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Anti-Amyloid Beta scFvs Ameliorate Amyloid Beta-Induced Neurotoxicity in Drosophila

Permanent Link: http://ufdc.ufl.edu/UFE0045610/00001

Material Information

Title: Anti-Amyloid Beta scFvs Ameliorate Amyloid Beta-Induced Neurotoxicity in Drosophila
Physical Description: 1 online resource (43 p.)
Language: english
Creator: Mathur, Krishanu
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: alzheimers -- drosophila -- neurodegeneration -- scfv
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Alzheimer’s disease (AD) is a debilitatingneurodegenerative disorder characterized by the presence of amyloid plaques andneurofibrillary tangles. With no known cure at present, investigations intodifferent therapeutic strategies have produced varying results. Use ofmonoclonal antibodies targeted against amyloid beta has proven to be anefficient therapy strategy in animal models and has the potential to betranslated into clinically relevant strategies. Replacing these antibodies withengineered antibodies, such as single chain variable fragments (scFvs), hasallowed researchers to reap the efficacy of monoclonal antibodies whileminimizing associated side effects. For my masters thesis, I investigate theeffectiveness of two anti-amyloid beta scFvs, scFv-Ab9 and scFv-213, inarresting amyloid beta-induced neurodegeneration in a transgenic Drosophila disease model. I generatedstable fly lines expressing two different anti-amyloid beta scFvs andeffectively showed that these antibodies partially rescue flies from amyloidbeta neurotoxicity. The rescue efficacy was additively enhanced byco-expression of both scFvs. This work highlights the application of Drosophila based disease models in theefficient screening and validation of new therapeutic antibodies againstamyloid beta neurotoxicity. These new, and more effective anti-amyloid betascFvs can then be investigated further in rodent models and moved towardsclinical trials.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Krishanu Mathur.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Ormerod, Brandi K.
Local: Co-adviser: Fernandez-Funez, Pedro.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045610:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045610/00001

Material Information

Title: Anti-Amyloid Beta scFvs Ameliorate Amyloid Beta-Induced Neurotoxicity in Drosophila
Physical Description: 1 online resource (43 p.)
Language: english
Creator: Mathur, Krishanu
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: alzheimers -- drosophila -- neurodegeneration -- scfv
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Alzheimer’s disease (AD) is a debilitatingneurodegenerative disorder characterized by the presence of amyloid plaques andneurofibrillary tangles. With no known cure at present, investigations intodifferent therapeutic strategies have produced varying results. Use ofmonoclonal antibodies targeted against amyloid beta has proven to be anefficient therapy strategy in animal models and has the potential to betranslated into clinically relevant strategies. Replacing these antibodies withengineered antibodies, such as single chain variable fragments (scFvs), hasallowed researchers to reap the efficacy of monoclonal antibodies whileminimizing associated side effects. For my masters thesis, I investigate theeffectiveness of two anti-amyloid beta scFvs, scFv-Ab9 and scFv-213, inarresting amyloid beta-induced neurodegeneration in a transgenic Drosophila disease model. I generatedstable fly lines expressing two different anti-amyloid beta scFvs andeffectively showed that these antibodies partially rescue flies from amyloidbeta neurotoxicity. The rescue efficacy was additively enhanced byco-expression of both scFvs. This work highlights the application of Drosophila based disease models in theefficient screening and validation of new therapeutic antibodies againstamyloid beta neurotoxicity. These new, and more effective anti-amyloid betascFvs can then be investigated further in rodent models and moved towardsclinical trials.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Krishanu Mathur.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Ormerod, Brandi K.
Local: Co-adviser: Fernandez-Funez, Pedro.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045610:00001


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1 ANTI SCFVS AMELIORATE INDUCED NEUROTOXICIT Y IN DROSOPHILA By Krishanu Mathur A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Krishanu Mathur

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3

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4 ACKNOWLEDGMENTS Before I express gratitude toward the contributors of this work, I would like to dedicate this print space to the series of fortunate (and some unfortunate) events that transpired to give me the confidence and comprehension to finish this thesis. I would like to thank my family, without whom I would lack direction or motivation to explore my dreams. My d eepest gratitude to my principal investigators, Dr. Ferna ndez Funez and Dr. Rincon Limas for their undeterred attention and guidance towards my project. Over the course of this thesis, I had the good fortune to work alongside talented individuals, both exp erienced and newcomers from the Fernandez Funez/Rincon Limas lab. Their My heartfelt appreciation for my committee members, the time and energy they spent towards my th esis work ensured its timely completion. I would like to thank my friends, for them I can never feel blessed enough. I hope some of their passion and wisdom rubbed off on me during our time at the University of Florida. Sukhmani Bedi, my strongest critique and my warmest friend. Her critical inputs to my thesis helped me give it the form and flow I was desperately seeking. I would also like to make a special mention of the entire administrative and technical staff at the University of Florida. Their unti ring efforts ensured that my focus stayed on my work. Last but definitely not the least, thanks go out to denizens and patrons of the city of Gainesville, their affection and hospitality made Gainesville my home away from home.

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5 TABLE OF CONTENTS pa ge ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 6 LIST OF ABBREVIATIONS ................................ ................................ ............................. 7 ABSTRACT ................................ ................................ ................................ ..................... 8 INTRODUCTION ................................ ................................ ................................ ........... 10 Beta ................................ ................................ .. 10 Therapeutic Strat egies ................................ ................................ ............................ 12 Immunotherapy ................................ ................................ ................................ ....... 13 Single Chain Variable Fragment ................................ ................................ ............. 15 Project Aims ................................ ................................ ................................ ............ 16 MATERIALS AND METHOD S ................................ ................................ ...................... 18 Generation Of Plasmids Carrying Transgenes ................................ ....................... 18 Drosophila Strains ................................ ................................ ................................ .. 18 Fresh Eye Images ................................ ................................ ................................ ... 18 Field Emission Scanning Electron Microscopy (FESEM) ................................ ........ 19 I mmunohistochemistry (IHC) ................................ ................................ .................. 19 Cell Death Analysis ................................ ................................ ................................ 20 ................................ ................................ ........ 20 RESULTS ................................ ................................ ................................ ...................... 22 Generation Of Transgenic Flies Expressing Ant i ................................ .... 22 Induced Eye Phenotype ............................ 23 Anti Ab9 ................................ ................................ ............................. 24 Anti 213 ................................ ................................ ............................. 24 Generation Of Lines Expressing Both Anti Ab9 and 213 ........................ 25 Anti Induced Cell Death In The Eye ................................ ...... 26 ti ................................ ................................ .. 27 ................................ .............. 28 DISCUSSION ................................ ................................ ................................ ................ 35 LIST OF REFERENCES ................................ ................................ ............................... 39 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 43

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6 LIST OF FIGURES Figure page 3 1 llustration showing generation of stabl e stocks of Drosop hila carrying anti scFv gene ................................ ................................ ................................ ........... 29 3 2 Rescue in eye phenotype of transgenic flies by expression of scFv. ................. 30 3 3 llustration s howing generation of stable stocks of Drosophila carrying both scFv Ab9 and scFv 213 genes ................................ ................................ ........... 31 3 4 Co expression of scFv decrea ye imaginal disk of third instar larvae. ................................ ................................ ..... 32 3 5 development ................................ ................................ ................................ ....... 33 3 6 anti neuron periphe ry in the v entral nerve cord of third instar larvae. ................................ ................................ ......... 33 3 7 scFv. ................................ ................................ ................................ ................... 34 3 8 il deposits in adult fly brains ........ 34

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7 LIST OF ABBREVIATIONS AD Amyloid beta APP Amyloid p rotein precursor BSA Bovine serum albumin CAA Cerebral amyloid angiopathy DNA Deoxyribonucleic acid mAb Monoclonal antibodies NFT Neurofibrillary tangles PBS Phosphate buffered saline scFv Single chain variable fragment UAS Upstream activation sequence

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8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master Of Science ANTI SCFVS AMELIORATE INDUCED NEUROTOXICIT Y IN DROSOPHILA By Krishanu Mathur May 2013 Chair: Brandi Ormerod Co chair: Pedro Fernandez Funez Major: Biomedical Engineering characterize d by the presence of amyloid plaques and neurofibrillary tangles. With no known cure at present, investigations into different therapeutic strategies have produced proven to be an efficient therapy strategy in animal models and has the potential to be translated into clinically relevant strategies. Replacing these antibodies with engineered antibodies, such as single chain variable fragments (scFvs), has allowed researchers to reap the efficacy of monoclonal antibodies while minimizing associated side effects. For thesis, I investigate the effectiveness of two anti Ab9 and scFv induced neurodegeneration in a transgenic Drosophila disease model. I generated stable fly lines expressing two different anti effectively sh rescue efficacy was additively enhanced by co expression of both scFvs. This work highlights the application of Drosophila based disease models in the efficient screening and vali

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9 more effective anti moved towards clinical trials

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10 CHAPTER 1 INTRODUCTION A key histopathological characteristic of many neurodegenerative diseases is the accumulation of abnormally folded proteins. These misfolded proteins aggregate, either extra or intracellularly, leading to the disruption of neuronal functions and ultimately, neuronal cell loss (Selkoe et al., 2002). An example of one such debilitating dementia and the loss of at least one cognitive domain of the brain, AD is recognized as a neurologic deterioration, which inc reases sharply after the age of 65. nd Amyloid Beta At present, 5.4 million Americans are reported to be suffering from AD. This number is estimated to rise to 11 16 million by 2050. AD is currently the sixth leading cause of death in the US. While the number of deaths caused by other leading causes such as stroke, prostate cancer, and heart diseases has decreased over the last 2012). This increase in mortality rate is due to the lack of disease modifying treatments at this time. Neuropathological studies indicate the accumulation of misfolded extracellular amyloid plaques, consisting of misfolded amyloid beta, and intracellular neurofibrillary tangles (NFT), composed of hyper phosphorylated tau, as the major determinants of AD pathology (Katzman, 1986). Monomeric amyloid secretase, a type I glycosylated aspartyl protease, and secretase, a large protein complex (Burd ick et al., 1992). The heterogeneous cleavage activity of secretase

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11 N ), having different solubility, 40 dominates a s the major cleaved peptide species present extracellularly, along with 42 In AD, improper cleavage of APP at the C terminal results in 42 and partici pate towards oligomerization and fibrillation (Benilova et al., 2012). 42 peptides have been shown to cause toxicity to neurons in both, cell cultures and in vivo (Deshpande et al., 2006). In add ition, mutations in presenilin 1 and 2 genes encoding the catalytic site of secretes, cause early onset and aggressive forms of AD by increasing the 42 40 ratio (Kumar Singh et al., 2006). Mutations affecting the function or expression levels of AP P have been linked to increased neurodegeneration. For (Prasher et al., 1998). Also, transgenic mic e with mutant human APP show a time peptides or expression of mutant APP in tau transge nic mice leads to accelerated hyper phosphorylation of tau and tangle formation, both phenotypes associated with AD (Gotz et al., 2001; Lewis et al., 2001). hyper phosophorylatio n of tau and its aggregation in the cytoplasm. The toxic products of these misfolded proteins are the cause of neuronal dysfunction and neuron death,

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12 both hallmarks of AD. This neuronal death is found chiefly in the cerebral cortex of the brain. This loss in synapses leads to degeneration of the temporal and parietal lobes and part of the frontal cortex, leading to dementia. Earlier, in vivo studies and brain autopsies of AD patients linked the presence of amyloid fibrils to cell toxicity. However, these st udies ignored the weak correlation between the severity of dementia in AD patients and the relative density of amyloid plaques ( Nslund et al., 2000). Recently, Living rat long term potentiation in the hippocampus (Walsh et al., 2002). These studies consider large, polymeric A with smaller, potentially neurotoxic assemblies. Consequently, there is no unanimity on the precise identity of these toxic mediators of neurotoxicity or their cellular targets. This gap in knowledge has been an impediment in designing a strong and efficacious treatment strategy against AD. Therapeutic Strategies From our current understanding, AD is a neurodegenerative condition brought on and its clearance. Considering this idea, researchers are presently investigating various therapeutic strategies. The first or secretase. Recent studies are aimed towards identification of inhibitors that can target secretase and yet be small enough to cross the blood brain barrier. Potent membrane permeable inhibitors against secretase have been devised, but lack extensive testing in human pa tients due to the concerns that these inhibitors,

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13 which target the mutant form of presenilin, may interfere with signaling by Notch proteins and other cell surface proteins (Wong et al., 2004). Lower incidence of AD was also correlated to long term use of cholesterol lowering drugs such as statins. However, the suggested mechanism for influence of cholesterol on APP is poorly underst ood chelation of Ca 2+ /Zn 2+ mouse model (Cherny et al., 2001). Immunotherapy The use of antibody based strategies accelerate its clearance is gaining momentum. In 1996, researchers showed that antibodies directed towards the N in vitro (Solomon et al., 1996). Experiments cond ucted a year later displayed the ability of antibodies to disaggregate pre formed fibrils and, subsequently, induce previous studies, formation of amyloid plaques in t ransgenic mice over expressing APP confirmed these results and showed an association betwe en removal of plaques and improvement in cognition in an AD mouse model (Janus et al., 2000). The success of mmunization were halted after a few patients (~6% of the cohort) developed autoimmune meningoencephalitis (Gilman et al., 2005). The reason behind meningoencephalitis is believed to be abnormal brain infiltration of T lymphocytes initiated by the T cell ac

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14 Efforts have since then been directed towards developing safer active vaccines, such as those comprising of smaller antigen sequences to minimize toxicity mediated by cellular immunity. Alternatively, many researchers ar through the use of ready made antibodies, affords researchers greater control over sed in active immunization protocols, monoclonal antibodies (mAbs) used in passive vaccination are easier to produce and are more stable in vivo. Also, mAbs have zero risk of mediating the cellular immune response. Additionally, researchers can control the frequency and dosage of the antibody, making it a safer option in comparison to active immunization. The ease of producing high affinity and immunologically safe antibodies, which can be quickly moved into the clinic, has pushed passive immunization to th e forefront of AD therapies. Significant work was done to demonstrate reduction in amyloid load in AD transgenic mice after intraperitoneal injections of anti Despite the advantages of dosage control and high affinity for the target, other studies have recognized side effects with the administration of full mAbs. Administration of mAbs is linked to a risk of cerebral microhemmorrhage (Wang et al. 2010). Frequent he parenchymal regions of the brain to the blood vessels, a shift often associated with worsening of cerebral amyloid angiopathy (CAA) (Pfeifer et al., 2002). A limitation of this strategy is that a small fraction of peripherally administrated mAbs finds t heir way into the brain due to their poor blood

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15 accomplished by fragment crystallizable region (Fc) mediated phagocytosis, these Fc mediated functions of mAbs are not critical and do no t play a significant role in targeting replaced with engineered antibodies, a significant decrease in side effects and an increase in blood brain barrier penetration is reported. Single Chain Variable F ragment An alternative to full mAb passive immunotherapy is the use of single chain variable fragment (scFv) antibodies. ScFvs are monovalent, minimal antibodies synthesized as a single polypeptide chain comprising the variable lig ht domain linked to the variable heavy domain by a linker at the C terminus (Bird et al., 1988, Holliger and Hudson 2005). ScFvs, although lacking an Fc component, are as efficacious and specific as their parent mAb (Levites et al., 2006 a ). These engineer ed antibodies exhibit a shorter half life, do not activate microglia, show zero antibody induced cell toxicity or complement activation, and are easy to express and fold when expressed intracellularly ( Robert and Wark 2012; Marin Argany et al., 2011). Ove r the years, advances in phage techniques coupled with PCR technology have allowed scientists to create large scFv libraries. Using phage display, scFvs can either be displayed on the surface of the phage to allow further manipulations to increase binding properties or be released in soluble form (Skerra et al., 1991). Compared with other engineered antibodies, scFvs are the most common recombinant antibody format used in therapeutic studies against AD. Their use against Research showed that scFv 508F, derived from the IgM 508 antibody in vitro had similar anti aggregation and neuroprotective properties as its parent antibody (Frenekel et al., 2000). In 2010, a research study suggested the use

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16 of modified Asec 1 scFv to h 40 peptide ( Kasturirangan and Sierks 2010). Complementary to in vitro analyses, studies in animal models offer an scFv gene h holds an advantage over active immunization as it provides a stable and sustainable source of scFv in the host. Compared to passive immunization via antibody injections, gene based therapies overcome the hurdle of repeatedly administering expensive antib odies to attain the desired results. This makes scFv gene therapy a much safer and suitable strategy for the chronic treatment of AD. In 2006, three anti 9 was engineered from anti 16 mAb 9, scFv specific mAb 40.1, and scFv 42.2 was derived from anti specific mAb 42.2 (Levites et al., 2006a, Levites et al., 2006 b). scFv 9 has a binding epitope at the N 42.2 tested if the scFvs could prevent A scFvs were delivered via adeno associated virus to new born pups (P0), which were then allowed to age for 3 months, before examining for plaque loads. The investigators reported a significant decrease in Project A ims Work done on the anti significant neurodegenerat ion in the APP mouse model. Use of rodent models also

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17 limited the investigators from combining the antibodies to determine if they lead to better Using Drosophila as a disease model offers the advantage of following degenerative changes in the brain in a shorter period of time. In addition, the ease of manipulation that Drosophila genetics affords, researchers have an advantage with the number of antibodies that can be screened and the possibili ty to test antibody combinations for neuroprotection. My thesis project is an evaluation of the protective activity of two anti Drosophila disease model. I aim to document the efficacy of anti scFv 9 (referred as scFv Ab9) and scFv 42.2 (referred as scFv induced neurotoxicity. Furthermore, I want to examine the effect of c o expressing the two anti Drosophila genetics can be used for efficient screening of new antibodies based on accurate phenotypic screens and protein assays. Following which, the efficacy of olding can be validated in animal models and, subsequently, tested in human trials. Due to the conserved cellular mechanisms, disease therapies exhibit a high degree of resemblance between fly and mouse models (Wolfgang et al., 2005). This validates our us e of fly models in evaluating new therapeutic strategies in an economical and time efficient manner.

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18 CHAPTER 2 MATERIALS AND METHOD S Generation Of Plasmids Carrying Transgenes Anti The scFv fragments were released from clones pSecTag2b scFv Ab9 and pSecTag2b scFv 213. No changes to the DNA sequence of the scFvs were necessary to make them compatible for expr ession in Drosophila. Purified DNA of a pUAST plasmid was prepared in the Fernandez Funez/Rincon Limas lab. The cDNA of the anti were cloned into the plasmid using the restriction sites in the pUAST polylinker (Makridou et al., 2003). The resultin g constructs were sequenced to confirm insert identity and submitted to Rainbow Transgenic Flies, Inc. for injection into fly embryos. Drosophila Strains The following Drosophila carrying constructs were obtained from Bloomington Drosophila Stock Centre ( http://flystocks.bio.indiana.edu): Sco/CyO; MKRS/TM6B, yellow white (yw) UAS:LacZ, Kenyon cell specific OK107 Gal4, and eye specific GMR Funez/Rincon Limas lab. All crosses were reared on Jazz Mix medium ( Fisher Scientific) at 27 o C, unless otherwise stated. Fresh Eye Images For fresh eye images, one day old males were collected in Eppendorf tubes and stored at 80 O C for 30 min. Using a Leica DFC295 digital microscope adjusted to a magnification of 80x, ima ges were digitally captured as a Z stack and rendered together as a montage. Images were acquired and processed using the montage function on LAS software version 4.1, provided by Leica.

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19 Field Emission Scanning Electron Microscopy (FESEM) For SEM, whole f lies were dehydrated through an ethanol gradient of 50%, 70%, 80%, 90%, 95% and 100%. They were then treated with hexamethyldisilazane (HMDS, Electron microscopy sciences), dried overnight, placed on metallic stubs, and sputter coated at the Electron Micro scopy and Bio imaging Core (University of Florida). Flies were imaged using FESEM (S 4000, Hitachi High Technologies America, Inc) and images were digitally captured using Quartz PCI software at 200x and 1000x magnification. Immunohistochemistry (IHC) Ey e imaginal discs and ventral nerve cords were dissected in 1x PBS (phosphate buffered saline), fixed in 4% formaldehyde for 30 min, washed in a solution of 1x PBS and 0.1% TritonX 100 (PBS T), blocked for 30 min in 1% BSA (bovine serum albumin) and incubat ed overnight with primary antibody at 4 o C. After washing with PBS T, samples were blocked again with 1% BSA and incubated in secondary antibody for two hours at room temperature. Post incubation, samples were washed and mounted onto slides in Vectashield ( Vector Laboratories). Primary antibodies used: mouse anti myc (diluted 1:9,000, Cell Signaling Technology), mouse anti cleaved caspase3 (1:100, Cell Signaling Technology). Secondary fluorescent conjugated anti bodies: Anti rabbit Alexa 488 (1:200, Invitrogen by Life Technologies corporation) and anti mouse Cy3 (1:600, Jackson ImmunoResearch Laboratories, Inc). Fluorescence was detected on a Carl Zeiss ApoTome structured illumination microscope using 20x (0.8 num erical aperture) Carl Zeiss objective. Images were acquired using multiple channel acquisition mode and automatic exposure setting

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20 available on AxioVision software. The region of interest was cropped post capture using Adobe Photoshop. Cell Death Analysi s Each digitally captured eye imaginal disc was analyzed using anti cleaved caspase 3 antibody. Positive cells in each disc were manually counted and the data was analyzed by a one test and one way ANOVA using tatistical software. Statistical results were represented as mean SEM and asterisks were used to represent critical levels of significance. Detection Of Fi For thioflavin S assay, adult fly heads were dissected in 1x PBS solution, fixed for 30 min in 4% formaldehyde and washed in PBS T. Brains were then incubated for 10 min at room temperature in a freshly prepared, and filtered solution of 0.03% thioflavin S in 50% ethanol. After 10 min, brains were washed with 50% ethanol and then with PBS T. Brains were then mounted onto slides in Vectashield (Vector Laboratories). Thioflavin S levels in the brain were visualized using Carl Zeiss ApoTom e structured illumination microscope. Images were digitally captured as single stacks using single channel acquisition mode and constant exposure option on AxioVision with 20x (0.8 numerical aperture) and 63x (1.4 numerical aperture, oil) objectives. Image fibril deposits. The area of the brains positive for deposits, as determined by thioflavin S staining, was manually highlighted using the software, which then calculated the mean inten sity of signal. Mean intensity calculated by this analysis was assumed to have a

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21 For data analysis, the mean intensity values were compared using a two tailed test and one wa y ANOVA using Prism statistical software. Statistical results were represented as mean SEM.

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22 CHAPTER 3 RESULTS Generation Of Transgenic Flies Expressing Anti Eye color in flies is phenotypically determined by the expression of white gene and mu tations in this gene alter eye color from red to white (Morgan, 1910). We used the GAL4/UAS system in all Drosophila constructs as it allows precise control over gene activation. This bipartite system consists of the transcription activator Gal4 and the Up stream Activation Sequence (UAS), an enhancer that activates transcription when Gal4 binds to it. We started by inserting the cDNA derived from the two anti 42 scFv genes, Ab9 and 213, into a pUAST plasmid carrying a mini white + gene as reporter. The plasmids were then injected into embryos of yw flies, carrying a mutation in the white gene. Microinjection of the transgene leads to gene delivery in few somat ic cells and germ line precursor cells. The F0 generation was genetically mosaic and did not express the white gene in all somatic cells, and hence, all flies were white eyed. We collected males and females of this F0 generation and placed them in individu al vials. Next, we crossed the F0 flies individually with yw flies to help us screen for transformants (Fig.3 1). Three scenarios were observed in the progenies: Case one, all F1 flies in the vial were white eyed. This meant no transformants were present in the vial and all flies were discarded. Case two, some flies displayed eye color. If all the flies in the vial had a single eye color, it represented a single insertion event. We collected males with eye color for further crosses. Case three, flies displ ayed two or more eye colors. This represented multiple independent insertions. In such a case, we selected males with lighter eye color because they represented flies with single insertions.

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23 The F1 transformants had the anti chromosomes. The IV chromosome is small and mostly heterochromatic, therefore unlikely for DNA insertion. Balancer chromosomes are used in Drosophila genetics to prevent crossing over and they carry dominant markers, which makes it easier to screen for heterozygous mutations. We used CurlyO (CyO) and MKRS as balancer chromosomes for the II and III chromosomes, respectively. Both balancers are homozygous lethal. We crossed F1 males with females carrying balancers for chromosome II and III and the progenies were scanned for individuals bearing the gene for scFv ( w + ) and both the balancers. A self cross of males and females carrying both balancers was then setup for the selected proge nies. This constituted the stable transgenic lines (Fig.3 1). When compared to heterozygous individuals carrying the transgene and a balancer, homozygous individuals have a darker eye color (two copies of w + ). Since expression of transgene is dependent on location, all lines showed varying degree of eye color. Based on the intensity of the eye color and the presence of balancers, we mapped insertions onto either chromosome II or III. A total of eight lines were es tablished for flies expressing anti Ab9 and six for anti 213. Induced Eye Phenotype To test whether scFv Ab9 and 213 are active in vivo and to select lines that induced phenotype, we u in the eye through development and into adulthood. Compared to wild type Drosophila, neurotoxicity. The eye is smaller,

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24 highly disorganized, contain black necrotic spots and there is depigmentation over the ommatidia field. Overall, the eye looks glassy, has a darker color and a rough surface (Fig.3 2). Anti Ab9 To start, we set up independent crosses of all eight lines of scFv Ab9. Males of scFvs serves as a phenotypic screen, as we can select scFv lines based on their rescue of the Ab9 under the control of tissue specific driver, GMR Gal4. As a positive control, we From the progeny of the eight crosses, we selected F1 males and compared their Ab9 F16, and induced phenotype and, hence, were not investigated further. Lines scFv Ab9 M11, F7, M2, M27, F20, and F11 di splayed varying degree of rescue. Compared to the positive control, eyes looked larger and displayed lesser depigmentation. However, we could still observe few necrotic spots and the ommatidia were not completely organized. Amongst these six lines, we care fully compared eye phenotype through the stereoscope and selected scFv Ab9 M27 induced phenotype (Fig.3 2). Anti 213 To select for best rescue of eye phenotype amongs t the six lines carrying gene for scFv 213, we set up similar crosses between homozygous males from the six lines and

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25 control. Of the six F1 progenies, two lines, scFv 213 M 11, and M52 did not show any rescue in eye phenotype and, hence, were not taken up for further investigation. Amongst the remaining four lines, varying degrees of rescue were observed. The eye color was restored to a lighter shade of red, depigmentation d ecreased significantly, and the ommatidial field looked more organized. Yet, the eye size was smaller than a wild type fly and a few necrotic spots were visible. On comparison amongst the four lines, scFv 213 M63, M25, M29, and M59, scFv 213 M25 (insert ion on III chromosome) showed best rescue in eye phenotype (Fig.3 2). From the above results we can infer that the scFvs were correctly expressed, partially, individuals from A induced neurotoxicity. When compared between the two scFvs, scFv 213 (Fig.3 2). This difference in activity can be either linked to increased expression of scFv Ab9 relative to scFv 213 or can be attributed to higher affinity of scFv Generation Of Lines Expressing Both Anti Ab9 and 213 To determine if the two scFvs Ab9 and 213 had a synergetic effect of the two scFvs Ab9 and est lines, scFv 213 M25 and scFv Ab9 M27, to generate a new stock of flies carrying a copy of both the anti scFvs. To generate these stocks, we started by crossing homozygous males from the scFv 213 M25 strain with Sco/CyO; MKRS/TM6B strain females to introduce the dominant markers CyO and MKRS on chromosomes II and III, respectively (Fig.3 3). A

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26 similar cross was set up for scFv Ab9 M27 strain to introduce dominant markers Sco and TM6B on chromosome II and III, respectively. We then crossed F1 individu als from the two crosses to obtain flies bearing scFv Ab9 and CyO on chromosome 2 and Scfv 213 and TM6B on chromosome 3. With the low (1:16) probability of getting the desired progeny, we paid special attention to the cross to ensure no progeny was lost or miscounted. A self cross from the desired progeny of F1 generation was then setup to start a fresh, stable double transgenic line of flies (scFv Ab9+213). As expected, on comparing the eye phenotype of positive control to the scFv Ab9+213 flies, we observ ed significant rescue. There were no necrotic spots, the eye had a healthier red color and a natural oval shape. No depigmentation could be seen and ommatidial arrangement was more organized than the rescue by either scFv 213 or scFv Ab9. This corroborate s our hypothesis that the two scFvs do not block each Anti Induced Cell Death In The Eye After observing the phenotypic rescue by scFvs, we investigated whethe r this was induced neurodegeneration. To test chose the eye imaginal disc from a third instar larvae because the eye disc is easy to identify and gives a good estimation of tissue development in a small area. This allowed induced cell death. A few cells, not recruited for development, in the eye imaginal disc die during developme nt. However, this loss of cells does not affect the final morphology of an adult fly eye. scFv Ab9/scFv 213 flies. For positive control, I picked larvae from the cross between

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27 cleaved caspase 3. Staining of the positive control showed a high number of caspase 3 positive cells, i.e, cells undergoing apoptosis (Fig.3 4). Compared with that, express ion 213) resulted in a significant decrease in cell apoptosis (Fig.3 4, Fig.3 5). Additionally, when we co Ab9+213), a drastic decrease in cell death was observed, with the number of cells undergoing apoptosis being reduced by 75% (Fig. 3 5). This decrease in cell death associated with scFv activity helps explain the rescue in eye phenotype of adult flies, as seen in figure 3 2. Although the se results add support to our hypothesis that scFv induced neurotoxicity, we should corroborate these findings with alternative assays such as TUNEL staining (Tare et al., 2011). Rescue acti instar larvae. Ventral nerve cord is easily seen under the microscope and the neuron distribution al lows easy localization of the desired protein. We stained neurons isolated the 6E10 antibody and an anti myc antibody was used for scFvs (Fig.3 6). We detected that scFvs was consistent across the two scFvs and in the case of expressing both the scFvs together.

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28 iorate its neurotoxicity, it would also be interesting to investigate if the anti fibril deposits in rescued individuals Thioflavin S binds beta sheet rich structures and emits a green fluorescent signal. We used 1 2 day old female virgins derived from the cross between OK107 213, scFv Ab9 and scFv osits in the Kenyon cell region of the brain (Fig.3 7). For quantification, we correlated mean intensity of thioflavin S staining to deposition in the brain (Fig.3 8). ated form of amyloid beta and

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29 Figure 3 1. llustration showing generation of stable stocks of Drosophila carrying anti eration which were crossed with yw and further on with balancer flies to select transformants. Transformants expressed the white gene (w+) and were selected for further crosses to generate stable single insertion stocks carrying the anti

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30 Figu re 3 2. Rescue in eye phenotype of transgenic flies by expression of scFv. Comparison in eye phenotype of one day old male flies co with scFv (UAS 213; UAS Ab9 and UAS cZ) and negative control (UAS LacZ). All flies are under the GMR Gal4 driver and were grown in 27 O C. Top row shows fresh eyes and the bottom row shows scanning electron microscopy images of the same genotype as the fresh eyes on top. SEM images taken at 20 0x magnification. Insets are 1000x magnification of the ommatidia field

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31 Figure 3 3. Illustration showing generation of stable stocks of Drosophila carrying both scFv Ab9 and scFv 213 genes. The two best lines from each set of the anti both scFvs

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32 Figure 3 4. Co imaginal disk from third instar larvae were stained for myc tag on scFv (red channel) and caspase 3 activity (bright green spots in the green channel). Eye disks expressing scFv (UAS 213; UAS Ab9 and UAS ll death, represented by caspase 3 signal. Positive control is co negative control used is UAS lacZ fly. All protein expression is under GMR Gal4 driver and all larvae were incubated in 27 O C

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33 Figure 3 5 p<0.025, and ***p<0.0001 Figure 3 6. hery. Ventral nerve red channel) and anti and scFv localized around the cell visualized in side the cell. UAS protein expression is under OK107 Gal4 driver and all larvae were incubated in 27 O C

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34 Figure 3 7. not decrease with expression of anti scFv. Thiofla vin day old adult fly brains (n=5). No deposits were visible in the control (LacZ flies) while positive control (UAS Z) and anti 213; UAS Ab9 and UAS expression is under OK107 Gal4 driver and all flies were grown in 27 O C. The top row shows the whole fly brain imaged at 20x objective (0.8 NA) an d the Figure 3 8.

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35 CHAPTER 4 DISCUSSION The last decade has witnessed an intense effort towards the development of disease modifying therapeutic strategies for AD. The biggest impediment in this respo nsible for neuronal dysfunction and cell death (Benilova et al., 2012). While some in vivo studies conducted in APP transgenic mouse models link the discovery of fibrillar inducing neurotoxicity (Walsh et al., 2002). Despite the absence of an accepted molecular model for AD, therapeutic strategies aimed at neutralizing different forms of transgenic mic e (Wahrle et al., 2002, Solomon et al., 1996). reduce its toxicity (Bard et al., 2000). The unsatisfactory results from recent clinical trials testing two anti e challenges in exploring the full potential of passive immunization (Aisen, 2013). One of the major hurdles of these studies is the limited ability of full size antibodies to cross the blood brain barrier and accumulate at high levels in the brain. A stra tegy to overcome this limitation is the use of smaller, engineered antibodies such as scFvs. Easily manipulable and more stable than full size antibodies, scFvs can be produced rapidly and inexpensively in E. coli (Skerra et al., 1991). Additionally, admin istration of scFv unlike mAbs, has not been linked to CAA or microhemmorrhage The efficacy of scFvs is reflected in their rising use in diagnostics, breast cancer research, and AD. There is a continued effort towards producing stronger

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36 scFv variants poss essing higher affinity and specificity for its target protein ( Holliger and Hudson, 2005 ) Recent studies using APP transgenic mice showed that scFvs have anti associated neuro degeneration are still lacking. One critical deterrent for this gap in knowledge is the inability of APP transgenic mice to mimic neurodegeneration as observed in human cases of AD. Hence, to examine the efficacy of scFv Ab9 and 213 induced neu rodegeneration, we used Drosophila as our AD model. The scFvs under investigation, scFv Ab9 and 213 have been shown to bind in vitro and in APP transgenic mice (Levites et al., 2006b). In this report, we describe fo r the first time the efficacy of scFvs in abrogating induced neurodegeneration in a Drosophila AD model. Using the eye as a induced eye pheno type, as well as in significantly reducing induced cytotoxicity. On comparison, scFv toxicity than scFv 213. This observation could be attributed to either higher expression levels of scFv Ab9 or higher affinity toward deposits by thioflavin induced neurotoxicity. Although further investigation is required, we can speculate that the scFvs are acting by reco gnizing and binding misfolded oligomers of Argany et al., 2011).

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37 scFvs are not able to act on it. An alternative hypothesis is that the scFvs neutralize Drosophila lacks an adaptive immune system, there are no immune cells to clear these non toxic aggregates. To test these hypotheses, we will investigate different time points for scFv soluble and insoluble. In addition, we will also evaluate the phenotypic rescue in mushroom bodies degeneration, the learning and olfactory center of D rosophila co expressing anti mechanism of these scFvs will serve in identifying participating pathways and assist in recognizing toxic mediators of AD. Alt hough both the scFvs investigated in this study result in a partial rescue from support Cl oning and expression of these mammalian scFvs proved to be very efficient and required minimum sequence alteration: both the signal peptide and the Kozak sequence were compatible with expression in flies. Hence, these are encouraging results to use Drosoph ila as an in vivo platform to screen for anti activity that can later be tested in AD mouse models by viral mediated expression. The easy genetic manipulation of the fruit fly allowed us to bring the two scFvs onto the same flies. Using these flies, we report for the first time a synergetic activity of induced neurodegeneration. This novel strategy opens an exciting opportunity to explore different combinations of engineered antibodies, such as s or intracellular) or in

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38 different forms (oligomers or fibrils). This strategy can also be expanded into studying synergistic combinations of therapeutic agents for different neurodegenerativ e diseases. Our use of the Drosophila AD model to investigate the efficacy of anti induced neurodegeneration underscores the ability of a strong Drosophila disease model in investigating therapeutic strategies. Screening of therapeutic agents using an animal model provides advantages of understanding protein interactions and pathways in physiologically relevant tissues and is clearly a powerful choice over in vitro techniques (McKoy, 2012). The eye phenotype used in this report is a non invasive and time saving method to select therapeutic agents for their potential in ameliorating neurodegeneration. Site directed insertion of transgenes can serve to use the eye phenotype for comparing therapeutic agents. Conserved cellular mechanism bet ween humans and Drosophila in addition to the ease of transferring gene sequences between mammalian and fly systems, further justifies our use of Drosophila for our investigation. As a fast and reliable screen for scFvs and an efficient system to accurat ely compare the efficacy of scFvs and their combinations, Drosophila can be utilized to screen new therapeutic agents. The selected proteins can then be further investigated in rodent models for safety and robustness. Future studies applying such an approa ch will drive clinical research for the development of novel therapeutic strategies to treat neurodegenerative diseases.

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43 BIOGRAPHI CAL SKETCH Krishanu Mathur was born in Rajasthan, India. Raised in New Delhi, he grew up with a fondness for fables and a secret weakness for Mughali food. Encouraged by his teachers to take up engineering, Krishanu spent his undergraduate degree exploring the world of biotechnology at Netaji Subhas Institute of Technology, a top ranked engineering college in India. Spirited to learn more about advancements in disease treatment, Krishanu made a trip across the ocean to the Biomedical Engineering Department 2013, taking along with him invaluable lab experience and countless stories. Krishanu dreams of traveling the world one day as a respected professional in the field of biotechnology.