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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-12-31.

DARK ITEM
Permanent Link: http://ufdc.ufl.edu/UFE0044652/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-12-31.
Physical Description: Book
Language: english
Creator: Liu, Yue
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: Pharmacodynamics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Yue Liu.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Liu, Bin.
Electronic Access: INACCESSIBLE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044652:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044652/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-12-31.
Physical Description: Book
Language: english
Creator: Liu, Yue
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: Pharmacodynamics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Yue Liu.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Liu, Bin.
Electronic Access: INACCESSIBLE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044652:00001


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1 COMPLEX II OF THE MITOCHONDRIAL RESPIRATORY CHAIN IS TH E KEY MEDIATOR OF DIVALENT MANGA NESE INDUCED HYDROGEN PEROXIDE PRODUCTION IN MICROGLIA By YUE LIU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF F LORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Yue Liu

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3 To my parents and grandparents

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4 ACKNOWLEDG E MENTS I would like to begin my acknowledgement by thanking my advisor Dr. Bin Liu. Dr. Liu has been a big inspiration professionally and a very wise man in my life outside the scientific world. He is like a father or a friend with whom I can discuss anything in my life with and know he would be supportive. He is ver y close to my heart and it will remain so for the rest of my life. Next, I would like to thank Dr. David Barber and Dr. Scott Grieshaber who are both brilliant scientists. I had the honor to do part of my graduate research with them and have been impresse d with the way they think, t hey do research, and their warm hearted help. I would like to especially thank Dr. Scott Grieshaber for near the end of my PhD study. I would als o like to thank Dr. Barber and Dr. Grieshaber for reading my manuscripts and giving comment s for publication. I would like to thank my committee member Dr. Joanna Peris, who is the most fun, kind and sweetest person I have ever known During my rotation wi th her I was astonished at how brilliant ideas would flow out of her mind. Later in my graduate study, she has been a positive energy in my committee which magically gave me comfort in the most stressful exams. Her positive energy has also influenced the professors I applied to for postdoc positions. I would like to thank my committee member Dr. Dorette Ellis. Dr. Ellis cares about both my project and how I was doing in my personal life. She cheered me up and offered professional opinions when I had tough times with my project. She offered me warmth a nd comfort at the most difficult time in my personal life. She made me felt comfortable to open up and talk to her about any difficulties. I am so thankful that I have her with me during my study and in my life I am so blessed to have such a special group of brilliant scientists as my dissertation committee. In addition, I would also like to thank all the

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5 professors in the department for providing a friendly, equal environment to get my PhD degree. I would also like to thank the staff of the department office: Tammy Ridgeway, Donna Walko and Natalie Torres for their friendship and most importantly, their assistance with numerous paper works throughout my staying at the University of Florida. I would like to than k Dr. Ping Zhang for showing me how to do hydrogen peroxide assay. I would also like to thank my dearest friends Dr. James Kasper, Dr. Garima Dutta, Dr. Heera Sharma, and Rajiv Tikamdas for their friendship and support. They have been there through my ups and downs, happy moments and sad moments. They are the greatest treasure I found here during my PhD study. Special thanks to my boyfriend Robert Ng, who is the most loving, inspiring and diligent person that I know in my generation. He is the major daily d riving force for me to work hard and plan for the future. He tolerates me for who I am to make me happy and i n good mood to go to work every day And he is always available to help me when I needed him. I could not have done this without him. I would lik e to thank my parents: Weiguo Liu and Xiaohua Liu; and grandparents: Zhenfan Hou, Jiexin Liu and Yuqin g Geng for their unconditional love, understanding and support There are no words to describe the love that I feel in my heart from my family. At the e nd of my acknowledgements, I would like to dedicate this dissertation to my dearest grandfather Zhenfan Hou, whose loving memory would accompany me no matter where life takes me.

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6 TABLE OF CONTENTS P age ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 BACKGROUND AND INTRODUCTION ................................ ................................ 16 Manganese (Mn) Toxic ity ................................ ................................ ........................ 16 Mn Essentiality ................................ ................................ ................................ 16 Mn Levels in Food ................................ ................................ ............................ 17 Mn Neurotoxicity ................................ ................................ ............................... 17 Sources of Mn Exposure ................................ ................................ .................. 18 Occupational exposure ................................ ................................ .............. 18 Environmental ex posure ................................ ................................ ............ 20 Medical and others sources ................................ ................................ ....... 23 Diagnosis and Biomarkers for Manganism ................................ ....................... 23 Treatments ................................ ................................ ................................ ....... 24 ................................ ................................ ............................... 25 Definition and History ................................ ................................ ....................... 25 Symptoms and Diagnosis ................................ ................................ ................. 26 Dopaminergic Neurons ................................ ................................ ..................... 26 Etiology and Risk Factors ................................ ................................ ................. 27 Genetic factors ................................ ................................ ........................... 27 Environmental factors ................................ ................................ ................ 27 Treatment Options ................................ ................................ ............................ 28 Medication ................................ ................................ ................................ .. 28 Deep brain stimulation ................................ ................................ ............... 28 Gene therapy ................................ ................................ ............................. 29 Similarities and Differences Between Manganism and iPD ................................ .... 29 Symptoms and Neuropathology ................................ ................................ ....... 29 Molecular Mechanisms ................................ ................................ ..................... 31 Mechanism of Mn Neurotoxicity ................................ ................................ .............. 32 Mitochondrial Dysfunction ................................ ................................ ................ 32 Oxidative St ress ................................ ................................ ............................... 33 Reactive oxygen species ................................ ................................ ........... 33 Oxidative stress in manganism ................................ ................................ .. 35

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7 Effects of Mn on Neuronal Synapses ................................ ............................... 36 Dopaminergic synapses ................................ ................................ ............. 36 GABAergic synapses ................................ ................................ ................. 37 Glutamatergic synapses ................................ ................................ ............ 37 Glial mediated Inflammation ................................ ................................ ............. 39 Astroglia ................................ ................................ ................................ ..... 39 Microglia ................................ ................................ ................................ ..... 40 Gene Expression Studies ................................ ................................ ................. 41 Interaction with Iron Regulation ................................ ................................ ........ 41 Mn Uptake and Subcellular Distribution ................................ ................................ .. 42 Mn Homeostatic Control ................................ ................................ ................... 42 Mn Species in the Blood ................................ ................................ ................... 42 Toxicokinetics ................................ ................................ ................................ ... 44 Routes of Mn Uptake into the CNS ................................ ................................ .. 44 Mechanism of Mn T ransport across the BBB ................................ ................... 45 Transferrin (Tf) ................................ ................................ ........................... 45 Voltage dependent and store operated calcium channels (SOCCs) .......... 46 DMT1 ................................ ................................ ................................ ......... 46 Zip 8 and zip 14 ................................ ................................ ......................... 47 Mechanism of Mn Transport across the Blood CSF B arrier ............................. 47 Uptake by Neuronal and Glial Cells ................................ ................................ .. 48 Mn Axonal Transport ................................ ................................ ........................ 49 Mn Su bcellular Distribution ................................ ................................ ............... 49 Efflux of Mn from CNS ................................ ................................ ...................... 51 Specific Aims ................................ ................................ ................................ .......... 52 2 MATERIAL AND METHODS ................................ ................................ .................. 57 Materials ................................ ................................ ................................ ................. 57 Methods ................................ ................................ ................................ .................. 58 Rat HAPI M icrogl ial C ell C ulture ................................ ................................ ....... 58 Rat P rimary M ixed G lia and A stroglia C ulture s ................................ ................ 58 Preparation of Microglial Mitochondrial, Nuclear and Cytosoli c Fractions ........ 59 Mitochondrial Respiration Assay ................................ ................................ ...... 60 Measurement of H 2 O 2 P roduction ................................ ................................ .... 61 Measurement of Intracellular ROS Production ................................ ................. 62 Western B lot A nalysis ................................ ................................ ...................... 63 ICP MS Analysis ................................ ................................ .............................. 63 Confocal Microscopy and Image Acquisition ................................ .................... 64 Statistical Analysis ................................ ................................ ............................ 65 3 THE INVOLVEMENT OF GLIAL CELLS IN MN 2+ INDUCED OXIDATIVE STRESS ................................ ................................ ................................ ................. 69 Introduction ................................ ................................ ................................ ............. 69 Rationale ................................ ................................ ................................ .......... 69 Hypothesis ................................ ................................ ................................ ........ 70

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8 Experimental Design ................................ ................................ ............................... 70 Results ................................ ................................ ................................ .................... 71 Effect o f Mn 2+ on H 2 O 2 Release in Rat HAPI Microglial Cells, Rat Primary Mixed Glia and Astroglia ................................ ................................ ............... 71 Effect of Divalent Metals on H 2 O 2 Release in Rat HAPI Microglial Cells, Rat Primary Mixed Glia and A stroglia ................................ ................................ .. 71 Discussion ................................ ................................ ................................ .............. 72 Relative C ontribution of M icroglia vs. A stroglia to Mn 2+ induced H 2 O 2 R elease ................................ ................................ ................................ ......... 73 Oxidative S tress I nduced by D ivalent T ransition Metal s ................................ ... 73 4 THE INVOLVEMENT OF MITOCHONDRIA IN MN 2+ MEDIATED NEUROTOXICITY ................................ ................................ ................................ .. 81 Introduction ................................ ................................ ................................ ............. 81 Rationale ................................ ................................ ................................ .......... 81 Hypothesis ................................ ................................ ................................ ........ 82 Experimental Design ................................ ................................ ............................... 82 Results ................................ ................................ ................................ .................... 83 Mitochondria are the M ajor S ubcellular Site of Mn 2+ induced H 2 O 2 P roduction ................................ ................................ ................................ ..... 83 Complex II S ubstrate is M ore E fficient than C omplex I S ubstrates in S upporting the Mn 2+ induced H 2 O 2 P roduction ................................ ............. 84 ETC c omplex II P lays a M ore P rominent R ole t han C omplex I in Mn 2+ induced H 2 O 2 P roduction ................................ ................................ ............... 85 Discussion ................................ ................................ ................................ .............. 87 Mitochondria as the S ubcellular S ite of Mn 2 + induced ROS G eneration in M icroglia ................................ ................................ ................................ ........ 87 Involvement of ETC Complexes in Mn 2+ induced H 2 O 2 Release in Microglia ... 88 Complex II in M n 2+ induced H 2 O 2 Production ................................ ................... 89 5 THE SUBCELLULAR DISTRIBUTION OF MN 2+ IN MICROGLIA ......................... 100 Introduction ................................ ................................ ................................ ........... 100 Rationale ................................ ................................ ................................ ........ 101 Hypothesis ................................ ................................ ................................ ...... 102 Experimental D esign ................................ ................................ ............................. 102 Results ................................ ................................ ................................ .................. 102 Mn 2+ Influx into the Nucleus and Mitochondria in Microglia Assessed by Confocal Microscopy ................................ ................................ ................... 102 Quantitative Evaluation of Mn in Different Cell Compartments by ICP MS .... 103 Discussion ................................ ................................ ................................ ............ 104 6 SUMMARY AND FUTURE DIRECTIONS ................................ ............................ 113 Grand Conclusion and Relevance ................................ ................................ ........ 113 Major Limitation of Current Study ................................ ................................ ......... 115

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9 Future Directions ................................ ................................ ................................ .. 115 LIST OF REFERENCES ................................ ................................ ............................. 118 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 137

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10 LIST OF TABLES Table page 1 1 Typical levels of Mn in common food sources listed by Agency for Toxic Substances and Disease Registry ................................ ................................ ...... 56

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11 LIST OF FIGURES Figure page 1 1 Reactive oxygen species reactions.. ................................ ................................ .. 54 1 2 Mitochondrial electron transport chain. ................................ ............................... 55 2 1 Reaction of a mple x red to r esofufin. ................................ ................................ ... 66 2 2 Microglial cell fractionation and purification of mitochondrial and nuclear fractions. ................................ ................................ ................................ ............. 67 2 3 Effect of the presence of mitochondrial protein on H 2 O 2 determination. .......... 68 3 1 Experimental design of effects of Mn 2+ and other divalent metals on H 2 O 2 prod uction ................................ ................................ ................................ ......... 75 3 2 Effects of Mn 2+ on H 2 O 2 production in HAPI microglial cells, mixed glia and astroglia cultures. ................................ ................................ ............................... 76 3 3 Effects of divalent cations on H 2 O 2 production in HAPI microglial cells, mixed glia and astroglia cultures. ................................ ................................ .................. 77 ................................ ................................ ................................ ................... 78 3 5 E ffects of divalent metals at 100 M on H 2 O 2 production in HAPI microglial cells. ................................ ................................ ................................ ................... 79 3 6 Effect s of divalent metals on ROS produ ction in HAPI microglial cell. ................ 80 4 1 Mitochondrial ETC complexes inhibitors and substrates ................................ ... 92 4 2 Effect of Mn 2+ on H 2 O 2 production in mitochondrial, cytosolic and nuclear fractions of HAPI microglial cells. ................................ ................................ ....... 93 4 3 Verification of m itochondrial functionality and purit y of subcellular fractions. ..... 94 4 4 Substrate specificity for induced H 2 O 2 production from mitochondria. ....... 95 4 5 Effects of on mitochondrial complex II respiration. ................................ ..... 96 4 6 Effects of the ETC inhibitors and on H 2 O 2 production from mitochondria in the presence of complex II substrate. ................................ ............................. 97 4 7 Effects of the ETC inhibitors and on H 2 O 2 production from mitochondria in the presence of complex I substrates. ................................ ............................ 98

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12 4 8 Effects of TTFA, malate and malonate on the induced mitochondrial H 2 O 2 producti on in the presence of succinate. ................................ ................... 99 5 1 Mn uptake in microglia measured by fura red quenching assay. ...................... 108 5 2 Experimental design o f Mn 2+ subcellular accumulation in microglia analyzed by ICP MS. ................................ ................................ ................................ ....... 109 5 3 Effects of Mn 2+ on fura red in microglia. ................................ ........................... 110 5 4 Quenchi ng effect of 100 M Mn 2+ on fura red fluorescent signal in mitochondria and nuclei over time ................................ ................................ .... 111 5 5 Mn content in subcellular organelles of microglia. ................................ ............ 112 6 1 Microglia mediate Mn neurotoxicity via H 2 O 2 production from mitochondrial ETC complex II.. ................................ ................................ ............................... 117

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13 LIST OF ABBREVIATION S AA a ntimycin A CNS central nervous system DA dopamine DMEM ied eagle medium DMT divalent metal transporter ED TA ethylene diamine tetraacetic acid ETC electron transport chain FCCP carbonylcyanide 4 (trifluoromethoxy) phenylhydrazone GABA aminobutyric acid GSH glutat h ione HAPI highly aggressively proliferating immortalized rat microglia HBSS h ank's balanced salt solution HRP horse radish peroxidase H 2 O 2 hydrogen peroxide ICP MS inductively c oupled plasma mass spectrometry LDH lactate dehydrogenase PD ROS reactive oxygen species SOD superoxide dismutase TTFA thenoyltrifluoroacetone

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14 Abstract of Dissertation Presented to the Gra duate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy COMPLEX II OF THE MITOCHONDRIAL RESPIRATORY CHAIN IS TH E KEY MEDIATOR OF DIVALENT MANGA NESE INDUCED HYDROGEN PEROXI DE PRODUCTION IN MICROGLIA By Yue Liu December 2012 Chair: Bin Liu Major: Pharm aceutical Sciences Exposure to excessive levels of manganese (Mn) is associated with the disease Increased oxidative burden on neurons has been implicated in the pathogenesis of Mn neurotoxicity. We have recently reported that divalent Mn (Mn 2+ ) stimulates microglia, the resident immune cells of the brain, to release hydrogen peroxide (H 2 O 2 ) and the resulting microglial activation facilitates Mn 2+ induced neurotoxicity. The goal of the present study was to elucidate the mechanism of the Mn 2+ induced H 2 O 2 production in microglia. Our data demonstrated that H 2 O 2 release caused by exposure to low microm olar concentrations of Mn 2+ was a microglial specific response mediated by mitochondrial complex II. Exposure to Mn 2+ induced a significant release of H 2 O 2 from rat microglia but not astroglia. Subcellular fractionation studies revealed that Mn 2+ was capab le of inducing significant H 2 O 2 production in the mitochondrial, but not cytosolic or nuclear fractions prepared from microglia. Analysis of the relative contribution of mitochondrial respiratory chain complexes indicated that Mn 2+ induced mitochondrial H 2 O 2 production required

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15 the presence of complex II substrate succinate. In contrast, complex I substrates malate and glutamate failed to support H 2 O 2 production in the presence of Mn 2+ Furthermore, the succinate supported Mn 2+ induced H 2 O 2 production in mi croglia was abolished by pharmacological inhibition of complex II, but not complexes I and III, which suggest that mitochondrial complex II is a key mediator in Mn 2+ induced H 2 O 2 production in microglia. In order to elucidate the subcellular distribution of Mn 2+ in microglia, ICP MS and confocal microscopy were used. We report here that Mn 2+ at micromolar concentrations readily transports across the cellular membrane and enter both the mitochondria and nucleus. ICP MS studies with microglial subcellular fr actions revealed that the highest amount of Mn 2+ was found to be associated with the mitochondrial fraction compared with the other fractions (mitochondria > cytosol> nuclei> microsome). The information on Mn 2+ subcellular distribution in microglia will p rovide insights in elucidating the mechanism of Mn 2+ induced neurotoxicity and microglia induced neuroinflammation in this process. In summary, findings presented in this dissertation should advance our knowledge on the mechanisms by which Mn 2+ induces oxi dative stress and neurotoxicity.

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16 CHAPTER 1 BACKGROUND AND INTRO DUCTION Manganese (Mn) T oxicity Mn E ssentiality Mn is an element that exists ubiquitously on earth ; it is the twelfth most abundant element and fifth most abundant metal on earth, making u p about 0.1% of earth crust ( Turekian and Wedepohl, 1961 ) In human bodies, Mn serves as an essential nutrient that regulate s the metabolism of protein s lipid s and carbohydrate s ( Aschner and Aschner, 2005 ) Mn is crucial for a variety of biological functions including development, bone formation, immune response and reproduction ( Aschner and Aschner, 2005 ) Mn is an important cofactor for many enzymes including Mn superoxide dismutase (Mn SOD), glutamine synthetase (GS), arginase, and pyruvate carboxylase. GS is present predominantly in t he matrix of astroglia and functions to convert glutamate to glutamine ( Takeda, 2003 ) Mn SOD is an antioxidant enzyme located in the mitochondrial matrix to protect mitochon drial DNA from oxidative damage ( Carl et al., 1987 ) GS is the most abundant manganoprotein in the brain and thus plays an imp ortant role in the extracellular glutamate scavenging system. It is generally believed that 80% of Mn content in the brain is associated with GS ( Aschner and Aschner, 2005 ) Pyruvate carboxylase is another astrocytic enzyme that is involved in glucose metabolism. The activity of this enzyme is important for the central nervous system ( CNS ) glucose metabolism as glucose is a primary energy so urce for the brain ( Wedler, 1993 ) Mn deficiency is not typically reported in hu man since Mn exists in many food sources. Mn deficiency can be induced in rat s by feeding low Mn diet. The result ing phenotype is

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17 associated with seizure, bone malformation, and reduc ed fertility, indicating Mn play s a role in maintaining normal neuronal functions ( Aschner and Aschner, 2005 ) Mn L evels in F ood Diet is t he major source of Mn intake to the general population. The metal is present naturally in many food sources, with the highest levels found in nuts, leafy vegetables, grains and animal products (Aschner and Aschner, 2005). Typical levels of Mn in common foo d sources have been listed by Agency for Toxic Substances and Disease Registry ( ATSDR, 2008 ) in Table 1 1. The range for Mn daily intake through diet fr om eating typical Western and vegetarian diets has been estimated to be 0.7 10.9 mg ( Greger, 1999 ) Mn N eurotoxicity Exposure to elevated levels of Mn, either from ingestion or inhalation, can re sult in a neurological disorder known as manganism. Manganism was first described by James Couper ( Couper, 1837 ) in 5 workers exposed to Mn oxide; it is characterized by similar movement symptoms as those observed in idiopathic Parkin symptoms of manganism typically develop in two stages. At the early stage, patients with manganism suffer from mainly psychiatric disturbances such as anxiety, fatigue, depression, loss of appetite, hallucination s violent and comp ulsive behaviors ( Mena et al., 1967 ) With progression of the disease, patients start to manifest locomotor symptom s from extrapyramidal circuits, such as rigidity, bradykinesia, tremor, dystonia, and gait disturbances ( Cersosimo and Koller, 2006 ) The motor dysfunctions of manganism closely resemble those of iPD.

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18 Sources of Mn E xposure Mn overexposure can occur acutely and sub chronic ally by inhalation of high levels of Mn containing dust or fume s under occupational settings. Mn overexposure can also occur from environme ntal exposure (low level long term) to Mn from the use of Mn containing gasoline additive methylcyclopentadienyl manganese tricarbonyl (MMT) and drinking from water sources contaminated with industrial Mn. Low level long term exposure to Mn represents a th reat to the public health. In addition, Mn exposure has also been reported under certain medical settings in patients with dysfunctions in controlling normal Mn homeostasis. Occupational e xposure Occupational exposure is the major cause of Mn intoxication in human s and represents a huge workplace safety issue in the United States. Mn is widely used in a myriad of industries. Various manganese compounds can be used to produce: dry cell batteries (MnCl 2 ), glass (MnO 2 ), ceramics, fungicides (MnSO 4 ), disinfect ants and preservative s for flowers and fruits (KMnO 4 ) and fungicide s (organomanganese compounds). Cases of Mn toxicity have been reported in mining ( Rod riguez Agudelo et al., 2006 Montes et al., 2008 ) ferroalloy smelting ( Kaji et al., 1993 Mergler et al., 1994 Bast Pettersen et al., 2004 ) welding ( Bowler et al., 2006 ) dry cell battery factories ( Bader et al., 1999 ) and glass ceramics manufactures ( Srivastava et al., 1991 ) Mn overexposure mainly affects the brain, the lung and the reproductive organs. Notably, neurological deficits start to appear at lower doses before other deficits occur in individuals exposed to Mn. Therefore the federal government developed regulation s on Mn according to the levels that cause neurological dysfunction The Occupational Safety and Health Administration (OSHA) set a legal limit of 5 mg/m 3 Mn in air averaged

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19 over an 8 hour work day ( OSHA, 1998 ) However, it is r ecommended that Mn levels in occupational settings should not exceed 0.03 mg/ m 3 in the respirable dust ( OSHA, 1998 ) The guidelines on Mn safety concentrations are based on several studies by Roels et al. ( Roels et al., 1987 ) in which workers exposed to 1 mg/m 3 Mn oxide in a factory were found to exhibit a slight increase in frequency of weakness and tremor, and decreased scores on psychomotor tests (simple reaction time, short term memory, eye hand coordination and hand steadiness). Later, the same group reported in 92 male workers exposed to Mn dioxide dust in a dry alkal ine battery factory Mn exposed workers performed significantly worse in simple reaction time, eye hand coordination and hand steadiness relative to the control worker s with similar work schedules and workload ( Roels et al., 1992 ) The Mn concentration was estimated to be 215 g/m 3 in respirable dust with exposure duration range from 0.2 to17.7 years (average 5.3 years) ( Roels et al., 1992 ) In a longitudinal follow up study, Roels et al. ( Roels et al., 1999 ) observed that Mn concent ratio ns were directly, and inversely correlated with performance on a test measuring eye hand coordination. In 1994, Merglar and colleagues reported that 145 workers from a ferroalloy factory in Canada were observed for adverse effects to the exposure of 0 .014 11.48 m g /m 3 (mean 1.186 mg/m 3 ) Mn in the total dust for an average of 16.7 years. Mn exposed workers showed decreased performance on tests of motor function, and exhibited significantly lower levels of cognitive flexibility ( Mergler et al., 1994 ) Lastly in a study on 58 workers exposed to 0.027 0.27 mg/m 3 Mn (total dust) for 1 28 years (mean, 13 years) in a ferroalloy factory in Italy, investigators ( Lucchi ni et al., 1995 ) reported these workers exhibited decreased

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20 neurobehavioral performance (finger tapping, symbol digit, digit span, and additions tests) and cognitive abilities. Environmental e xposure Level of ambient Mn is closely associated with the p roximity to industrial Mn sources ( Santos Burgoa et al., 2001 ) Mn levels in the air have been reported to range from 220 300 ng/ m 3 near industrial sourc es, whereas Mn levels in areas without Mn industrial sources have been reported to range from 10 70 ng/m 3 ( Barceloux, 1999 ) In previous studies on the general population living in a Mn mining district in Mexico, the average Mn levels in the air was reported to be 420 ng/m 3 ( Rodriguez Agudelo et al., 2006 Montes et al., 2008 Hernandez Bonilla et al., 2011 ) These researchers found a significant correlation of Mn exposure in the air and motor de ficit in the general population ( Rodriguez Agu delo et al., 2006 ) However, Mn levels in the blood of people living in Mn mining district (10 g/L) were not significantly increased compare d to normal (4 15 g/L) ( Rodriguez Agudelo et al., 2006 Montes et al., 2008 ) In a study on a children population between 7 and 11 years old in Mexico, researcher s reported subtle negative association of Mn exposure on motor speed and coordination ( Hernandez Bonilla et al., 2011 ) In a recent study condu cted in Ohio, resident s living close to a larg e ferro and silico Mn smelter did not have significantly increased blood Mn levels ( Kim et al., 2011 ) However, assessment on fine motor skills on participates suggested that the Mn exposed group showed significantly higher postural sway scores under eyes open conditio ns ( Kim et al., 2011 ) Environmental exposure of Mn has been a rising concern due to the introduction of MMT into gasoline as an octane booster ( Kaiser, 2003 Finkelstein, 2007 Walsh, 2007 )

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21 MMT is an organic Mn compo und manufactured by Ethyl to substitute lead as an antiknock agent. MMT was first approved for use in Canada in 1976 at a concentration of 18 mg/L of gasoline ( Kais er, 2003 ) It was banned in the US from 1976 for the concern of Mn expos ure to the general public, but E regulation in 1995 and as a result, MMT was approved for use in US at a concentration of 8.3 mg/L of gasoline. Af ter combustion, a fraction of Mn combustion products of MMT ( Mn sulfate, Mn phosphate and Mn oxide ) are released in to the atmosphere ( Ressler et al., 1999 Zayed et al., 1999 ) Bioavailability and toxicity of these Mn compounds are related to the ir solubility in the order of Mn sulfate > Mn phosphate > Mn oxide. The United States Environmental Protection Agency ( USEPA ) estimated the background ambient concentration of Mn in urban areas to be 40 ng/m 3 based on the measurement of 102 cities in the US ( USEPA, 1990 ) However, other studies have reported Mn concentrations in the air range from 5 10 ng/m 3 ( Lynam et al., 1999 Rodriguez Agudelo et al., 2006 ) The amb ient Mn levels have been studied in Canada where MMT has been used since 1976. The use of MMT in Montreal was not found to significantly increase ambient Mn concentration in a study conduct by the manufacture company of MMT Ethyl Corporation ( Lynam et al., 1999 ) Another study by Bolte et al. ( 2004) reported ambient Mn levels near the expressway i n Montreal (25 ng/m 3 ), was significantly higher than rural area (5 ng/m 3 ). However, the levels of Mn in the blood from human subject s were not significantly different between city and rural areas ( Bolte et al., 2004 ) In addition, Loranger and colleagues found ambient Mn levels were significantly correlated with traffic density ( Loranger et al., 1994 ) The current minimal risk level (MRL) of Mn in respirable dust is 40 ng /m 3 for chronic inhalation exposure

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22 (365 days or more) ( ATSDR, 2008 ) Based on current data, t he concern over MMT use is legitimate because Mn emission fro m gasoline combustion produces soluble Mn species in the air with higher bioavailability in human bodies. The accumulation of Mn from MMT and industri al sources may set the stage for the brain to be more sensitive to later insult s, thus more susceptible to develop movement abnormalities and Parkinsonism. Drink ing from water sources that are contaminated with high concentrations of Mn represents another environmental source of Mn exposure to the general population. USEPA guideline s ( USEPA, 2004 ) In 1941, Kawamura reported Mn exposure in people drinking from a well contaminated with Mn from a buried dry battery factory in Japan ( Kawamura, 1941 ) Areas such as Bangladesh have been reported with problems with high Mn levels in drinking water. A recent study found Mn in drinking water dose dependently cause low cognitive and verbal skill s in Bangladeshi children ( Wasserman et al., 2006 ) Mn absorption and retention are high during infancy due to the immaturely developed intestinal and biliary system s ( Keen et al., 1986 Bell et al., 1989 ) A cross sectional epidemiological study in infants exposed to wat er Mn greater than or equal to /L are associated with an elevated mortality risk during the first year of life compared to unexposed infants in the control group ( Hafeman et al., 2007 ) Overexposure of Mn can also occur from consuming soy based infant formulas ( Lonnerdal, 1994 Krachler and Rossipal, 2000 ) Infant formulas have been reported to ( Collipp et al., 1983 Ljung and Vahter, 2007 ) 70 fold higher than that in human breast ( Dewey et al., 1991 USEPA, 1997 )

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23 Medical and o thers s ources Mn homeostasis is tightly control led by absorption from the GI tract and excretion from the bile. Certain sub populations with defects in organs controlling Mn homeostatic are at higher risk for Mn exposure. For example, patients with chronic liver failure ( Hauser et al., 1994 Krieger et al., 1995 ) receiving total parenteral nutrition ( Fell et al., 1996 Bertinet et al., 2000 ) chronic iron deficiency ( Boojar et al ., 2002 Kim, 2005 ) and individuals with inheritable defects of Mn homeostasis ( Tuschl et al., 2008 ) have been reported to develop manganism. In addition, rapid onsets of manganism have been reported in drug addicts receiving intravenous injection of methcathinone (ephedrine) contaminated with potassium permanganate ( de Bie et al., 2007 Meral et al., 2007 Sanotsky et al., 2007 Sikk et al., 2007 ) Diagnosis and B iomarkers for M anganism As mentione d in the symptoms section, typical manganism symptoms resemble those of iPD, making it difficult to diagnose. Magnetic resonance imaging (MRI) represents a sensitive and non invasive method to analyze Mn concentrations in the brain ( Cersosimo and Koller, 2006 ) Mn is paramagnetic a nd detectable in MRI because the metal has unpaired electrons in level 3d ( Josephs et al., 2005 ) Mn is capable of shortening the relaxatio n time of the T1 ( Josephs et al., 2005 ) The MRI will give hyperintense signals in the basal ganglia structure in manganism patients but normal signal in the PD patients. Although symptoms of manganism are usually progressive, if patients are treated before any structural damage to the neurons occur, there is hope for reversing the neurological disturbances observed at early stage s of Mn intoxication. Therefor e, assessment of manganism at early time points using reliable biomarkers is extremely

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24 important for the treatment and prognosis of this disease Currently, Mn intoxication can be tested by examining Mn levels in the blood, urine and hair ( Aschner et al., 1999 ) Blood Mn concentration is believed to reflect the current burden of Mn to the body ( Aschner et al., 1999 ) The Mn levels in the blood, urine and hair usually go back to normal several months after exposure ( Aschner et al., 1999 ) This make s the diagnosis dif f to Mn is terminate d Urinary Mn is relatively v ariable and unreliable compared to blood Mn levels because the majority of Mn is excreted by the bile in to feces ( Klaassen, 1974 ) However, urinary Mn testing can be used as an indicator for the effectiveness for chelation therap y since in patients it has been reported that Mn excretion doubled after ethylene diamine tetraacetic acid (EDTA) treatment ( Jiang et al., 2006 ) Treatment s Chelation therapy has proved to be effective in treating metal intoxications and is the primary treatment option for Mn exposure. Chelators such as EDTA and its salt compounds can bind metal ions, and together the chelator and metal complex will be excre ted by the urinary and/or biliary routes. CaNa 2 EDTA has been shown to decrease liver and brain Mn levels in Mn intoxicated rats ( Kosal and Boyle, 1956 ) decrease Mn induced dopamine autooxidation in vitro ( Nach tman et al., 1987 ) and increase excretion of Mn via the urinary route in humans ( Herrero Hernandez et al., 2006 ) In addition to EDTA, a drug proved to treat tuberculosis has emerged to treat Mn toxicity. Sodium para aminosalicylic acid (PAS) is an antibacterial drug that has carboxyl, hydroxyl and amine groups in its structure, making it an ideal metal chelator. PAS was first tested in rats exposed t o Mn ( Tandon et al., 1 975 ) Later human cases also showed effectiveness of PAS for treating manganism ( Jiang et al., 2007 )

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25 Another therapeutic treatment option for manganism is levo dopa, the gold standard treatment for PD ( Lu et al., 1994 ) However, clinical examinations may show initial improve ment of extrapyramidal symptoms ( Mena et al., 1970 ) The effect of levodopa can decrease after a short period of time ( Huang et al., 1993 ) The decreased effect of levodopa treatment on manganism patients i s due to the fact that dopaminergic neurons stay large ly intact in Mn intoxicated brain. The lack of prolonged response from levodopa is also used as a criterion to distinguish manganism from iPD. Moreover, it has been argued that the administration of lev odopa in manganism can be counter effective as Mn catalyzes dopamine auto oxidation to generate quionones and semiquinons, free radicals that can cause oxidative stress to proteins, lipids and DNA ( Graham, 1984 Lloyd, 1995 ) In summary, m anganism and iPD are di stinct disorders. To better distinguish between these two conditions, it is necessary to briefly describe the general pathology and symptoms of iPD. D isease Definition and H istory primarily from the degeneration of nigrostriatal dopaminergic neurons. PD is the second m ost common neurodegenerative disease affecting more than 1 millio n people in the United States. With aging of the population, this number is anticipated to be doubled by 2025. PD was first described by an English physician James Parkinson in his book a sh aking palsy in 1817 ( Parkinson, 2002 ) The path ological hallmarks of PD are formation of intracellular protein aggregates Lewy bodies (alpha s ynuclein protein aggregates) and massive loss of dopaminergic neurons in both the subs tantia nigra par compact a and striatum.

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26 Symptoms and D iagnosis There are two categories of PD symptoms: movement related and psychiatric. Movement symptoms include resting tremor, rigidity, bradykinesia, akinesia (difficulty to initiate movement), and pos tural instability. Psychiatric features include depression, anxiety, sleep disorder, dementia, etc. The diagnosis of PD is based on the identification of 2 of the 3 cardinal symptoms of PD including bradykinesia, rigidity and rest ing tremor. In addition, movement problems of PD almost always start from one side of the body, and are more severe than the other side of the body throughout the disease. Sustained response to dopaminergic medications is another diagnostic marker for PD. Further more using radiol abeled ligands binding to dopamine transporters at the pre dopaminergic neuronal terminals and using functional imaging techniques such as positron emission tomography and single photon emission computed tomography can be used for direct visualization of d opaminergic neuronal integrity ( Ray and Strafella, 2012 ) Dopaminergic N eurons Dopamine is a monoamine neurotransmitter that belongs to the catecholamine family. It is an important modulatory neurotransmitter synthesized in the dopaminergic neurons. T he synthesis of dopamine starts from hydroxylation of tyrosine by tyrosine hydroxylase to L dihydroxypheylalanine (levodopa), which can be transformed to dopamine by dopa decarboxylase or aromatic L amino acid decarboxylase (AADC). Dopamine can be converte d to norepinephrine by dopamine beta hydroxylase. Dopaminergic neurons are located in the substantia nigra, the ventral tegmental area and the hypothalamus in the CNS. Dopaminergic neurons project their axons into different areas in the brain to form speci fic pathways. There are 4 major dop aminergic

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27 pathways in the CNS: n igrostriatal pathway, mesolimbic pathway; mesocortical pathway and tuberoinfundibular pathway. In nigrostriatal pathway, neurons project from the substantia nigra into the striatum. The neu rodegeneration of the nigrostriatal pathway underlies iPD development ( Bjorklund and Dunnett, 2007 ) Etiology and R isk F actors The e tiology of PD is unknown. Oxidative stres s, gliosis and mitochondrial dysfunction have all been implicated in PD development. A 25% inhibition of mitochondrial complex I has been reported in the post mo rtu m PD brain ( Mizuno et al., 1989 ) However, whether these events are primary or secondary to neurodege ne ration is still a matter of debate. In sporadic PD, aging is the biggest risk factor of PD. The average age of onset for PD is around 60 years when abo ut 50 60% of dopaminergic neurons are degenerated. Genetic f actors In familial PD, onset can be as early as in the 20s. Several genes have been identifie d to cause PD. Their protein products include alpha synuclein, parkin, ATP13A2, DJ1, PTEN induced puta tive kinase 1 (PINK1), and leucine rich repeat kinase 2 (LRRK2) ( Lesage and Brice, 2009 ) However, the familial form of PD only accounts for about 5 10 % of the total PD cases. More than 90 % of t he total PD cases are sporadic and have a late onset around 60 years of age. The lat e onset of PD is believed to result from the interplay of a genetic predisposition and environmental factors. Environmental f actors Environmental toxicants have been shown to increase the risk of developing PD. Other than Mn, environmental exposure to pest icides ( rotenone, paraquat and maneb),

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28 herbicide (agent orange), and organochlorine pesticides dieldrin and lindane ( Mao et al., 2007 Mao and Liu, 2008 Sharma et al., 2010 ) have been shown to induce dopaminergic degeneration and devel opment of PD like movement symptoms ( de Lau and Breteler, 2006 Spivey, 2011 Tanner et al., 2011 ) Treatment O ptions Medication Levodopa is the gold standard for PD treatment for more than 30 years. It is converted to dopamine in dopaminergic neurons by dopa decarboxylase. For the purpose of preventing levodopa from being metabolized before crossing the BBB, peripheral dopa decarboxylase inhibitors such as carbidopa and benser azide typically pre scribed with levodopa. Another enzyme that degrade s levodopa is catechol O methyltransferase, inhibitors for this enzyme is also prescribed with levodopa. Dopamine agonists are a second class of drugs for PD treatment They work by bind ing to dopaminergic post synaptic receptors. However dopamine agonists are not as effective and have more side effects than levodopa treatment. Monoami ne oxidase inhibitors (MAO I) are another class of drugs used to treat PD. MAO I increase dopamine level s in the basal ganglia by blocking its metabolism mediated by monoamine oxidase B (MAO B). The reduction in MAO B activity results in increased dopamine concentrations in the striatum. However, these drugs only provide relie f from symptoms of PD but do not slow down or stop the neurodegeneration process. ( Simola et al., 2010 Kincses and Vecsei, 2011 ) Deep b rain s timulation Deep brain stimulation is a relative ly new method that was discovered in the 1990s. It is used in advanced stage PD patients who have limited response to

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29 dopaminergic medications or are suffering from severe side effects from the medications ( Bronstein et al., 2011 Peron and Dondaine, 2012 ) It requires implanting an electrode into the thalamus, glob us pallidus o r subthalamic nucleus areas in the brain and a stimulator under the collarbone to send impulses into the electrode ( Amick and Grace, 2006 ) The patients usually experience immediate r elief from PD symptoms after the surgery. However, deep brain stimulation surgery bears risks associated with the invasive surgical procedure such as inflammation. Some patients may suffer from cognitive problems after the surgery ( Kluger et al., 2012 ) Gene t herapy Viral vectors have been exp lored to deliver genes to replenish the loss of dopamine input to striatum. Adeno associated virus serotype 2 (AAV2) or recombinant AAV have been tested in clinical trials to deliver therapeutic genes into the PD patients. A phase II clinical trial study u sing bilateral surgical infusion of AAV2 GAD has reported efficacy and safety ( LeWitt et al., 2011 ) Clinical trials are underway for other genes of promises such as glutamic acid decarboxylase (GAD), AADC glial cell line derived neurotrophic factor (GDNF) and neurturin. Similarities and D ifference s B etween M anganism and iPD Symptoms and N europathology Patients with manganism have motor dysfunctions overlapping w ith those of idiopathic PD (iPD) Both diseases are chronic progressive disorders that deteriorate over years. Although locomotor symptoms appear to be similar in manganism and iPD, patients with manganism have milder resting tremor and stand in a more upr ight position ( Chu et al., 1996 McMillan, 2005 ) Manganism patients manifest a unique cock like walk and loss o f balance, which are different from that of iPD. Differences in

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30 the symptoms observed in patients with manganism and iPD may be due to these two diseases affect ing different areas of the brain. iPD patients have a clear cut degeneration of dopaminergic ne urons in the substantia nigra par compacta, whereas in manganism, substantia nigra par compacta dopaminergic neurons have been largely shown to be structurally and functionally intact ( Olanow, 2004 ) The initial and central site of injury in manganism has been reported to be the globus pallidus (GP) the site where Mn accumulate s according to the MRI scan. In severe cases of manganism, neurodegeneration has been associated with GABA neurons in the GP ( Perl and Olanow, 2007 ) The basal ganglia are the center for motor control in human brain; it consists of four nuclei of different neuronal populati ons: striatum (caudate and putamen), substantia nigra, GP and subthalamic nucleus ( Stocco et al., 2010 ) There are three neurotransmitters in the basal ganglia: gluta mate, GABA and dopamine. Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS respectively Dopamine is a modulatory neurotransmitter. The striatum receives input from the cerebral cortex and sends inhibitory neurot ransmitter GABA output to other components of the basal ganglia. The GP neurons receive input from the striatum, and sends GABA to a number of motor related areas. The substantia nigra receives GABA input from the striatum, while the substantia nigra sends dopamine input to the striatum. The subthalamic nucleus receives input from the cortex and GP externa, and produces glutamate output to GP interna and the substantia nigra. The inhibitory and excitatory pathways of the basal ganglia depend on precise regu lation of multiple neurotransmitters, making the similarities between sy mptoms of manganism and iPD not surprising ( Wu et al., 2012 )

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31 Another distinction between these two diseases are Lewy bodies. O ne of the pathological hallmarks of PD, Lewy bodies are rarely found in manganism patients brain ( Olanow, 2004 ) Distribution of Mn in normal brain is heterogeneous, with the highest concentrations found in the basal ganglia struct ures ( Perl and Olanow, 2007 ) Upon Mn overexposure, elevated Mn levels have been reported in the basal ganglia and basal forebrain structures including the GP, striatum, tha lamus, and frontal corte x. The neuropathological hallmarks of manganism are neurodegeneration and gliosis in the se brain regions ( Bikashvili et al., 2001 ) Molecular M echanisms iPD i s a multifactorial disease that involves genetic and environmental factors. Genes that have been reported to contribute to iPD development include alpha synuclein, parkin, ATP13A2, DJ1, PTEN induced putative kinase 1 (PINK1), and leucine rich repeat kinase 2 (LRRK2 ) (Lesage and Brice, 2009). Previous studies suggest mutations in parkin and ATP13A2 might also predispose an individual to Mn toxicity by affecting Mn transport mechanism. Parkin is a protein that contributes to hereditary and sporadic PD develo pment; it is encoded by the PARK2 gene ( Kitada et al., 1998 Lesage and Brice, 2009 ) Parkin is a component of ubiquitin E3 ligase complex, with the function to mediate proteasomal degradation for divalent metal transporter 1 (DMT1) ( Roth et al., 2010 ) Mutations in this gene are associated with autosomal recessive juvenile PD ( Kitada et al., 1998 Lesage and Brice, 2009 ) In manganism, DMT1 has been shown to play a role in Mn uptake into the body and the CNS. Dysfunction in DMT1 proteasome degradation pathway would increase DMT1 levels in the cell and result in increased Mn uptake, thus render ing individuals with this gene mut ation at higher risk for Mn intoxication.

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32 ATP13A2 (PARK9) is a gene identified in early onset and hereditary cases of PD; it encodes a transmembrane P type transport ATPase 13A2 A recent study in yeast has repo rted an ortholog of human ATP13A2 can prot ect again Mn toxicity and deletion of this gene sensitized yeast cells to exposure of manganese ( Chesi et al., 2012 ) The m echanism of ATP13A2 protection of Mn toxicity is unknown. However, this finding implies a genetic link between Mn toxicity and iPD. The mutant ATP13A2 might be a Mn transport playin g a role in mediating Mn homeostasis and delivery into the CNS ( Gitler et al., 2009 Chesi et al., 2012 ) Mutat ions in genes associated with early onset iPD may also underlie the individual susceptibility observed in the development and progress of manganism In summary, manganism can be distinguished from iPD anatomically by the initial injury site in the brain and pathologically by the different neurodegenerative mechanisms. However, whether Mn exposure set s the stage for future PD development is still a question to be addressed. Mechanism of Mn N eurotoxicity Considerable eff ort has been made to elucidate the m echanism of Mn induced neurodegeneration in the past two decades. There are several mechanism s that are believed to take part in manganism. Mitochondrial D ysfunction ATP for the body. Mitochondrial dysfunction has been associated with a wide range of diseases including PD and other neurodegenerative diseases. There is mounting evidence linking mitochondria dysfunction to Mn neurotoxicity. For example, m itochondria are general ly believed to be the major subcellular site for Mn accumulation

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33 ( Liccione and Maines, 1988 ) probably via the calcium (Ca) uniporter ( Gavin et al., 1999 ) Mn is also capable of increasing mitochondrial Ca 2+ concentration by inhibiting both the sodium dependent and independent Ca 2+ exporter ( Gavin et al., 1999 ) This leads to the activation of the permeability transition pore (PTP) and mitochondrial dysfunction ( Gavin et al., 1990 ) Once inside the mitochondria, Mn can induce ATP depletion, mitoch ondrial membrane potential disruption and increased ROS production ( Aschner et al., 1999 ) Oxidative phosphorylation is performed by the mitochondrial electron transport chain (ETC) located in the inner mitochondrial membrane. The ETC is composed of 5 large e nzyme complexes named NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), ubiquinone cytochrome c oxidoreductase (complex III), cytochrome oxidase (complex IV) and ATPase (complex V). Each complex contains a number of redox center s that f unction to transport electrons along the ETC. Specifically, Mn has been shown to interfere with oxidative phosphorylation by inhibiting F1 ATPase and complex I at higher concentration ( Gavin et al., 1992 1999 ) In addition, Mn has been reported to inhibit complex II, III, V activity, which eventually lead s to decrease d ATP production ( Zwingmann et al., 2003 Milatovic et al., 2007 ) Mn has been shown to exert a preferential effect on aconitase and ot her iron sulfur p roteins in which iron acts as a cofactor in the ir active catalytic center such as complex I and complex II ( Zheng et al., 1998 ) Oxidative S tress Reactive o xygen s pecies Reactive oxygen species (ROS) are a series of oxygen derived oxidants that can damage macromolecules such as proteins, lipids and carbohydrates of the cells. ROS

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34 are intimately linked to energy production as they are produced as byproducts of norma l oxidative phosphorylation in the mitochondrial ETC When the production of ROS overwhelms the intracellular antioxidant defense system, oxidative stress ensue. Common ROS include superoxide, hydrogen peroxide and hydroxyl radical (Figure 1 1) Single ele ctron reduction of oxygen gives rise to superoxide ( O 2 ) which is the progenitor ROS. O 2 subsequently produces hydrogen peroxide (H 2 O 2 ) by the antioxidant enzyme superoxide dismutase (SOD). Catalase and glutathione (GSH) are antioxidants that scavenge H 2 O 2 When intracellular antioxidant levels are low, H 2 O 2 can diffuse across the cellular membranes of the original cell and migrate to adjacent cells or organs to cause oxidative damage. H 2 O 2 is not a very reactive ROS by itself. However, when H 2 O 2 comes across iron, it can be converted to a highly reacti ve ROS hydroxyl radical by Fent on reaction. One explanation for why the nigrostraital DA neurons are particular ly susceptible in PD pathogenesis is explained by the higher content of iron in substantia nig ra and therefore more severe oxidative stress in this brain area. The mitochondrial ETC represents the primary intracellular ROS production machinery ( St Pierre et al., 2002 ) Mitochondria have an outer and inner membrane. The space between the out er and inner membrane is the i ntermembrane space. The ETC also called the respiratory chain is located on the inner membrane. The ROS are produced as byproduct s of normal respiration. Figure 1 2 shows the mitochondrial ETC complexes and ATP by oxidative phosphorylation. The electrons are provided by malate and glutamate from complex I or succinate from complex II. In normal respiration, electron transport along the E TC is coupled with proton pumping from the matrix to the

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35 i ntermembra ne space. The membrane potential formed by this proton gradient across the inner membrane drives ATP synthase to produce ATP. The production of ROS from the ETC is minimal under normal con ditions. However, under pathological conditions or in the presence of electron transport inhibitors, the flow of electrons along the ETC is disrupted. Subsequent ly, electrons escape from the ETC and react with oxygen to form O 2 thus the production of RO S from the ETC is substantially elevated. Other than the mitochondrial ETC, NAPDH oxidase represents another important intracellular ROS production system in phagocytic cells like the macrophages. NAPDH (nicotinamide adenine dinucleotide phosphate) oxidas e is a mult i subunit membrane protein. It s normal function is to produce ROS from the o xidation of NADPH in the phago somes in order to kill foreign agents. NADPH oxidase complex is composed of subunits gp91 phox, p22phox, p40phox, p47phox, p67phox. The ROS produced by this enzyme have been suggested to contribute PD pathogenesis in animal models ( Wu et al., 2003 ) In addition, mutations in NADPH oxidase are associated with a hereditary disorder called chronic granulomatous disease ( Segal et al., 2000 ) Oxidative s tress in m anganism There is mounting evidence suggesting oxidative stress play s a n important role in Mn neurotoxicity. In rat s orally exposed to Mn for 7 days, increased striatal concentrations of antioxidants such as ascorbic acid and GSH have been reported, indicative of the presence of oxidative stress ( Desole et al., 1994 ) In rat s exposed to inhaled Mn sulfate, researchers have reported decreased GSH and increased metallothionine levels and marker of oxidative stress in the rat brain ( Dobson et al., 2003 ) Non human primate models exposed to Mn sulfate through inhalation has

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36 reported decreased GSH and increased metallothionine levels in frontal cortex and caud ate ( Erikson et al., 2007 ) Eff ects of Mn on N euronal S ynapses In normal brain, Mn plays a role in neuronal functions. Previous studies have shown that potassiu m evokes Mn release in rat amygdala ( Takeda et al., 1998b ) Intraneuronal axonal transport has been report ed as a mechanism for Mn uptake into the rat brain ( Sloot and Gramsbergen, 1994 Takeda et al., 1998c ) Dopaminergic s ynapses Most studies on Mn effe ct on neuronal circuitry have been done in dopaminergic neurons because of the similarity of PD and manganism in motor functions. In vitro experiments have shown that Mn can induce auto oxidation of dopamine to generate free radicals and subsequent oxidati ve damages ( Graham, 1984 Lloyd, 1995 ) Research in rodent models on dopamine biology in manganism has generated mixed results on conce ntration of dopamine, dopamine transporter s (DAT) and receptor s Studies using non human primate s ha ve confirmed the finding in human autopsy that the dopaminergic neurons are structurally intact after Mn exposure. In addition, Mn ex posure did not change dopamine levels ( Olanow et al., 1996 Struve et al., 2007 ) However, Mn decreased D2 li ke dopamine receptor level on postsynaptic neuronal terminals ( Eriksson et al., 1992 ) Recent studies on non human primate s provide more insights in dopamine dy sfunction in Mn neuro to xicity. Chen at al. ( Chen et al., 2006 ) reported acute Mn exposure produced a transient decrea se in DAT levels for a month in baboon. Also Mn can inhibit dopamine uptake in striatal synaptosome by reducing the available number of binding sites in DAT. In addition, another study reported the most

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37 dramatic change of Mn exposure on para meters of dopamine system was the decreased dopamine release f rom the DA presynaptic terminals ( Guilarte et al., 2008a ) GABAergic s ynapses The most prominent feature of manganism pathology is associated with high concent ration of Mn in the globus pallidus. Pallidal neurons are primarily GABAergic neurons, and they receive glutamatergic input from the subthalamic nuclei. GABA ( aminobutyric acid) is the major inhibitory neurotransmitter in the brain. Early studies in rat s have shown Mn treatment increase s GABA levels in the striatum ( Bo nilla, 1978 ) Further studies in rat s confirmed increase of striatal GABA concentrations and suggested Mn counteracted age dependent decline in glutamic acid decarboxylase in rat s chronically exposed to Mn ( Lai et al., 1981 Gianutsos and Murray, 1982 ) The function of glutamic acid decarboxylase is to convert glutamate to GABA. In contrast, other studies repor ted decreased brain GABA levels in rodent models ( Brouillet et al., 1993 ) Furthermore, a study show ed no effect of Mn on GABA levels in rat s ( Bonilla et al., 1994 ) Non human primate exposed to Mn by inhalation showed no alteration in GABA levels in their brain ( Struve et al., 2007 ) Another study reported no alteration in level of GABAa receptor in non human primates c hronically exposed to Mn oxide ( Eriksson et al., 1992 ) Glutamatergic s ynapses Since pallidal GABAnergic neurons receive glutamatergic projections from the sub thalamic nuclei, the action of glutamate has been investigated on Mn neurotoxicity. Glutamate is the major excitatory neurotransmitter in the brain. Glutamate has been proposed to play a role in Mn neurotoxicity since Mn has been reported to increase synap tic levels of glutamate by inhibiting glutamate transport, and the increased levels

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38 of glutamate can cause excito toxicity (Hazel and Norenberg 1997; Erikson and Aschner 2002). Increased glutamate concentration in rat brain s exposed to Mn has been reported in previous studies ( Lipe et al., 1999 Reaney et al., 2006 ) However, glutamate level has also been reported to be unchanged in rat brain ( Bonilla et al., 1994 ) A more thoroug h study by Zwingmann and colleagues reported in rat s exposed to Mn, glutamate levels increased in frontal cortex, but decrease d in the GP ( Zwingmann et al., 2007 ) In non human primate s exposed to Mn th r ough i nhalation for 13 weeks, Struve and colleagues reported Mn did not affect glutamate levels in the GP and s triatum ( Struve et al., 2007 ) In addition, Mn exposure caused increased mRNA levels and decreased protein levels of the two major astrocytic glutamate transporte r s: glutamate aspartate transporters and glutamate transporter 1 ( Erikson et al., 2007 ) Ano ther major finding is that glutamine synthetase (GS) level was decreas ed ( Erikson et al., 2007 ) GS is an important enzyme in glutamate regulation in the brain; its normal function is to convert glutamate to glutamine. Therefore, th e reduction of GS would lead to increase d glutamate and glutamate mediated excito toxicity Other studies support the hypothesis that glutamate plays a contributing role in Mn neurotoxicity by showing Mn can be taken up by the pallidal neurons through activ ated glutamate channels (Kannurpatii 2000). This result is supported by the fact that Mn induced damage can be blocked by the noncompetitive NMDA receptor antagonist, MK 801 (Brouillet 1993). In summary, due to the limited number of studies, the effects o f Mn exposure on neurochemistry and neuronal functions have only been answered inconclusively. The discrepancy from different studies are largely due to different animal species used,

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39 routes of Mn exposure, dose and duration of Mn exposure, and differences in brain regions analyzed. Glia l mediated I nflammation In addition to neurons, the human brain is composed of similar number of non neuronal cells known as glia. They have long been assume d to play a passive role mainly providing physical and nutrition al support for the neurons. However, recently, this dogma has been overturned and neuroscientists now recognize an active role of glia in modulating brain neurotransmission circuitry. Glia account for half of the brain cell population and 90% of the volume of the CNS parenchyma ( Azevedo et al., 2009 ) The glia/neuro n ratio varies across different areas of the brain. For example, the basal ganglia where PD and manga nism pathology takes place, has an extremely high ratio of glia and neuron (11:1). In contrast, the cerebral cortex and cerebellum have glia/neurons ratios of only about 3.76 and 0.23 respectively ( Azevedo et al., 2009 ) Different types of glia l cells have been identified, including: astroglia, microglia, oligodendrocyte, schwann cell, and radial glia. In human and experimental models of manganism, the presences of activated astroglia and microglia have been reported by ultrastructural study ( Bikashvili et al., 2001 Hazel l et al., 2006 ) Astroglia Astroglia make up 70% of brain volume. Under physiological conditions, they play important function to provide trophic and structural support for the neurons. In metal neurotoxicity, astroglia have been reported to function li ( Tiffany Castiglion and Qian, 2001 ) Wedler et al ( Wedler et al., 1989 ) reported that chicken astroglia can concentrate Mn 50 fold higher than that in the medium. Aschner et al ( Aschner et al., 1992 ) suggest ed rat astroglia have a more efficient uptake mechanism

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40 for Mn The astrocyte might be the fi rst target of Mn in the CNS as researchers found decreased glial fibrillary acidic protein (GFAP) and S100beta when the neuronal functions were intact ( Henriksson, 2000 ) The disruption of normal astrocytic func tion has been postulated to contribute to Mn neurotoxicity through n itric oxide (NO) production. Indeed, astroglia have been reported to release inflammatory factors such as NO that is mediated by inducible NO synthase in response to Mn treatment ( Spranger et al., 1998 ) The upregulated inflammation has been reported to be mediated by nuclear factor kB ( NfkB ) and mitogen activated protein kinases ( MAPKs ) ( Liu et al., 2005 ) Microglia Microglia are the resident immune cells of the brain. They are the key players mediating neuro in flammation which is initiated either by the pr esence of foreign agents or neuronal demise ( Liu, 2006 ) Microglial activation is characterized by mo rphological changes, cell proliferation, migration to the lesion site and expr ession of inflammatory factors ( Liu, 2006 ) Chronic neuroinflammation can contribute to neurodegeneration by releasing a wide range of neurotoxic factors. Previous studies have shown that Mn treatment induce microglia to produce ROS and reactive nitrogen species ( Chang and Liu, 1999 Filipov et al., 2005 Zhang et al., 2007 Zhang et al., 2009 Zhang et al., 2010 ) In addition, Mn treatment has also been shown to potentiate lipopolysaccharide ( LPS ) induced cytokines (int erleukin 1 beta and TNF alpha) and prostaglandin E2 ( PEG2 ) production from microglial cell cultures ( Chang and Liu, 1999 Filipov et al., 2005 Zhang et al., 2007 Zhang et al., 2009 Zhang et al., 2010 ) Microglial activation in the substantia nigra has been reported in rats and non human primates exposed to Mn ( Zhao et al., 2009 Verina et al., 2011 ) Notably, t he distribution of mic roglia is

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41 heterogeneous across different brain regions with the highest proportion in the midbrain area containing the basal ganglia ( Lawson et al., 1990 Kim et al., 2000 ) lending additional support to their contributing role in manganism pathogenesis. Gene E xpression S tudies A number of studies have explored the genome wide screening to identity novel genes or pathways involved in Mn neurotoxicity in cells from rodent and human origins. Sengupta et al ( Sengupta et al., 2007 ) s howed in a genome wide study on cultured human astroglia, Mn treatment induc ed upregulation of inflammation and downregulation of DNA replication and repair. Another gene expression study on Mn treated mice brains reported upregulation of S100 beta and its protein. S100 beta is primarily expressed in astrocytes. Also in the same stud y, downregulation of neurofilam ent subunit genes were detected ( Baek et al., 2004 ) A r ecent study in non human primate s orally exposed to Mn, a number of genes and pathways related to cell cycle regulation, DNA repair, apoptosis, ubiquitin proteasome system, protein folding, cholesterol homeostasis, axonal/vesicular transport, and inflammat ion have been discovered ( Guilarte et al., 2008b ) Notably the researchers found the most upregulated gene was amyloid like protein (APLP1). Dif fuse amyloid beta plaques were detected in the frontal cortex of Mn treated macaques. Other brain gene expression of apoptosis, protein folding and degradation, inflammation and axonal/vesicular transport was affected ( Guilarte et al., 2008b ) These studies provide insights into the molecular events and pathways that are disturbed in Mn neurotoxicity. Interaction with I ron R egulation In the human body, Mn and iron (Fe) share common carriers and transport er systems. For example, Fe transporters such as transferrin and divalent metal

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42 transporter (DMT1) have been shown to mediate Mn uptake as well. Thus low Fe in the system would put them at increased risk for Mn toxi city. Indeed, in rodent models, Fe deficient diet s resulted in elevated Mn intake and oxidative stress in the brain and motor function deficits ( Fitsanakis et a l., 2009 ) In Fe deficient human subpopulation, over 7% of Mn can be orally absorbed versus only 3% by normal individuals. In young women who consumed low Fe diet, Mn absorption has been reported to increase. Fe deficiency is the most prevalent single nu tritional deficiency in world, especially in developing countries. It is estimated that about 2 billion people have Fe deficiency. Infants, children and pregnant women are the most vulnerable subpopulations. In addition, patients with a hereditary disease call hemochromatosis carry a mutation in the human hemochromatosis protein ( HFE ) gene The normal function of this protein is complex with transferrin receptors and lower Fe uptake ( Griffiths et al., 2000 ) As a result of the mutant HFE gene, transferrin receptor is deregulated in hemochromatosis patients and the Fe and Mn uptake will be increased into the body. Mn U ptake and S ubcellular D istribution Mn H omeost atic C ontrol Under physiological conditions, Mn absorption and excretion are tightly control led in the body. 1 5% of Mn in the diet is absorbed via the gastrointestinal (GI) tract. More than 95% of Mn is excreted in the bile ( Davis et al., 1993 ) Since only a small portion of Mn can be absorbed from the GI tract, Mn overexposure via ingestion is typically not found Mn S pecies in the B lood Mn mainly assumes 2 different valen ce states in the human body: +2, +3 ( Archibald and Tyree, 1987 ) Mn 2+ is stable in aqueous environment between pH 2 7.5.

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43 Mn 3+ is unstable at physiological pH and can undergo disproportionation to Mn 2+ and Mn 4+ ( Archibald and Tyre e, 1987 ) It is believed that about 80% of Mn in the plasma is bound to transferrin in the plasma introduced orally or intravenously in the rat ( Davidsson et a l., 1989 ) Ultrafiltration study of rat serum suggested that more than 70% of Mn in blood was protein bound ( Rabin et al., 1993 ) In circulation, Mn 2+ is believ ed to globulin and albumin ( Aisen et al., 1969 ) The globulin and albumin bound Mn 2+ cannot cross intact blood brain bar rier (BBB). Size exclusion chromatography suggested about 55% of Mn in blood is bound to albumin or transferrin, 30% of Mn in blood was bound to low MW species, such as Mn citrate (3.5%) and inorganic Mn (94%) ( Nischwitz et al., 2008 ) Another study reported 84% of Mn in blood was albumin bound, hexahydrated ion account for 6.4%, Mn bicarbonate is 5.8%, Mn citrate and Mn transferrin were 2 and 1% respectively ( Harris and Chen, 1994 ) However, another study reported that Mn transferrin complex is the predominant species of Mn in blood plasma ( Davidsson et al., 1989 ) All Mn 3+ are expected to bind transferrin, which can bind to its receptor on a variety of cell surfaces. In normal conditions, about 30% of tra nsferrin b inding site s are occupied by Fe 3+ leaving 70% of the bin ding site s available to bind Mn 3+ Transferrin binds Mn 3+ rather tightly and transferrin and transferrin receptor mediated endocytosis has been shown to contribute to Mn transport into the brain via the BBB and the blood cerebrospinal fluid (BCF) barrier.

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44 Toxicokinetics Normal levels of Mn in the brain tissue average approximately 0.25 g/g wet weight ( Takeda, 2003 ) Normal ranges of Mn levels are about 4 15 g/L in blood, 1 8 g/L in urine, and 0.4 0.85 g/L in serum ( USE PA, 2004 ) Routes of Mn U ptake into the CNS Physiological Mn concentrations in brain tissues range from 2 8 M ( Pal et al., 1999 ) The two barrier systems between the blood and the brain parenchyma, i.e. the BBB and the blood CSF barrier, protect the bra i n from a variety of insults and pose an obstacle for drug delivery for neurological disorders. However, Mn can readily enter the CNS from the blood compartment. Three different routes have been described for Mn transport into the brain. First is by crossing the BBB through the endothelial cells on the blood vessel ( Aschner et al., 2002 Aschner and Dorman, 2006 ) Second is by crossing the blood CSF barrier through the choroid plexus ( Bradbury, 1997 Wang et al., 2008 ) Third is via the olfactory nerve and trigeminal nerves directly to the brain during inhalation ( Brenneman et al., 2002 Dorman et al., 2002 ) Which route Mn employs to enter the brain largely depends on the source of exposure. O ccupational exposure to Mn which involves inhalation of Mn containing dusts of fumes would go through the olfactory bulb located in the nasal mucosa and employs the retrograde axonal transport mechanism into the brain. The lung is the first organ exposed to Mn, and the pulmonary epithelial linin g take up Mn and deposit it in the lymph or blood. Otherwise, Mn can be cleared from the lung by mucocilliary elevator and be delivered into the GI tract, where Mn will be taken up via the s ame fashion as the ingested Mn. In the case of Mn exposure by ingestion, Mn uptake takes place in the

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45 digestive system. At GI tract pH 2, Mn complexes can be dissolved and Mn will be release d from foods by digestion in the gastric juice. Mn will be subsequ ently absorbed by the microvilli into the blood via transporter proteins. The mechanism of Mn transport a cross the BBB has been extensively investigated. The BB B forms around 4 months after bir th in humans. During embryonic and early postnatal periods, the BBB is absent or leaky, exposing the infant to higher levels of Mn in the brain. There are several transporter systems implicated in Mn active transport and facilitated diffusion Mechanism of Mn T ransport across the BBB While the uptake of Mn through the olfactory pathway bypasses the blood brain barrier (BBB), there are other routes of Mn transport across the BBB to get access to the CNS. BBB is a highly restrictive structure. Anatomically, BBB consists of tight junctions between blood vascular endot helial cells, a basement membrane of collagen, collagen like protein, pericytes (surround 30% of the endothelial cells), and astrocyte foot processes ( Yokel, 2009 ) Mn readily crosses the BBB by carrier mediat ed processes as Mn 2+ ion, Mn citrate, and Mn transferrin. Several transporter mechanisms have been shown to mediate this process, including Tf receptor mediated endocytosis; DMT1; voltage dependent and storage mediated calcium channels; and ZIP proteins. N o unique transporter for Mn has been identified so far. Transferrin (Tf) Tf has a MW of 77,000 Da. The large size renders it incapable of crossing membranes without a carrier. Tf Mn complex can bind to Tf receptors located on cellular surface of BBB endoth elial cells and form an endocytosis vesicle. Then enzymes in the vesicle will digest the Tf Mn complex, and the V ATPase drops the pH to 5.5, reducing

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46 Mn 3+ to Mn 2+ which can transport into the cell by DMT1 on the endosomal membrane. However, experiment us ing hypotransferrinemic mice which only produce less than 1 % of Tf, the transport of 54 Mn into the CNS and into glial cells were not affected compare to the control animals ( Dickinson et al., 1996 Takeda et al., 1998a ) These studies suggest that Mn uptake into CNS and glial cells were not dependent on Tf. Consistent with this notion, a study has reported tha t after Mn wa s intravenously injected into experimental animals, it was rapidly transport ed into the brain via non transferrin related pathways, the transport was reported to be saturable ( Takeda et al., 20 00 ) Voltage d ependent and s tore operated c alcium c hannels (SOCCs) Mn 2+ and Ca 2+ have the same charge and relative size, suggestive of Ca 2+ channels might mediate brain Mn uptake. SOCC functions to allow Ca 2+ entry from extracellular fluid when intracell ular storages of Ca 2+ are low. The role for Ca 2+ uptake process mediating brain Mn uptake was supported by in situ brain perfusion technique that the Ca 2+ concentration in the perfusate is negatively correlated with Mn 2+ transport into the rat brain ( Crossgrove and Yokel, 2005 ) There is also evidence that Ca 2+ inhibitors, reduced temperature, and divalent cations can decrease Mn uptake into human hepatocarcin oma cells (Finley 1998). The influx of Mn through calcium channels has to be initiated by external stimulus, such as membrane potential and Ca 2+ waves. Ca 2+ channels hav e also been reported to mediate Mn 2+ uptake at neuron terminals. DMT1 DMT1 is a high af finity membrane transporter with non selective affinity for divalent cations ( Gunshin et al., 1997 ) The transporter is identified at the BBB and has been sugges ted to be a candidate for Mn uptake cross the BBB ( Burdo et al., 2004 Thompson et al., 2007 ) The iron resp onse element (IRE) on DMT1 mRNA functions to

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47 upregulate the level of DMT1 during Fe deficiency ( Garcia et al., 2007 ) However, in Mn transport experimen ts using B elgrade rats, which do not express functional DMT1, the transport of Mn cross the BBB was not changed ( Crossgrove and Yokel, 2004 ) Although DMT1 might not pl ay an essential role in Mn transport at the BBB, another study reported that in Belgrade rats the uptake of Mn in olfactory nerve, reticulocyte, and intestinal Mn absorption are lower than heterozygous and normal rats ( Thompson et al., 2007 ) The physiological importance of DMT1 in Mn uptake into the brain remains controversial. Zip 8 and z ip 14 Zip transporter proteins are member s of the solute carrier 39 metal transporter family. There are 14 members in this family and they are highly conserved across rodent and human ( Eide, 2004 ) It has been shown that in mouse fetal fibro blast, z ip8 has a high affinity for Mn ( He et al., 2006 ) The Km of z ip 8 for Mn 2+ is close to physiological concentrations of Mn determined in tissues and cell lines ( He et al., 2006 ) However, it is yet to be determined whether z ip proteins are functional at the BBB and in intact animal model. It is estimated that only abo ut 50% of transporters on BBB have been identified (Pardridge, 2003). The discovery of more BBB ion channels and transporter s may aid the understanding Mn transport in the CNS cross the BBB in the future. Mechan ism of Mn T ransport across the B lood CSF B ar rier Mn can be transport ed across the blood CSF barrier by active transport ( Schmitt et al., 2011 ) PET scanning of manganism brain has report ed 54 Mn concentra te in the choroid plexus after intravenous injection. In addition, previous studies have demonstrated that 54 Mn distribution in the brain is essentially the same after

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48 intracerebroventricular injection and intravenous injection of 54 MnCl 2 ( Takeda et al., 1994 ) The role o f transferrin in Mn transport across the CSF was examined by injecting the transferrin bound 54 Mn into the ventricle, which showed that the transferrin bound Mn was not transported into the brain parenchyma ( Takeda et al., 2000 ) Mn species in the CSF have been qualified before, report a composition of 56% Mn 2+ ion, 20% Mn citrate, 15 % Mn phosphate in human CSF ( Nischwitz et al., 2008 ) Uptake by N euronal and G lial C ells As discussed above, different Mn spec i es can be transported into the brain extracellular fluid from the BBB endothelial or blood CSF barrier epithelial cells. In the brain parenchyma, transfer rin is produced and secreted into the interstitial fluid from the oligodendrocytes ( Connor et al., 1990 ) Neurons and glia have been shown to express transferrin receptors on their plasma membranes ( Connor, 1994 Moos, 1996 ) Therefore, transferrin bound Mn can be taken up into the cells by receptor mediated endocytosis. The transport of transferrin bound Mn complex has been confirmed in vitro using neuroblastoma cell cultures ( Suarez and Eriksson, 1993 ) However, it is reported that transferrin level s in brain extracellular fluid is less than 0.25 M It is likely that most of transferrin sit es are bound by Fe 3+ leaving no sites available for Mn 3+ binding ( Bradbury, 1997 ) Most Mn species in the brain has been suggested to be a low MW compound, likely to be Mn citrate ( Michalke et al., 2007 ) Glial cells can also uptake Mn via a non transferrin mediated mechanism as observed in glia cells from the hypotransferrinemic mice, most likely mediated by DMT1 ( Takeda et al., 1999 ) DMT1 is expressed in high densi ties in hippocampal pyramidal and granule cells, cerebellar granule cells, the preoptic nucleus and pyramidal cells of

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49 the piriform cortex ( Gunshin et al., 1997 ) It is likely DMT1 mediate s the uptake of Mn ion and low molecular weight ligand bound Mn into these brain cells. Mn A xonal T ransport In previous studies aim ed to address Mn transport in neural circuit, 54 MnCl 2 was injected intrastriatally and intranigr ally in the rat brain, and 54 Mn has been detected in the rat substantia nigra and striatum respectively ( Sloot and Gramsbergen, 1994 Takeda et al., 1998c ) This study demonstrated Mn axonal transport in the dopaminergic nigro striatal pathway and the GABAergic striato nigral pathway. The transport of Mn from the striatum to the substantia nigra can be suppressed by the injection of an axonal transport inhibitor, colchicine ( Takeda et al., 1998c ) In addition, Mn transport in the glutamatergic terminals has been studied using high K + stimulation. High K + stimulated Mn release from glutamatergic neuronal terminals is strongly correlated with increased glutamate in the extra cellular fluid ( Takeda et al., 2002 ) Mn release was mediated through sodium channel s as indicate d by sodium channel inhibiter tetrodotoxin ( Takeda et al., 2002 ) Mn has also been shown to decre ase glutamate uptake via the glial glutamate transporters, im ply ing Mn exposure might result in increased glutamate levels in the extracellular fluid and subsequent excitatory neurotoxicity might be involved in Mn neurotoxicity ( Ha zell and Norenberg, 1997 ) Mn uptake from the olfactory bulb into the CNS is also mediated by retrograde axonal transport from olfactory endothelial cells along first order neurons and second order neurons to the striatum of the brain ( Gianutsos et al., 1997 ) Mn S ubcellular D istribution Not only is the di stribution of Mn in the brain heterogeneous, the subcellular distribution of Mn is also non uniform. As a n important cofactor, Mn is needed for many

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50 enzymes for activity. Previous studies conducted by Gunter group in search of Mn speciation in cells especially in the mitochondria has failed to identify Mn 3+ in the mitochondria in neur o ns or astroglia except for a Mn SOD signal ( Gunter et al., 2006 ) suggest ing that Mn 2+ may be the predominant toxic species in the cells. The interaction of Mn and mitochondria are well documented. In addition to producing ROS, Mn has been reported to inhibit oxidative phosphorylation, decrease ATP production, open mitochondri al permeability transition pore and release cytochrome C from the mitochondrial intermembrane space into the cytosol ( Aschner et al., 1999 ) Therefore, it was generally accepted that mitochondria are the preferential site of Mn subcellular localization. A number of older studies have shown accumulation of Mn in the mitochondria after Mn treatment in vivo after tissue fractionation ( Maynard and Cotzias, 1955 Miller et al., 1975 Liccione and Maines, 1988 Gavin et al., 1999 Lai et al., 1999 ) In contrast, recent studies using specific brain cells (neurons, astroglia, epithelia and endothelia) in vitro and in vivo re ported that mitochondria play a less important role in Mn distribution in these cell types and that the nuclei ( Kalia et al., 2008 Morello et al., 2008 ) or the Golgi apparatus ( Carmona et al., 2010 ) sequester significant amounts of Mn Apart from the different methodologies used in these studies, the inconsistent result on Mn subcellular localization suggest the possibility that Mn subcellular distribution varies among different cell types. Despite the central role of microglia in neur oinflammation, the subcellular localization of Mn has never been studied in microglia. The information on Mn subcellular distribution can provide insight into both the biological function and mechanism of cytotoxicity of Mn.

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51 Efflux of Mn from CNS There are no established mechanisms for Mn export from t he CNS or mammalian cells. Studies o n cultured chick glial cells have shown carrier mediated Mn efflux ( Wedler et al. 1989 ) Ferroportin is a cyt oplasmic Fe exporter. A recent study has demonstrated that in inducible human embryonic kidney (HEK293T) cell model, ferroportin expression can be induced by Mn with decreased Mn cytotoxicity and accumulation, presumably by e xporting Mn outside the cells ( Yin et al., 2010 ) Another study reported that the export of Mn by ferroportin was inhibited by a low extracellular pH and by incubat ion in a high K + medium ( Madejczy k and Ballatori, 2012 ) Mn efflux out of the brain is likely to be diffusion mediated. This conclusion was drawn from comparing Mn efflux to a BBB impermeable reference compound ( Kakee et al., 1996 ) Recently studies aiming at developing a physiologically based pharmacokinetic model of inhaled Mn in rats demonstrated that half life of brain Mn influx was several fold shorter than the half life of brain Mn efflux, suggesting that Mn influx into the brain is much rapid than Mn efflux out of the brain ( Nong et al., 2008 ) The brain Mn efflux is consistent with lower Mn elimination from the brain compared to other tissues The brain half life of Mn has been studied by 54 Mn radiotracer in rodent and non human primate models. In 16 different r at brain regions, half life of Mn was estimated to range from 51 to 74 days ( Takeda et al., 1995 ) In non human primate s limited decrease s of Mn in the cerebral cortex was observed in 278 days after peripheral injection ( Dastur et al., 1971 ) Half life of the inhaled Mn in macaque brain was reported to range from 223 to 267 days ( Newland et al., 1987 ) Whole body and brain half lives of Mn in human have been estimated to be 15 37.5 and 37 62 days determined after single dose of intravenous injection ( Cotzias et al., 1968 )

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52 Specific A ims O verexposure to manganese (Mn) results in a Parkinsonian like move ment disorder called manganism. Neurodegeneration in manganism patients is most prominent in the globus pallidus of the basal ganglia structure ( Olanow, 2004 ) Previous studies in rats and non human primates exposed to Mn have shown that m icroglial activation and oxida tive damage are increased in the brains of these animals, changes that have been suggested to increase neuroinflammation and neuronal damage ( Zhao et al., 2009 Verina et al., 2011 ) In agreement with this, we have shown that the reactive oxygen species (ROS) released from rat microglia facilitate neuronal cell death in microglia neuron co cultures ( Zhang et al., 2009 ) However, i t remains unclear how excessive Mn 2+ induces ROS production in microglia. Lack of this knowledge hinders development of therapeutic interventions fo r treatment of manganism and other neurodegenerative diseases. The main objective of this dissertation is to determine how Mn 2+ induces ROS production in microglia. It is generally believed that mitochondrial electron transport chain (ETC) is major intra cellular site of ROS production. Our central hypothesis is that the mitochondria in microglia are responsible for ROS production in the presence of Mn 2+ The rationale for the proposed research is that understanding how Mn 2+ induces ROS production in micro glia will lead to development of innovative and specific pharmacological approaches to treat manganism in particular, and parkinsonism in general. We test ed our central hypothes is by pursuing the following three specific aims: 1. The involvement of astroglia and microglia in Mn 2+ induced H 2 O 2 production Our hypothesis is that microglia are the major extracellular source of H 2 O 2 in the brain treated with Mn 2+ This aim is accomplished by comparing H 2 O 2 levels in rat

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53 microglia, astroglia and mixed glia culture s in response to Mn 2+ treatment. In order to test the specificity of Mn 2+ to induce H 2 O 2 production, we also compared the ability of a number of divalent transition metals (Mn 2+ Cu 2+ Cd 2+ Co 2+ ,Ni 2+ Zn 2+ Fe 2+ ) to induce H 2 O 2 release in microglia and as troglia. 2. The involvement of mitochondria in Mn 2+ induced H 2 O 2 production in microglia. Our working hypothesis is that mitochondrial electron transport chain ( ETC ) is critical for Mn 2+ induced H 2 O 2 production in microglia because ETC complexes contain redox centers capable of producing ROS. This aim is accomplished by subcellular fractionation of rat microglia and measuring H 2 O 2 production in the mitochondria, nuclear, and cytosolic fractions in response to Mn 2+ treatment. D ifferent electron donors and speci fic electron transport inhibitors are used to identify the molecular mechanism of Mn 2+ induced H 2 O 2 production in microglial mitochondria. 3. The subcellular distribution of Mn 2+ in microglia. In this aim, inductively coupled plasma mass spectrometry (ICP MS) and confocal microscopy of fura red fluorescence quenching assay are employed to identify the subcellular accumulation site for Mn 2+ in microglia. The comparison between mitochondria and nuclei are emphasized specifically. The results from this study will elucidate the mechanism by which Mn 2+ induces ROS production in microglia and will provide new information about how mitochondria and the ETC complexes contribute to this process. These results are expected to identify potential new targets for treatment of manganism and will increase our understanding of oxidative stress mediated by neuroinflammation in parkinsonism.

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54 Figure 1 1. Reactive oxygen species reactions. Single electron reduction of oxygen gives rise to superoxide ( O 2 ), which is the progeni tor ROS (Equation 1) Superoxide give rise to hydrogen peroxide (H 2 O 2 ) catalyzed by superoxide dismutase (SOD) (Equation 2) Catalase and glutathione (GSH) are antioxidants that convert H 2 O 2 to H 2 O (Equation 3 and 4 ). H 2 O 2 can be converted hydroxyl radical by Fe 2+ (Equation 5).

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55 Figure 1 2. Mitochondrial electron transport chain Elect r ons enter the ETC through either NADH ( nicotinamide adenine dinucleotide ) or FADH 2 ( flavin adenine dinucleotide ) Hydrogen ions are pumped from the matrix into the intermembrane space, building up a membrane potential that is used by ATPase to produce ATP. Electrons can escape from the redox centers in the ETC to form ROS.

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56 Table 1 1. Typical levels of Mn in common food sources listed by Agency for Toxic Su bstances and Disease Registry ( ATSDR, 2008 ) : Type of food Range of mean concentrations (mg/kg) Nuts and nut products 18.21 46.83 Grains and grain prod ucts 0.42 40.70 Legumes 2.24 6.73 Fruits 0.20 10.38 Fruits juices and drinks 0.05 11.47 Vegetables and vegetable products 0.42 6.67 Desserts 0.04 7.98 Infant foods 0.17 4.83 Meat, poultry, fish and eggs 0.10 3.99 Mixed dishes 0.69 2.98 Condime nts, fats and sweeteners 0.04 1.45 Beverages (including tea) 0.00 2.09 Soups 0.19 0.65 Milk and milk products 0.02 0.49

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57 CHAPTER 2 MATERIAL AND METHODS Materials Dulbecco's modified Eagle's medium (DMEM) was purchased from Mediatech (Manassas, VA). H eat inactivated fetal bovine serum (FBS) Amplex UltraRed reagent 5 (and 6) chloromethyl 2 ,7 dichlorodihydrofluorescein diacetate (CM H 2 DCFDA), and horseradish peroxidase (HRP) were obtained from Invitrogen (Carlsbad, CA). Phenol red free Hank's bala nced salt solution (HBSS) was obtained from Hyclone (Logan, UT). Poly D lysine, Mn chloride, cupric sulfate and sucrose were obtained from Fisher Scientific (Fair Lawn, NJ). Cobalt chloride was from Merck (Whitehouse Station, NJ). Superoxide dismutase ( SOD) and catalase were from EMD Biosciences (San Diego, CA). H 2 O 2 (3%) was from Cell Technology (Mountain View, CA). Complete mini protease inhibitor cocktail tablets were from Roche (Indianapolis, IN). 12 mm number 1.5 borosilicate glass coverslips were obtained from sigma (St. Louis, MO). 30% Ultrex II hydrogen peroxide, ultra pure nitric acid and ultrapure water were from Baker (Phillipsburg, NJ). MitoTracker green, fura red AM, PowerLoad concentrate and ionomycin were from molecular probes (Carlsbad, CA). The Bradford protein assay reagents and bovine serum albumin (BSA) were from Bio Rad Laboratories (Irvine, CA). Anti LDH, anti Histone H1, anti cytochrome c oxidase subunit III (COX III) antibodies, and HRP conjugated donkey anti goat secondary antib ody were from Santa Cruz Biotechnology (Santa Cruz, CA) HRP conjugated goat anti rabbit secondary antibody was from Cell Signaling Technology (Beverly, MA). SuperSignal West Dura chemiluminescence reagent and Micro BCA protein assay kit were from Pierce

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58 (Rockford, IL). Percoll was from GE Healthcare Sciences (Piscataway, NJ). All other reagents were from Sigma (St. Louis, MO). Methods Rat HAPI M icroglial C ell C ulture Rat highly aggressively proliferating immortalized (HAPI) microglial cells ( Cheepsunthorn et al., 2001 ) were cultured in DMEM containing 5% FBS, 50 U/ml penicillin and 50 g/ml streptomycin at 37 C in a humidified environment with 5% CO 2 a nd 95% air as previously described ( Zhang et al., 2007 Dutta et al., 2012 ) For treatment, HAPI cells were seed ed at 1 10 5 /well in 96 well culture plates one day before treatment. Rat P rimary M ixed G lia and A stroglia C ulture s Primary m ixed glia and astroglia cultures were prepared from the brains of 1 day old Fisher F344 rat pups following our previous described protocols ( Zhang et al., 2007 Zhang et al., 2010 ) Briefly, whole brain tissues were harvested, triturated and seeded at 1 10 5 /well in poly D lysine coated 96 well culture plates (for assays) or 2 10 7 /flask in poly D lysine coated 150 cm 2 culture flasks (for harvesting astroglia). Cultures were maintained at 37 C and 5% CO 2 in DMEM/F12 supplemented with 10% F BS, 2 mM l well plates that contained approximately 15% microglia and 85% astroglia were used for experiments 2 weeks after seeding. Astroglial cultures were prepared by a mild mechanical shaking (2 h) to remove microglia from the mixed glia cultures followed by passaging of the microglia depleted cultures. After the fifth passage, astroglia were seeded at 5 10 4 /well i n poly D lysine coated 96 well plates and grown overnight in DMEM with 10% FBS before

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59 treatment. Immunostaining for cell type specific markers indicated a purity of > 98 % for astroglia. For experiments, cultures were seeded in 96 well culture plates All procedures involving animals were approved by University of Florida Institutional Animal Care and Use Committee. Preparation of M icroglial M itochondrial, N uclear and C ytosolic F ractions by differential centrifugation as previously described ( Frezza et al., 2007 ) Cells were seeded in 100 mm culture dishes and grown to confluence before use. All subsequent procedures were performed at 4C. Cells were washed 3 times with phosphate buffered saline (PBS) and scraped into 350 l/dish of isolation buffer (IB) (250 mM sucrose, 10 mM HEPES, 1 mM EGTA, 2 mM PMSF, 0.1% bovine serum albumin (BSA), and protease inhibitor cocktail, pH7.4). Cells were homogenized in a Dounce homogenizer with tight pestle for 40 strokes and > 75 % of the cells were disrupted as confirmed by Trypan blue exclusion. Cell h omogenate was transferred into 1.7 ml centrifuge tubes and centrifuged at 800 g for 10 min. Th e supernatant was saved and the pellet containing the nuclear fraction and unbroken cells was resuspended in IB and homogenized again for 10 20 strokes to achieve a disruption rate of > 90%. The homogenate was centrifuged again at 800 g for 10 min. The p ellet was saved as the crude nuclear fraction. The supernatant was pooled and centrifuged at 10,000 g for 10 min. The resulting pellet was saved as the crude mitochondrial fraction and the supernatant was saved as the crude cytosolic fraction. The mit ochondrial pellet was further washed twice in suspension buffer (SB) (250 mM sucrose, 10 mM HEPES, 0.1% BSA, pH7.4) and centrifuged at 10,000 g for 10 min. The final crude mitochondrial pellet was resuspended in SB.

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60 Crude mitochondrial fraction was fu rther purified using a discontinous Percoll gradient as described ( Leister and Herrmann, 2007 ) The crude mitochondrial pellet prepared as describe d above was resuspended in 1 ml of ice cold 12% Percoll prepared in IB and carefully layered on a discountinous Percoll gradient of 20% (3 ml) and 40% (1 ml) in a 5 ml ultracentrifuge tube. Following centrifugation for 10 min at 30,000 g, the mitochondr ial fraction that existed as a thin white band between the 20% and 40% Percoll was carefully retrieved and diluted with 8 volumes of IB. After two washes by centrifugation at 17,000 and 7000 g for 10 min each, the final pellet was resuspended in 100 l of SB. The crude nuclear fraction was further enriched using the sucrose cushion method ( Cox and Emili, 2006 ) Briefly, the crude nuclei pellet was resuspended in 4 ml of IB and carefully layered on top of a sucr ose cushion (61% in IB, 1 ml). After centrifugation at 80,000 g for 30 min, the nuclear pellet was resuspended in the SB and washed twice by centrif ugation at 800 g for 10 min. The final nuclear pellet was resuspended in 100 l of SB. The crude cytosolic fraction was further centrifuged at 100,000 g for 60 m in to obtain purified cytosol. Protein concentration was determined using the BCA protein assay reagents with BSA as standard and the mitochondrial preparation wa s immediately used for assays. Aliquots were saved for Western blot analysis for marker protei ns. Mitochondrial R espiration A ssay Mitochondrial o xygen consumption was measured at room temperature using a YSI 5300 O 2 monitor (Yellow Spring Instrument, Yellow Springs, Ohio) coupled with a HP3395 integrator ( Hewlett Packard Company, Palo Alto, CA) Mitochondrial

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61 preparation (0.25 mg protein) was kept in a buffer consisting of 120 mM KCl, 10 mM HEPES, 2 mM MgCl 2 2 mM KH 2 PO 4 0.1% BSA, pH7.4, 1 ml). Uncoupled respiration was initiated by adding 5 M of carbonylcyanide 4 (trifluoromethoxy) phenylhydraz one (FCCP) in the presence of substrates (5 mM glutamate + 2. 5 mM malate + 5 mM succinate). In the complex II respiration using succinate ( 5 mM ) as the substrate, Mn 2+ ( 10, 100 and 800 M ) was added before and after the addition of ADP to test if Mn 2+ had an effect on mitochondrial respiration. Measurement of H 2 O 2 P roduction The amount of H 2 O 2 released from cells into the culture media was determined using the fluorescent probe Amplex UltraRed in the presence of HRP as previously describ ed ( Zhang et al., 2007 ) Briefly, cells in 96 well culture plates were washed three times with HBSS and then treated with divalent metals in HBSS (100 l/well) The amount of H 2 O 2 w as determined in the presence of 50 M Amplex UltraRed and 1 U/ml HRP. Fluorescence intensities were measured at 530 nm (excitation) and 590 nm (emission) using the Synergy HT plate reader (Bio Tek Instruments, Winooski, VT). The concentrations of H 2 O 2 in the samples were calculated based on a standard curve generated from known concentrations of H 2 O 2 standard run alongside the samples. To determine the H 2 O 2 production by microglial subcellular fractions, (10 g) were mixed with 50 M Amplex UltraRed and 1 U / ml HRP in an assay system containing 250 mM sucrose, 10 mM HEPES, 0.1% BSA (pH7.4 ) in a final volume of 100 l. The ETC inhibitors used were 10 M rotenone, 10 M antimycin A, 5 m M malonate, 10 M thenoyltrifluoroacetone (TTFA), 1 M myxthothial, 1 mM azide and 1 M oligomycin. Concentrations of the inhibitors were chosen to achieve a complete inhibition of specific electron transport sites in each complex based on reports in the literature ( Barja and

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62 Herrero, 1998 ) The respiratory substrates used were malate (2.5 mM) and glutamate (5 mM) for complex I and succinate (5 m M) for complex II respectively. Production of H 2 O 2 was initiated by the addition of substrates, and the fluorescence was monitored over a period of 30 min. H 2 O 2 production w as calculated following a previously described method ( St Pierre et al., 2002 ) Briefly, each experimental condition and standard w as performed in the absence and presence of subcellular fraction proteins to establish the magnitude of interference b y the assay components. Mitochondrial proteins caused a mild quenching of Amplex UltraRed fluorescence. Typical 4 point H 2 O 2 standard curves determined with and without the addition of mitochondria are shown in Figure 2 1 Concentrations of H 2 O 2 were firs t calculated based on the standard curves in the presenc e of each subcellular protein. Then H 2 O 2 production in the absence of protein (background signals from inhibitors and substrates) was calculated based on the control standard. The final H 2 O 2 concentr ation of each treatment condition was obtained by subtracting the concentration measured in the absence from that measured in the presence of subcellular proteins. The amount of H 2 O 2 production in the reaction system was calculated over the 30 min period. Measurement of I ntracellular ROS P roduction The production of ROS was determined with CM H 2 DCFDA as previously described ( Zhang et al., 2009 ) Briefly, cells in 96 well culture plates were treated with HBSS or divalent metals At the end of treatment, cultures H 2 DCFDA in HBSS. Fluorescence intensities w ere determined at 485 nm (excitation) and 530 nm (emission) using a Synergy HT plate reader.

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63 Western B lot A nalysis To solublize membrane proteins, the P ercoll purified mitochondrial and sucrose cushion purified nuclear fractions were incubated with Trito n X 100 (0.5% final concentra tion in SB) for 30 min on ice. The mixture was then briefly sonicated with two 5 second bursts and centrifuged for 20 min at 20,000 g at 4 C The supernatant was saved and protein concentration was determined using BCA reag ents with BSA as a standard ( Zhang et al., 2007 ) Cytosolic and detergent solublized mitochondrial and nuclear p roteins (50 g) were separated on 10% polyacrylamid e gels and transferred to nitrocellulose membrane. Following blocking at room temperature for 1 h with blocking solution [5% nonfat milk in PBS 0.1% Tween 20 (T PBS)], the blots were incubated overnight at 4C with primary antibodies against cytochrome c, cytochrome c oxidase subunit III, LDH, or histone H1 (all di luted 1:1000 in 3% BSA T PBS). Bound primary antibodies were detected by incubation with appropriate HRP conjugated secondary antibodies in blocking solution for 1 h at room temperature. The memb ranes were developed using the SuperSignal West Dura chemiluminescent reagent and recorded with the BioRad ChemiDoc XRS digital imaging system and the Quantity One image analysis software. ICP MS A nalysis 2+ prepared in HANKS for 6 hours. Then the cells were fractionated following the procedure as described above. Samples fro m each fraction were frozen at 80 C until analysis. Method for ICP MS ana lysis was modified from Barber et.al (2005). Briefly, cytosol, of concentrated ultra

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64 hydrogen peroxide was added to further digest the samples at 110 C for 60 mins. analysis. Thermo Electron Xseries ICP MS was used to conduct the analysis with indium as an internal standard. Mn concentration in each fraction was determined from a standard curve of Mn based on the mass and charge ratio 55 signal. The detection limit of this method is 0.002 ppb. Confocal M icroscopy and I mage A cquisition HAPI microglial cells were grown on 12 mm num ber 1.5 borosilicate glass coverslips coated with Poly L lysine in 6 well plate. Cells were washed twice in calcium free HBSS buffer supplemented with 10 mM HEPES, 2 mM L glutamine succinate to support healthy mitochondrial function in live cells. To increase loading of fura fura red AM before staining. Then we mix this mixture in 1ml label ing solution containing 200 nM mitoTracker green. The final concentration of fura red AM in the labeling solution was 10 M. Cells were incubated in the labeling solutions for 1h at 37C. Thus the cells were washed with HBSS twice and then incubated at ro om temperature for another 30min to allow the dye to undergo complete de esteration. Mn 2+ solution was prepared as 100 x by dissolving MnCl 2 in distilled deionized water (dd H 2 O 100 x Mn 2+ solutions in dd H 2 lcium free HEPES buffer and added to the cells on the coverslip. Mn 2+ (0, 1, 2.5, 5, 7.5, 10, 15, 20, 100 red. The images of cells were taken right after the addition of Mn 2+ solution. Images were recorded using a spinning disk confocal system connected to a Leica DMIRB microscope with a 63 oil immersion objective, equipped with a Photometrics

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65 cascade cooled EMCCD camera, under the control of the open source software www.micromanager. org/) (Knowlton et al., 2011). Images were processed using the image analysis software IMAGEJ (http://rsb.info.nih.gov/ij/). Images of fura red were acquired at excitation wavelengths of 491 nm with emission wavelength of 695 nm. Images o f mitoTracker green were acquired at excitation of 488 nm and emission of 512 nm. Ten regions were chosen for image from each Mn 2+ treatment condition for fura red signal. Statistical Analysis Data were analyzed for significance by one way analysis of va riance (ANOVA) followed by Fisher's PLSD post hoc analysis using the JMP version 8 softw are (SAS Institute, Cary, NC). A p value of < 0.05 was considered statistically significant.

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66 Figure 2 1. Reaction of a mplex r ed to r esofufin. Resorufin emits f luorescence at 590 nm upon excitation at 530 nm. This reaction is ca talyzed by horse radish peroxidase (HRP) (m odified from Invitrogen)

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67 Figure 2 2. Microglial cell fractionation and purification of mitochondrial and nuclear fractions. HAPI microglia were fractioned into mitochondrial, nuclear, and cytosolic fractions through differential centrifugation. Nuclei and mitochondria were further purified by sucrose cushion and Percoll gradients.

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68 Figure 2 3 Effect of the presence of mitochondr ia l protein on H 2 O 2 determination. Known amounts of H 2 O 2 standard (0 4 M) were assayed in the presence and absence of 10 g mitochondria l proteins

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69 CHAPTER 3 THE INVOLVEMENT OF G LIAL CELLS IN MN 2+ INDUCED OXIDATIVE ST RESS Introduction Microglia and astroglia are major glial cells of the brain. Astroglia provide neurotrophic factors for neurons and maintain homeostasis of th e central nervous system (CNS) Microglia are the resident brain immune cells that can be activated rapidly in response to t he presence of infectious agents, toxins or neuronal demise. Activated glial cells release proinflammatory factors and free radicals, which represent threats to adjacent neurons ( Liu, 2006 ) Among the reactive oxygen sp e c i is charged thus cannot easily diffuse to extracellular environment to harm adjacent can be converted to H 2 O 2 by SOD, and H 2 O 2 is readily released to the extracellular space to harm other cell types in the CNS. In this aim, we will compare the ability of microglia and astroglia to release H 2 O 2 in response to the treatment of low micromolar concentrations of MnCl 2 We hypothesize that microglial H 2 O 2 release is more prominent compared to that of astroglia. In order to test the specificity of Mn to induce H 2 O 2 production, we will also compare the ability of a number of divalent transition metals ( Mn 2+ Cu 2+ C d 2+ Ni 2+ Co 2+ Zn 2+ Fe 2+ and Hg 2+ ) to induce H 2 O 2 release in microglia and astroglia. The amount of H 2 O 2 released will be measured by Amplex UltraRed probe. Rationale Microglia, astroglia and neuron account for the major cell population of the CNS. Unde r Mn intoxication, microglia and astroglia undergo gliosis and release inflammatory factors, including ROS and cytokines. The increased amount of extracellular ROS leads to oxidative stress related neuronal damage. The three cell cultures used in this stud y

PAGE 70

70 are rat microglial HAPI cells, rat primary mixed glia and rat primary astroglia. HAPI is an immortalized pure rat microglial cell line. The primary rat mixed glia culture contains approximately 15% microglia and 85% astroglia. Primary rat astroglia are m ore than 98% pure. Low micromolar range of Mn 2+ doses were chosen for the reason that we are interested to see the effect of Mn 2+ to induce H 2 O 2 release at low level exposure. In addition, Mn 2+ at this range of concentration has been shown to facilitate mi croglial mediated neurotoxicity ( Zhang et al., 2007 Zhang et al., 2009 ) Hypothesis (i) H 2 O 2 release induced by Mn 2+ is more prominent in microglia than astroglia. (ii) Mn 2+ is more effective than other commonly found divalent metals in inducing H 2 O 2 release in microglia. Experimental D esign For each treatment, a stock solution (10 mM) of metals was freshly prepare d in dd H 2 O HAPI microglial cells, primary mixed glia l and primary astroglial cells were seeded into 96 well plate On the day of treatment, cultures were washed three times with warm HBSS and treated with a working solution of increasing concentrations o f Mn 2+ (1, 10, 30 M), or 10 M of each divalent tran sition metal (Mn 2+ Cu 2+ Cd 2+ Co 2+ Ni 2+ Zn 2+ Fe 2+ Hg 2+ ) in HBSS for 6 h ours in a volume of 100 l per well. The amount of H 2 O 2 released from cells into the culture media was determined using Amplex UltraRed in the presence of HRP (Figure 3 1)

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71 Results Effect of Mn 2+ on H 2 O 2 R elease in R at HAPI M icroglial C ells, R at P rimary M ixed G lia and A stroglia Figure 3 2 illustrates that 0 Mn 2+ induce d increasing amount of H 2 O 2 rele ase from HAPI and primary mixed glial c ells. However, no significant H 2 O 2 release from primary astroglia was detected with 0 Mn 2+ treatment. Notably, in HAPI and primary mixed glial cells, 30 M Mn 2+ failed to produce more H 2 O 2 Mn 2+ indicating a saturating effect of H 2 O 2 release with concentration of Mn 2+ higher than 10 M thus most subsequent studies used 10 uM concentration to elucidate mechanism of Mn 2+ induced H 2 O 2 release. The result presented in Figure 3 2 implies that astroglia are not capable of releasing H 2 O 2 i n response to low micromolar Mn 2+ treatment, suggesting that microglia are the major source of extracellular H 2 O 2 release by Mn 2+ stimulation in the CNS. Effect of D ivalent M etal s on H 2 O 2 R elease in R at HAPI M icroglial C ells, R at P r imary M ixed G lia and A st roglia To determine whether divalent transition metals were all capable of inducing H 2 O 2 release in microglia, we treated HAPI microglial cells with 10 M of commonly found divalent transition metals Mn 2+ Cu 2+ Cd 2+ Ni 2+ Co 2+ Zn 2+ Fe 2+ and Hg 2+ for 6 h. As shown in Fig ure 3 3 treatment with 10 M of Mn 2+ induced a 6 fold greater release of H 2 O 2 than that in control cells. Under the same conditions, Cd 2+ Ni 2+ Co 2+ Zn 2+ and Fe 2+ did not have significant effects and Cu 2+ induced a slight increase in H 2 O 2 release from HAPI microglial cells ( Figure 3 3 ). Treatment with 10 M of Hg 2+ resulted in significant toxicity as evidenced by significant cell membrane blebbing and vacuolar cell bodies compared to the cells in the control cultures (Fig ure 3 4 ). In a ddition, cells

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72 treated with 100 M of the indicated metals exhibited a similar profile in H 2 O 2 release compared to cells treated with 10 M of the same metals ( Figure 3 5 ), similar to the lack of concentration dependence between 10 and 30 M Mn 2+ observed in our previous study ( Figure 3 2 ). In addition to HAPI microglial cells, we determined the effects of divalent metal ions on H 2 O 2 release in rat primary mixed glia and rat primary astroglia. When mixed glia cultures were exposed to Mn 2+ (10 M, 6 h), p roduction of H 2 O 2 was 5 fold greater than that in the control ( Figure 3 3 ). However, under the same conditions, other divalent metals tested were ineffective in inducing H 2 O 2 release. In primary astroglia, none of the divalent metals tested induced a sig nificant H 2 O 2 release ( Figure 3 3 ), indicating that microglia, but not astroglia in mixed glia culture were the source of Mn 2+ induced H 2 O 2 release. These results indicated that Mn 2+ but not the other divalent metals tested were capable of inducing H 2 O 2 r elease from microglia but not astroglia. Discussion This study in rat microglia, mixed glia and astroglia demonstrate d that the microglia were the major source of Mn 2+ induced H 2 O 2 release and that this effect wa s specific for Mn 2+ but not a common featur e of divalent transition metals. This conclusion wa s supported by the following observations: (a) Mn 2+ induced significant increase s in H 2 O 2 production in the microglia and mixed glia, but not astroglia ; (b) The treatment of other divalent cations (Mn 2+ C u 2+ Cd 2+ Ni 2+ Co 2+ Zn 2+ Fe 2+ Hg 2+ ) failed to induce significant H 2 O 2 product ion in any of the three cell cultures.

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73 Relative C ontribution of M icroglia vs. A stroglia to Mn 2+ induced H 2 O 2 R elease Microglia are the primary source of ROS generation in the CNS ( Liu, 2006 ) We have previously shown that microglia facilitate Mn 2+ induced dopaminergic neuro toxicity induced by and microglia originated ROS is a key contributor to the enhanced neurotoxicity ( Zhang et al., 2009 ) Astroglia are a less robust source of RO S production compared microglia ( Liu et al., 2003 ) Their key function is to provide neurons with nutritional and structural support ( Milatovic et al., 2007 ) In the context of metal induced neurotoxicity, astrocytes are believed to be the storage site for metals in the CNS ( Tiffany Castiglion and Qian, 2001 ) Previous studi es have reported increased intracellular O 2 production in astrocytes induced by Mn 2+ ( Liao et al., 2007 ) in agreement with our results on intracellular ROS produ ction in rat primary astroglia treated with Mn 2+ However, little H 2 O 2 was released by astrocytes following Mn 2+ treatment. In this study, no significant H 2 O 2 release was observed in astroglia treated with low micromolar concentrations of Mn 2+ (Fig ure 3 2 ). This lack of significant H 2 O 2 release may be related to the observations that astrocytes possess higher concentration of antioxidant enzymes such as GSH compared to neurons and microglia ( Knott et al., 1999 Teismann and Schulz, 2004 ) Therefore, as a homeostatic function, superoxide produced inside astroglia might be readily neutralized by high levels of a ntioxidant leaving little for conversion into H 2 O 2 and release into the extracellular space to harm vulnerable bystanding neurons. Oxidative S tress I nduced by D ivalent T ransition M etal s Divalent transition metals have been closely associated with oxidative stress. For example, Cu 2+ Fe 2+ and Zn 2+

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74 disease and PD for their effects on the induction of oxidative stress ( Collen et al., 2003 Chang et al., 2010 Gasparova et al., 2010 ) In this study, we found that among the divalent metals tested, only Mn 2+ cau sed a significant release of H 2 O 2 from rat microglia (Fig ure 3 3 ) with Cu 2+ had a slight increase (P=0.0935; n=4). In hepatocytes, Cu 2+ and Cd 2+ have been reported to cause a rapid production of intracellular ROS, depletion of the antioxidant GSH, and cyt oxicity ( Pourahmad and O'Brien, 2000 ) We hav e also detected significant increase in intracellular ROS from both HAPI microglia and mixed glia culture in response to treatment with 10 M of Mn 2+ Cu 2+ and Fe 2+ (Fig ure 3 6 ). It remains to be determined why Cu 2+ and Fe 2+ treatments do not cause increased release of H 2 O 2 from rat microglia. One possibility could be related to the activity of mitochondrial specific enzyme Mn superoxide d ismutase (Mn SOD). Mn SOD activity has been shown to be elevated by the exogenously added Mn 2+ ( Culotta et al., 2006 ) Fe 2+ has been demonstrated to substitute Mn 2+ in the Mn SOD, leaving the enzy me in an inactivated form ( Whittaker, 2003 ) which c ould result in a much subdued dismutation of i ntracellular superoxide to H 2 O 2

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75 Figure 3 1. Experimental design of effects of Mn 2+ and other divalent metals on H 2 O 2 production HAPI microglia primary mixed glia and astroglia were seeded and cultured to confluence in 96 well plate. Cells were wa shed with warm HBSS for three times and then treated with vehicle (HBSS) or metals solutions in HBSS for 6 hours. After treatment, Amplex UltraRed was used to detect H 2 O 2 production in the culture media.

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76 Figure 3 2 Effects of Mn 2+ on H 2 O 2 pr oduction in HAPI microglial cells, mixed glia and astroglia cultures. Cells in 96 well plates were treated for 6 h at 37C in HBSS as a control or indicated concentrations of MnCl 2 The amounts of H 2 O 2 released into the media were then determined by Amplex UltraR ed Results are expressed as a percentage of the amount of H 2 O 2 released from cells in the control group for each cell type Results are mean SEM of 3 4 experiments. *, P<0.05 ; **, P<0.005 compared to the control.

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77 Figure 3 3 Effects of di valent cations on H 2 O 2 production in HAPI microglial cells, mixed glia and astroglia cultures. Cells in 96 well plates were treated for 6 h at 37C in HBSS as a control (C) or 10 M of indicated divalent metals. The amounts of H 2 O 2 released into the media were then determined Results are expressed as a percentage of the amount of H 2 O 2 released from cells in the control group for each cell type Results are mean SEM of 3 4 experiments. **, P<0.005 compared to the control.

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78 Figure Mn 2+ Hg 2+

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79 Figure 3 5 E ffects of divalent metals at 100 M on H 2 O 2 production in HAPI m icroglial cells. Cells were treated for 6 h in HBSS ( C ) or 100 M of indicated divalent metals. The amounts of H 2 O 2 released into the media were then detected. Results are expressed as a percentage of the amount of H 2 O 2 released from control cells. Result s are mean SEM of 3 5 experiments. **, P<0.005 compared to the control.

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80 Figure 3 6 Effect s of divalent metals on ROS produ ction in HAPI microglial cell. Cells were treated for 6 h with HBSS (C) or 10 M of the indicated divalent metals. ROS product ion was detected using DCF DA. Results are mean SEM of 3 5 experiments. **, P<0.005 compared to the control.

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81 CHAPTER 4 THE INVOLVEMENT OF M ITOCHONDRIA IN MN 2+ MEDIATED NEUROTOXICITY Introduction In Chapter 3 we observed significant H 2 O 2 release from mic roglia in response to Mn 2+ treatment. I n this Chapter, our main goal is to elucidate the mechanism of Mn 2+ induced H 2 O 2 production in microglia We are first interested to determine which subcellular site is responsible for Mn 2+ induced H 2 O 2 There are tw o potentially important subcellular sources of ROS production site in microglia: NADPH oxidase and mitochondrial electron transport chain (ETC) We have previously tested the effects of NADPH oxidase inhibitors to block Mn 2+ induced H 2 O 2 production The l ack of effect by NADPH oxidase inhibitors suggest ed that NADPH oxidase does not play a role in Mn 2+ induced H 2 O 2 production in microglia Rationale (1) Except for NADPH oxidase, m itochondria l ETC is another intracellular source for ROS production. We are interested to find out if Mn 2+ induced H 2 O 2 production is originated from the mitochondria l ETC (2) To determine which ETC complex Mn interacts with to production ROS. In the mitochondrial ETC, c omplex I and complex II are the electron entry points. In t his study, glutamate and malate will be used as complex I substrates. Succinate will be used as complex II substrate. The use of specific respiratory substrates can help to differentiate electron flow pathways upon Mn interference. The presence of ETC comp lex inhibitors can significantly enhance ROS production by blocking electron flow at the inhibition site. In this study, we will use

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82 ETC inhibitors to identify which complex is responsible for Mn 2+ induced H 2 O 2 production. Hypothesis (1) M itochondria are t he major subcellular s ite of Mn 2+ induced H 2 O 2 release in microglia. (2) Mn 2+ induces H 2 O 2 production by interfering with the electron transport chain complexes. Experimental D esign W e fractionate d HAPI microglia into nuclei, mitochondria and cytosol by di fferential centrifugation. To obtain pure preparations from each subcellular compartment, we performed P ercoll gradients and sucrose cushion to obtain pure mitochondrial and nuclear fractions (Figure 2 2). The purities of each subcellular fraction w ere ass essed by immunoblot analysis by mitochondrial specific marker cytochrome c oxidase subunit III, cytosolic specific marker lactat e dehydrogenase, and the nuclear marker NOX III. The coupling of mitochondrial oxidative phosphorylation and ATP production was examined by respiration curves using oxygen probe. To determine H 2 O 2 release induced by Mn 2+ of protein from each subcellular fraction will be incubated with indicated concentrations of Mn 2+ or specific ETC complexes inhibitors. We carried out exten sive screening with various ETC inhibitors ( Figure 4 1): c omplex I: rotenone; complex II: malonate; complex III: antimycin A, myxthothial; complex IV: azide and complex V: oligomycin. The abilities of these ETC inhibitors to produce ROS by themselves were compared with the combination of the inhibitor and Mn 2+ If any of the ETC inhibitors has modulatory ef fect on Mn 2+ produced ROS or vic e versa, the Mn 2+ interaction site on the ETC should be identified.

PAGE 83

83 period of 30 mins. ETC i nhibitor or Mn 2+ mitochondrial protein. Mitochondria l respiratory substrates (5 mM glutamate, 2.5 mM malate and 5 mM s ucci nate) are added into each fraction to initiate the reaction by providing electron flow for the mitochondrial ETC (Figure 4 1) Results Mitochondria are the M ajor S ubcellular S ite of Mn 2+ induced H 2 O 2 P roduction As shown in F i gure 4 2 when mitochondrial respiratory substrates (5 mM glutamate + 2.5 mM malate + 5 mM succinate) were present, 10 M Mn 2+ induced a significant H 2 O 2 production in the mitochondrial fraction. However, Mn 2+ did not induce significant H 2 O 2 production in the cytosolic or nuclear fra ctions (Fi gure 4 2 ) under the same conditions. As positive controls, rotenone and antimycin A, known inhibitors of mitochondrial ETC complex I and III respectively ( St Pierre et al., 2002 ) induced robust H 2 O 2 production in mitochondria, but not in cytosol or in nuclei (Fi gure 4 2 ). To confirm that the mitochondrial preparations isolated from microglia were functionally viable, we measured respiration rate usin g oxygen consumption assay. As shown in Fi gure 4 3 A, addition of FCCP, an oxidative phosphorylation uncoupler, c aused a 6 fold increase in the rate of respiration in the mitochondrial fraction (Fi gure 4 3 A). This result, together with the aforementioned responses to ETC inhibitors (Fi gure 4 2 ) demonstrates that the coupling between respiration and phosphorylation was intact in the isolated mitochondria. The purity of subcellular fractions was assessed by immunoblot analysis for organelle specific

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84 markers As shown in Fi gure 4 3 B mitochondrial marker proteins cytochrome C and COX III were detected only in the mitochondrial, but not in cytosolic and nuclear fractions. Similarly, LDH and histone H1 were detected only in the cytosolic and nuclear factions respectively (Fi gure 4 3 B ), indicating that the subcellular fractions prepared from HA PI microglia were highly pure. Complex II S ubstrate is M ore E fficient than C omplex I S ubstrates in S upporting the Mn 2+ induced H 2 O 2 P roduction To determine the underlyin g mechanism of Mn 2+ induced H 2 O 2 production by microglial mitochondria, we supplemented mitochondrial proteins with either the combination of glutamate and malate (complex I substrates), or succinate (complex II substrate), and measured production of H 2 O 2 by mitochondria in the presence of Mn 2+ rotenone or antimycin A. The amount of H 2 O 2 produced by mitochondria respiring on malate and glutamate was very low and was not increased by the addition of Mn 2+ (Fig ure 4 4 ). In contrast, the amount of H 2 O 2 prod uced by mitochondria respiring on succinate was considerably elevated compared to that on malate and glutamate, consistent with the notion that succinate drives ROS production from complex I by reverse electron transfer ( Liu et al., 2002 ) More importantly, this succinate supported H 2 O 2 production was further elevated in the presence of 10 and 100 M Mn 2+ (Fig ure 4 4 ). As expected, r otenone induced significant H 2 O 2 production by mitochondria respiring on complex I substrates and antimycin A induced significant H 2 O 2 production in the presence of either substrates (Fig ure 4 4 ). Mn 2+ at 100 M induced a comparable amount of H 2 O 2 production in the presence of succinate as that induced by antimycin A (Fig ure 4 4 ). These data show that complex I substrates,

PAGE 85

85 glutamate and malate, support e d minimal rates of H 2 O 2 production by microglial mitochondria in the absence or presence of Mn 2+ In contrast, complex II substrate, succinate, afford ed significant H 2 O 2 production and Mn 2+ further promote d H 2 O 2 production by microglial mitochondria (Fig u re 4 4 ). The effect of Mn 2+ on complex II activity was investigated by respiration in the presence of succinate. As shown in Figure 4 5 A, Mn 2+ at 10 and 100 M exerted minimal effects on mitochondrial O 2 consumption. At 8 00 M Mn 2+ slightly enhanced mit ochondrial O 2 consumption. Furthermore, Mn 2+ at 10 100 and 800 M failed to alter mitochondrial state 3 respirations in the presence of ADP (Figure 4 5 B). These results suggested that the increased H 2 O 2 production induced by Mn 2+ was not due to increase d utilization of succinate. ETC c omplex II P lays a M ore P rominent R ole t han C omplex I in Mn 2+ induced H 2 O 2 P roduction The observation that complex I and II substrates differentially support ed Mn 2+ induced H 2 O 2 production prompted us to further investigat e mechanism of Mn 2+ induced ROS production in the ETC. For this purpose, we tested the effects of various electron transport inhibitors on Mn 2+ induced H 2 O 2 production in the presence of complex I or complex II substrates. In the presence of succinate, t he amount H 2 O 2 produced by mitochondria was significantly increased in the presence of complex III inhibitors antimycin A and myxothiazol ( Figure 4 6 ). In contrast, complex II inhibitor malonate decreased the succinate supported H 2 O 2 production to baselin e ( Figure 4 6 ). Addition of Mn 2+ (10 M) to mitochondria respiring on succinate significantly increased the amount of H 2 O 2 produced ( Figure 4 6 ). This succinate supported

PAGE 86

86 and Mn 2+ elevated H 2 O 2 production was significantly reduced only by co treatment wi th malonate, but not by rotenone, antimycin A, myxothiazol, azide or oligomycin ( Figure 4 6 ). These results indicate d that the electrons which g a ve rise to superoxide production (consequently H 2 O 2 production) in response to Mn 2+ treatment enter ed the ETC at at the level of complex II. Consistent with this notion blockade of complex II activity abolishe d Mn 2+ induced H 2 O 2 production by microglial mitochondria ( Figure 4 6 ) When the effects of the inhibitors and Mn 2+ on H 2 O 2 production in mitochondria sup plemented with complex I substrates glutamate and malate was determined, t he amount of H 2 O 2 produced by mitochondria respiring on malate and glutamate was very low ( Figure 4 7 ). Addition of Mn 2+ (10 M) did not increase baseline H 2 O 2 production from the m itochondria l ETC ( Figure 4 7 ). Addition of rotenone, antimycin A and myxothiazol to mitochondria respiring on malate and glutamate induced significant increases in the amount of H 2 O 2 generated ( Figure 4 7 ). However co treatment of 10 M Mn 2+ with the ET C inhibitor s did not significantly alter the profile for the effects of the inhibitors alone on H 2 O 2 productions ( Figure 4 7 ). These results suggested that complex I was not a contributor to the Mn 2+ induced H 2 O 2 production in microglial mitochondria To gain insight into the ROS generating site in complex II responsible for the Mn 2+ induced H 2 O 2 production we tested the effect of three site specific complex II inhibitors. Malonate and malate are analogues of succinate which inhibit complex II by bindin g to the flavin binding site (site II F ). TTFA is a specific inhibitor of ubiquinone binding site in complex II (site II Q ). As shown in Figure 4

PAGE 87

87 8 malonate and malate significantly decreased the Mn 2+ induced H 2 O 2 production whereas TTFA failed to preven t H 2 O 2 production induced by Mn 2+ These results suggested that Mn 2+ induced H 2 O 2 production was generated from site II F in complex II. Discussion This study in HAPI microglia demonstrate d that the mitochondrial ETC was the major intracellular site of Mn 2+ induced H 2 O 2 production, and that this effect wa s mediated principally through ETC complex II. This conclusion wa s supported by the following observations: (a) Mn 2+ induced a significant increase in H 2 O 2 production in the mitochondrial, but not nuclear and cytosolic fractions from microglial cells; (b) The presence of a complex II substrate (succinate) but not that of complex I (malate and glutamate) was required for the Mn 2+ induced H 2 O 2 production in microglial mitochondria; and (c) Complex II inhibit or malonate and malate inhibited the Mn 2+ induced H 2 O 2 production by competing with succinate for the active sites on complex II. Mitochondria as the S ubcellular S ite of Mn 2+ induced ROS G eneration in M icroglia The second goal of our study was to identify the subcellular site of Mn 2+ induced H 2 O 2 generation in microglia. In this study, isolated microglial mitochondria we re an efficient producer of free radicals only in the presence of ETC substrates. With succinate as the substrate, formation of H 2 O 2 incr eased by several folds and Mn 2+ further increased the succinate driven H 2 O 2 formation (Fig ure 4 4 ). On the contrary, nuclear fraction from microgia was incapable of generating significant quantities of H 2 O 2 in the absence or presence of Mn 2+ A

PAGE 88

88 number of previous studies have suggested that Mn 2+ can interfere with the mitochondrial oxidative phosphorylation, repspiration and ATP production ( Galvani et al., 1995 Gavin et al., 1999 Chen et al., 2001 Zwingmann et al., 2003 Milatovic et al., 2007 Gunter et al., 2010 ) Mn 2+ has been reported to preferentially accumulate in either mitochondria ( Gunter et al., 2009 ) or nuclei in astroglia and neurons ( Kalia et al., 2008 Morello et al., 2008 ) Whether there is one predominant subcellular organelle for Mn 2+ storage/enrichment or different profiles exist for different cell types remains to be resolved. Although the subcellular compartmentation, if any, of Mn 2+ in microglia has not been determined, our results suggest that the accumulation of Mn 2+ in the mitochondria may be more directly related to the enhanced H 2 O 2 production and release in microglia Involvemen t of ETC Complexes in Mn 2+ induced H 2 O 2 Release in Microglia Complex III inhibitors antimycin A and myxothiazol are known to be capable of inducing H 2 O 2 production ( St Pierre et al., 2002 ) Antimycin A and myxothiazol inhibit the center i and Rieske iron sulfur center of complex III, respectively ( St Pierre et al., 2 002 ) The inhibition afforded by antimycin A facilitates the formation of semiubiquinone radical which is highly unstable and readily gives rise to superoxide formation at complex III center o ( St Pierre et al., 2002 ) In this study in the presence of complex I substrates, rotenone and antimycin A produced significantly higher amounts of H 2 O 2 ( Figure 4 7 ). Myxothiazol increased H 2 O 2 production to a similar level as that of rotenone, suggesting that H 2 O 2 produced by myxothiazol was most probably generated at the same site as that for rotenone at complex I. In the presence of succinate,

PAGE 89

89 antimycin A produced the highest amount of H 2 O 2 This result is in ag reement with a previous study which reported that complex III (when inhibited by antimycin A) plays the central role in producing H 2 O 2 from mitochondria from brain homogenate ( Chen et al., 2003 ) In this study, in the presence of complex I substrates, Mn 2+ at 10 M failed to induce significant H 2 O 2 production from the mitochondria ( Figure 4 4 ). This result suggests that complex I is not the site of Mn 2+ induced ROS p roduction in forward electron transport from complex I towards downstream complexes in the ETC. One the other hand, in the presence of complex II substrate succinate, Mn 2+ at the same concentration induced considerable H 2 O 2 production from the mitochondri a ( Figure 4 4 ). A ddition of rotenone was not able to inhibit this effect, demonstrating that Mn 2+ induced H 2 O 2 production was not through reverse electron transfer from complex II back to complex I. However, at a higher concentration (100 M), Mn 2+ alone induced a slight increase in H 2 O 2 production in the presence of complex I substrates although the increase was not s tatistically significant ( Figure 4 4 ). Mn 2+ at this concentration may begin to have an effect on complex I. Notably, complex II inhibitor malonate was the only inhibitor that decreased the Mn 2+ induced H 2 O 2 production supported by succinate ( Figure 4 6 ) The respiration data sugges t that Mn 2+ induced H 2 O 2 production was not due to increased electrons influx from complex II (Figure 4 5 ) Complex II in Mn 2+ induced H 2 O 2 Production Mitochondrial complex II [succinate dehydrogenase (SDH)] catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol. The enzyme is composed of four subunits: A, B, C and D. SD HA contains a

PAGE 90

90 flavin binding site (site II F ) and SDHB has 3 iron sulf u r clusters. SDHC and SDHD are hydrophobic and serve to anchor SDH to the mitochondrial inner membrane. Complex II was not usually considered to be a major producer of ROS in the mitoch ondrial ETC. However, a very recent study ( Quinlan et al., 2012 ) has demonstrated that complex II can be an important source of ROS both during forward electron transfer and reverse electron transfer. This finding seem to corroborate well with our findings in this study that complex II played a key role in Mn 2+ induced H 2 O 2 production The precise mechanism governing how Mn 2+ interacts with complex II to cause an increased ROS production remains to be determined. However, earlier s tudies have rep orted that mutations in SDHC and cytochrome b in complex II produce significantly elevated amount of O 2 ( Ishii et al., 2005 Slane et al., 2006 ) In addition, Mn 2+ has been shown to have an inhibitory effect on mitochondrial iron sulfur containing enzyme, and this effect is most likely due to the competition of Mn 2+ with the Fe 2+ binding sites ( Chen et al., 2001 ) We speculate that Mn 2+ may destabilize iron sulfur clusters containing SDH B to cause a configuration change of SDH, which has been shown to give rise to ROS formation ( Lemarie et al., 2011 ) Previous studies have proposed that Mn 2+ could interact with each one of the complexes of the E TC to induce ROS formation ( Galvani et al., 1995 Gavin et al., 1999 Chen et al., 2001 Malecki, 2001 Zhang et al., 2004 ) These observations might have been complicated by different methodologies and systems emp loyed. Firstly, most of the studies have used a wide range of Mn 2+ concentrations of (5 M 1000 M) in different systems (neuron, astrocyte,

PAGE 91

91 whole brain tissue) ( Chen et al., 2001 Chen and Liao, 2002 Zwingmann et al., 2003 Zhang et al., 2004 Milatovic et al., 2007 ) Secondly, those studies have measured ETC complex activity instead of ROS production. Our study was aimed at identifying the specific site and mechanism of action in microglial mitochondrial ET C for ROS production by low micromolar concentration of Mn 2+ that may be relevant to elevated environmental exposure to Mn. Therefore, our findings may help gain significant insight into the complex interactions among various cells in the brain in the pat hogenetic process of Mn neurotoxicity.

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92 Figure 4 1. Mitochondrial ETC complexes inhibitors and substrates Inhibitor used to induce ROS production from the ETC were rotenone (complex I) malonate (complex II), antimycin A and myxthothial (complex III) azide (complex IV) and oligomycin (complex V) M alate / glutamate and succinate are specific electron donors for complex I and II respectively They are used to establish the substrate specificity for induced H 2 O 2 production.

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93 Figure 4 2 Effect of Mn 2+ on H 2 O 2 production in mitochondrial, cytosolic and nuclear fractions of HAPI microglial cells. Ten g of subcellular fractions were incubated for 30 min at 37C with 10 M of Mn 2+ rotenone or antimycin A in the assay buffer. Substrates (5 mM gluta mate, 2.5 mM malate and 5 mM succinate) were added to initiate the reaction. Results are normalized against mitochondrial fraction supplemented with the respiration substrates. Data are mean SEM of 3 5 experiments performed in duplicate. *, P<0.05, ** P<0.005 compared to the control (no substrates). B. Effect of on H 2 O 2 production in sucrose cushion purified nuclei from HAPI microglial cells. Ten g of protein were incubated with 10 M of rotenone or antimycin A in the assay buffer for 30 min at 37C. Substrates (5 mM glutamate + 2.5 mM malate + 5 mM succinate) were added to initiate the reaction. Results are normalized against nuclei alone. Data are mean SEM of 3 experiments performed in duplicate.

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94 Figure 4 3 Verification of m itocho ndrial functionality and purit y of subcellular fractions. ( A ) Mitochondrial respiration. Respiration was determined using a buffer contain ing 250 g mitochondrial protein and respiration substrates (5 mM glutamate + 2.5 mM malate + 5 mM succinate). Maxi mum respiration was initiated by the addition of M FCCP. ( B ) Immunoblotting analysis Subcellular fractions ( 50 g proteins ) were immunoblotted for the presence of cytochromes c and anti COX III ( mitochondrial marker proteins), LDH (cytosolic marker), and h istone H1 ( nuclear marker) Results are r epresentative of three separate experiments.

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95 Figure 4 4 Substrate specificity for induced H 2 O 2 production from succinate (suc) or glutamate/malate (M/G) were incubated with 1, 10 or H 2 O 2 production was then determined as described in the Methods. Data are mean SEM of 3 8 experiments performed in duplicate. *, P<0.05; **, P<0.005 compared to control.

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96 Figur e 4 5 Effects of on mitochondrial complex II respiration. (A, B) mitochondrial respiration was initiated by the addition of succinate. was added at increasing concentrations to the mitochondria. ADP was added to further stimulate state 3 respir ations.

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97 Figure 4 6 Effects of the ETC inhibitors and on H 2 O 2 production from mitochondria in the presence of complex II substrate. Mitochondrial proteins (10 g) were incubated with 10 M of and/or the indicated inhibitors. H 2 O 2 productio n was determined as described in the Methods. Data are mean SEM of 3 5 experiments performed in duplicate. *, P<0.05; **, P<0.005 compared to control (mitochondria + suc); #, P<0.05 compared to 10 M treatment.

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98 Figure 4 7 Effects of the ETC i nhibitors and on H 2 O 2 production from mitochondria in the presence of complex I substrates. This experiment was performed in the same manner as that described for Figure 4 6 except that malate/glutamate were used as the substrates. Data are mean S EM of 3 8 experiments performed in duplicate.

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99 Figure 4 8 Effects of TTFA, malate and malonate on the induced mitochondrial H 2 O 2 production in the presence of succinate. Mitochondria proteins (10 g) were incubated with 10 M with and without TTFA (10 M), malate (2.5 mM) or malonate (5 mM). H 2 O 2 production was then determined as described in Methods. Results are expressed as a percentage of H 2 O 2 production in the presence of 5 mM succinate. Results are mean SEM of 3 4 experiments. *, P<0.05; **, P<0.005 compared to the control.

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100 CHAPTER 5 THE SUBCELLULAR DIST RIBUTION OF MN 2+ IN MICROGLIA Introduction Mitochondria play a critical role in mediating Mn induced neuronal damage; however, it is still unclear if they are th e preferred subcellular site for Mn sequestration. For example, several studies using whole rat brain showed that mitochondria sequester more Mn than other intracellular organelles ( Maynard and Cotzias, 1955 Miller et al., 1975 Liccione and Maines, 1988 G avin et al., 1999 Lai et al., 1999 ) In contrast, studies of specific brain cells including neurons, astroglia, epithelia and endothelia, reported that mitochondria play an insignificant role in Mn sequ estration in these cell types and that nuclei ( Kalia et al., 2008 Morello et al., 2008 ) and golgi apparatuse s ( Carmona et al., 2010 ) sequester mo re M n compared to mitochondria Together, the results of these studies suggest that the subcellular distribution of Mn varies among brain cell types. The subcellular distribution of Mn has not been determined in microglia. Given the importance of microgli a in mediating neuroinflammation and of mitochondria in Mn induced neurodegeneration, we hypothesized that the primary site of Mn accumulation in microglia is the mitochondria. To test this hypothesis, we used confocal microscopy to examine Mn distributio n in live microglia by Mn quenching Fura red signal. In addition, we used inductively coupled plasma mass spectrometry (ICP MS) to examine Mn concentrations in cell fractionations of rat microglia.

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101 Rationale A number of earlier studies report that Mn 2+ res ides in the mitochondria in animals. However, recent studies argue that nuclei are the major Mn 2+ subcellular location both in vivo and in neuron as well as astroglia cultures. Given this controversy, we are interested in identifying the subcellular accumu lation site for Mn 2+ in the microglial cells. We d eveloped Mn quenching assay of f ura red AM to test the subcellular localization of Mn 2+ The cell permeant AM ester form of f ura red effectively localizes into mitochondria and nucleus. Fura red binding Mn 2+ with a high affinity (Kd=). Mn 2+ bound to fura red will quench the f ura red signal. The mitochondria are labeled with mitoT racker green. The quenching of f ura red signal co localized with mitoTracker green will demonstrate sequestration of the ion by t he mitochondria. The same rule will apply for the nucleus. An advantage of this method is that we can monitor the cells in real time for spatial distribution in live cells. Powerload was u sed to increase the loading of f ura red into the cells by dispersing the indicator in the loading medium. The ionophore Ionomycin was used to transport Mn 2+ ion across the membrane systems. After we demonstrated that mitochondria and (or) nucleus can sequester Mn 2+ we wa nt to quantify the amount of sub cellular accumulati on of Mn 2+ by inductively coupled plasma mass spectrometry (ICP MS). ICP MS is a very sensitive method to assess metal content. Cells are first incubated with Mn 2+ and then lysed and fractionated into nucleus, mitochondria, cytosol and mitosome. The cell l ysate are analytically diluted and the amount of metals can be assessed

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102 in the solution, providing absolute values for Mn uptake into each subcellular organelles expressed versus total protein concentration. Hypothesis Either mitochondria or nuclei are the major subcellular accumulation site for Mn 2+ Experimental D esign Fura red quenching assay: To determine Mn 2+ subcellular distribution, we first loaded the microglia with Ca 2+ fluorescent indicator fura red AM, then exposed the cells to varying concentrat ions of MnCl 2 (0 20 M). The rationale is that fura red signal will be quenched in the cell compartments that Mn 2+ can get access to (Figure 5 1). ICP MS: HAPI cells plated in 10020 mm dishes will be incubated with 10 M or 100 M Mn 2+ for 6 hours. After incubation, cells were washed with ice cold PBS for three times. For the total uptake, control cells will be scrabbed off in 350 l PBS per dish and centrifuged at 800 g for 10 min at 4 C. The cell pellet will be analyzed as total Mn uptake. For subcell ular analysis, cells will be scrabbed off in 350 l isolation buffer. Nuclei, mitochondria and cytosol will be fractionated following the procedure of differential centrifugation. Mn uptake in each fraction will be quantified by ICP MS (Figure 5 2). Result s Mn 2+ I nflux into the N ucleus and M itochondria in M icroglia A ssessed by C onfocal M icroscopy To determine Mn 2+ subcellular distribution, we first loaded the microglia with Ca 2+ fluorescent indicator fura red AM, then exposed the cells to varying

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103 concentra tions of MnCl 2 (0 20 M). The rationale is that fura red signal will be quenched in the cell compartments that Mn 2+ can get access to. Figure 5 3 A shows a representative field of cells loaded with fura red. Co staining of the mitochondrial dye mitoTracke r green was performed to pinpoint fura red signal in the mitochondria (Figure 5 3 B). The nucleus was identified by the ultra structural morphology. Quenching of fura red by increasing concentration of Mn 2+ was performed in the presence (Figure 5 3 D, 1F) or absence (Figure 5 3 C, 1E) of ionomycin, which is an ionophore that facilitates the transport of metal ions across the plasma membrane. In the absence of ionomycin, Mn 2+ showed quenching effect on fura red signal at the lowest dose used in the experiment ( 1 M), and the plateau of fura red quenching curves was reached at around 10 M of Mn 2+ in both nucleus and mitochondria (Figure 5 3 C, 1E). The addition of ionomycin further enhanced the quenching effect at lower doses of Mn 2+ (1 10 M) in both the nucleus and mitochondria (Figure 5 3 D, 1F). The quenching of Mn 2+ over 20 min was assessed in the presence of 100 M of Mn 2+ (Figure 5 4 ). The effectiveness of Mn 2+ to quench fura red suggests that Mn 2+ is readily transported into both the nuclei and mitochondria in rat microglia. Quantitative E valuation of Mn in D ifferent C ell C ompartments by ICP MS After we treated microglia with 10 M or 100 M of MnCl 2 for 6h, we fractionated the cells into mitochondrial, nuclear, microsomal (vesicles reformed from ER fragmen ts after ultracentrifugation) and cytosolic fractions, and used ICP MS to test the levels of Mn in each fraction. In control cells, the Mn content in all four fractions was in the range of 0.006 0.023 ng/mg protein without exogenous Mn 2+ treatments (Figure 5 5 A). When microglia were treated with 10

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104 M Mn 2+ the Mn contents significantly increased in all compartments, with mitochondria being the highest (0.044 ng/mg). When microglia were treated with 100 M Mn 2+ the mitochondrial (0.238 ng/mg) and cytosoli c (0.141 ng/mg) protein fraction accumulated higher amounts of Mn 2+ than the nuclear (0.029 ng/mg) and microsomal (0.0471 ng/mg) protein fractions, suggesting the mitochondria can accumulate more Mn 2+ In resp ect to fold increase (Figure 5 5 B), after 10 M Mn 2+ treatment, microsome and cytosol each had 3.1 and 3.0 fold increase in Mn content. Mitochondria had 2.0 fold increase and nuclei had the lowest increase, only 1.5 fold. After 100 M Mn 2+ treatment, fold increase was highest in the cytosol fra ction (1 1.7 fold). The m itochondria and microsome fraction each had a 9.5 and 8.8 fold increase respectively. Whereas the lowest fold increase was found in the nuclei (4.6 fold) compared to the control. Discussion In this study, we have shown that Mn 2+ influx int o the nucleus and mitochondria of microglial cells using fura red quenching assay. The treatment related influx of Mn 2+ was evident in both the nuclei and the mitochondria. In addition, we quantified the levels of Mn in each microglial subcellular fraction using ICP MS. Our results indicate that Mn 2+ can readily enter microglial cells, and can subsequently be sequestered by both the mitochondria and nuclei. The visible light excitable fura red enables us to visualize Mn 2+ quenching fura red signal in live m icroglia. The ability of Mn 2+ to quench fura red signal has been demonstrated in previous studies ( Wu and Clusin, 1997 ) In addition, Kwakye and colleagues recently developed a new approach to measure extracted cellular Mn level based on the quenching effect of Mn on fura

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105 disease cell model ( Kwakye et al., 2011 ) Our results suggest that after Mn 2+ incubation, fura red signals in both the mitochondria and nucleus were dramatically decreased ( Figure 5 3 ), indicating a rapid uptake mechanism of Mn 2+ into both organelles. Notably, this method detects chelatable Mn 2+ in cell compartments. The subcellular level of M n was quantitatively evaluated by ICP MS. ICP MS is a technique with high sensitivity, and it detected highest Mn concentration in the mitochondrial and cytosolic f raction s less in the nuclear and microsomal fraction s (Figure 5 5 ). However, there is a po ssibility that some Mn was lost during the washing step s Notably, ICP MS results represent the tightly bound form of Mn in each subcellular fraction. Since Mn is an essential cofactor for a variety of endogenous enzymes, there is a baseline amount of Mn i n each fraction. Mn has been shown to interact with mitochondrial proteins, such as Mn SOD and ETC complexes ( Gavin et al., 1999 ) Several studies have reported i nteraction of Mn with nucleotides of DNA, RNA, and ribos omes in vitro ( Jouve et al., 1975 Vogtherr and Limmer, 1998 ) Our results demonstrating Mn subcellular distribution in microglia is more in consiste nce with results from older studies using whole brain tissue. This might in part be due to same method ( cellular fractionation and ICP MS) used by us and other experiments to assess Mn content. Alternatively, the subcellular localization of Mn could vary a mong different brain cell types. Therefore, the recent findings in neurons and astroglia that nuclei and Golgi apparatus as the primary site of Mn localization might suggest that in other brain cell types like microglia,

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106 mitochondria might sequester more M n to compensate for the low level of Mn in neuronal and astroglial mitochondria. One potential mechanism of Mn neurotoxicity is via disrupting mitochondrial function. The subcellular localization of Mn in the mitochondria is also consistent with many prev ious studies demonstrating disturbances in mitochondrial ETC complexes after Mn treatments. or example, Gavin and colleagues ( Gavin et al., 1992 ) showed Mn 2+ inhibits th e rat brain mitochondrial F1ATPase. They also showed that Mn uptake into the mitochondria was mediated by calcium uniporter, energized the by internally negative membrane potential. G alvani et al. ( Galva ni et al., 1995 ) reported that MnCl 2 treatment in a neuronal cell line PC12 inhibits the ETC complex I, and later the same group further reported that Mn inhibits the ATPase at very low concentrations and inhibits complex I at higher concentrations ( Maynard and Cotzias, 1955 Miller et al., 1975 Liccione a nd Maines, 1988 Gavin et al., 1999 Lai et al., 1999 ) Additionally, Malecki ( Malecki, 2001 ) demonstrated a dose dependent loss of mitochondrial membrane potential and complex II activity in rat primary neuron cultures. Zhang et al. ( Zhang et al., 2004 ) reported decreased enzymatic activity from complex I through complex IV in the mitochondrial ETC. The inhibition of electron transport chain complexes can lead to increased production of reactive oxygen species and oxidat ive damage to mitochondrial DNA. Our previous study supports a key role of mitochondrial complex II (succinate dehydrogenase) in releasing significant amounts of hydrogen peroxide from microglia, which contributes to Mn neurotoxicity ( Liu manuscript in prep ) This idea is supported by a previous study

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107 showing Mn preferentially inhibits mitochondrial [Fe S] containing enzymes ( Zh eng et al., 1998 Chen et al., 2001 ) Decrease in Fe content has been known to occur under Mn overload in rats chronically exposed to Mn ( Zheng et al., 1999 ) One possibility is that Mn competes with Fe for transporters (transferrin, DMT 1); replaces iron in protein binding sites, and causes dysregulation of Fe metabolism. The efflux of Mn from the mitochondria has be en shown to be mediated by Na + independent mechanism, energized by the pH gradient ( Gavin et al., 1990 ) In conclusion, this present study provides evidence that in rat microglial cell, mitochondria are capable of accumulating Mn subcellularly. This information aids the understanding of mechanis m of Mn induced neurotoxicity.

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108 Figure 5 1. Mn uptake in microglia measured by fura red quenching assay. HAPI microglial cells were grown on 12 mm number 1.5 borosilicate glass coverslips coated with Poly L lysine in 6 well plate. Cells were washed twice in calcium free HBSS buffer Cells were l oaded with fura red AM and mito Tracker green. F ura red AM accumulates in different compartments in the cell. AM groups are cleaved from fura red by esterase activity. Cells were treated with different concentrations of MnCl 2 Mn 2+ quenches th e fura red signal in cellular compartments it can enter. If Mn 2+ enters mitochondria, mitochondrial fura red signal will decrease. Fluorescence signal was acquired by confocal microscope.

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109 Figure 5 2 Experimental design of Mn 2+ subcellular accumula tion in microglia analyzed by ICP MS. HAPI cells were plated in 10020 mm dishes and cultured to confluence. Cells were washed with PBS for three times and then incubated with 10 M or 100 M Mn 2+ for 6 hours. After incubation, cells were washed with ice c old PBS for three times and fractionated into mitochondria, nuclei, cytosol and microsome following the procedure of differential centrifugation. Mn content in each fraction was quantified by ICP MS

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110 Figure 5 3 Effects of Mn 2+ on fura red in microgl ia. Fluorescent images of microglia loaded with fura red (A) and co stained with fura red and mitoTracker green (B). Quenching effect of Mn 2+ on fura red fluorescent signal in mitochondria in the absence (C) and presence (D) of ionomycin. Quenching effect of Mn 2+ on fura red fluorescent signal in nuclei in the absence (E) and presence (F) of ionomycin.

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111 Figure 5 4 Quenching effect of 100 M Mn 2+ on fura red fluorescent signal in mitochondria and nuclei over time Nuclei Mitochondria

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112 Figure 5 5 Mn con tent in subcellular organelles of microglia. Cells were treated in HANKS(control), 10 M or100 M of Mn 2+ for 6 h and the levels of Mn 2+ was measured by ICP MS. Values are ex pressed as mean SEM of ng Mn/mg of protein (A) and fold increase compared to th e control (B) of 4 in dependent experiments. *, **, P< 0.05, 0.005 compared to control in each organelle.

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113 CHAPTER 6 SUMMARY AND FUTURE D IRECTIONS Grand C onclusion and R elevance Under certain occupational, medical and environmental settings, overexposure to Mn results in a Parkinson's disease like movement disorder called manganism. The pathology of manganism is characterized by neurological dysfunctions in the basal ganglia structure of the brain. The basal ganglia are located in the midbrain region and ar e particularly enriched in microglia, the resident immune cells of the CNS. One possible mechanism by which Mn 2+ induces neurotoxicity is through increasing microglia derived inflammatory factors, such as cytokines and free radicals that can harm adjacent neurons. We have previously shown that addition of Mn 2+ to neuron glia co cultures induced a dramatic increase in the ROS production from glial cells that results in enhanced neuronal damage ( Zhang et al., 2009 ) Given that the microglia derived ROS can potentially harm neurons, the first goal of this study was to characterize the effects of Mn 2+ on H 2 O 2 production in rat microglia and astroglia. H 2 O 2 is formed vi a dismutation of superoxide free radical and is a major contributor to oxidative damage. We report that low micromolar concentrations of Mn 2+ increased H 2 O 2 release from rat microglia and primary mixed glia. In contrast, rat primary astroglia failed to pro duce H 2 O 2 in response to the same range of Mn 2+ Examination of other common divalent transition metals demonstrated that the induction of H 2 O 2 release was specific to Mn 2+ Our next goal was to decipher the mechanism of ROS production in microglia. We f ractionated rat microglia into mitochondria, nuclei and cytosol using differential centrifugation and downstream P ercoll and sucrose gradients purification. We found

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114 that Mn 2+ treatment dramatically increased H 2 O 2 production in the mitochondrial fraction c ompared to baseline. No increase in H 2 O 2 production was observed in the nuclei and cytosol fractions treated with Mn 2+ Based on the results of this experiment, we then focused on identifying the electron transport chain (ETC) complexes responsible for Mn 2 + induced H 2 O 2 production in mitochondria. The mitochondrial ETC is composed of five complexes. The complexes transport electrons through a series of redox reactions to produce ATP. When the ETC is inhibited, electrons escape from the ETC to react with oxy gen, producing ROS. The involvement of ETC complexes in Mn 2+ induced H 2 O 2 production was investigated by screening ETC inhibitors and substrates. We report that complex II plays a key role in Mn 2+ induced H 2 O 2 production. Mitochondria play a critical ro le in mediating Mn 2+ induced neuronal damage; however, it is necessary to characterize how much Mn 2+ can be sequestered by the mitochondria in microglia. A couple of recent studies have reported that nuclei are the preferential subcellular site to accumula te Mn 2+ in neurons and astroglia. The subcellular distribution of Mn 2+ has not been studied in microglia. Therefore, in our next goal of this study, we sought to elucidate the subcellular distribution of Mn 2+ in microglia. We report here that micromolar c oncentrations of Mn 2+ readily transported across the cellular membrane and entered both the mitochondria and nucleus. ICP MS studies with microglial subcellular fractions revealed that mitochondrial proteins accumulated higher amount of Mn 2+ than the cytos olic, nuclear and microsomal fractions.

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115 Overall, this present study provides evidence that microglia readily release H 2 O 2 in presence of Mn 2+ supporting the idea that Mn 2+ induce neurotoxicity by activating microglia, which pose oxidative damage to adja cent neurons. These findings provide insights in elucidating the mechanism of Mn 2+ induced neurotoxicity as well as mechanism of ROS producti on from the mitochondrial ETC. Major L imitation of C urrent S tudy A major drawback of this study is that we used on ly cell cultures to predict Mn effect on neurotoxicity and neurodegeneration. For the effect of divalent metals to induce ROS from cells, we isolated primary rat mixed glia and astroglia. For the mitochondrial experiment, we used an immortalized rat microg lial cell line HAPI. Although in vitro system provide great insight into microglial mediated neurotoxicity, the findings have to be further confirm in animals to validate the role of ETC complex II in ROS production and neuroinflammation induced by Mn 2+ F uture D irections For future studies, I would love to use another alternative method of oxidative stress to support our ROS production data, such as fluorescent labeling for mitochondrial superoxide production, mitochondrial morphology, membrane potential e tc. I would love to further study the interaction between Mn 2+ and complex II. In order to test our hypothesis that Mn 2+ inhibits complex II by substituting Fe 2+ in the iron sulfur center, we can first try to purify complex II from isolated mitochondria In order to obtain higher yield of complex II, bacterial expression system and protein purification can be performed as well ( Suraveratum et al., 2000 ) T hen test if Mn 2+ treatment can decrease iron content in complex II by ICP MS. We can also do computational simulation to

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116 predict potential Mn 2+ binding sites on complex II and how the binding would change complex II conformation to produce ROS. We can vali date the identified binding sites by mutating amino acids residues on complex II to test if the mutation would affect complex II Mn 2+ binding and ROS production. Our result can be strengthened by knocking down complex II by RNAi in microglia cells to test if less complex II would decrease Mn 2+ induced oxidative stress. If complex II knockdown is lethal to the cells, we can try to do an inducible knockout. We can also obtain RNAi cell library of microglia, test Mn 2+ treatment to identify genes and pathways i nvolved in Mn 2+ induced ROS production, to see if correlations could be identified with complex II mediated ROS production pathway. The role of complex II and microglia as a mediator for Mn 2+ neurotoxicity should be tested in animal models (conditionally knockdown in microglia) for final validation.

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117 Figure 6 1. Microglia mediate Mn neurotoxicity via H 2 O 2 production from mitochondrial ETC complex II. Microglia are the major source of Mn 2+ induced H 2 O 2 release; and mitochondria are the major intr acellular site of H 2 O 2 production, which was centrally mediated by ETC complex II.

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137 BIOGRAPHICAL SKETCH Yue Liu was born in Tangshan, Hebei pr ovince, China to Weiguo Liu and Xiaohua Liu. She was raised in Tangshan and after completing her high school from there, she went on to obtain a B achelor of Science in b iotechnology from Beijing Forestry University, Beijing. Afterwards, she was fascinated by the techniques and knowledge one can obtain from biology to contribute to medicine, she determined to continue higher educa tion in biomedical science. S he pursued a Master of Science in b iopharmaceuticals under Dr. Larry Wakelin in school of biomedical sciences from University of New South Wales in Sydney, Australia. Then she joined the PhD program in the Department of Pharma ceutical Sciences at the University of Florida working under Dr. Bin Liu Upon graduation from the University of Florida, she inten ds to continue her scientific career in academia.