria during oxidative phosphorylation und er normal physiological conditions. O2 can be converted to hydrogen peroxide (H 2 O 2 ), hypochlorous acid (HOCl; by myeloperoxidase) or peroxynitrite (ONOO ; by interaction with O2 ). O2 and other oxygen derived intermediates capable of modifying organic mo lecules are referred to as reactive oxygen species (ROS). ROS act by direct DNA damage. Oxidative attack of DNA produces 8 hydroxyguanine, urea and thymine glycol. Increased levels of 8 hydroxyguanine and 8 hydroxy 2 deoxyguanosine have been observed in PD brains. Also lipid peroxidation and alteration of cellular signaling can occur. Free radicals attack on double bonds of unsaturated fatty acids such as linoleic and arachidonic acid that leads to the formation of toxic breakdown products like 8 hydroxy 2, 3 nonenal (HNE),
41 malondialdehyde and acrolein. Elevated levels of HNE and malondialdehyde are seen in the brains of PD patients (F. C. Fang, 2004) H 2 O 2 is a small uncharged non radical ROS that can freely diffuse through the cellular membrane and c an help in cellular signaling. H 2 O 2 can damage DNA directly through its reaction with metal binding histones leading to oxidation of nucleotides. C atalases present in the cytosol; glutathione peroxidase localized in mitochondrial matrix, cytosol and outer membrane ; and peroxiredoxins can detoxify hydrogen peroxide. Hydroxyl ions generated by the fenton reaction cause maximum DNA damage owing to their high reactivity with the mitochondrial and nuclear DNA. These reactive oxygen species (ROS) are detoxified by various antioxidant proteins, which are encoded by nuclear genes. Thus, mitochondria to nucleus signaling must occur to detect and regulate superoxide generation (P. Storz, 2006) Superoxide is generated a t complex I and complex III of the electron transport chain (ETC) located in the inner membrane of mitochondria. From the site of generation it is released into the mitochondrial matrix and the intermembrane space. O2 present in the intermembrane space passes through the mitochondrial per meability transition pore located in the outer mitochondrial membrane. Voltage dependent ani on cha nnel (VDAC), a component of the mitochondrial permeability transition pore, allows O2 to leak into the cytoplasm. Manganese superoxide dismutase (MnSOD) present in the mitochondrial matrix and Cu/Zn SOD present in the cytoplasm detoxify O2 at both the locations. MnSOD or SOD2 activation is tightly regulated by the transcription factors FOXO3a and NF kB. Cysteine containing kinases or phosphatases located at the mitochondrial membrane or
42 matrix serve as sensors of increased ROS production and relay i nformation for transcription of nuclear antioxidant proteins. The protein tyrosine phosphatase PTEN located on the outer mitochondrial membrane is reversibly inactivated by H 2 O 2 (S. R. Lee et al., 2002) Its oxidation and inact ivation leads to the activation of mitochondrially located tyrosine (Src, which regulates NF kB activation and indirectly SOD2 transcription ) or serine threonine kinases ( PKC, PKD, Raf, akt). The m itochondrial matrix protein p32 plays a role in the interaction be tween mitochondria and nucleus by binding to PKC and PKD. C Jun N terminal kinase (JNK) is another ROS induced mediator of signaling. Mitochondrial Dysfunction and Oxidative S tress Several lines of evidence implicate mitochondrial dysfunction and oxidativ e stress in PD. Post mortem studies show involvement of oxidative da mage in PD Oxidative stress may be generated due to mitochondrial dysfunction or DA metabolism and can generate ROS which can compromise neuronal integrity. Complex I defect of mitochond rial ETC is most common in PD patients (P. M. Keeney et al., 2006) Impaired complex I makes neurons more susceptible to glutamate excitotoxicity (T. V. Votyakova and I. J. Reynolds, 2001) Furthermore, a single nucleotide polymorphism in NADH dehydrogenase 3 of complex I significantly reduced the risk of PD development in caucasians thus provid ing genetic evidence that mitochondria can modify PD pathogenesis (J. M. van der Walt et al., 2003) Both environmental agents and genetic manipulations contribute to respirat ory chain defects. PD cybrids (engineered cell lines containing PD mitochondria) are more susceptible to MPP + induced cell death (A. R. Esteves et al., 2008) Rotenone, another complex I inhibitor can preferentially damage DA neurons and form lewy body
43 inclusions (J. T. Greenamyre et al., 2003) Tr ansgenic mice in which activity of Cu/Zn superoxide dismutase (SOD) was increased were resistant to MPTP (S. Przedborski et al., 1992) Further, knockout mice without glutathione peroxidase were shown to be more sus ceptible to MPTP (P. Klivenyi et al., 2000) One of the mechanisms linking mitochondrial dysfunction to oxidative stress is the MPTP induced release of cytochrome c from dysfunctional mitochondria into cytoplasm. B ax is a proapoptotic protein present in the cytosol that translocates to the outer mitochondrial membrane and induces the opening of mitochondrial voltage dependent anion channel (VDAC) and release of cytochrome c into the cytosol. Transgenic knockouts of Bax were less susceptible to MPTP induced DA neurodegeneration (M. Vila et al., 2001) Another proposed mechanism involves neuronal hyperpolari zation induced by ROS. ATP sensitive potassium channels (Katp) control D A release in the striatum and ROS act as physiological regulators of Katp channels in DA neurons (L. Bao et al., 2005) A key study showed that genetic invalidation of a pore forming subunit of Katp protected DA neu rons from chronic low dose administration of MPTP (B. Liss et al., 2005) ATP depletion signals Katp channels to open. This causes potassium efflux and a state of membrane hyperpolarization which lowers the electric al activity of the cell and energy consumption. This makes DA neurons functionally silent and reduces the release of striatal DA. Mitochondrial ROS operate as activators of Katp channels in the DA neurons when ATP levels are not affected (L. Bao et al., 2005) Reduced excitability of the neurons promotes translocation of Bax from the cytosol to mitochondria thereby activating the apoptotic pathway.
44 Mutations in DJ1 or PARK 7, proteins that protect against oxidative stress mainly in mitochondria are shown to be involved in familial PD (P. P. Michel et al., 2006) Complex I inhibition both in vitro and in vivo leads to the formation of lewy bodies synuclein aggregation is a downstream consequence of mitochondrial dysfunction (D. J. Moore et al., 2005) synuclein k/o mice are resistant to MPTP mediated neurodegeneration (W. Dauer et al., 2002) synuclein in mediating deleterious effects of mitochondrial defic it On the basis of studies in mice and drosophila, parkin links mitochondria with the ubiquitin proteasome system (UPS) (J. C. Greene et al., 2003) Oxidative stress generated due to the mitochondrial dysfunction can promote S nitrosylation of parkin which can impair its ubiquitin ligase activity and its neuroprotective function (T. M. Dawson and V. L. Dawson, 2010) Thus, inhibition of ubiquitination of parkin substrates can contribute to PD pathogenesis. PINK1 can offer partial protection f rom mitochondrial dysfunction induced by proteasomal inhibition (E. M. Valente et al., 2004) Combined involvement of neuronal toxins and genes in promoting mitochondrial defects and oxidative damage is a very common phenomenon. In aged non human primates, MPTP administration leads to the formation of filamentous intracellular synuclein (N. W. Kowall et al., 2000) Parkin is selectively nitrosylated in the brains of MPTP treated mice in a nitric oxide dependent manner and also in humans with sporadic P D (T. M. Dawson and V. L. Daw son, 2010) Neuromelanin The most highly pigmented cells in human brain are DA neurons of the SN and noradrenergic neurons of the locus coeruleus. These neurons have high levels of
45 neuromelanin. In PD patients neuromelanin concentration in the SN drops t o less than 50% compared to control subjects (L. Zecca et al., 2002) Due to its unstable catechol ring, DA is non enzymatically oxidized by molecular oxygen to form H 2 O 2 and o quinone (A. Hermida Ameijeiras et al., 2004) O quinone undergoes a series of oxidation reactions to form a black polymeric insoluble pigment called neuromelanin. Enzymatically DA is deaminated by monoamine oxidase (MAO) to form H 2 O 2 and 3,4 dihydroxyph enylacetic acid (DOPAC) which is methylated by catechol O methyl transferase (COMT) to form homovallinic acid (HVA). It is well known that excess of DA in the cytosol (due to a defect in VMAT2 which packages DA into vesicles immediately after its synthes is and helps to release it into synaptic cleft) can lead to lipid peroxidation and neuronal damage due to the formation of quinones and semiquinones. Neuromelanin is proposed to be protective of such DA mediated oxidative damage by targeting damaged organe lles for lysosomal degradation under normal physiological conditions (D. Sulzer et al., 2000) Thus, oxidative stress in PD could be a result of a breakdown of this DA/neuromelanin biochemistry. MPP + paraquat, a number of metals like Zn, Cu, Mn and mainly Fe can b ind to neuromelanin. It is s peculated that neuromelanin can reduce the toxic effects of these metals and neurotoxicants by sequesteration. However, some studies suggest that this reservoir actually serves to slowly release toxicants over time. By binding t o iron it can actually promote the fenton reaction in cases of increased oxidative stress such as PD. In some cases of sporadic PD and MPTP intoxication extraneuronal melanin granules are seen which are phagocytosed by microglia and are re ported to be resp onsible for their activation (H. Wilms et al., 2003)
46 Impairment of Ubiquitin Proteasome System Emerging evidence suggests that impairment of the ubiquitin proteasome system (UPS) and protein mishandling may also u nderlie the pathogenesis of familial and sporadic PD (R. Betarbet et al., 2005) (D. J. Moore et al., 2003) Structural and functional deficits in the synuclein in the DA neurons are seen in sporadic cases of P D (D. M Branco et al., 2010) (K. S. McNaught et al., 2002) Improvement in the pathologic al features of transgenic flies expressing both normal and mutant synuclein by chaperones supports the role of protein mishandling in PD. Moreover, lewy bodies seen in PD also stain for chaperones implicating their role in disease pathogenesis. Genes encoding Parkin and UCHL 1 within the UPS in familial PD also support the notion that dysfunctional UPS may be a contributing factor to DA neuronal demise. Interestingly, DA neuronal death caused by the synuclein is rescued by co expression of human parkin (L. Petrucelli et al., 2002) However, this protection offered by parkin against the synuclein, but instead through a rescue of the impaired UPS. Parkin is shown to i nteract synuclein interacting protein synphilin 1 and through this interaction promote s the formation of lewy bodies that are marked for clearance by UPS (K. L. Lim et al., 2005) DA neurons are highly vulner able to proteasome inhibition (Y. Yang et al., 2003) synuclein is shown to inhibit the proteasome and increase the sensitivity of cells to proteasome inhibitors. Based on the vicious interplay between various factors implicated in PD, a model for PD pathogenesis is proposed. If complex I inhibition is central to PD pathogenesis, it synuclein aggregation
47 which can bind and inhibit protea some. This would lead to abnormal accumulation of ubquitinated proteins and their reduced clearance by UPS, ultimately resulting in DA neuron death (T. M. Dawson and V. L. Dawson, 2003) Neuroinflammation Neuroinfl ammatory mechanisms contribute significantly to the cascade of events leading to neuronal degeneration. These mechanisms comprise microglial activation, lymphocytic infiltration and astrogliosis. Neuroinflammation is not merely a consequence of neurodegene ration and might be actually involved in the progression of neuronal damage by production of deleterious inflammatory molecules (M. J. LaVoie et al ., 2004) A recently appreciated neuropathological feature of PD is the presence of glial response in all areas of the brain showing neurodegeneration. This field has drawn considerable attention over the past decade as accumulating data suggest that glia l activation is a pivotal contributor to DA neurodegenera tion in PD and we will elaborate on this aspect for the purpose of this paper (Y. Du et al., 2001) (G. Dutta et al., 2008) (H. M. Gao et al., 2003a) Glia and Neurodegeneration Glia comprise the major cell population in the adult human brain, and have been traditionally associated with providi ng support for neurons. Glia biology came into its own when researchers showed that glia are critical for the development of the nervous system and are key player s in neurode generation. Glia regulate the brain vasculature and BBB, modulating ischemia and m igraine. They are also important in the repair of neurons after injury and may play a role in the pathology of neurodegenerative disorders. Multiple Sclerosis is a demyelinating disorder caused by a malfunction of oligodendrocytes. Glial scarring is recogn ized as one of the important mechanisms that
48 reduces proper myelination in adult animals and contributes to the disease (R. H. Miller and S. Mi, 2007) Glia are also important in Amyotrophic Lateral Sclerosis (ALS) Spinocerebellar A Mutated proteins seen in these d isorders invoke microglial responses Glia release toxic compounds which can damage vulnerable neuronal populations. These findings raise the possibilit y of replacing damaged glia by stem cell therapy for treating neurodegenerative diseases (S. A amodt, 2007) Astrocytes have been known to contribute to cerebrovascular disorders like brain ischemia, which is often caused by stroke. Astrocytic glycogen stores can provide energy to deprived neurons but can also p arti cipate in lactic acidosis induced brain damage by releasing excitatory neurotransmitters like glutamate (D. J. Rossi et al., 2007) Since astrocytes are connected by gap junctions to form networks, t hey can also spread the ischemia induced damage to nearby neurons in close vicinity to the site of injury. Neuropathic pain is a neuroimmune disorder involving interaction between gli a, immune cells and neurons (M. Costigan et al., 2009) Blocking the signaling pathways between neuronal and non neuron al cells could be beneficial in disease prevention. However, a number of challenges need to be overcome for successful intervention. Firstly, there is a need to unravel the factors or signals that actually promote these neuron glia/immune interactions. Se condly, segregating the beneficial signals in normal physiology from the dangerous ones in pathological conditions is imperative to avoid fatal mistakes.
49 Microglia are the resident macrophages of the immune system in the brain They have a complex role in that they can either protect or damage the neurons depending on their mode of activation and specific location (U. K. Hanisch and H. Kettenmann, 2007) (H. Kettenmann et al., 2011) Popular belief is that microglia are chronically engaged in repair mechanisms for minor neuronal insults, and the clinical disease is observed only upon failure of these repair efforts. Fully activated microglia are detrimental to neurons, but other intermediate reactive states may actually help in neuronal survival by releasing neurotrophic factors or by clearing excess glutamate from t he extracellular space (M. A. Lynch, 2009) (A. M. Fontainhas et al ., 2011) Microglia mediated neurodegener ation is a primary area of research in relevance to PD. Understanding neuron microglia interplay will help in modulating the disease upstream of microglial activation by elucidating signaling molecules driving this microglial activation. Moreover, pharmacological modulation of PD involving use of inhibitor molecules that direct microglial activation in addition to anti inflammatory drugs acting downstream of microglial activation can tackle the disease at two separa te levels and henceforth may offer a more robust system of disease treatment. In lieu of this hypothesis we will delve into the normal and pathological functions of microglia in neurodegeneration especially PD and then branch into neuron microglia interpla y and the proposed signals regulati ng this mechanism in detail in C hapter 3. Microglia Pio del Rio Hortega introduced the concept of microglia as a cellular element of the CNS in a book chapter in 1932. In this article he postulated the following: Microg lia are mesodermal in origin, have amoeboid morphology and enter the brain during early development. They transform into a branched ramified morphology in the mature brain.
50 They have a capacity to proliferate, migrate and phagocytose. After a pathological event, microglia assume an amoeboid phenotype and participate in removing infec tious agents or cellular debris It is extremely fascinating that all his assumptions are actually valid age. In 1878, Carl From mann described that glia chan ge morphology. Their soma ta become larger and cellular processes become shorter and less numerous upon activation. In 1960s George Kreutzberg developed the facial nerve model which allowed studying microglial responses to injury in tissues with an intact B lood Brain Barrier (BBB) (K. Blinzinger and G. Kreutzberg, 1968) Microglial cells are derived from progenitor cells that are mesodermal or mesenchymal in origin. These cells originating from th e bone marrow migrate from the blood as monocytes and have an amoeboid morphology. Studies show that the exchange of microglia in the normal undist urbed brain is minimal as the CNS is protected by BBB (B. Ajami et a l., 2007) D amage to the BBB increases monocytic infiltration into the brain and their transform ation into microglia (A. Mildner et al., 2007) Thus in a healthy intact brain microglia exist as a stable population Chan et al, showed that from the middle of first trimester and throughout the early part of second trimester there is an embryonic invasion of cells giving rise to microglial population (W. Y. Chan et al., 2007) Hence, there are atleast two populations of microglial cell s: one drived from the myeloid cells and the other derived from fetal macrophage s that is related to the amoeboid microglial population (P. Rezaie et al., 2005) After invading the brain parenchyma, microglial cells assume a ramified morphology. TGF
51 factors and cytokines released from astrocytes are some of the candidates believed to be involved in promoting the ramified phenotype of microgli a (T. Schilling et al., 2001) In the healthy mature CNS, microglia l cells have a ramified morphology. Any disturbance or alteration in brain homeostasis like infections, trauma, ischemia or toxicants can invoke ra pid profound changes in microglial cell shape, gene expression and functional behavior, a term summarized as microglial activation or reactive microgliosis. Microglia become amoeboid and actively proliferate and migrate to the site of injury (D. Davalos et al., 2005) Activated microglia show increased expression of cell surface markers and release chemoattractive and immunomodulatory molecules that can recruit immune cell populations into the CNS. Proinflammatory s ubstances released upon microglial activation include TNF 6 and NO. Nevertheless, microglia also produce neurotrophic factors and physically associate with endangered neurons. In vitro, microglial cells do not have the typical ramifie d morphology. They show different shapes ranging from spindle, rod or amoeboid versions with short thick processes but morphological transformation can be induced by treatment with microglia activating inflammogens like LPS. Activated cells acquire a bi or tripolar spindle or rod shape morphology depending on the nature of insult (P. T. Nelson et al., 2002) (P. Rezaie et al., 2002) Morpho logy is not always the best reflector of reactive phenotype of microglia (D. S. Markovic et al., 2009) Some gene inductions or activities occur in the absence of morphological transformations in microglia. Since CN S is an immune privileged organ, microglial activation is a highly regulated process. There are different stages of microglial activation depending on the molecular, morphological and functional characteristics. Fully activated microglia
52 present themselves like macrophages. Regional variations in glial populations are also seen in the CNS. M icroglia may not be a homogenous population. A study showed that CA1 region of the hippocampus responded differently compared to the CA3 region to NMDA receptor induced excitoto xicity. T his variation was due to the heterogeneity of microglial cells in these areas. TREM 2 (triggering receptors expressed on myeloid cells 2), normally expressed by qui escent microglia show variation in their expression in microglia not only f rom region to region but also within (U. K. Hanisch and H. Kettenmann, 2007) The c urrent view of micro glia is that in a healthy CNS they are constantly surveying t he neuronal environment with their motile processes (A. Nimm erjahn et al., 2005) (U. K. Hanisch and H. Kettenmann, 2007) This shows that actually there are no periods of microglial inactivity at any time (H. Kettenmann et al., 2011) Hence microglial activation is not an all or none response with a fix ed uniform output. It is a graded response which may start as a defense oriented process to ward off the source of neuronal damage and convert to a repair oriented function and finally assume a phagocytic role. In chronically a ctivated states of microglia their neurodegenerative effects by virtue of release of proinflammatory factors take precedence over their neuroprotective effects. Microglia can be identified in the human and animal brains by a number of intracellular or c ell surface associated markers Surface expressed glycan moieties are recognized by isolectin B 4 (W. J. Streit and G. W. Kreutzberg, 1987) Antibodies against IbA1 protein (involved in calcium homeostasis) are very popular for visualizing microglia (Y. Imai and S. Kohsaka, 2002) The expression levels of many molecules such as
53 Mac 1, CD11b, IbA1, HLA DR (antigen presenting glycoprotein) increase with microglial activation (D. Ito et al., 1998) (J. Ma et al., 2003) Major histocompati bility cla ss II (MHCII) molecules are only expressed by activated microglia. However, all of these microglial markers also stain for perivascular macrophages present in CNS. Expression of CD4 5 positive cells is low in parenchymal microglia. Infiltrating macrophages on the other hand seem to have a high CD45 expression. More recently, highly selective antigens like Glucose transporter 5 (GLUT5) for immunolabeling human microglia have been introduced (Y. Horikoshi et al., 2003) However, GLUT5 is not selective for rodent microglia and might st ain other CNS macrophages. Antibodies against keratin sulfate epitope 5D4 can be useful for such discrimination in rats (A. Bertolotto et al., 1998) Genetically modified animals expressing fluorescent proteins under the control of microglia expressed factors like CX3CR1 have recently been developed as microglia indicating mice (S. Jung et al., 2000) Normal microglial cells exhibit properties of low resting membrane potential and low voltage gated membrane currents. They express inward rectifying K + currents predominantly (S. Chung et al., 1999) Activated microglial cells express both inward and outward rectifying K + channels. A question that continues to persist to date is what happens to a previously activated microglia? It is believed that postactivated microglia may remain indistinguishable from resting microglia morphologically. However, experienced cells could behave differently upon the next challenging encounter. This could either have some prote ctive effect where by former activation makes cells more prepared to react next time or it could influence the likelihood of developing neurodegenerative diseases
54 by virtue of epigenetic changes and gene alterations that occurred in the cell during first at tack. Microglial A ctivation and PD Three different types of research provide evidence for microglial activation in PD: epidemiological, animal models and cell culture. Epidemiological studies investigatin g the effects of anti inflammatory agents should be used as a proof of concept for involvement of microglia in PD. If inflammatory process seen in disease pathology is protective of neuronal injury, use of anti inflammatory agents should exacerbate the disease. If it is just involved in clearing debris, dr ug administration should induce no change in pathology of PD. If however, microglia are indeed involved in the disease progression, use of anti inflammatory drugs should be neuroprotective (P. L. McGeer and E. G. McG eer, 2008) Population based studies have reported that regular NSAID users have a 55% less incidence of PD compared to non users (H. Chen et al., 2003) In another study, ibuprofen reduced the risk of PD by 35% (X. Gao et al., 2011) Animal models and in vitro cell culture studies also illustrate that DA neurons a re highly vulnerable to microglia mediated inflammatory attack. Rotenone and 6 OHDA animal models of PD are based on administration of oxidizing compounds tha t are selectively toxic to DA neurons. LPS model of PD is a very popular animal model for microgli al activation in synuclein animal models are also of particular interest. LPS model Lipopolysaccharide (LPS) is a bacterial endotoxin and a potent stimulator of peripheral immune cells including macrophages and monocytes (M. A. Dentener et al., 1993) (A. E. Medvedev et al., 2000) (S. Sanlioglu et al., 2001) LPS, in association with
55 a soluble LPS binding protein (LBP), binds to the plasma membrane anchored CD14 that relies on its association with Toll like receptors (such as TLR 4) to transduce signals across the plasma membrane. Subsequent activation of various kinases and transcription factors results in upregulation of gene transcription for a variety of proinflammatory factors and free radical generating enzymes. LPS induced activation of microglia results in the production and accumulation of neurotoxic factors which impact dopaminergic neurons that are particularly vulnerable to oxidative stress. In contrast to its potent stimulating activity towards immune cells, LPS does not seem to have a direct deleteri ous effect on neurons (they lack TLR 4 receptors) (S. Lehnardt et al., 2003) making it an excellent choice to study inflammation mediated dopaminergic neu rotoxicity in PD (Liu et al., 2002) Supranigral injection of LPS in adult rat brain resulted in rapid microglial activation (6h) and a dose and time dependent degeneration of nigrostriatal pathway (2 3 days later). Non DA neurons in the SN and DA neurons in the adjacent VTA region were sp ared of the LPS induced d amage (Castano et al., 1998) (B. Liu et al., 2000b) Inflammation mediated preferential degeneration of the SNpc DA neurons was confirmed by studies that employed fluorogold retrograde labeling of the striatonigral DA pathway and demonstrated that LPS supranigral injection resulted in the degeneration of SNpc but not VTA DA neur ons (M. M. Iravani et al., 2002) Successful demonstration of preferential degeneration of the nigrostriatal DA pathway induced by a bout of neuroinflammation prompted scientists to examine a potentially more rele vant and significantly less intense neurotoxic dose of LPS that could mimic PD in humans. In the establishment of chronic infusion model, LPS stored
56 in an osmotic mini pump was delivered at a constant flow rate of 0.5 l/hour and a dose rate of 5 ng/hour to a supranigral site of rat brain (H. M. Gao et al., 2002) DA neurodegeneration was not observed in the first two weeks when LPS was being infused whereas time dependent microglial activation occurred in the same time frame. Significant DA degeneration was observed only after 4 6 weeks peaking at 8 10 weeks (H. M. Gao et al., 2002) (M. K. McCoy et al., 2006) By 8 10 weeks after the start of LPS infusion, approximately 60% of TH ir SNpc neurons had disappeared. DA neurons of the VTA and non DA neurons such as GABAergic neurons, on the other hand, remained mostly unaffected (H. M. Gao et al., 20 02) (M. K. McCoy et al., 2006) Prenatal exposure of LPS to gravid female rats produced pups with reduced number of SN DA neurons and increased vulnerability to DA neurotoxins like 6 OHDA and rotenone (Z. Ling et al., 2004) (Z. D. Ling et al., 2004) MPTP m odel Accidental discovery of MPTP as a DA neurotoxin in a group of heroin addicts has already been discussed above. Autopsy of bra ins 20 years after exposure exhibited microglial activation similar to that observed in PD cases (J. W. Langston et al., 1999) In non human primates, prenatal administration of MPTP produced a sustained inflammatory response in SN of an 18 year old monkey that was inje cted with the drug 11 years ago (M. E. Emborg, 2007) However, no lewy body pathology was seen either in humans or monkeys after MPTP administration. In mice, microglial activation was seen in the striatum 24 h post treatment with MPTP and sustained for 14 days. Astroglia were activated 24 h later (B. Liu, 2006) (A. Czlonkowska et al., 1 996) However, significant DA neurodegeneration occurred only after 7 days of MPTP administration. In a couple of other studies microglial activation was detected as early as 12 h and
57 subsided to control levels in 1 week. Astroglial activation occurred at a later time point and was sustained until the end of study (21 days) (G. T. Liberatore et al., 1999) Expression of glial activation associated genes, TNF ncreased significantly 2 4 h following MPTP i njection whereas DA neurodegeneration did not ensue until later time points (K. Sriram et al., 2006a) Immunohistochemical and gene array studies show an increased expression of IL 6, TNF idase complex subunits in MPTP models of PD. Alpha synuclein model synuclein is a major component of the synuclein is an activator of microglia is of great interest. In mixed neuron glia cultures extracellular agg synuclein activated microglia and exacerbated DA neurodegeneration (W. Zhang et al., 2005) This microglia mediated neurotoxicity was dependent on the activation of NADPH oxidase and ROS generation. It was further reported that PHOX induction was linked to the direct activation of Mac 1 receptor located on microglia. Overall, all these models of PD clearly suggest that sustained microglial activation could be responsible for PD. N euroinflammatory res ponses from microglia can contribute significantly to the progression of DA neurodegeneration in PD. Proinflammatory Factors and PD Activation of microglia leads to an increase in the production of cytokines like TNF 6, activation of NADPH oxidase and induction of iNOS and COX 2. A number of microglia derived factors and activation associated enzymes including but not limited to IL 2, IL 4, IL 6, COX 2, iNOS and TNF CSF, str iatum and SN of human PD brains (B. Liu, 2006) SN of PD brains show
58 increased lipid peroxidation, elevated levels of iron, reduced levels of antioxidant enzymes and defects in mitochondrial complex I, all of which suggest the involvement of oxidative stress and ROS generation in DA neuronal demise. Animal models of PD also show an increase in various proinflammatory cytokines like TNF 6 and induction of iNOS, and NADPH oxidase. Detection of these factors and enzymes in PD models before the appearance of DA neurodegeneration strongly suggest that microglia derived factors contribute to the amplification of downstream neurona l damage (B. Liu, 2006) To understand the contribution of individual proinflammatory and neurotoxic factors in the nigrostriatal degeneration we will focus on in vitro and in vivo animal studies. Cytokines Cytokines bind to their cell surf ace receptors and induce or repress expression of certain genes and their transcription factors. TNF inflammatory cytokines in the repertoire of activated microglia derived factors. TNF synthesized as a type II transmembrane trimeric protein (tmTNF) which is cleaved by TNF olTNF) (M. K. McCoy and M. G. Tansey, 2008) SolTNF induces inflammation by acting on TNF receptor 1 which is highly expressed in nigrostriatal dopaminergic neurons and thus makes DA neurons more vulnerable to TNF (M. K. McCoy et al., 2006) TNF which leads to the activation of NF kB, a transcription factor that controls the expression of numerous genes. The TNF receptor associated factor 2 (TRAF 2) activates kinases like JNK, MAPK and MEKK which might be involved in further downstream signaling
59 TNF leads to apoptosis. IL 1 signaling involves activation of IL 1 receptor associated kinases called IRAK 1 and IRAK 2 which cause myriad of downstream effects like induction of AP 1 (a transcription factor), activation of JNK, P38 MAPK and NF kB. Homozygous deletions of both TNF conferred resistance to MPTP mediated microglial and astroglial activation and subsequent DA neurodegeneration (K. Sriram et al., 2006b) (K. Sriram et al., 2002) Deletion of the gene for TNF loss of striatal DA and was ineffective again st SN DA cell loss (B. Ferger et al., 2004) A number of other studies showed that genetic deletion of either TNFR1 or TNFR2 or TNF the subacute and ch ronic MPTP PD models (K. Sriram et al., 2006b) but not in acute models (B. Ferger et al., 2004) Polymorphism in IL d ecrease d age of onset of sporadic PD (M. Nishimura et al., 2000) Mice lacking IL 18 (a member of IL 1 PD (S. Sugama et al., 2004) IL 6 has been reported to act as an immunomodulatory cytokine. It has both pro and anti inflammatory activity as seen in t he IL 6 knockout models of MPTP (L. M. Bolin et al., 2002) (J. Van Snick, 1990) Further studies are needed to clarify the role of cytokines in neurod egeneration NADPH o xidase A significant source of extramitochondrial reactive oxygen species (ROS) production during inflammation is NADPH oxidase (PHOX). PHOX i s a multimeric enzyme composed of gp91phox, p22phox, p47phox, p67phox and p40phox subunits. In
60 the resting microglia PHOX is inactive because the transmembrane subunits gp91phox and p22phox are separated from the rest of their cytosolic subunits. Upon micr oglial activation p47phox becomes phosphorylated and the entire cytosolic complex translocates to the membrane, where it associates with gp91phox and p22phox forming a functionally active PHOX enzyme capable of r educing oxygen to superoxide and other secon dary oxidants. PHOX is an antimicrobial system of phagocytic cells. Defects in the gp91phox subunit of NADPH oxidase are seen in chronic granulomatous disease which increases the susceptibility of host to infections by various microorganisms (F. Meissner et al., 2010) Postmortem analysis of SN in the PD brain samples shows high levels of gp91phox subunit (D. C. Wu et al., 2003) 6 OHDA administration induced significant produ ction of ROS and microglial activation in rats. Levels of gp91phox and p41phox were also elevated (J. Rodriguez Pallares et al., 2007) NADPH oxidase inhibitor apocyanin could attenuate microglial activation and red uce the levels of both transmembrane subunits of PHOX. Gene deletion of gp91phox provided significant protection against the loss of DA neuronal cell bodies as well as the striatal DA levels in MPTP model of PD (V. Anantharam et al., 2007) Nitric o xid e s ynthase (NOS) The nitric oxide synthase (NOS) family consists of three isoforms: nNOS (type1), iNOS (type2) and eNOS (type3). nNOS and eNOS are constitutively expressed and require the formation of Ca 2+ calmodulin complex for their activation wherease iNOS works independently of Ca 2+ signaling. All three isoforms need co factors such as haem, tetrahydrobiopterin and NADH for their activity. nNOS is abundant in brain areas like cerebral cortex, nucleus accumbens, st riatum, hippocampus (CA1 and dentate gyrus) and hypothalamus. It has also been found in astrocytes and cerebral blood
61 vessels. eNOS is expressed in cerebral endothelial cells where it regulates cerebral blood flow. eNOS has also been found in the rat astro cyte cultures. Levels of iNOS in the CNS are low but can be induced in astrocytes and microglial cells following inflammation, viral infection or trauma. NOS family of enzymes is responsible for synthesis of nitric oxide, NO. These enzymes catalyze the co nversion of L arginine to citrulline plus NO in the presence of oxygen. Polymorphism in nNOS and iNOS genes is seen in human PD brains (C. Lev ecque et al., 2003) Both nNOS and iNOS have been in implicated in the MPTP mediated DA demise in PD (S. Przedborski et al., 1996) (Y. Itzhak et al., 1999) Mice lacking iNO S were less sensitive to MPTP induced loss of SN DA neurons. However, no effect of gene deletion on MPTP mediated microglial activation or striatal DA loss was seen (G. T. Liberatore et al., 1999) Partial rescue of the striatal DA levels was seen in mice lacking nNOS and nNOS inhibitors were successful in rescuing the DA neurons and striatal DA in both mice and baboons (P. Hantraye et al., 1996) (S. Przedborski et al., 1996) Pretreatment with L NAME, a non specific NOS inhibitor improved striatal DA levels and locomotor abnormalities in 6 OHDA as well as LPS treated rats (M. K. Barthwal et al., 2001) Nitric oxide (NO) and PD The signalling molecule NO is a neuromodulator and has both neuroprotective and neurodegenerative roles. It is involved in various functions like neurotransmission, regulation of blood vessel tone and immune response. In the CNS it is involved in mainte nance of synaptic plasticity and regulation of sleep, appetite and body temperature. In the PNS, it regulates non adrenergic and non cholinergic relaxation of
62 smooth muscles. NO can interact with many intracellular targets resulting in stimulatory or inhib itory output signals. NO S nitrosylates caspase 3 and NR1 and NR2 subunits of NMDA receptors thus regulating apoptotic cell death (Y. B. Choi et al., 2000) Through the cGMP pathway NO activates cyclic AMP res ponsi ve element binding protein (CREB) and Akt, both of which are neuroprotective in nature. If a cell is in a pro oxidant state NO can undergo oxidative reductive reactions to form toxic compounds (reactive nitrogen species, RNS) which cause cellular damage. N O can react with superoxide anions (produced by iNOS in inflammatory conditions, or nNOS in case of excitotoxic ity) to form peroxynitrite which has strong oxidant properties. Soluble guanylyl cyclase (sGC), a cytosolic haem containing protein is activate d by the binding of NO and catalyzes the transformation of GTP to cGMP. cGMP has downstream effectors like protein kinase G and cyclic nucleotide gated channels. Through these pathways NO causes smooth muscle relaxation and neurotransmission. Term nitros ative stress is associated with the cellular damage caused by NO and RNS. Several studies suggest that peroxynit rite formed by the reaction between NO and superoxide might be responsible for cellular damage seen in neurodegenerative disorders. NO has been shown to activate both the constitutive and inducible forms of cyclooxygenase which are upregulated in PD brains (V. Mollace et al., 2005) Formation of nitrotyrosine, a marker of nitrosative stress is documented in PD patients. Matrix metalloproteinase 9 (MMP9) wh ich causes neuronal apoptosis in DA neurons is S nitrosylated by NO (Z. Gu et al., 2002) Yao et al and Chung et al demonstrated that S nitrosocysteine derived NO is able to nitrosylate parkin, and thereby reduce its
63 protective function (D. Yao et al., 2004) (K. K. Chung et al., 2004) NO also promotes S nitrosylation of GAPDH. S nitrosylated GAPDH binds to SIAH1, another E3 ubiquitin ligase. This complex translocates into the nucleus to induce apoptosis (M. R. Hara et al., 2006) NO is also known to S nitrosylate PDI, protein disulfide isomerase which is involved in the formation of disulfide b onds and proper protein arrangement (T. Uehara et al., 2006) In the brains from PD patients this protective role of PDI is lost. Cyclooxygenase COX 2, which is normally expressed in low levels in the DA neurons is seen to be upregulated in PD in both human and animal models (M. de Meira Santos Lima et al., 2006) Knockout of COX 2 offered significant DA neuroprotection in the MPTP mouse models (S. Hunot et al., 2004) Celecoxi b, a selective COX 2 inhibitor decreased st riatal microglial activation and COX 2 and iNOS expression in MPTP and 6 OHDA models (S. Hunot et al., 2004) (R. Sanchez Pernaute et al., 2004) Dopaminergic cell loss was also significantly attenuated with celecoxib. NSAIDs such as aspirin, salicylate and indomethacin protected against microglial activation, DA neuronal loss and striatal DA levels in MPTP treated mice (N. Aubin et al., 1998) (K. P. Mohanakumar et al., 2000) Motor C isease Signals from cerebral cortex are processed through the basal ganglia thalamocortical motor circuit and ret urn to the same area via feedback pathway. Two pathways exist in the basal ganglia ci rcuitry : Direct pathway Output from the striatum directly inhibits GPi (globus pallidus interna) and SNr (substantia nigra reticulata). Striatal neurons containing D1 rece ptors (activate adenylate cyclase and increase cAMP) constitute the direct pathway an d project to the GPi/SNr; The indirect pathway Inhibitory signals from striatum are sent to GPe (globus pallidus externa) and from GPe to STN (subthalamic nucleus). Striat al neurons
64 containing D2 receptors (inhibit adenylate cyclase activity and decrease cAMP) constitute the indirect pathway and project to GPe. The STN exerts excitatory (glutamatergic) influence on GPi/SNr. The GPi/SNr sends inhibitory signals to the ventra l lateral nucleus of the thalamus. DA is released from the SNpc neurons to activate the direct pathway and inhibit the indirect pathway. In PD, decreased striatal DA stimulation decreases the inhibition of GPi/SNr via the direct pathway. Through the indir ect pathway, decreased DA inhibition causes increased inhibition of GPe resulting in disinhibition of STN. Increased STN output increases activity of GPi/SNr which in turn inhibits thalamus. Thus, thalamus can no longer send excitatory signals to the cere bral cortex to initiate movement and hence there is a suppression of movement. Treatment Levodopa Over 40 years since its introduction levodopa (L DOPA) remains the drug of choice for treatment of PD symptoms. Unlike dopamine which cannot cross the BBB, L DOPA can easily access the brain and metabolize to dopamine. However, to prevent its catabolism peripherally by dopa decarboxylase it is always administered with a peripheral decarboxylase inhibitor, carbidopa. In the brain L DOPA is converted to dopamine by decarboxylase which is stored in the DA neurons of substantia nigra. There is a satisfactory response to the initial treatment for around 1 5 years. As the disease progresses effects of L DOPA begin to diminish after each dose resulting in (T. Huynh, 2011) This problem is believed to be ca used by pulsatile stimulation of the striatal DA receptors. This can be overcome by increasing the frequency of drug administration or by changing to a
65 controlled release form. However, PD asso ciated postural instability, dementia and autonomic dysfunction are unresponsive to L DOPA therapy. Dopamine A gonists Dopamine agonists like bromocriptine were introduced in the 1970s and they directly activate DA receptors in the absence of DA. Apomorphi ne, the oldest DA agonist has high toxicity when given orally and thus has to be administered subcutaneously. It has a rapid and short duration of action and is used mostly as a New dopamine agonists pramipexole and ropinirole are selective for the D2 family of receptors. These DA agonists are recommended as the first line monotherapy in early stage PD, to delay L DOPA treatment and its related side effects. MAO B and COMT I nhibi tors Monoamine oxidase B inhibitors (MAO B) decrease the oxidative metabolism of DA in the brain and thereby increase nigrostriatal DA levels. These include selegiline, lazabemide and rasagiline. Several potential antioxidative and antiapoptotic neuroprote ctive effects of the MAO B inhibitors have been suggested. The enzyme activity of catechol O methyltransferase (COMT) considerably reduces the concentration of L DOPA in the blood that is available to enter the brain. Inhibition of COMT extends the plasma half life of L DOPA and stabilizes its plasma levels (C. B. Levine et al., 2003) This is achieved by COMT inhibitors li ke tolcapone and entcapone. Both these classes of inhibitors are often taken as monotherapy or in combination with each other or L DOPA, thereby helping to alleviate the wearing off effect of long term levodopa therapy.
66 Anticholinergics Anticholinergic me dications like benztropine, procyclidine and trihexylphenidyl were used long before L DOPA or DA agonists were developed. They mainly relieve tremors and stiffness in PD patients. Their use is limited by anticholinergic side effects like dry mouth, blurry vision and worsening of confusion in PD patients. Hence, they are not recommended for patients above 65 years of age. Alpha 2 Adrenergic A natgonists Dyskinesia is an apparently unavoidable consequence of long term L DOPA treatment. Dopamine receptor agoni st monotherapy only delays the progression of dyskinesia. To date, functional neurosurgery has been the most effective way to overcome levodopa associated dyskinesia. Pharmacologically, amantadine, NMDA receptor antagonist can offer some relief as an anti dyskinetic drug. A key abnormality underlying levodopa induced dyskinesia is the overactivity of direct striatal pathway and alpha 2 receptor stimulation. Levodopa essentially acts by in creasing brain noradrenaline in addition to dopamine. This can be modu lated by administration of adrenoceptor antagonists or inhibitors of dopamine hydroxylase, an enzyme that converts dopamine to noradrenaline in noradrenergic neurons (S. F. Leibowitz and C. Rossakis, 1979) Over time, use of le vodopa leads to desensitization of striatal DA receptors to dopamine and hence d yskinesia ensues Thus, Alpha 2a and 2c adrenergic receptor antagonists like idazoxan and fipamezole have shown to reduce levodopa induced dyskinesia and extend the duration of action of levodopa. Adenosine A2A Receptor A ntagonists A2a receptors are located on the striatal medium spiny neurons and modulate the release of GABA. A2a antagonists affect the release of acetycholine from striatal
67 cholinergic interneurons and release dopamine from the nigrostriatal tract. A2a antagonist KW 6002 produced long lasting improvement in motor activity without the development of dyskinesia in MPTP primate model of PD. Surgical T argets: Ablation and Deep Brain Stimulation Increased glutamater gic signals from the putamen have m ade GPi a target for ablation and deep brain stimulation (DBS). Both unilateral and bilateral pallidotomies have been performed in PD patients. Even though there were improvements in tremors and dyskinesia, these patients suffered from irreversible deterioration of speech and cognition. Thalamus and STN are also potential surgical targets for treating tremors in some countries but due to the severity of side effects their removal is not very common in United States. Gener ally deep brain stimulation (DBS) is preferred over ablation because it has the same effect while being reversible and adjustable. Thalamic stimulation significantly improves tremors of the contralateral limb without the adverse effects on speech and cogni tive functions. However, in many cases there is no marked improvement in activities of daily living as there is no effect on bradykinesia (J. E. Ahlskog, 2001) Bilateral subthalamic stimulation is more successful in alleviating all the principal symptoms of tremors, rigidity and bradykinesia but depression, psychiatric problems and occulomotor defects can ensue as a result of inhibition of STN limbic areas (M. Zabek et al., 2003) Al though effective, DBS requires careful postoperative adjustment and battery replac ement every 3 5 years Occasionally patients have to undergo emergency surgeries following rebound symptoms that emerge due to failure of the stimulator. Also, worn out c ases with insufficient
68 therapy usually do not respond well to surgery. Moreover this is a very expensive procedure due to the long term need for patient management (A. Gray et al., 2002) Gene Therapy Three gene based approaches can be used to elucidate the alteration in physiological functions of a prot ein encoded by a specific gene: 1. Gene transfer approach in which a wild type copy of the mutated gene is intr oduced ; 2. RNA modification therapy in which mRNA encoded by a mutant gene is targeted ; 3. Stem cell therapy in which human stem cells are used to replace damaged cells Gene t ransfer a pproach Gene transfer approach is a good choice when a disease is due to los s of f unction (recessive) mutations. Administration of a wild type gene would restore the normal function and reverse disease phenotype. Two methods can be use d to introduce wild type genes: Ex vivo: where a cell population from the patient is genetically altered to express the wild type gene and introduced back into the patient ; Both these approaches require two key components: a gene expression cassette consisting of a w ild type gene (transgene, normally cDNA of the wild type gene lacking transgene) that allow the gene to be expressed in transfected cells, and a delivery system to introduce the wild type genes into cells For successful delivery of the gene of interest the expression cassette must either integrate into the genome of the host or remain extrachromosal ie it should be able to enter the nucleus of the cell It is imperative to main tain a balance between the expression levels of the transgene; it
69 should be high enough to alleviate disease phenotype but low enough to prevent an autoimmune response. RNA m odification Dominant mutations in genes cannot be rescued simply by introducing a wild type gene as in these cases the encoded protein can assume new functions called gain of function mutations. Also, in some cases of dominant mutations the mutant proteins can interfere with functions of wild type proteins, called dominant negative mut ations. In such scenarios turning off the expression of the mutant gene that encodes the mutant protein would be the need of the hour. Most commonly used approaches for targeting RNA transcribed from the dominant gene include RNAi, ribozymes and anti sense oligonucleotides. However, most of these approaches are relatively new and pose challenges in S tem c ell t herapy Stem cell therapy is the modern day equivalent of organ transplantatio n. Stem cells can be embr yonic or somatic in nature Em bryonic stem cells are derived from inner cell mass of blastocyst and are capable of unrestricted cell divisons and are pluripotent. Ethical concerns regarding use of human embryos and technical difficulties in culturing them are impediments to their clinical application. Somatic stem cells are derived from a specific group of cells within an adult tissue and are more restricted in the type of cell type they can assume after cell division. Gene T herapy for PD The loss of dopam ine input to striatum lead researchers to explore the possibility of dopamine replacement. Studies conducted in rodent and primate PD models
70 demonstrate that tyrosine hydroxylase gene transfer into striatum increases local levels of endogenous L DOPA and h elps in improving motor symptoms (D. Kirik et al., 2002a) T yrosine hydroxylase synthesizes L DOPA and is the rate limiting enzyme in dopamine synthesis. L DOPA is then coverted to dopamine by aromatic amino acid decarboxylase. Gene therapy involves using viral vectors like adenovirus, lentivirus and adeno associat ed virus. GTP cyclohydrolase which synthesizes tetrahydrobiopterin cofactor used in dopamine synthesis and vesicular monoamine transporter which regulates synaptic dopamine are some other gene based therapies that have shown successful improvements in PD s ymptoms in animal models (T. Bjorklund et al., 2010) (Q. Chen et al., 2005) However, concerns regarding the long term unregulated product ion of dopamine have limited the translation of these studies clinically. Delivery of growth factor genes into brain is another area of high promise. Growth factors can promote growth of degenerating neurons and also protect neurons from toxicity. Deliver y of glial derived neurotrophic factor (GDNF, a member of TG F superfamily) gene into striatum and subtantia nigra of rodent and non human primates increased levels of dopamine and reversed functional deficits following MPTP treatment (A. H. Schapira, 2005) Since there is a loss of GABAergic inputs to STN in PD resulting in exe cessive glutamatergic drive to GPi/SNr, AAV associated delivery of glutamic acid decarboxylase gene into STN significantly improved motor de ficits in rat PD models (J. Luo et al., 2002) Another surgical strategy that has been employed in PD treatment is direct infusion of drugs into the brain. In lesioned animals GDNF administration rescued the loss of DA neurons and increased levels of tyrosine hydroxylase and dopamine.
71 However, an initial human trial of GDNF infused into the ventricular system showed severe side effects with no significant improvements in motor functions (J. G. Nutt et al., 2003) Site of drug delivery with insufficient penetration of GDNF into target brain areas was speculated to be the problem. Later, in another phase I study GDNF infused directly into the putamen of five PD patients resulted in a 30% increase in striatal DA levels with no significant adverse effects (S. S. Gill et al., 2003) In order to bypass the suboptimal distribution of the growth factor in target brain areas, a group of researchers genetically engineered human neural progenitor cells (hNPC) which secreted GDNF. Transplantation of these engineered cells in the nigral and st riatal areas of brains of three monkeys exposed to MPTP showed an increase in tyrosine hydroxylase and VMAT2 positive fibers as well as a reduction in clinical rating score for PD (M. E. Emborg et al., 2008) This m ay be a possible alternative for intracerebral trophic factor delivery for PD. GABAa agonists like muscimol injected into PD brains reduced bradykinesia and improved behavioral abnormalities (M. S. Baron et al., 2002 ) However the effects were shortlived and required conti nuous infus ion to maintain sustained improvements Fetal Tissue Transplantation Fetal cell transplantation has been introduced as a treatmen t option based on the premise that fetal neuronal cells p ossess the capacity to regene rate the damaged DA neurons in patients with PD. DA neuronal cells are obtained from the brainstem of aborted embryos/fetuses aged 6 15 weeks post conception. In a placebo controlled trial although uptake of fluorodopa increase d on PET scan, clinical improvements did not necessarily correlate and were more profound in younger patients (M. S. Baron et al., 2002) Dyskinesia similar to that seen with levodopa treatment ensues in most of the
72 transplantation PD cases after a few years. A lot more work is needed to ensure targeted transplantation while overcoming associated dyskinesia. Stem Cells The question of whether fetal transplantation provides adequate number of dopaminergic cells paved the way for embryonic stem cells as a treatment option. Expression of Nurr1 protein (differentiates mid brain precursors into DA producing neurons) in embryonic stem cells increased their survival rate while improving parkinsonian symptoms and electrophysi ological properties in rat model of PD (M. A. Lee et al., 2002) Embryonic stem cells can also be engineered to deliver growth factors as already mentioned above. GDNF expressing neural stem cells prevented degenera tion of DA neurons in 6 OHDA mouse models and also improved amphetamine induced turning behavior (P. Akerud et al., 2001) Stem cell therapy although promising is also controversial. Social and ethical issues limit the supply of available tissues. A complete understanding of the integration of stem cells into neural
73 CHAPTER 2 EXPERIMENTAL PROTOCO LS Cell C ulture Rat IR B3AN27 (N27) dopaminergic (DA) neuronal cells, derived from SV40 large T antigen immortalized fetal E12 mesencephalon, retain key characteristics of DA neurons (E. D. Clarkson et al., 1998) (A. G. Kanthasamy et al., 2008) (P. Zhang et al., 2009) These cells express tyrosine hydroxylase, DA transporter and DA metabolite HVA and produce significant amounts of dopamine. N27 DA cells were grown in RPMI 1640 supplemented with 10% heat inactivated fetal bovine se rum (FBS), 50 U/mL penicillin and 50 g/mL streptomycin (Ne uronal Maintenance Media) at 37 C in a humidified environment with 5% CO 2 and 95% air. B35 cortical neuronal cells were de rived in 1974 from nitrosoethyl urea (NEU) induced tumors of neonatal rat central nervous system (C. A. Otey et al., 2003) These cells have been used to study signaling ted with 10% heat inactivated FBS 50 U/mL penicil lin and 50 g/mL streptomycin. Rat HAPI (highly aggressive and proliferative) microglial cells obtained from mixed glial cultures of 3 day old rat brain were grown in DMEM s upplemented with 5% FBS, 50 U/mL penicillin and 50 g/mL streptomycin (Micr oglial Maintenance Med ia) at 37 C and 5% CO 2 These cells are enriched for microglia and possess characteristics similar to microglia/ brain macrophages providing a good model to study microglia (P. Cheepsunthorn et al., 2001) (P. Zhang et al., 2007) (H. Mao et al., 2007b)
74 Cell Viability Assay The effect of toxicants on the viability of N27 and B35 cells was determined using the MTT [3 (4,5 dimeth ylthiazol 3 yl) 2,5 diphenyl te trazolium bromide] assay. Cells were seeded at a density of 510 4 cells/well in a 24 well plate for a day to become 80 90% confluent. Cells were then treated for 24 h with ne uronal maintenance media (500 L /well) alone [vehicle con trol for 1 methy 4 phenylpyridinium (MPP + )], dieldrin (MP Biomedicals, Solon, OH), or MPP + (Sigma, St. Louis, MO). Stock solutions of dieldrin (10 mM, ethanol) and MPP + (10 mM, PBS) were prepared fresh each time before treatment. At the end of treatment MTT reagent (5 mg/mL, 50 L /well) was added and the cells were incubated for 45 mins. Later, the entire media was removed and cellular formazan crystals formed were dissolved by adding 500 L dimethyl sulfoxide (DMSO) /well and shaking the plate on a sha ker for 10 minutes. 150 L of solution was added to a 96 well plate in triplicate from each well and cell viability was assessed by a Synergy HT multi mode microplate reader (BioTek Instruments, Winooski, VT) read at 550 nm. Preparation of N27 Neuronal Conditioned M edia (CM) N27 or B35 neuronal cells were seeded (4 X 10 6 cells/dish) in 10 cm culture dishes and grown to 80% confluence in Neuronal Maintenance Media. To prepare CM, cells were rinsed (3X) with serum free RPMI 1640 (SF RPMI) and then treated for desired t ime intervals with SF RPMI (7 mL /dish) containing vehicle or toxicant. For microglial activation assay, neuronal cultur e supernatants (i.e., CM, ~14 mL ) for each treatment condition (2 sister dishes/condition) were collected, combined and ce ntrif uged for 10 min at 250 x g at 4 C. No appreciable number of floating N27 cells were observed under microscope before the harvest of culture supernatants in any treatment conditions
75 and no cell pellets were observed following centrifugation. The resu lting supernatant was then c oncentrated (at 4 C) to ~500 L using the Amicon Ultra 15 centrifugal concentrators (3 KDa cutoff; Millipore, Billerica, MA). No detectable amount of cystatin C was found in the filtrate by Western blot analysis (data not shown ). The protein concentration of CM was determined with the DC protein assay reagents (BioRad, Hercules, CA) using bovine serum albumin (BSA) as standard. Aliquots of concentrated CM (normaliz ed by protein concentration) were immediately added to microgli al cultures to determine the effect on microgl ia (see below) or stored at 80 C for further analysis. For proteomic study, neuronal supernatants (2 dishes/condition) were collected and centrifu ged for 10 min at 250 x g and 4 C. The resulting supernatant was mixed with s ix volumes of cold acetone ( 20 C). The mixture was kept over night at 20 C and then centrifuge d for 60 min at 10,000 x g at 4 C. The resulti ng pellet was resuspended in 1mL of cold acetone and transferred to a microcentrifuge tube. Afte r two washes with cold acetone, t he pellet was dissolved in 50 L iTRAQ Dissolution Buffer (Applied Biosystems, Carlsbad, CA) and stored at 20C. Protein concentration was determined using the micro BCA reagents (Pierce, Rockford, IL) and BSA as standard Isobaric Tags for Relative and Absolute Q uantitation (iTRAQ) Proteomic analysis of neuronal CM was performed using the Applied Biosystems (D. S. Barber et al., 2007) Briefly, 50 g of acetone precipitated proteins dissolved in Dissolution Buffer were incub ated with Reducing Reagent (2 L ) f or 1 h at 60 C followed by a 10 min incubation at room temperature with Cysteine Blocking Reagent (1 L ). Proteins were then digested by incubation overnight at 37C with trypsin (1:50 ratio). Digested proteins from each treatment condition were labeled with distinct iTRAQ tag reagent and then
76 combined. The combined sample was separated into 10 fractions using a strong ion exchange column connected to a HPLC. Each fraction was then analyzed by reversed phase LC MS MS (QS TAR, Applied Biosystems) MS data were analyzed using Scaffold (Proteome Software Inc. Portland, OR) for protein identification and determination of relative abundance. Gene Ontology (Go) analysis was performed to determine the cell pathway relevance of the proteins. Microglial A ctivation HAPI cells were seeded in 12 well culture plates (2.5 x 10 5 cells/well) and grown to near confluence in Microglial Maintenance Media. Prior to treatment, cells were serum star ved for 1 h in SF DMEM (H. Mao et al., 2007a) Afterwards, N27 CM (100 g protein, <150 L ) or equal volume of SF RPMI (as control) was added to HAP I cells in fresh SF DMEM (1.5 mL /well final volume). At the end of treatment, aliquots of HAPI cell culture media (75L per well, in tri plicate) were saved for the determination of levels of nitrite and cytokines. HAPI cells were washed twice in PBS and lysed in a sample bu ffer (50 mM Tris HCl, pH 6.8, 1 mM EDTA, 2% SDS, and protease imhibitors) for immunoblotting analysis. Nitrite A ssay NO production in HAPI cell supernatant (as nitrite) treated with neuronal CM was assessed by adding 75 L of Greiss reagent (sulphanilic acid solution in phosphoric acid and N (1 naphthyl ethylenediamine dihydrochloride) to HAPI supernatant collected in triplicate in a 96 well plate. A standard curve was made using sodium nitrite. The plate was kept in dark at room temperature for 10 minutes for color development (purple colored azo compound) after which it was read at 540 nm using Synergy HT multi mode microplate reader.
77 DCF Assay for ROS D etection The effect of N27 neuronal CM on microglial ROS production was determined using a fluorescent probe 5 (and 6) chloromethyl 2, 7 dichlorodihydrofluorescein diacetate (CM H 2 DCFDA). HAPI cells (5 10 4 cells/w ell) were seeded in 96 well culture plate and grown to near confluence in Microglial Maintenance Media. Prior to treatment, cells were serum starved for 1 h in SF DMEM. Afterward s, N27 CM (10 g protein, <10 L ) or equal volume of SF RPMI (as control) wa s added to HAP I cells in fresh SF DMEM (100 L /well final volume). At the end of treatment, cells were rinsed (3X) with phenol red incubated for 1 h with 5 M CM H 2 DCFDA in HBSS (100 L /well). D CF DA can easily enter the cell due to its high lipophilicity and gets reduced to DCF by cellular esterases. Reaction with ROS, mainly hydrogen peroxide generates fluorescence which can be measured at 485/530 nm (excitation/emission) using a Synergy HT mu lti mode micro plate reader. ROS production was expressed as relative fluorescence units normalized to total protein content in each well. Protein Assay Immediately after fluorescence intensity measurement for ROS, cells w ere rinsed twice with PBS. 100 L of 2N sodium hy droxide was added to each well and kept overnight at 20 C. 10 L of 2N HCL was added to 10 L of sample for neutralization and diluted further to a linear range of the assay using distilled water. Protein concentration was determined using BCA kit from Pierce with BSA as standard. Samples were incubated at 37C for 2 h. Absorbance was read at 562 nm using Synergy HT microplate reader (BioTek Instruments).
78 Immunoblotting Proteins (35 g of HAPI cell lysate or 15 g N27 CM) were resolved on 7.5% (for iNOS) or 12 16% (for cystatin C) Tris glycine gels (Bio Rad) and transferred, electrophoretically, to nitrocellulose membranes. The transfer time was increased to 1.5 h for a 16% gel (as opposed to 1 h for 7.5% and 1.15 h for 12.5% gels) to ensu re proper transfer of higher molecular weight proteins. The membranes were briefly stained with Ponceau S (0.1%) to visually confirm equal protein loading and proper transfer. This is a rapid and reversible staining method for locating protein bands. Afte r blocking for 1 h in 5% non fat dry milk in T PBS (0.05% Tween 20 in PBS), the membranes were incubated overnight at 4C with a mouse monoclonal anti iNOS antibody (1:2500, BD Biosciences, San Jose, CA) or a rat polyclonal anti cystatin C antibody (1:1000 Millipore) diluted in 3% BSA T PBS. Bound primary antibodies were visualiz ed by incubating the membrane for 1 h with horseradish peroxidase conjugated goat anti mouse/rabbit secondary antibodies diluted 1:5000 for iNOS and 1:2500 for cystatin C detectio n in 5% non fat dry milk in T PBS, followed by incubation with SuperSignal West Dura chemiluminescent reagents (Pierce). Chemiluminescent substrates for HRP conjugated antibodies consist of a stable peroxide buffer and an enhanced luminal solution. When t he blot was incubated with a 1: 1 ratio of the two reagents, a chemical reaction generated light. Images were recorded with the BioRad ChemiDoc XRS system and analyzed with the BioRad Quantity One software. Band intensities for iNOS were normalized to tha actin detected by stripping and reprobing the membranes with an anti actin antibody following our previously described protocol (P. Zhang et al., 2007)
79 Immunodepletion Immunodepletion of cystatin C from N2 7 CM was performed using the Invitrogen Protein G coated Dynabeads. Magnetic separation using these beads helped in faster and gentler recovery of the protein, less consumption of antibody and reproducible results. Protein G is a component of cell wall and is produced by streptococcus strain. It binds to Fc region of most immunoglobulins (IGs). Briefly, N27 CM (500 g, ~200 L) was incubated with 20 L of anti cystatin C antibody or, as a control, a polyclonal anti g lial fibrillary acidic protein (GFAP) ant ibody (DAKO Carpinteria, CA) for 2 h at 4 C on a rotator. Afterwards, Pro tein G coated Dynabeads (100 L ) were was hed twice magnetically using 1 mL of citrate phosphate buffer (ph 5) each time. Basically, the beads were initially resuspended to ge t a hom ogenous suspension. 100 L of beads were taken in a microcentrifuge tube and kept on the magnet. The beads stuck to the magnet and the supernatant was discarded. The tube was removed from the magnet and 1mL of citrate phosphate buffer was added. The suspen sion was resuspended by pipetting a couple of times and the same step was repeated. After the second wash the N27 CM and antibody mixture was added to the beads and the complex was kept on an orbital platform shaker for 45 min at room temperature. The pro tein antibody bead complex was pulled down magnetically. Immunodepleted N27 cell CM was evaluated for ability to activate HAPI microglia (see above). The clearance of cystatin C in the N27 cell CM was confirmed by Western blot analysis for cystatin C. Im munofluorescence The neurotoxicity potential of N27 CM treated microglial CM was determined by immunofluorescence analysis for caspase 3. N27 DA cells were seeded in 8 well Lab 4 cells/well and grown for 24 h in
80 Neuronal Maintenance Media. Supernatants from HAPI microglial cells previo usly treated with N27 CM (250 L /cham ber well) were mixed with 250 L of SF RPMI and incubated with N27 cells for 24 h. At the end of treatment, cells were rinsed with PBS and fixed with 4% formaldehyde solution for 10 minutes to retain the structure and location of the cellular protein. Keeping cells in formaldehyde solution for more than 15 minutes could result in cross linking the protein to the extent that antigen retr ieval might be needed to ensure free access of the antibody to the protein. Cells were permeabilized at 4C overnight with cold 70% ethanol. Ethanol helped to dehydrate the cells thereby precipitating the cellular proteins and causing the cells to adhere t o the surface of the microplate facilitating access to the antigen. Then, cells were blocked by incubation for 1 h with blocking solution (3% heat inactivated goat serum, 0.1% Triton X100 in PBS). Cleaved casepase 3 was detected by incubation for 1 h with a rabbit monoclonal anti cleaved caspase 3 antibody (1:1000 in blocking solution, Cell Signaling Technology, Beverly, MA) at room temperature. Bound primary antibody was determined by incubation with Alexa 568 conjugated goat anti rabbit secondary antibo dy (1:400 in blocking solution, Molecular Probes). Cells were mounted with Vectashield containing DAPI for nuclear staining. Fluorescence images were recorded and analyzed with a Zeiss fluorescence inverted microscope and Axiovision imaging software. Nu mber of cleaved caspase 3 positive and DAPI positive cells in fields of 350 x 275 m were visually counted under the fluorescence microscope. ELISA Amounts of cytokines, TNF measured using enzyme linked immunoso rbent assay reagents from R&D Systems (Minneapolis, MN) as previously described (P. Zhang et al., 2009) Briefly microtitre
81 plates were coated with capture antibody diluted in PBS. 100 L was added to each well and incubated overnight. Next day the extra capture antibody was removed by manually inverting the plate and washing it thrice using wash buffer. After the last wash the plate was carefully dried by blotting it a couple o f times on paper towels. 300 L of rea gent diluent (1% BSA in PBS) was added to block any remaining sites on the PVC plate not covered by capture antibody and kept for 2 h Again the plate was washed three time s with wash buffer. Later, 50 L of standard or samp le was added to the plates and f inal volume was made upto 100 L using reagent diluent. The plate was kept overnight at 4C. After washi ng the plate three times, 100 L of biotinylated detection antibody diluted in reagent diluent was added and the plate was kept for 2 h at room tempera ture. Later, 100 L of streptavidin HRP was added to the plate for 20 minutes in dark. After repeatin g the washing step again, 100 L of substrate was added to detect the biotin streptavidin complex attached to HRP. The plate was kept in dark for 5 10 minu tes for the color to develop. 50 L of stop solution was added to stop the reaction further after the color was completely developed. Absorbance was read using a spectrophotometer at 450 and 570 nm. Deglycosylation N 27 cells were seeded in 10 cm dishes a t a density of 410 6 for 24 h to attain 75 80% confluency. The cells were treated with 7 m L /dish of SF RPMI alone or 30 M dieldrin for 24 h. The neuronal supernatant was collected in centrifugal filters (Amicon Ultra 15, 3KDa, Millipore) an d concentrated to approx. 500 L Concentrated neuronal supernatant was subjected to a second round of concentration to achieve a volume of ~15 20 L using microcon centrifugal filter units (Ultracel YM 3, Millipore). Protein concentration was determined using Bio Rad D C kit. Non denaturing protocol for
82 deglycosylation was followed for both immunoblot analysis for cystatin C and functional assays analyzing protein activity. Deglycosylation was performed using the deglycosylation kit that contains N glycanase F (PNGase F) sialidase A and O protocol. Briefly, super concentrated N27 cell CM (75 g protein) was incubated for 6 h with 10 L of 5x reaction buffer and 1 L of individual or combination s of the deglycosylation en zymes to a final volume of 50 L Deglycosylated N27 CM (50 g) was added to each well of HAPI microglial cells and incubated for 12 h to determine the effect on microglial activation. Fifty and 15 g of deglycosylated N27 CM w ere used for microglial activation and immunoblot analysis for c ystatin C respectively. Statistical Analysis Statistical significance was determined using an analysis of variance (ANOVA) m (SAS Institute, Cary, NC). A p value of <0.05 was considered statistically significant.
83 Figure 2 1 Experimental Design Immortalized rat N27 DA neuronal cells were treated with MPP + or dieldrin to induce significant yet modest reduction in viabilit y comp ared to vehicle treated cells. Conditioned media (CM) were collected and subjected to proteomic analysis to obtain profiles of proteins released from toxicant injured N27 DA neurons. Following antibody based immuno depletion of specific proteins ide ntified in the proteomic profile analysis, the ability of CM to activate immortalized rat HAPI microglial cells and induce DA neurotoxicity was determined.
84 Figure 2 2. Deglycosylati on by enzymatic treatment Deglycosylating enzyme PNGase F acts on aspa ragine linked oligosaccharides. O glycanase can remove O linked sugars without any modifications in serine/threonine residues. However, it is ineffective if these sugars are capped by sialic acid and this require s treatment with another deglycosylating enz yme; sialidase A. Cst3 is the gene encoding for protein cystatin C. Non glycosylated isoform of the protein has a molecular weight of 13 14 KDa.
85 CHAPTER 3 DOPAMINERGIC NEURONS RELEASE SIGNALS THAT ACTIVATE MICROGLIA Introduction is a progressive debilitating neurodegenerative disorder which is pathologically characterized by the loss of dopaminergic neurons in the substantia nigra. This neurodegeneration gives rise to the typical symptoms of remor, rigidity and bradykinesia. Imagine flexing biceps and triceps (antagonistic muscle groups) at the same time and the rigidity resulting from it. The picture becomes much grimmer as the subjects age. M any causes leading to the disease have been descri bed but the complete etiology remains elusive This may be due to the fact that a multitude of factors, both environmental and genetic, can contribute to the development of disease Genetic causes include synuclein (A. C. Belin and M. Westerlund, 2008) (R. Kruger et al., 1998) leading to the familial form of PD. Idiopathic or non genetic PD on the other hand is more complex and genetic susceptibility is still undescribed. Sporadic or idiopathic PD has been linked to environmental factors (l ike toxins and drugs) based on the evidence that MPTP, a side product in the chemical synthesis of mep e ridine caused PD in humans (J. W. Langston et al., 1983) A substantial role of pesticides in causing dopaminergic neurodegeneration has also been reported (J. P. Hubble et al., 1993) Microglia are the immune system of the CNS. CNS is immune privileged in the sense that it is mainly inaccessible to peripheral immune components which can only penetrate the blood brain barrier in a few situations like trauma and some other conditions (infectious, vascular or meningeal contingencie s). In the absence of the peripheral immune system, microglia prevent the CNS from harmful agents and clear
86 the dead cells, debris & and metabolic waste. However, the functions of microglia are much diverse. Microglia appear in the CNS concomittantly with the advent of neurogenesis during nervous sytem development (A. Bessis et al., 2007) (I. Dalmau et al., 1997) and play a significa nt role in programmed cell death of neurons during this period (J. M. Schwab and H. J. Schluesener, 2004) (L. Pont Lezica et al., 2011) This programmed cell death (PCD) phenomenon is linked to many products of microg lia. It can release TNF (F. Sedel et al., 2004) nitric oxide (C. C. Chao et al., 1992) and IL which cause apoptosis. Other factors released from microglia are also implicated in neuronal death during development including cathepsin B (P. J. Kingham and J. M. Pocock, 2001) re active oxygen species (M. L. Block et al., 2007) glutamate (D. L. Taylor et al., 2003) etc. Cathepsin B was identified to be the cause of death to hippocampal neurons. Programmed cell death during development and microglial acquisition & activation are also well connected temporally (F. Sedel et al., 2004) It is noteworthy that similar factors were found to be involved in microglia mediated neuronal death in disease. Microglia also seems to regulate synaptic morphology and function in adult CNS. The functions mentioned above are essential for maintaining health and homeostasis in the CNS. The question howe ver remains that what signals ask for microglial activation at the precise temporal stage. There is accumulative evidence that neurons constantly inform microglia, the principal players in brain innate immunity, about their status and are capable of contro healthy brain microenvironment. Disappearance of these signals causes microglia l
87 signals are i nduced upon stress for a defined microglial activation program (K. Biber et al., 2007) her be membrane bound or released or both. Some of the off signals identified till date are CD22, CCL21 and fractalkine (A. E. Cardona et al., 2006) (E. K. de Jong et al., 200 5) which are released from neurons and they bind to their respective receptors present on microglia. Other signals like CD200, (H. Ohnishi et al., 2005) are expressed on neuronal surface and they bin d to their receptors on microglia when neurons and microglia come in close contact. Regulator of G protein signaling 10 (RGS 10) has recently been iden tified as a negative regulator of reactive microgliosis by inhibition of NF kB pathway (J. K. Lee et al., 2011) Thus, these off signals are responsible for co discussed further, the role of microglia in disease should be elaborated. Although, microglia are necessary for g ood health of the CNS, their chronic activation could be detrimental. Microglial activation is seen in most neurodegenerative (J. C. Schlachetzki and M. Hull, 2009) PD (Y. Ouchi et al., 2005) and amyotrophic lateral sclerosis (J. S. Henkel et al., 2009) It is only now clear that microglial activation precedes neurodegeneration in these disorders. Also, numerous studies carried out using cell culture and animal models point to the role of microglial activation in neuronal da mage. Microglial activation has a special role in PD as it leads to DA neurodegeneration in SNpc and striatum. As in development, reactive m icroglia in the adult brain produce a variety of cytokines, chemokines and reactive oxygen species which lead to de ath of dopaminergic neurons. All these factors work in cohort to exaggerate neuronal misery with one factor helping the other factor in crime. Foremost, microglial interleukins like IL
88 p regulated in order of minutes post exposure to LPS or Interferon gamma (J. B. Koprich et al., 2008) The whole repertoire of signaling pathways activated by IL IL 1R in m icroglia can increase the formation of several mediators like ROS, RNS (reactive nitrogen species), PGE2, NO and some anti inflammatory cytokines. On the other hand, TNF neurotoxicity, increases intracellular ROS generation which in turn inhibits the protective effects of NF kB and further leads to sustained JNK activation. Moreover, the role of NF kB is controversial as on one hand it increases expression of MnSOD (superoxide dismutase) & some antiapototic pr oteins, and on the other, it is shown to enhance cyclooxygenase 2 expression and thus promote inflammation (M. G. Tansey and M. S. Goldberg, 2009) (M. K. McCoy and M. G. Tansey, 2008) Interleukins and LPS ca n also directly activa te NF kB in addition to TNF it can induce apoptosis in neurons and TNFR / mice show significantly lower DA neurodegeneration in SNpc (K. Sriram et al., 2002) Enzyme NADPH oxidase produces superoxide molecules in activated microglia and causes a massive build up of oxidative stress. Reactive oxygen species like superoxides, peroxynitrite, hydrogen peroxide (which can generate hydroxyl radicals and superoxides) are hig hly unstable molecules which can be toxic to neurons and cause damage to lipids, proteins and DNA. ROS can activate ASK1 (apoptosis signal regulating kinase) leading to apoptosis via the JNK pathway. NADPH (PHOX) knockout mice are protected from SNpc DA n eurodegeneration (W. Zhang et al., 2004) Furthermore, Nitric oxide produced by iNOS which is expressed in activated microglia serves as a toxic molecule as well as a marker
89 for microglial activation. NO reacts with superoxide to form peroxynitrite which is highly reactive and damaging. It can cause nitrosylation of various proteins and can induce PARP mediated cell death (A. A. Pieper et al., 1999) PARP signals apoptosis inducing factor (AIF) translocation from mitochondria to nucleus which in turn causes DNA fragmentation, membrane flipping and nuclear condensation independent of caspases. Alpha synuclein deposits in lewy bodies also appear to be nitrated b y peroxynitrite and iNOS & NO is found in CSF samples of PD patients (E. Paxinou et al., 2001) Overall, reactive microglia appears to cause a tremendo us inflammatory response which becomes neurotoxic. Although it is now clear that activated microglia could be detrimental to neurons, it is still ambiguous how it is activated in the first place. Microglia could be activated by foreign antigens such as LPS or internal factors. It is clear that microglia being macrophages would be attracted to the site of injury through a process known as chemotaxis. Chemotaxis refers to the chemical substances produced by the damaged cells which attract macrophages and othe r immune cells. Such signals signals described signals that activate microglia in different neurodegenerative conditions still remain to be discovered. A disturbance or loss of balance bet neurodegenerative diseases is responsible for the unwanted effects of microglia upon neurons. Under normal physiological conditions both signal classes work together to control beneficial or detrimental microglial functio ns and thus contribute to double edged sword of microglial activation (K. Biber et al., 2007) The a im of this paper was t o identify proteins released from mildly injured DA neurons that were involved in microglial activation and could serve as potential
90 lasses to gain better understanding of the vicious cycle of neuron microglia interplay. It is well documented that chronic microglial activation is associated with neuronal death. Multiple downstream inflammatory p athways take precedence over the neuropro tective functions of microglia. This eventually leads to further neuronal injury and ultimately neuronal demise. Delineation of the entire repertoire of neuronal signals activating microglia can jumpstart the development of inhibitor molecules to regulate microglial activation and thus prevent its downstream neurotoxic effects. In this paper we established a system to study neuron microglia interplay and to characterize secreted neuronal factors. We used a cell culture based model and mass spectrometry bas ed proteomic analysis to identify factors released from MPP + and dieldrin injured N27 DA neurons. Among the proteins that showed increased release was cystatin C, an extracellular cysteine protease inhibitor Results Toxicity of D ieldrin and MPP + on N 27 DA N eurons To induce injury to DA neurons, we used MPP + the active metabolite of the well characterized DA neurotoxin MPTP and the organochlorine pesticide dieldrin that is known to cause DA neurodegeneration (J. W. Langston et al., 1983) (H. Sharma et al., 2009) We first determined treatment conditions that would inf lict only modest reduction in cell viability as evidenced b y MTT assay. As shown in Fig. 3 1 A, treatment of N27 DA neuronal cells for 24 h with 0 100 M MPP + resulted in a concentration dependent reduction in cell viability, with 5% and 11% reduction obs erved for cells treated with 30 and 100 M MPP + respectively, compared to vehicle treated control cells. When N27
91 cells were treated for 24 h with 0 30 M dieldrin, an 8% and 14% reduction in cell viability was observed for cells treated with 10 and 30 M dieldrin, respectively (Fig. 3 1 B). Treatment for 6 h with 30 M dieldrin resulted in a 5% red uction in cell viability (Fig. 3 1 B). Soluble F actors Released F rom Neurotoxicant Injured Dopamine Neurons I nduce Microglial A ctivation Next, we prepared co nditioned media (CM) from N27 DA cells treated for 24 h with vehicle (CM V ), 30 M MPP + (CM M ) and 30 M dieldrin (CM D ). CM V CM M and CM D were added to the HAPI microglial cultures to determine their effect on the induction of iNOS, a marker for microglial activation (B. Liu et al., 2002) Compared to CM V neuronal CM M and CM D induced a significant upregulation of microglial iNOS protein expression and production of nitrite, a stable metab olite of NO (Fig. 3 2 A & 3 2 B). W e looked at different time points (8 h, 12 h, 24 h and 48 h) for induction of iNOS and established a bell shape curve for cystatin C mediated iNOS activation (data not shown). Since iNOS activation was at its peak at 12 h, we chose this time point for trea ting microglia with neuronal CM. Lack of a D irect E ffect of Neurotoxicants on Microglial A ctivation In contrast, direct treatment of HAPI microglial cells with comparable concentrations (1 30 M) of dieldrin or MPP + failed to induce iNOS upregulation (Fig. 3 3) and nitrite production (data not shown), while marked iNOS upregulation was observed with LPS, a potent stimulator of immune cells (B. Liu, 2006) (G. Dutta e t al., 2008) These results suggest that soluble factors released from DA neurons injured by exposure to DA neurotoxin MPP + or pesticide dieldrin were capable of augmenting microglial activation.
92 Proteomic Profiling of Soluble Factors Released From Neuro toxicant I njured DA N eurons To determine the identity and relative abundance of soluble factors released from injured DA neurons, we collected CM from N27 cells treated with vehicle (CM V ) or 30 M dieldrin (CM D ) for 6 h, a time point when cell viability s tarted to be affected and subjected it to a mass spectrometry based technique, iTRAQ. More than two hundred proteins were identified (data not shown) Pathway analysis of the identified proteins indicated a strong relevance to PD pathogenesis, cellular ca tabolism and biosynthesis, stress responses and signaling pathways (Table 3 1). Cystatin C Levels Increase in Toxicant Treated DA N eurons To identify neuronal factors potentially involved in the CM induc ed microglial activation (Fig. 3 4), we focused on th e proteins that exhibited increased abundance in CM from injured neurons. Of the >200 proteins identified in the CM from dieldrin treated N27 cells, the relative abundance of seven proteins was 2 3 times higher compared to that in CM fro m vehicle treated cells (Table 3 2). Of the 7 proteins, we started by exploring the potential role of cystatin C which was reported to enhance interferon gamma induced peritoneal macrophage activation in vitro (K. H. Frendeus et al., 2009) Western blot analysis of concentrated CM collected from N27 cells treated with 30 M dieldrin for 6 h showed a significantly augmented level of cystatin C compared to CM V (Fig. 3 4 A), consistent with that found with iTRAQ analysis of acetone p re cipitated CM proteins (Table 3 2). Cystatin C level was further increased in CM from N27 cells treated for 24 h with 30 M dieldrin (Fig. 3 4 A). Moreover, increased release of cystatin C was not limited to dieldrin injured N27 cells; treatment with MPP + (30 M, 2 4h) caused a significant eleva tion of cystatin C in CM (Fig. 3 4 B).
93 Discussion In this study we use an in vitro approach to recapitulate the essence of neuronal signals in regulating microglial functions. Literature is replete with the importanc e of neurons in activating microglia either by contact by virtue of membrane bound ligands or through secreted proteins. However, only a handful of studies have successfully demonstrated the complete loop of endogenous neuronal signals activating microglia and this activated microglia in turn being detrimental to neuronal health. We use pure DA neuronal (N27 cells) and microglial cell lines (HAPI cells) to show the distinct role of neurons and glia in this neuron microglia interplay. For example, extracellu lar synuclein activates microglia in primary mesencephalic neuron glia cultures and leads to enhanced DA neurotoxicity. Microglial synuclein and activation of NADPH oxidase were responsible for t his synuclein (W. Zhang et al., 2005) (A. D. Reynolds et al., 2008) synuclein is impli cated in microglial activation, it would be interest ing to see a similar effect of synuclein isolated from midbrain neuronal CM. That would show an endogenous molecular pathway common to many forms of neurodegeneration that bidirectionally links CNS inf lammation to neuronal death. In our study we use non toxic dose s of MPP + and dieldrin to inflict m ild injury to DA neurons (Fig. 3 1). We show that CM from these neurons is capable of activating microglia as evidenced by iNOS inductio n and nitrite product ion (Fig. 3 2 A a nd 3 2 B). We further confirm that this microglial activation is not an effect of the neurotoxicants by treating HAPI cells directly with different concentrations of MPP + and dieldrin (Fig. 3 3). Thus, we use a simple system to recapitulat e the neuron microglia cycle upstream of
94 microglial activation. The results (Fig. 3 2) very clearly point towards the involvement of proteins being released from injured DA neurons in activating microglia. Mixed neuron glia cultures albeit a closer repres entation of human physiology often times fail to distinguish the contribution of different brain cell types in the progression of neurodegeneration. Moreover, some proteins are released by both neurons and glia but contribute differently to a physiological or pathological process For synuclein knockout mice microglia display a basally reactive phenotype and secrete elevated levels of proinflammatory cytokines TNF 6 compared to wild type cells (S. A. Austin et al., 2006) synuclein or aggre synuclein has been reported to activate microglia (W. Zhang et al., 2005) Since we are particularly interested in signals released from dopaminergic neurons cuss in the next few paragraphs about the factors that have been reported in literature. These include MMP 3 which is released from apoptotic neurons and goes on to activate microglia (Y. S. Kim et al., 2005) Indee d MMP 3 / mice were significantly protected against MPTP induced dopaminergic neurodegeneration and showed minimal ROS production in substantia nigra and striatum (Y. S. Kim et al., 2007) It was also reported tha t MMP 3 induced ROS production and microglial activation in cell cultures and these activated microglia were capable of inducing widespread neuronal apoptosis in mixed neuron glia cultures which was attenuated when microglial NADPH oxidase was knocked out (Y. S. Kim et al., 2005) Similarly heat shock protein 60 was shown to be released from ruptured neurons which activated TLR4 receptor on microglia leading to its activation. HSP60
95 induced neurotoxicity was establis hed to be caused by NO overproduction and it was attenuated when microglia expressed a defective form of TLR4. However, this study was performed using primary cultures from frontal cortices and neurodegenerative effects were not established on dopaminergic neurons (S. Lehnardt et al., 2008) Micro calpain or calpain, a cytosolic calcium dependent protease was identified in the CM of MPP+ injured N27 cells. This neuronal CM activated microglia in mixed neuron glia c ultures. Addition of recombinant calpain showed a similar effect on microglia and also caused downstream DA neurodegeneration through NADPH oxidase mediated superoxide production (S. Levesque et al., 2010) In th is paper we use proteomics (iTRAQ) to unravel the identity of proteins present in N27 DA neuronal CM that were activating microglia. We found >200 proteins whose levels varied compared to control. Since we were interested in neuronal signals activating mic roglia we focused on 7 proteins which increased significantly (Table 3 2). Based on literature search we chose cystatin C as a potential candidate. Cystatin C belongs to the growing cystatin superfamily of cysteine protease inhibitors that have potential r oles in tumorigenesis, regulation of matrix metalloproteinases, kidney filtration, immune modulation and neurodegeneration (J. Ochieng and G. Chaudhur i, 2010) Members of family 1 cystatins (also called stefins) are normally unglycosylated small proteins that predominantly reside intracellularly. Family 3 cystatins (i.e., kininogens) are usually glycosylated large intravascular proteins. Cystatin C i s a member of type 2 cystatins that are slightly larger than family 1 cystatins in molecular mass. Some of the family 2 cystatins are glycosylated. Type 2 cystatins are mostly extracellular. Cystatin C is a constitutively secreted protein found primarily in CSF,
96 seminal plasma, urine, synovial fluid and blood plasma. Hydrophobic leader sequence of 26 amino acids (AA) is cleaved off during its secretion to release a 120AA mature protein (A. J. Barrett, 1986) (A. Anastasi et al., 1983) Perhaps the most well studie d involvement of cystatin C in pathological conditions is the association of urine cystatin C levels with kidney filtration capacities (F. Bonomini et al., 2009) Interestingly, increased levels of cystatin C have also been suspected to contribute to the atherosclerotic process and the development of cardiovascular diseases especially in patients with chronic kidney disord ers (N. Taglieri et al., 2009) In the brain, cystatin C is upregulated primarily in glial cells in the hippocampal regions of epileptic rats (T. J. Pirttila et al., 2005) In this study, we report that neurotoxin inflicted mild injury to DA neurons facilitates the release of DA neuronal cysta tin C into the CM (Fig. 3 4). This increased release of DA neuronal cystatin C is not a result of compromised cell membrane integrity as indicated by a lack of increase in extracellular levels of lactate dehydrogenase, an indicator of cell membrane breakage (data not shown), but rather a part of the secretome of the >200 proteins with strong correlatio n with pathways related to 1). We will be looking at the involvement of neuronal cystatin C in mediating m icroglial activation in C hapter 4 Based on our iTRAQ data and immunoblot results we hypothesize that cystatin C facilita tes microglial activation. Our approach of combining proteomic analysis with cell culture models may be a highly effective means in identification of key mediators of progressive neurodegeneration in complex and often idiopathic neurological disorders incl uding PD.
97 Figure 3 1. Effect of neurotoxicants MPP + and dieldrin on viability of N27 DA neuronal cells. N27 DA cells were treated for 24 h with vehicle or indicated concentrations of MPP + (A) or for 6 h or 24 h with indicated concentrations of dield rin (B). Cell viability was determined with MTT assay and expressed as a percentage of that of vehicle treated control cells. Results are mean SEM of 3 5 experiments performed in triplicate. *, p < 0.05 compared to the control.
98 Figure 3 2. CM fro m toxicant injured DA neurons induce microglial iNOS upregulation. CM collected from N27 DA neuronal cells treated for 24 h with vehicle (CM V ), 30 M MPP + (CM M ) or 30 M dieldrin ( CM D ) were used to treat HAPI microglial cells. Levels of iNOS protein (A) were determined at 12 h following actin. Nitrite production (B) was determined 12 h following CM treatment. Results are mean SEM of 3 experiments. and **, p < 0.05 and 0.005, compared to CM V treated HAP I microglial cells.
99 Figure 3 3. Effect of direct treatment with dieldrin or MPP + on microglial iNOS upregulation. HAPI microglial cells were t reated for 24 h with vehicle indicated concentrations of dieldrin or MPP + or LPS (10 ng/mL ) and iNOS prote in expression was determined. Results are representative of two experiments.
100 Figure 3 4 Neurotoxic assault promotes DA neuronal cystatin C release N27 DA neuronal cells were treated with vehicle or 30 M dieldrin for 6 or 24 h (A) or 30 M MPP + for 24 h (B). CM were collected and proteins (15 g) were resolved on a 12.5% polyacrylamide gel under standard electrophoresis conditions. The levels of cystatin C were then determined by immuno blotting. Results are expressed as band intensities normaliz ed to vehicle treated control and are mean SEM of three experiments. and **, p < 0.05 and 0.005 compared to the control respectively.
101 Table 3 1.Pathway analysis of identified proteins Number of genes in the pathway Pathway Actual Expected P v alue Parkinson disease 12 1.2 0 <0.005 Glycolysis 7 0.84 <0.005 Blood coagulation 5 0.54 <0.005 Cytoskeletal regulation by Rho GTPase 6 0.98 <0.005 p53 pathway 7 1.55 <0.005 FGF signaling pathway 6 1.26 <0.005 Pentose phosphate pathway 2 0.07 <0.05 Serine glycine biosynthesis 2 0.11 <0.05 FAS signaling pathway 3 0.35 <0.05 De novo purine biosynthesis 3 0.43 <0.05 Integrin signalling pathway 6 1.86 <0.05 Plasminogen activating cascade 2 0.18 <0.05 DNA replication 3 0.5 0 <0.05 Cadherin signaling pathway 5 1.52 <0.05 De novo pyrimidine deoxyribonucleotide biosynthesis 2 0.24 <0.05 Huntington disease 6 2.2 0 <0.05 De novo pyrmidine ribonucleotides biosythesis 2 0.25 <0.05 PLP biosynthesis 1 0.03 <0.05
102 Table 3 2. Proteins with increased abund ance following dieldrin treatment Accession No Protein Name Ratio IPI00230837.4 14 3 3 protein / 2.23 IPI00471889.6 Annexin A5 2.28 IPI00231801.3 Cystatin C 2.31 IPI00475503.2 Dynein, axonemal, heavy polypeptide 11 2.56 IPI00476899.1 Eukaryotic translation elongation factor 1 2 2.57 IPI00567665.1 38 kDa protein 3.17 IPI00387868.2 Ischemia responsive 94 kDa protein 3.26
103 CHAPTER 4 ROLE OF DOPAMINERGIC CYSTATIN C IN MICROG LIAL ACTIVATION AND DOWNSTREAM NEUROTOXI CITY Introduction One of the potential mechanisms of action underlying neurotoxicant induced progressive dopaminergic (DA) neurod development may involve a reciprocal interaction between DA neurons and brain immune cells (P. L. McGee r and E. G. McGeer, 2004) Early stage exposure to low levels of neurotoxicants may only cause mild damage to midbrain DA neurons that are known to be particularly vulnerable to stress (B. Liu, 2006) Injured DA neurons release neuronal fac tors, some of which can trigger a reactive activation of brain immune cells, especially microglia that are particularly abundant in the midbrain (B. Liu et al., 2000a) Activated microglia release a variety of pro inflammatory and neurotoxic factors that include nitric oxide (NO), reactive oxygen species (ROS) and cytokines including tumor necrosis factor alpha (TNF ) and interleukin 1beta (IL 1 ). Under chronic microglial activation t hese factors impact on DA neu rons to exacerbate DA neurotoxicity and more importantly, creat e a self amplifying vicious cycle of neuronal injury and microglial activation that fuels progressive DA neurodegeneration leading to the eventual development of symptomatic PD. Therefore, id entification of factors released from injured DA neurons and elucidation of their involvement in DA neurodegeneration should help uncover key mediators and critical events in the initiation and promotion stages of PD pathogenesis Previously we have ident ified a cysteine protease inhibitor, cystatin C as one such potential candidate. We saw a significant increase in levels of cystatin C in toxicant injured DA neurons both by mass spectrometry and immunoblotting. In this study, using
104 cystatin C antibody bas ed targeted immunodepletion, we determine the potential involvement of cystatin C in microglial activation and DA neurotoxicity in vitro. The relevance of our findings on the role of cystatin C in DA neurodegeneration is discussed. Results Absence of Cyst atin C From N euronal CM D A ttenuates CM D Induced Microglial A ctivation To decipher the role of cystatin C in the CM induced microglial activation, we determined the effect of cystatin C immunodepletion on microglia. Cystatin C depleted CM D was significant D or CM D with mock immunodepletion in the induction of microglial iNOS expressio n and nitrite production (Fig. 4 1 A & 4 1 B). As shown in Fig. 4 2, cystatin C was effectively removed from CM D following immunodepletion with an antibody against cystatin C, but not GFAP (Mock), an astrocyte specific protein. Cystatin C Depletion D ecreases Microglial ROS P roduction and C yto kine R elease Immunodepletion of cystatin C from CM D reduced microglial ROS production and cytokine release significantly over mock depeleted or non depleted CM D (Fig. 4 3 A, B & C). It is well documented that microglial activation precedes DA neurodegeneration in PD and could play a major role in neuronal damage by induction of iNOS, release of cytokin es and generation of oxidative stress (B. Liu, 2006) Hence, we used these markers to elucidate the role of cystatin C in microglial activation. Cystatin C is an Important Contributor to Microglial A ctivation C aused by CM M Loss of microgl ia activating capacity was not limited to cystatin C depleted CM D Microglial activation, as determined by iNOS expression and nitrite production was
105 markedly less robust when treated with CM from MPP + injured N27 cells (CM M ) following cystatin C immunode pletion compared to that treated with CM M without cystatin C immunodepletion or w ith mock immunodepletion (Fig. 4 4 A and 4 4 B). Neuronal Cystatin C A ctivat es M icroglia and Amplifies Downstream N eurotoxicity To determine the contribution of cystatin C to microglial activation mediated DA neurotoxicity, we used CM D with and without cystatin C immunodepletion to stimulate HAPI microglia and 12 h later applied supernatants from the HAPI microglia to N27 DA neurons. Twenty four h later, the N27 cells were fix ed and immunostained for activated caspase 3 for apoptosis and D API for nuclear staining (Fig. 4 5). No primary antibody was used as a negative control for background fluorescence (Fig. 4 5 A). Significant increase in the number of caspase 3 positive N27 DA neuronal cells was observed in cultures treated with supernatants from CM D activated microglia compared to that from CM V activated microglia (Fig. 4 6). However, supernatants from microglia stimulated with CM D devoid of cystatin C exhibited markedly re duced ability to induce caspase 3 activation in N27 cells (F ig. 4 6). These results indicate that cystatin C released from injured DA neurons is a key contributor to the induction of microglial activation and subsequent neurotoxicity. Recombinant Cystatin C Does N ot Activate M icroglia Next, we tested the effect of direct treatment with recombinant rat or human urine cystatin C on microglial activation. Incubation of HAPI microglial cells with either preparat ions of cystatin C (0.67 1 g/mL ) or the buffer vehicle for 24 h did not cause any detectable iNOS protein upregulation or nitrite production compared to the SF DMEM treated control cells, while LPS (in DMEM 1% FBS) caused a robust iNOS upregulation and nitrite production (Fig. 4 7 ).
106 Discussion In this study, we demonstrate that cystatin C released from neurotoxicant injured DA neurons is a key contributor to the induction of microglial activation and exacerb ation of DA neurodegeneration. This conclusion is supported by the following observations. First significantly increased release of cystatin C is identified by proteomic analysis and verified by immunoblot analysis (shown in C hapter 3) Second, immunodepletion of cystatin C markedly reduces the ability of CM from neurotoxicant injured DA neurons to activate microglia to upregulate iNOS, produce ROS and release proinflammatory cytokines (Fig. 4 1, 4 3 & 4 4) Third, immunodepletion of cystatin C consequently reduces activated microglia induced DA neurotoxicity as determined by the increase in caspas e 3 positive DA neurons (Fig. 4 5 & 4 6) Cystatin C is mostly a secreted protein and we focused on this extracellular cystatin C in our study. We found that the levels of secreted proteins in toxicant (dieldrin or MPP + ) treated CM were greater (~970g 5g, from two 10cm sister dishes) than vehicle treated CM (~780g 5g) Since we used equal protein loading for all our experiments we were loading significantly less volume of drug treated CM compared to control. This could imply that the actual concen tration of cystatin C in that specific volume was a lot less than our estimated protein concentration unless cystatin C was one of the most abundant sec retory proteins actin loading control could not be used for normalizing protein band intensity as it is mainly an intracellular protein. We used Ponceau S staining instead, to look at the bands on western blot. The r ole of cystatin C in inflammation and neurodegeneration has been conflicting T reatment of human monocytes and mouse periton eal macrophages with LPS and IFN reduced secretion of cystatin C in a dose dependent manner. It was postulated that
107 this could have an implication in pathophysiology of inflammation as cystatin C is a potent inhibitor of cathepsin B, an enzyme released by lysosomes durin g imflammation a nd phagocytosis (A. H. Warfel et al., 1987) some studies have sho wn that cystatin C reduces beta amyloid plaque formation and protects neurons against amyloid toxicity (W. Mi et al., 2007) (B. Tizon et al., 2010a) In vitro, increased cellular ex pression of cystatin C seems to be part of a defensive mechanism employed by cultured neurons under stress conditions (B. Tizon et al., 2010b) However, a number of findings have questioned the above mentioned anti imflammatory and neuroprotective role of cystatin C. Cystatin C was shown to augment inflammatory response by potentiating interferon induced activation of macrophages in culture (K. H. Frendeus et al., 2009) It has been implicated in apoptosis of neuronal cells both in vitro and in vivo (A. Nagai et al., 2005) Recently, it was shown that this neuronal apoptosis was mediated through a ctivation of JNK dependent pathway (X. Liang et al., 2011) Moreover, a nimal models have reported a d irect re lationship between temporal expression of cystatin C in glial cells and neuronal cells of hippocampus (T. J. Pirttila et al., 2005) (K. Lukasiuk et al., 2002) (G. X. Ying et al., 2002) Histological assessment of mo dels of transient forebrain ischemia showed similar results where cystatin C immunoreactivity was increased in morphologically degenerative pyramidal neurons (D. E. Palm et al., 1995) Cystatin C K/O mouse showed di minished brain damage after induction of global ischemia (T. Olsson et al., 2004) have show n that cystatin C colocalizes with amyloid beta precursor protein and may be
108 involved in its processing and aggregation (G. Vattemi et al., 2003) Deletion of cysta tin C in knockout mice results in an enhanced amyloid beta degradation. While in mice overexpressing cystatin C, no effect on cathepsin activity was observed, deletion of cystatin C results in an increased cathepsin B activity (B. Sun et al., 2008) (A. Nagai et al., 2005) This suggests that different mechanisms are responsible for the results obtained in transgenic mice compared to knockout mice. Point mutation of exon 2 in c ystatin C gene has also been implicated in hereditary cerebral angiopathy with amyloidosis (A. Palsdottir et al., 1988) In this study, for the first time we unravel the role of dopaminergic neuronal cystatin C in inducing microglial activation in relevance to PD. Direct intra nigral application of human cystatin C was shown to reduce 6 hydroxydopamine (6 OHDA) induced DA neurodegeneration in rat substantia nigra (L. Xu et al. 2005) This neuroprotective effect of cystatin C was attributed to its possible inhibition of cathepsin H, a cysteine protease whose levels were elevated in both striatum and SN following injury. The g lycosylation status of cystatin C seems to influen ce its neuromodulatory functions and could be be responsible for microglial activation by an alternate signaling pathway independent of the cathepsin axi s. In our study, both rat recombinant and human urine cystatin C failed to directly induce microglial a ctivation (Fig. 4 7 ). There could be multiple reasons for the failure of pure protein. First, recombinant cystatin C might not be folded properly as it was made in bacteria and bacteria do not have all the required chaperones Second reason could be that cystatin C needs a co factor for inducing microglial activation And lastly it could be that cystatin C needs to be post translational ly modified for inducing microglial activation The bacterial recombinant
109 protein and human urine cystatin C were non gly cosylated. As already pointed out, c ystatin C can exist in both glycosylated and non glycosylated forms. Infact multiple glycoforms of the protein have been reported (A. Dahl et al., 2004) So, the next logical step discuss in detail about the present understanding on the role of glycosylated cystatin C in the CNS and our findings on the importance of glycosylation for microglia activa ting capacity of cystatin C in C hapter 5.
110 Figure 4 1. Effect of cystatin C depletion from CM D on microglial iNOS upregulation (A) and nitrite production (B). HAPI microglial cells were treated for 12 h wit h CM v CM D CystC ID, CM D or CM D Mock ID. T he levels of iNOS protein (A) and production of nitrite (B) were determined. Results are mean SEM of three experiments. *, p < 0.05 co mpared to cells treated with CM V ; +, p < 0.05 co mpared to cells treated with CM D
111 Figure 4 2. Immunoblot to confir m successfu l removal of cystatin C from CM D Top Panel, immuno blot shows effectiv e removal of cystatin C from CM D with an antibody against cystatin C ( CM D CystC ID) but not GFAP ( CM D Mock ID); Bottom Panel, Ponceau S staining of the nitrocellulose membr ane for total protein loading (15 g).
112 Figure 4 3. Effect of cystatin C immuno depletion on CM D induced microglial ROS production and cytokine release. HAPI microglial cells were treated for 12 h with CM V CM D CM D CystC ID or CM D Mock ID. Microgli al ROS production ( A ) and release of TNF (B) and IL 1 ( C ) were determined. Results are mean SEM of three experiments. *, p < 0.05 compared to cells treated with CM V ; +, p < 0.05 compared to cells treated with CM D
113 F igure 4 4. Effect of cystatin C immuno depletion on the ability of CM f rom MPP + treated DA neurons to induce microglial iNOS upregulation. HAPI microglial cells were treated for 12 h with CM V CM M CystC ID, CM M or CM M Mock ID. Levels of iNOS protein ( A ) and nitrite production ( B ) were determined. Results are mean SEM fro m three experiments. ,* and **, p < 0.05 and p<0.005 compared to cells treated with CM V respectively ; +, p < 0.05 compared to cells treated with CM M
114 Figure 4 5. Immuno depletion of cystatin C reduces the ability of DA neuronal CM to cause microg lia mediated neurotoxicity. A a nti caspase 3 antibody was omitted to demonstrate the specificity of the immunostaining. B HAPI microglial cells were treated for 12 h with CM V CM D CM D CystC ID or CM D Mock ID Supernatants from treated HAPI microglial cells were then collected and applied to N27 DA neuronal cells. Twenty four h later, N27 cells were immunostained for cleaved caspase 3 and counter stained with DAPI for nuclei.
115 Figure 4 6. Quantification of DA neurotoxicity. N27 DA neuronal cells w ere treated with supernatants from neuronal CM treated HAPI microglial cells as described for Figure 4 5 In addition, as controls, N27 DA cells were treated for 24 h with microglial CM ( SF DMEM exposed to microglia for 24 h), SF RPMI DMEM (1:1) and SF RP MI (base neuronal medium). Following immunofluorescence staining, the number of cleaved caspase 3 positive cells was counted and normalized against that of DAPI positive cells. Results are mean SEM from three experiments. *, p < 0.05 compared to cells treated with supernatant from CM V treated microglial cells; +, p < 0.05 compared to cells treated with supernatant from CM D treated cells.
116 Figure 4 7. Rat recombinant or human urine cystatin C fails to induce microglial iNOS upregulation. HAPI micro glial cells were treated for 24 h with indicated amounts of recombinant rat cystatin C (CystC R ) or cystatin C purified from human urine (CystC H ). As controls, HAPI cells were treated with vehicle (buffer), DMEM 1% FBS and SF DMEM. LPS (10 ng/mL ) was used as a positive control. The levels of iNOS protein and nitrite production were determined. Results are from one representative experiment with similar results obtained in two other experiments.
117 CHAPTER 5 GLCOSYLATION STATUS OF CYSTATIN C IS IMP ORTANT F OR ITS MICROGLIA ACTIVATING POTENTIAL Introduction In Chapter 4 we saw that neuronal cystatin C is an important mediator of microglial activation. Its removal from neuronal CM attenuated microglial activation significantly. However, recombinant non glyco sylated rat and human cystatin C failed to show an effect on microglia directly. It is reasonable to assume that non glycosylated cystatin C by itself is incapable of inducing immune cell activation as evidenced by the requirement of the priming effect of interferon gamma (IFN ) on recombinant cystatin C induced macrophage activation (K. H. Frendeus et al., 2009) Similarly, only the glycosylated rat cystatin C, but not its N linked deglycosylated form, nor a synthetic peptide containin g the N carbohydrate moiety nor the non glycosylated chicken cystatin C which is highly homologous to rat cystatin C, has been shown to act as a co factor for fibroblast growth factor 2 induced neural stem cell proliferation (P. Taupin et al., 2000) It appears that glycosylated and non glycosylated cystatin C have distinctively different roles in neurodegeneration In this study we show that our neuronal cystatin C is glycosylated and glycosylated cystatin C f rom t oxin injured DA neurons plays an important role in mediating microglial activation and exacerbation of neurotoxicity Using a simplified in vitro cell culture apporoach we also show that D A neuronal cystatin C possess es unique qualitative and quantitative properties in its ability to ind uce microglial activation when compared to that from microglial cells and cortical neurons Before going into the results relevance to cystatin C.
118 Glycosylation is an enzymatic process that attaches glycans to proteins, lipids and organic molecules. It is a form of post translational modification. Majority of proteins synthesized in the rough endoplasmic reticulum (rER) undergo glyc osylation. Glycosylation helps in proper folding of proteins, provides stability to molecules and also aids in cell receptor or immune recognition. A ribosome in the cytosol begins synthesizing a protein until a signal recognition particle (SRP) recognizes an ER signal sequence which is a 5 10 hydrophobic amino acid sequence located at the N terminus of the nascent protein. SRP binds to the signal peptide allowing the ribosome SRP complex to bind to SRP receptor located on rER. SRP is a regulatory GTP prote in. The signal sequence is cleaved off within the lumen of the ER and the protein is now packaged into transport vesicles coated with COPI move and fuse with the plasma membrane and release the protein through exocytosis into the extracellular space. Majority of rER resident proteins are retained in th e ER through a retention motif located at the end of the protein sequence. There are different types of glycosylations. N linked glycosylation begins with the addition of a 14 sugar precursor to an amide molecule of asparagine in the polypeptide chain of the target protein. The structure of this precursor contains 3 glucose, 9 mannose and 2 N acetylglucosamine molecules. This 14 sugar unit is assembled in the cytoplasm and ER. Through a series of reactions, this branched chain i s transferred to a carrier molecule called dolichol and then it is transferred to the appropriate point on the polypeptide chain as it is translocated into the ER lumen. The oligosaccharide chain is attached through oligosaccharyltransferase to asparagine occurring in the tripeptide
119 sequence Asn X Ser or Asn X Thr where X could be any amino acid except proline. After attachment and proper folding of the protein, the 3 glucose residues are removed from the chain and the protein can be exported from the ER. T he glycoprotein thus formed is transported to golgi where the mannose residues are removed. Further removal of mannose residues leaves a core structure containing 3 mannose and 2 N acetylglucosamine residues which may be further elongated by sialic acid, N acetylglucosamine, N acetylgalactosamine, fucose or neuraminic acid. N linked glycans are extremely important in proper protein folding, cell cell interactions and targeting of lysosomal enzymes. N linked oligosaccharides may contribute 3.5 kDa or more to the mass of a glycoprotein. O linked glycosylation occurs at a later stage of protein processing in the golgi apparatus. It involves the addition of N acetyl galactosamine to serine or threonine residues (core structure) through specialized enzymes. A com mon theme in O linked glycosylation is the addition of polygalactosamine units to core structures formed by repetitive addition of galactose and N acetyl glucosamine units. Polylactosamine chains on O linked glycans are capped by the addition of sialic aci d. Sialic acid is a generic term for N or O substituted derivatives of neuraminic acid. They bear a negative charge via a carboxylic acid group which helps in water retention on the surface of the cell. They are normally expressed as terminal carbohydrat es on the glycoconjugates of eukaryotic cells. Sialylation serves a variety of functions like cell adhesion, signal recognition, virus infection and regulation of biological stability of a protein. The presence of sialic acid affects both the mass and char ge of a protein.
120 Results Neuronal Cystatin C is U nique Studies have shown that cystatin C can exist as a glycoprotein of multiple forms (A. Dahl et al., 2004) The lack of an effect of non glycosylated rat recombin ant and human urine cystatin C on microglial iNOS upregulation prompted us to determine whether the cystatin C in CM D from N27 DA neuronal cells was glycosylated. An extended resolution (16% SDS PAGE, 5 h compared to the usual separation condition of 12.5 % S DS PAGE in 1 h, Fig. 3 4 ) revealed that N27 DA neuronal cystatin C appeared as 4 bands: the predominant 17, 16 and the m inor 18 and 14 KD bands (Fig. 5 1 ). Treatment of CM D with deglycosylation enzyme s N g lycosidase F (PNGase F), O glycanase, or sialid ase A resulted in differential band patterns (Fig. 5 1 ) For instance, PNGase F treatment resulted in the disappearance of the 18 KD a band. Sialidase A treatment seemed to have caused the disappearance of the 17 KD a band and the shift of the 18 KD a band. Treatment with O glycanase did not result in a detectable change in the band mobility (Note that an air bubble formed during protein transfer caused the distortion in the lane. Results from two other experiments showed no changes in the band intensities ). Further treatment with both sialidase A and O glycanase or the combination of all three enzymes primarily led to the disappearance or reduction in the 17 and 18 KD a bands and the increase in inten sity of the lower bands (Fig. 5 1 ). O G lycosyla tion and S ial ylation are Important for Cystatin C Mediated Microglial iNOS U pregulation We next determined the effect of treatment with deglycosylation enzymes on the ability of CM D to activate micr oglia. As shown in Fig. 5 2 O glycanase or sialidase A treatm ent significant ly reduced CM D induced iNOS upregulation and nitrite production
121 although interestingly, the combined treatment with both enzymes was not more effective than either one alone (Fig. 5 2 A & B). Treatment with PNGase F or the combination of P NGase F sialidase A and O glycanase did not cause a reduction, and instead an augmentation, though statistically not significant, in CM D induced iNOS expression and nitrite production (Fig. 5 2 A & B) Removal of O G lycosyl and Sialic Acid Linkages Reduc e C ystatin C Induced M icroglial Cytokine P roduction In addition to a decrease in iNOS expression similar effect of deglycosylation was observed for CM D induced microglial release of TNF and IL 1 (Fig. 5 3 A & B ). Removal of O glycosyl or sialic linkages reduced CM D induced cytokine production CM D In contrast, treatment of CM D with PNGase F alone or all 3 enzymes combined increased microglial TNF and IL 1 release (Fig. 5 3 A & B). Differential Levels of Cystatin C in Neuronal and Glial Cells Under Normal Growth Conditions and Serum D eprivation To investigate whether the microglia activating ability was unique to the DA neuronal CM that contained elevated levels of cystatin C with a distinct glycosylation profile, we first wanted to compare the levels of cystatin C in the FBS containing maintenance media (CM 10% FBS for N27 and B35 cells, CM 5% FBS for HAPI cells) and serum free media (CM SF) that was exposed to respective ce lls for 24 h. As shown in Fig. 5 4, a faint cystatin C band was detected in media of N27 DA cells that were grown for 24 h in maintenance media (RPMI 1640 containing 10% FBS). The level of cystatin C increased > 3 fold in serum free media that were exposed to N27 cells for the same time period (Fig. 5 4). In the mean time, under the same conditions, cystatin C
122 was barely detectable in either the normal growth (DMEM 10% FBS) or serum free media that were exposed for 24 h to the B 35 c ortical neuronal cells (Fig. 5 4). In sharp contrast, the level of cystatin C in the media (DMEM 5% FBS) of normally growing HAPI microglial cells dwarfed that of N27 cells and it did not further elevate significantly in serum free media that were expo sed to HAPI microglia (Fig. 5 4). Cystatin C from challenged microglia does not activate microglia After having established that HAPI microglial cells were abundant producers of cystatin C we wanted t o determine w hether CM from these cells under stress wa s capable of inducing microglial activation For this, we used CM prepared from serum starved HAPI microglial cells and treated fresh cultures of HAPI cells. As shown in Fig. 5 5 A addition of 100 g HAPI CM proteins, similar to that used for N27 CM D di d not result in microglial iNOS upregulation. Immunoblot analysis showed that the amount of cystatin C in HAPI CM was five times that of CM from N27 DA cells cult ured in serum free media (Fig. 5 5 B). Neuronal and Microglial Cystatin C are D istinct fro m Each O ther Deglycosylation studies of HAPI microglial cystatin C indicated a differential profile from that of N27 DA neuronal cells. For instance, CM from HAPI microglial cells contained cystatin C that existed as a predominant 14 KD a band, less promin ent 16 KD a and 13 KDa band s and a v ery minor 18/19 KDa band (Fig. 5 6 ). Treatment of HAPI CM with PNGase F resulted in the disappearance of the 18 KD a band while s ialidase A treatment caused a reduction in the intensity of the 18 KD a band and near disappea rance of the 16 KD a band (Fig. 5 6 ) Treatment with O glycanase did not seem to have an impact on the 3 bands of the microglial cystatin C (Fig. 5 6 ) Band migration pattern following treatment with combination of sialidase A and O glycanase
123 was similar to that with sialidase A treatment alone Treatment with all three enzymes led to the disappearance of the 18/19 KD a ban d and the redu ction of the 16 KDa band (Fig. 5 6 ). CM fr om B35 Cortical Neuronal Cells C annot Upregulate M icroglial iNOS Besides serum starved microglia, we also determined the effect of CM from toxicant injured B35 cortical neurons on microglial activation. As shown in Fig. 5 7 A, mild yet significant reductions in cell viability were observed in B35 cells treated for 24 h with 30 and 5 0 M dieldrin (with 6.8 and 9.5% reductions respectively). Treatment of HAPI microglia with CM from dieldrin injured B35 cortical neuronal cells had no effect on HAPI microglial iNOS protein expression (Fig. 5 7 B). B35 Cortical Neurons Release Significan tly L ess Cystatin C Compared to N27 DA N eurons The level of cystatin C from B35 neurons in serum free media was only ~15% of that from N27 DA neurons under the same conditions and no significant increase was observed in B35 neurons treated for 2 4 h with 30 M dield rin (Fig. 5 8 ). These results indicate that DA neuronal cystatin C with distinct glycosylation modifications is a key contributor to neuronal injury induced microglial activation. Discussion In Chapter 4 we saw that non glycosylated recombinant cy statin C failed to induce microglial iNOS expression. Using a combination of deglycosylating enzymes, in this study we demonstrate that N27 DA neuronal cy statin C is glycosylated (Fig. 5 1) and that this glycosylation status is an important contributor to neuronal CM D mediat ed microglial activation (Fig. 5 2 and 5 3). Furthermore, compared to non DA neurons, DA neurons release cystatin C more readily in response t o the same toxicant treatment
124 (F ig. 5 8). DA neuronal cystatin C and microglial cystatin C, on the other hand differ in molecular mass especially that for the abundant species and i n glycosylation profiles (Fig. 5 1 & Fig. 5 6). Although c ystatin C is found in the cerebrospinal fluid (CSF) (A. J. Barrett, 1986) it remains to be determined how various types of neurons and non neuronal cells qualitatively and quantitatively contribute to th e presence of cystatin C in CSF under physiological and pathological conditions. This may be particularly the case when the presence and levels of cystatin C were determined using ELISA assays based on antibodies that, b y design, recognize cystatin C mole cules from vario us tissues in a given species. In this study, using pure populations of neuronal and glial cells, we have observed that under normal growth conditions, a far larger quantity of cystatin C was found in the CM of HAPI microglia than that from N27 DA neurons while the release of cystatin C from B35 neuron s was barely detectable (Fig. 5 4 ). In addition, cystatin C was present in high abundance in the CM of normally growing C6 glioma astrocytic cells ( data not shown ). This observation although based on in vitro studies suggests that, under physiological conditions, glial cells may be a major contributor to the presence of cystatin C in the CSF. Neurons appear to be a minor contributor to cystatin C released into the extracellular space under nor mal conditions O ur study has demonstrated that dopaminergic neurons that are known to be particularly vulnerable to stress (P. Jenner, 2003) but not non dopamine neurons or microglia, significantly augmented the secretion of cystatin C un der stress conditions su ch as serum deprivation (Fig. 5 4 ). More important ly cystatin C released from N27 DA neurons is different from HAPI microglia in molecular species and glycosylation profile (Fig. 5 1 & Fig. 5 6) and these
125 differences may be critica l to determining the involvement of cystatin C in microglial activation and the exacerbation of DA neurotoxicity. Our comparative studies suggest that cystatin C released from toxin injured DA neurons seems to possess certain unique characteristics in it s ability to induce microglial activation. First, the amount of cystatin C released from toxin injured B35 cortical neurons is a magnitude lower than that from N27 DA neurons under same toxic assault (30 M dieldrin for 24 h ) (Fig. 5 8 ). It is worth noti ng that B35 cortical neurons were less susceptible than N27 midbrain DA neurons to dieldrin induced damage (Fig. 5 7 A and Fig. 3 1 B) analogous to the innate vulnerability of midbrain DA neurons to toxic insult (P. Jenner, 2003) This ma rkedly reduced capacity to release cystatin C upon injury may explain the inability of CM from B35 cortical neurons to induce microglial activation (Fig. 5 7 B ). Second, HAPI microglial cells are capable of releasing a fair amount of cystatin C under stre ss (Fig. 5 5 B ). However, the molecular species and glycosylation modifications of microglial cystatin C (Fig. 5 6 ) seem to be quite different from that of DA neuronal cystatin C (Fig. 5 1 ). These differences may underlie, at least in part, the lack of a bility, in perhaps an autocrine manner, of cystatin C rich CM from HAPI microglial cells to i nduce its own activation (Fig. 5 5 A) Third, the existence of multiple species of cystatin C has been described (A. Dahl et al., 2004) Here we show that glyco modifications are important in mediating the induction of microglial activation b y DA neuronal cystatin C since the non glycosylated recombinant rat and human urine cystatin C could not produce any effect (Fig. 4 7 Chapter 4 ). Furthermore, certain glyco modifications such as O and sialic acid linked residues may be more
126 relevant than N linked residues to the microglia activating ability of DA neuronal cystatin C (Fig. 5 2 & Fig. 5 3 ). Our findings on the microg lia activating properties of glycosylated cystatin C released from mildly injured DA neurons derived from the midbrain region may be highly relevant to the pathogenesis of PD. Loss of midbrain nigrostriatal DA neurons is the pathological underpinning of P D. In addition to the innate vulnerability of the nigrostriatal DA neurons to damage, the midbrain region is highly enriched in microglia and the expression of proinflammatory and oxidative genes (D. C. Duke et al., 2007) (W. G. Kim et al., 2000) (L. J. Lawson et al., 1990) Therefore, upon a toxic insult, nigrostriatal DA neurons may be preferentially damaged. Cystatin C released from injured DA neurons can readily induce the activation of microglia that are abundant in the region. It is important to understa nd that once microglia is activated, it causes neurodegeneration by activation of multiple pathways which makes it difficult for devising therapeutic strategies. This creates a vicious cycle of neuronal injury and microglial activation with released DA neu ronal cystatin C as one of the contributors to the process that may underlie the progressive DA neurodegeneration in PD. Thus, shifting focus on factors which activate microglia is important as this would prevent detrimental microglial activation altoget her. Although many factors might be involved in microglial activation, intervention of upstream pathways would always prevent downstream amplification. Validation of findings in animal PD models and human studies and continued deciphering of the detailed m echanism responsible for the neuronal release and microglia activating properties of cystatin C should open a new avenue in our search for therapeutic targets and diagnostic biomarkers for PD.
127 Figure 5 1. Cystatin C released from toxin injured DA neu rons is glycosylated. Neuronal CM D were treated with buffer (CM D ) or PNGase F (CM D PNG), sialidase A (CM D Sial), O glycanase (CM D O gly), combination of Sialidase A and O glycanase (CM D Sial + O gly) or that of PNGase F, Sialidase A and O glycanase (CM D PNG + Sial + O gly). Treated proteins (15 g) were resolved, under extended electrophoresis conditions, on a 16% polyacrylamide gel followed by immunoblotting with the anti cystatin C antibody. Cystatin C showed four bands of 18 17, 16 and 14 KD respect ively as depicted by arrows. Results are from one representative experiment with similar results obtained in two other experiments.
128 Figure 5 2. Glycosylat ion status influences microglia activating potential of secreted DA neuronal cystatin C. DA neuro nal CM D treated with buffer control (none) or de glycosylation enzymes as described for Fig. 5 1 were used to treat HAPI microglial cells. Twelve h later, the levels of iNOS protein ( A ), nitrite production ( B ) were determined. Results are mean SEM from three experiments. +, p < 0.05 compared to buffer treated CM D (none).
129 Figure 5 3. O linked glycosylation and sialylation of DA neuronal cystatin C are important for microglial cytokine production. DA neuronal CM D treated with buffer control (none) or de glycosylation enzymes as described for Fig. 5 1 were used to treat HAPI microglial cells. Twelve h later, the levels of TNF (A) and IL experiments.
130 1 3.80.17 1 1.40.19 Figure 5 4. Comparison of the abundance of cystatin C in normal growth media and serum free media. N27 DA neurons, B35 cortical neurons and HAPI microglial cells were grown in serum c ontaining media or serum free me dia for 24 h. Equal volume (14mL ) of media were passed through 30 KDa centrifugal filters to remove large serum proteins inclu ding albumin. The filtrate (13mL ) was then concentrated using 3 KDa cen trifugal concentrators to 0 .5 mL each. Equal volumes (12 L for N27 and B35 cells and 3 L for HAPI cells) of concentrated normal or serum free media were then used for immunoblotting for cystatin C. Results are mean SEM from three experiments. Numbers at the bottom indicate cysta tin C band intensity normalized against that in media from cells grown in normal maintenance media. *, p < 0.05 compared to normal maintenance media.
131 Figure 5 5. Lack of an effect on microglial activation by cystatin C released from microglial cells. A, CM from HAPI microglial cells grown for 24 h in SF DMEM ([CM(HAPI)] were concentrated and used to treat fresh cultures of HAPI microglia for 24 h. As controls, HAPI cells were treated with SF DMEM, DMEM 5% FBS and LPS. Levels of iNOS protein were the n determined by immunoblotting. B, CM from N27 DA neuronal and HAPI microglial cells grown for 24 h in serum free media were collected, concentrated and then assayed for the amounts of cystatin C by immunoblotting (16% gel). Due to the high abundance of cystatin C in HAPI cell CM detected in pilot studies, 3.5 g of CM proteins from N27 and HAPI cells were used for comparison. The intensities of the cystatin C bands of HAPI cells were normalized against that of N27 cells. Results are mean SEM from thr ee experiments. **, p < 0.005 compared to N27 cells.
132 Figure 5 6. Microglial cystatin C has a different composition from neuronal cystatin C. CM from HAPI microglial cells were treated with buffer (none), PNGase F (PNG), sialidase A (Sial), O glyca nase (O gly), combination of Sialidase A and O glycanase (Sial + O gly) or combination of PNGase F, sialidase A and O glycanase (PNG + Sial + O gly). Treated proteins (3.5 g) were resolved (16% gel) and immunoblotted with the anti cystatin C antibody. C ystatin C showed four bands of 18/19, 16, 14 and 13 KD respectively as indicated by arrows. Results are from one representative experiment with similar results obtained in two other experiments.
133 Figure 5 7. C ystatin C from toxicant injured B35 corti cal neuronal cells fails to induce microglial iNOS upregulation B35 neuronal cells were treated for 24 h with vehicle or indicated concentrations of dieldrin (A). Cell viability was determined with MTT assay and expressed as a percentage of that of vehi cle treated control cells. Results are mean SEM of 3 experiments performed in triplicate. *, p < 0.05 compared to the control. ( B ) CM from B35 cells treated (24 h) with vehicle (CM V ) or 30 M dieldrin (CM D ) were used to treat HAPI microglial cells. SF DMEM, DMEM 5 %FBS and LPS were used as controls. Levels of iNOS protein were determined 12 h later Results are from one experiment with similar results obtained in two other experiments.
134 Figure 5 8. Effect of dieldrin treatment on cystatin C re lease in B35 cortical neurons. CM from B35 cells treated for 24 h with serum free media alone or that containing 30 M dieldrin were assayed for the amounts of cystatin C by immunoblotting (35 g protein, 16% gel). CM from N27 DA neurons (35 g protein) w as included in the same experiment as a reference. The intensity of the B35 cystatin C bands was normalized against that of N27 cells. Results are mean SEM from three experiments. **, p < 0.005 compared to N27 cells.
135 CHAPTER 6 OVERALL SUMMARY AND C ONCLUSIONS Neuroinflammation is a common theme unifying majority of neurod egenerative diseases like ALS, Multiple S (PD) and is characterized by microglial activation (R. F. Pfeiffer, 2009) Research in the last decad e has provided conclusive evidence supporting the precedence of microglial activation over neurodegeneration. Currently, focus on neuronal signals involved in regulating microglial phenotype has gained tremendous momentum with the last few years revealing factors like HSP60, MMP 3, synuclein (S. Lehnardt et al., 2008) (S. A. Austin et al., 2006) (Y. S. Kim et al., 2005) (S. Levesque et al., 2010) I n vivo knockout studies of these f actors individually have been successful in reversing PD pathology in animals to a ce rtain extent. However, none of these signals have shown enough promise to extr apolate their DA modulatory effects to clinically relevant settings. Considering the multifactorial etiology of PD it would be really difficult to modulate the disease significantly by targeting one specific neuronal factor. Hence, we believe that a more potent treatment strategy would be to target multiple neuron al signals with different mechanisms for initiating cross talk with microglia so that the compensatory mechanisms covering up for the lack of one molecule in the pool of neuronal factors can no longer come into limelight. Our experimental research added a novel factor, cystatin C to the list of neuronal signals mediating microglial activation. We used an in vitro cell culture based approach for the entire study. Furthermore, we worked with pure DA neuronal cells (N27) and microglial (HAPI) cells as they r epresented a more homogenous and abundant source of neuronal and non neuronal cells. Since neuron microglia dialogue becomes more
136 prominent in cases of neuronal injury we used neurotoxicant MPP + and pesticide dieldrin to inflict injury to DA neurons and ex perimentally elucidate the role of released proteinaceo us factors in activating microglia In C hapter 3 we saw that toxicant injured N27 DA neurons released soluble factors that were capable of inducing microglial iNOS upregulatio n and nitrite production ( Fig. 3 2). Using iTRAQ we successfully revealed the composition of this neuronal CM. We saw a number of factors whose levels were dysregulated upon drug treatment in comparison to control CM. Since our goal was to elucidate microglia activating signals we only concentrated on neuronal proteins whose levels went up sig nificantly after injury (Table 3 2). Cystatin C was chosen as one of the candidate proteins based on the available literature on its role in neurodegeneration. An alternative experiment c ould h ave been fractionation of the supernatant to isolate a fraction that retained microglia activating potential followed by iTRAQ to identify proteins in that fraction. Instead we preferred to evaluate the whole extracellular secretome as microglia activating potential could lie with a complex of proteins instead of a sin gle one which could be lost upon fractionation. As already discussed the protein is a cysteine protease inhibitor and is implicated as a diagnostic biomarker for kidney filtration disease (F. Bonomini et al., 2009) Its role in neurodegeneration is elusive and no consensus has been reached as to whether it is protective or damaging of DA neurons in relevance to PD. The various glycosylated forms of cystatin C add another wrinkle to this confusion as studies report mitogenic activity of the glycosylated protein acting as a co factor for FGF 2 (P. Taupin et al., 2000) and promoting macrophage activation in response to IFN gamma (K. H. Frendeus et al., 2009) Rat CM enriched with FGF 2 and glycosylated cystatin C was
137 also shown to suc cessfully proliferate adult neuronal stem cells derived from human post mortem brains (T. D Palmer et al., 2001) In C hapters 4 and 5 we highlight the involvement of DA cystatin C in microglial activation for the first time. Using immunodepletion studies we show that cystatin C null CM has a tremendously reduced capacity for activating microg lia in terms of iNOS expression (Fig. 4 1 A & 4 4 A), nitrite production (Fig. 4 1 B & 4 4 B), ROS production (Fig. 4 3 A) and cytokine release (Fig. 4 3 B & C). Evaluation of ROS production after treating microglial cells with NADPH oxidase inhibitors lik e apocyanin would confirm the involvement of the enzyme complex in producing ROS. We go a step further and complete the loop of neuron microglia interplay by demonstrating the downstream neurotoxic potential of HAPI microglial CM initially primed with cys tatin C replete /null neuronal CM as evidenced by immunofluorescent staini ng for cleaved caspase 3 (Fig. 4 5 & 4 6). Since cystatin C is abundantly released by both neurons and glia we wanted to confirm the involvement of neuronal paracrine signaling in ind ucing microglia and thereby eliminating the possibility of microglial cystatin C activating itsel f. Failure of non glycosylated recombinant and human urine cystatin C protein in activating microglia (Fig. 4 7) further compelled us to look into the composit ion of neuronal and microglial cystatin C. Deglycosylation studies and band migration pattern of the various deglycosylated isoforms of neuronal and microglial cystatin C on immunoblots made a very clear distinction in the glycosylation profile of cystatin C from the two cell types (Fig. 5 1 & 5 6). This could be the reason for the inability of microglial CM t o induce self activation (Fig. 5 5 A). O linked glycosylation and sialylation of neuronal cystatin C seemed to be of particular importance in regulati ng
138 microglial activation (Fig. 5 2 & 5 3). On the other hand, removal of N linked carbohydrate moieties increased expression of iNOS and production of nitrite and cytokines pointing towards its role in maintaining qui escent microglial states (Fig. 5 2 & 5 3). However our data was not statistically significant for a conclusive result. Using comparison based studies we also show that glia cells secrete cystatin C at a much higher level than DA or cortical neuronal cells (Fig. 5 4 & Fig. 5 5). Thus, based on a simplistic cell culture study we can speculate that the contribution of glia to the presence of cystatin C in CSF might be strikingly higher than other cell types. Cystatin C released from cortical neuronal cells did not produce any microglial act ivation (Fig. 5 7 B) which showed that cystatin C from DA neurons was specific in activating microglia and thus strengthened the already known fact of heightened innate vulnerability of DA neurons to toxic insults (P. Jenner, 2003) Significantly low levels of cystatin C in CM from cortical neurons compared to that from DA neurons could also be responsible for its failure in activating microglia (Fig. 5 7). Hence, using cell culture system we show that glycosylated DA neuronal cystatin C is a pivo tal player in mediating microglial activation and neurotoxicity. A major drawback of this study was that we could not procure glycosylated cystatin C in its pure form and all the experiments were an indirect indication of involvement of cystatin C in medi ating microglial activation Other proteins in the neuronal CM were also getting delgycosylated in addition to cystatin C using the deglycosylating enzymes. Similarly, even after removing cystatin C from CM we could not attribute the microglia activating p otential to it solely. The probability of other neuronal proteins acting as activator signals cannot be ignored.
139 It would be interesting to see the effect of cystatin C on microglial activation in mixed neuron glia cultures. Based on the current understan ding from our data we can expect to see an increased sensitivity of microglia to extracellular cystatin C in the surrounding neuronal environment. Moreover, non glycosylated recombinant cystatin C could be effective in activating microglia in the presence of other neuronal factors which could be acting in cohort with the protein t o induce microglial activation. Immunodepletion data can be strengthened further by using siRNA to knockdown cystatin C in neuronal CM and evaluating the microglia activating capac ity using activation specific markers. Immunoprecipitation of the protein using non denaturing conditions followed by mass spectrometry can be helpful in determining if cystatin C exists in combination with other proteins as a complex. Functional studies e lucidating the role of this isolated protein complex in microglial activation can shed more light on its mechanism of action. Inhibitor drugs that attenuate cystatin C signaling pathway can strengthen the role of cystatin C in regulating glia l morphology. Cystatin C could act by neutralizing or inhibiting cathepsins. Cystatin C is a cysteine protease inhibitor which blocks the action of cathepsins. Cathepsins B and D have established roles in inflammataion and mediating neuronal apoptosis. It is possible t hat imbalance between cystatin C and cathepsins in our system could lead to microglial activation. For the same reason, cathepsin levels can be monitored by immunoblotting both in drug treated neuronal cultures and in microglial cultures pre and post treat ment with neuronal CM. Also, other proteins whose levels increased in neuronal CM like annexin 5 an d ischemia responsive protein (Table 3 2) should be considered for future studies. Finally culmination of the study to in vivo animal models in terms of ne w inhibitor molecules for
140 cystatin C mediated microglial activation signaling would be the ultimate test for the therapeutic potential of our novel finding.
141 Figure 6 1. N euron microglia interplay. N eurotoxicants like MPP + and dieldrin injure DA neuron s to release protei naceous factors like Cystatin C MMP synuclein. T hese factors act on microglia to activate it and propel a downwards cycle of microglial activation mediated release of proinflammator y factors and cytokines like TNF 1 B y virtue of these toxic factors activated microglia can promote further neuronal injury and ultimately cause neuronal death. M odulation of neuronal factors activating microglia in addition to anti inflammatory agents can be an effective treatment strategy.
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174 BIOGRAPHICAL SKETCH Garima Dutta was born in New Delhi, India to Arun kumar Du tta and Veena Dutta. She got inte rested in pursuing a career in p harma cy after completing a two year d iploma in p harmacy course from the Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR), India. She was amongst the top five candidates who go t admitted direc tly to second year of Bachelor of Pharmacy in DIPSAR. Followin g successful completion of her b achelor s education she came to the University o f Florida to obtain her PhD in p harmacodynamics. She worked in the lab oratory of Dr Bin Liu on the intends to continue her career as a postdoc toral fellow in the laboratory of Dr Marie Francoise Chesselet at UCLA California and ultimately get a permanent position in academia.