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Using Drosophila to Evaluate the Neurotoxicity and Misfolding Induced by Two Pathogenic Mutations on Helix-3 of the Prio...

Permanent Link: http://ufdc.ufl.edu/UFE0045560/00001

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Title: Using Drosophila to Evaluate the Neurotoxicity and Misfolding Induced by Two Pathogenic Mutations on Helix-3 of the Prion Protein
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Pattamatta, Amrutha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: d201n -- drosophila -- helix-3 -- mutation -- neurodegeneration -- prion -- q211p
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: Prion diseases are a rare group of neurodegenerativedisorders caused by the misfolding of the Prion protein (PrP). Currently thereare no treatment options available for these diseases. Hence, a mechanisticunderstanding of Prion diseases will provide useful cues to design appropriatetherapeutic interventions. Alpha helix 3 (HC) plays a central role in thestability of PrP and mutations on HC have debilitating effects on its structureand function.  Research thus far hasfocused on the behavior of only some of these mutant PrP’s in vitro. For my thesis, I utilized two pathogenic mutations (D201N and Q211P) to understand the molecular pathways that govern PrPneurotoxicity, in Drosophila. D201N causes the disruption of aconserved capping box at the beginning of HC and Q211P introduces a helical break. We identified conformational abnormalitiesin PrP-D201N that induced the formation misfolded aggregates accumulating incellular compartments in the flies. Interestingly, in older flies, PrP-D201Nacquired an alternate C-terminal topology which, coupled with proteinaggregation, produced neurotoxicity. PrP-Q211P also formed a spontaneouslymisfolded species but did not induce neurotoxicity in the flies.  These results signify that while themutations are located in close proximity on HC, the changes they produce inprotein folding are distinct from each other. Therefore, each of the mutationhas its own method of pathogenesis. Future drug development strategies wouldinvolve the identification of a pathway common to all the mutations that failsvery early in development leading to the formation of a spontaneously misfoldedspecies.
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 Amrutha Pattamatta.
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

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045560:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045560/00001

Material Information

Title: Using Drosophila to Evaluate the Neurotoxicity and Misfolding Induced by Two Pathogenic Mutations on Helix-3 of the Prion Protein
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Pattamatta, Amrutha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: d201n -- drosophila -- helix-3 -- mutation -- neurodegeneration -- prion -- q211p
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: Prion diseases are a rare group of neurodegenerativedisorders caused by the misfolding of the Prion protein (PrP). Currently thereare no treatment options available for these diseases. Hence, a mechanisticunderstanding of Prion diseases will provide useful cues to design appropriatetherapeutic interventions. Alpha helix 3 (HC) plays a central role in thestability of PrP and mutations on HC have debilitating effects on its structureand function.  Research thus far hasfocused on the behavior of only some of these mutant PrP’s in vitro. For my thesis, I utilized two pathogenic mutations (D201N and Q211P) to understand the molecular pathways that govern PrPneurotoxicity, in Drosophila. D201N causes the disruption of aconserved capping box at the beginning of HC and Q211P introduces a helical break. We identified conformational abnormalitiesin PrP-D201N that induced the formation misfolded aggregates accumulating incellular compartments in the flies. Interestingly, in older flies, PrP-D201Nacquired an alternate C-terminal topology which, coupled with proteinaggregation, produced neurotoxicity. PrP-Q211P also formed a spontaneouslymisfolded species but did not induce neurotoxicity in the flies.  These results signify that while themutations are located in close proximity on HC, the changes they produce inprotein folding are distinct from each other. Therefore, each of the mutationhas its own method of pathogenesis. Future drug development strategies wouldinvolve the identification of a pathway common to all the mutations that failsvery early in development leading to the formation of a spontaneously misfoldedspecies.
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 Amrutha Pattamatta.
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: UFE0045560:00001


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1 USING DROSOPHILA TO EVALUATE THE NEUROTOXICITY AND MISFOLDING INDUCED BY TWO PATHOGENIC MUTATIONS ON HELIX 3 OF THE PRION PROTEIN By AMRUTHA PATTAMATTA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL F UL FILLMENT OF THE REQUIREMENTS FOR TH E DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Amrutha Pattamatta

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3 To Mom, Dad and Pritham

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4 ACKNOWLEDGMENT Acknowledgment, it is said would bring in recognition to the acknowledged. But these few lines written here are not with that purpose but, to express in a small way, a sense of gratitude to all concerned. At the outset, I would like to thank Dr. Pedro Fernandez Funez, for trusting m y ability as a student and a researcher, for accepting me in the lab, for supporting me financially, for understanding my working style and motivating me accordingly, and for leading me by example. I am affirmative that the lessons I have learnt from him, will help me in the sunniest and the gloomiest of days. I am extremely grateful to Dr. Pedro Fernandez Funez and Dr. Diego Rincon Limas, for being relentless sources of guidance and support and for providing all of us with an excellent atmosphere for doin g research. I am thankful to my committee members Dr Brandi K Ormerod and Dr Benjamin Keselowsky for their encouraging words, thoughtful criticism, time and attention during busy semesters. I feel extremely lucky to have had the opportunity to work al ongside several wonderful c olleagues. Special thanks to Jonatan, for teaching me more than the realms of a textbook, for unobtrusively looking over my shoulder, for being my noise absorber and for teaching me the most important thing about Spanish culture, to work hard all day, party harder all night and return to work the next day to keep it going. I would also like to thank Yan, Alfonso and Kurt for all the technical help and support throughout my stay here. My gratitude also goes to Swati, for being my f riend, colleague and sound board, most often all at the same time Krishanu, for teaching me how beautiful our work lives are with the c raziest music in the background and Jose, for cracking me up in the hardest of days and for loving my cooking. I would a lso like to thank Deanna for saving my soul with the dissections just when I bega n to feel like a mass murderer, Sally,

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5 Barbara, Stefanie and Brittney for keeping everything lively and going. I am very deeply indebted to everyone at the PFF DRL lab for making my work zone, my comfort zone. Gainesville has been a home away from home in India and I would like to thank all the people who have made this happen. Siddhesh, Mini, Chinmay and Rachna for listening to me endlessly, for late night coffees, study to gether sessions, for cooking amazing food and for relentlessly pampering me because after all I am the youngest! Thank you is in order also to Suguna, Vijay, Bhargav and Giri for introducing me to the best Friday nights ever. Life was a lot simpler on Satu rday mornings. To all the members of 1122, for being my family and for teaching me the most valued lessons of friendship. Unfortunately, I put an end to the tradition that lasted us 4 years. My housemates Jolin, Rachna, Radhika, Rahul and Nalanda, for easi ng out the transition from house to home. I would also like to thank my friends from home Madhvi, Akshaya, Divya, Vishnu, Iswarya, Harish and Aandal for shaping me to be the person I am today, for loving me and supporting me despite being miles away. My s incerest gratitude to my family, for all their faith and support. I thank my father for believing in me and knowing better than me that I would do well for myself, my mother for unconditionally supporting me and for having faith that I can complete her unf inished dream, my brother for being my friend, my foe and for always showing me the right path effortlessly and Preethi for always listening to me and providing useful inputs. I hope I did you proud. Last but definitely not the least, I would like to thank Shantanu, for his endurance and patience (I can be quite difficult!), for driving me to the lab on weekends and holidays, for being stable and rock solid and most importantly for being my best friend and confidant. I owe this thesis to each and every one of you.

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6 TA BLE OF CONTENTS page ACKNOWLEDGMENT ................................ ................................ .............................. 4 LIST OF TABLES ................................ ................................ ................................ ...... 8 LIST OF FIGURES ................................ ................................ ................................ .... 9 LIST OF ABBREVIATIONS ................................ ................................ ..................... 10 ABSTRACT ................................ ................................ ................................ ............. 11 CHAPTER 1 INTRODUCTION ................................ ................................ .............................. 13 Prion Diseases ................................ ................................ ................................ .. 13 Infectious PrD ................................ ................................ ............................. 13 Inherited PrD ................................ ................................ .............................. 13 Prion Protein ................................ ................................ ................................ ..... 14 Structure of PrP ................................ ................................ .......................... 15 Biosynthesis of PrP ................................ ................................ .................... 15 Signatures of PrP and Role in Pathogenesis ................................ .................... 16 Methionine Mutations in PrP Pathogenesis ................................ ...................... 17 Rationale Behind the Current Study ................................ ................................ 18 Polar Substitution: Aspartic acid to Asparagine (D202N, human numbering) ................................ ................................ .............................. 18 Non Polar Substitution: Glutamine to Proline (Q2 12P, human numbering) 19 Drosophila Models for Proteinopathies ................................ ............................. 20 Life Cycle of Drosophila ................................ ................................ ............. 20 Dros ophila for Neurodegenerative Diseases ................................ .............. 21 Yeast GAL4/UAS system ................................ ................................ ........... 21 Overall Objective and Goal ................................ ................................ ............... 22 Objective ................................ ................................ ................................ .... 22 Hypothesis ................................ ................................ ................................ .. 22 Goal ................................ ................................ ................................ ............ 23 2 MATERIALS AND METHODS ................................ ................................ .......... 24 Genetic Manipulation of Drosophila ................................ ................................ .. 24 Gal4 Lines ................................ ................................ ................................ .. 24 Generation of UAS Lines ................................ ................................ ............ 24 Behavioral Studies ................................ ................................ ............................ 26 Viability ................................ ................................ ................................ ....... 26 Climbing Assay ................................ ................................ ........................... 26 Biochemical Assays ................................ ................................ .......................... 27

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7 Tissue Homogenates ................................ ................................ ................. 27 Western Blot ................................ ................................ ............................... 27 Antibodies ................................ ................................ ................................ ... 28 Glycosylation Assa y ................................ ................................ ................... 28 Proteinase K (PK) Assay ................................ ................................ ............ 28 Immunoprecipitation with 15B3 ................................ ................................ .. 29 Immunohistochemistry and Microscopy ................................ ............................ 30 Dissections of Larval and Adult Brains ................................ ....................... 30 Antibodies ................................ ................................ ................................ ... 31 Microscopy ................................ ................................ ................................ 31 Statistical Analysis ................................ ................................ ............................ 31 3 RESULTS ................................ ................................ ................................ ......... 32 PrP D201N had Decr eased Survival Rates in Comparison to PrP WT ............ 32 Locomotor Dysfunction was Induced by PrP D201N but not by PrP Q211P .... 33 Neuropathology of the Mushroom Bodies ................................ ......................... 34 Quantification of Kenyon Cells ................................ ................................ .......... 36 Abnormal Cellular Distribution of PrP D201N and PrP Q211P ......................... 38 Biochemical Analysis of MoPrnp Revealed Differences in PrP glycofo rms ....... 40 Resistance of PrP D201N to Complete Digestion of N linked glycosylation ..... 41 Amino Acid Substitutions Introduce Conformational Abnormalities .................. 43 PrP D201N Acquires Partial Resistance to PK and an Alternate Topology ...... 48 4 DISCUSSION ................................ ................................ ................................ ... 51 Aspartic Acid to Asparagine: The Structural Journey ................................ ........ 52 Helix Breaks due to Proline does not Induce Neurodegeneration ..................... 55 Conclusi on ................................ ................................ ................................ ........ 57 LIST OF REFERENCES ................................ ................................ ......................... 58 BIOGRAPHICAL SKETCH ................................ ................................ ...................... 63

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8 LIST OF TABLES Table page 3 1 Climbing index of PrP WT and mutant PrP ................................ .................. 33

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9 LIST OF FIGURES Figure page 1 1 Mutations in the Prnp gene that ............... 14 1 2 Globular domain of PrP ................................ ................................ ............... 15 1 3 Conserved role o f aspartic acid on position 202 ................................ ........... 19 1 4 Structural changes due to the Q212P mutation.. ................................ .......... 20 2 1 Genetic manipulation of Drosophila ................................ .............................. 25 3 1 PrP D201N induced stunted viability ................................ ........................... 32 3 2 PrP D201 N induces locomotor dysfunction ................................ .................. 34 3 3 Anatomy of the mushroom body projections in the fly brain ......................... 3 5 3 4 Neuropathological anatomy of mushroom body proje ctions in PrP WT and mutant PrP ................................ ................................ ................................ .... 36 3 5 Anatomy of the Kenyon cells. ................................ ................................ ....... 37 3 6 Quantification of the surface a rea occupied by the Kenyon cells.. ................ 38 3 7 Subcellular distribution of PrP in larvae ................................ ........................ 39 3 8 Aggregation of PrP in the Kenyon cells. ................................ ....................... 40 3 9 Expression of MoPrP WT, MoPrP D201N, MoPrP Q211P and negative control UAS LacZ in flies ................................ ................................ ............. 41 3 10 PrP D201N displays aberrant glycosylatio ns ................................ ................ 43 3 11 Epitopes specific for the conformational antibodies. ................................ ..... 45 3 12 Conformational antibodies reveal aberrant protein fold in PrP D201N and PrP Q211P ................................ ................................ ................................ ... 46 3 13 Immunostaining of Kenyon cells with 6H4 ................................ ................... 47 3 14 Immunoprecipit ation of misfolded PrP in old fl ies of the PrP WT and PrP D201N ................................ ................................ ................................ .......... 48 3 15 Proteinase K resistance ................................ ................................ ................ 50

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10 LIST OF ABBREVIATIONS C Cysteine D Aspartic acid EndoH Endoglycosidase H GFP Green fluorescent protein GSS Gerstmann Straussler Scheinker HA, H B, HC Helices A, B, and C of Prion protein L Lysine M Methionine N Asparagine P Proline PrP Prion protein PrD Prion disease PNGase F Peptide N glycosidase F PK Proteinase K PrP Sc Scrapie ( Infectious ) PrP Q Glutamine S Serine STE Stop transfer effector TMD Transmembrane domain UAS Upstream activating sequence

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science USING DROSOPHILA TO EVALUATE THE NEUROTOXICITY AND MI SFOLDING INDUCED BY TWO PATHOGENIC MUTATIONS ON HELIX 3 OF THE PRION PROTEIN By AMRUTHA PATTAMATTA May 2013 Chair: Brandi K Ormerod Co chair: Pedro Fernandez Funez Major: Biomedical Engineering Prion diseases are a rare group of neurodegenerative disorders caused by the misfolding of the Prion protein (PrP). Currently there are no treatment options available for these diseases. Hence, a mechanistic understanding of Prion diseases will provide use ful cues to design appropriate therapeutic interventions. Alpha helix 3 (HC) plays a central role in the stability of PrP and mutations on HC have debilitating effects on its structure and function. Research thus far has focused on the behavior of only so me of in vi tr o For my thesis, I utilized two pathogenic mutations ( D201N and Q211P) to understand the molecular pathways that govern PrP neurotoxicity, in Drosophila D201N causes the disruption of a conserved capping box at the beginni ng of HC and Q211P introduces a helical break. We identified conformational abnormalities in PrP D201N that induced the formation misfolded aggregates accumulating in cellular compartments in the flies. Interestingly, in older flies, PrP D201N acquired an alternate C terminal topology which, coupled with protein aggregation, produced neurotoxicity. PrP Q211P also formed a spontaneously misfolded species but did not induce neurotoxicity in the flies. These results signify that while the mutations are locate d in

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12 close proximity on HC, the changes they produce in protein folding are distinct from each other. Therefore, each of the mutation has its own method of pathogenesis. Future drug development strategies would involve the identification of a pathway commo n to all the mutations that fails very early in development leading to the formation of a spontaneously misfolded species.

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13 CHAPTER 1 INTRODUCTION Prion Diseases Prion disease (PrD) is a collective name given to a rare group of fatal neurodegenerative d isorders caused by the modification of the endogenous Pr ion protein (PrP ) (1) Creuzfeldt Jakob disease (CJD), Gerstmann Strussler Scheinker (GSS) syndrome, kuru, fatal familial insomnia (FFI) (in humans), bovine spongiform encephalopathy (BSE, in cows), scrapie (in sheep), and chronic wasting disease (CWD, in elk and deer) are examples of PrD across different species (2) The occurrence of PrD can be sporadic, familial, or infec tious but almost always inv olves the conversion of PrP to abnormally folded isoforms Infectious PrD are commonly thought to arise from the transmission of an infectious isoform of PrP denoted as PrP Sc (scrapie). PrP Sc favors the formation of insoluble fibrillar aggregates up of these induces the (3) Other neuropathological changes inc lude astrogliosis with the absence of a local or global inflammatory response (4) The time interval between the disease onset and diagnosis can vary from 4 10 years. But once diagnosed, there is rapid degeneration of the brain often without any systemic changes leading to death in less than a year in most cases ( US Centers for Disease Control, 01 26 2006, retrieved 02 28 2010) Inherited PrD Inherited Prnp gene on chromosome 20 that encode s PrP. Currently, approximately 40 autosomal dominant mutations have

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14 been reported in the Prnp gene that cause the human disease (5) Combined, i nherited Figure 1 1 lists the various reported mutations resulting in different manifestations of inherited PrD. In humans, mutations to the Prnp gene are linked to fCJD, GSS syndrome and FFI (6) In cell culture, pathogenic PrP mutations have been shown to influence its structure, processing and stability, causing misfolding and aggregation (7 9) Figure 1 1 Mutations in the Prnp open reading frame of human Prnp gene is composed of the signal peptide ( terminal region. The GPI anchor is attached to the C terminal domain (not shown in figure, figure not drawn to scale) Prion Protein he accumulation and deposition of abnormal PrP (10,11) PrP is synthesized predominantly in the neurons and the glia of the CNS (12) The biological function of PrP is not fully understood, but it has been reported to be associated with a variety of functions su ch as, regulation of neuronal proliferation (13) synaptogenesis (14) signaling (15) and cell adhesion (16) H owever the role of PrP in ne urodegeneration is well established.

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15 Structure of PrP In humans, PrP is composed of 253 amino acid residues before post translational modification and 209 amino acid residues after the removal of the signal peptide (SP) (17,18) It has a molecular mass of 27 29 kDa an d is composed of three helices (HA, HB, HC two sheets (S1, S2 one highly conserved disulfide bond (Cys179 on HB and Cys214 on HC), two facultative N linked glycosylation sites ( N 181 and N 197), an unstructured N terminus, and a globular C terminus (Figure 1 2 ). Also, the N terminal region is composed of two domains conserved across species: five repeats of an octameric amino acid forming the octapeptide repeat (OR) region and a hydrophobic region known as the transmembrane domain (TMD) (19) Figure 1 2 Globular domain of PrP. 3 Dimensional structure of the globular domain of sheets (green) and loops connecting the secondary structures. Biosynthesis of PrP PrP is found on the surface of the cells, attached to the membrane by virtue of a gly cophosphatidyl inositol (GPI) anchor (20) PrP is synthesized in the rough

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16 endoplasmic reticulum, where post translational modifications include cleavage of the signal peptide, addition of oligosacc harides to the N linked glycosylation sites, formation of the disulfide bond between HB and HC, and finally the attachment of the GPI anchor. PrP moves to the cell surface from the ER transiting through the Golgi complex. In the Golgi, post translational m odifications convert the oligosaccharides to complex sugars. PrP on the cell surface is constantly recycled and replenished with the help of endosomes that internalize the protein from the surface (21) Signatures of PrP and R ole in P athogenesis The rate of incidence of hu man CJD had reached 1 per million of the population per annum (WHO, 2011). However, the BSE outbreak in the 1980s led to the death of 400,000 cattle, 176 Britons and 50 other people worldwide. Global health concerns thus sti mulated a lot of research on i nfectious PrP. PrP Sc associated with infectious characteristically contain a higher proportion are resistant to proteases and form amyloid like aggregates. However, PrP Sc is not typically present in suggesting that other PrP isoforms may also cause disease (22) Studies on the translocation of PrP at the ER lumen in cell free translational system s revealed that PrP may exist in more than one topological form (23,24) Although PrP synthesized in the ER an d anchored to the cell membrane is completely digested by proteases, other topological isoforms are protease protected. N terminal ( Ntm ) PrP is p artially translocated, with the N terminus in the ER lumen and the C terminus in the cytosol. The C terminus of C terminal ( Ctm ) PrP is in the lumen of the ER whereas its N terminus is in the cytosol. Alterations to the TMD (Ala113 to Ser135), the hydrophilic domain (Lys1 04 to Met112), and the SP, direct the formation of these topological isoforms. On proteolysis, Ntm PrP has a size of 14 kDa and Ctm PrP has a

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17 size of 18 kDa. Ct m PrP is attached to the cell surface through the GPI a nchor and is inserted into the membrane through the TMD on PrP (25) PrP A117V a human pathogenic mutation causing GSS syndrome misfolds into a Ctm PrP isoform both in vitro and in vivo Although the mechanism by which Ctm PrP induces direct or indirect neurodegeneration is still unknown, the isolation of large a mounts of Ctm PrP from mice carrying this mutation has provided evidence to the existence of a pathogenic topology of PrP in familial conditions (25,26) No evidenc e exists for the pathogenic nature of Ntm PrP. Methionine M utations in PrP P athogenesis The globular domain made up of HA, HB, HC and several stabilizing long and short range hydrophobic interactions, is believed to play a central role in prion pathogenesis HC is a highly conserved, rigid and mutations have been reported on HC because of its dominating role in the stability of the protein Thus, perturbation s in HC may destabilize the helix leading o misfolding and neurotoxicity. An in vitro study on the stabilizing interactions of the globular domain indicated that the conserved methionine residues a t position 206 and 213 on human PrP play an important role in the native folding of the globular domain and the formation of the stabilizing interactions with HA (27 29) It was also reported that the oxidation of M206 on HC leads to PrP misfolding. No clinical mutations have been reported at these positions. However to test the instability conferred to HC because of the stabilizing role played by M20 5 and M21 2 ( mouse numbering) these mutations were introduced and studied in Drosophila (Sanchez Garcia et al., 2012 submitted) Synthe tic replacement of the methionine residues by serine induced dramatic disturbances in the protein leading to misfo lding and aggregation The M205 M21 2 S double mutant flies acquired

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18 neurodevelopmental defects in the mushroom bodies (the learning center i n flies). PrP M205 S M 2 1 2 S was hyperglycosylated and formed a topological isoform of PrP CtmPrP (Sanchez Garcia et al., 2012 submitted) T he M>S substitution s caused the g lobular domain to collapse because of the failure of interactions between HB a n d HC with HA and the perturbation of the disulfide bond s between the c ysteine residues caus ed a failure of the oxidative folding (29) Rationale behind the C urrent S tudy Disruption of conserved stabilizing interactions causes instability of PrP leading to misfolding, as is seen in the case of the methionine mutants. For this study, we identified two pathogenic mutations on HC th at are reported in clinical cases. These mutations were modeled into Drosophila to study the in vivo behavior of the protein. Polar Substitution: Aspartic acid to Aspar a gine (D202N, human numbering) PrP is a highly conserved across species. In particular, HC contains stabilizing interactions which maintain the integrity of the globular domain namely: (i) the N capping box between Ser/Thr X X Glu/Asp (X any amino acid) at the beginning of HC (F igure 1 3 ) (ii) the 4 flanking the N capping box, and (iii) the conserved ionic bond between th e side chains of Glu200 and Lys 204 (30) G2 00L is a known pathogenic mutation and is linked to fCJD (31) The capping box and hydroph obic staple motif function in parallel during protein folding by directing the formation of HC. The presence of these two conserved regions accelerates protein folding (32,33) S ubstitutions in the N capping box (T199A, D202N) le d to instab ility of HC, causing lower helical propensity. In general, synthetic removal of the capping box stabilizing motifs in proteins causes the collapse of the folding pathway leading to temperature sensitive mutants that show inability to refold at physiological temperatures

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19 (34) I n an in vitro study on D202N >60% of Pr P is retained in the ER and the glycoform pattern displays an abundance of di and monoglycosyla ted patterns rather than the un glycosylated form. The aggregates thus formed were detergent insoluble (35) PrP D201N has however never been tested in vivo Figure 1 3 Conserved role of aspartic acid on position 202. D202 (human numbering) on HC, forms a con served capping bond with T199 on HC. The capping box along with other local stabilizing forces plays a critical role in the spatial orientation of HC and its interactions with other helices (30) Non Polar Substitution: Glutamine to Proline (Q212P, human numbering) The glutamine residue at position 212 is located centrally i n HC, in close proximity to Cys 214 that forms a conserved disulfide bond with Cys179 on HB. Gln212 also forms a hydrogen bond with Thr216 (36) Rep lacement of a polar neutral amino acid (glutamine) by a non polar neutral amino acid (proline) is hypothesized to introduce a kink in the structure of the helix creating a strain between the stabilizing interac tions on HC. In general, Pro mutations have be en reported to cause helix breaks (37,38) Upo n introduction of Pro212, HC turns around Pro to form thermodynamically

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20 unfeasible interactions with Glu211 (36) F igure 1 4 ). Predominant changes also occur in the hydrophobic cluster l ea ding Phe175 to twist away from S 2 and HB. As a consequence, the protein shows increased accessibility to solvents, causing faster destabilization. In cell culture, it has been reported that Q212P accumulates and forms aggresomes in cytosol upon proteasom al inhibition. Q212P however has never been modeled in vivo (39) Figure 1 4 Structural changes due to the Q212P mutation. Q212 (human numbering) is located centrally on HC. The Q212P mutation causes a change in polarity of H C introducing a local kink in the structure and eventually braking HC at (36) Drosophila M odels for P roteinopathies Life C ycle of Drosophila Wild type Drosophila melanogaster or fruit fly has characteristic red compound eyes, yell ow thorax with striped lines, long hair on the thorax and a pair of intertwined transparent wings Thomas Hunt Morgan in 1900 discovered the use of Drosophila to

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21 study genetic mutations. takes about 10 days to grow. Drosophila is characteristically an ectothermic species, in that, the development depends on the temperature of incubation. During its developmental cycle, the fruit fly goes through three larval stages, with the third larval stage having a fully dev eloped nervous system. The adults fly emerges from the pupae after complete metamorphosis and differentiation Experiments in this study either use the third instar larva or the adult fly just after eclosion. Drosophila for N eurodegenerative D iseases Dros ophila is currently in use for studying various neurodegenerative diseases amyotrophic lateral sclerosis, several s pinocerebellar ataxia s non coding repeat expansions, and Prion diseases (40) The a dva ntages of using fl ies to model pathogenic mutations in disease co nditions include the following: (i) advances in molecular biology techniques enable ease of genetic manipulation in Drosophila (ii) short life make it a productive model for high throughput screens and proof of concept experiments (iii) low costs (iv) ability to perform reverse genetic engineering to rescue phenotypes in a short time span (v) to study the therapeutic effects of prophylactic c ompounds, small molecules towards drug discovery and lastly (vi) there is a wealth of knowledge available on the organization and function of the nervous system in the fly alongside information on the circadian rhythms, learning and memory and neural ci rcuitry making it easy to understand neuronal mechanisms due to mutations better. Yeast GAL4/UAS system The GAL4 UAS system was developed by Brand and Perrimo n in 1993 to study the expression of genes in Drosophila (41) The GAL4 gene composed of the yeast

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22 transcription activator protein Gal4 that specifically binds to t he upstream activation sequence (UAS) This is engineered into the flies c arrying the transgene to bind to the desired Gal4 to drive gene expression and protein synthesis. In this study three different Gal4 drivers were employed for the ubiquitous expression of the pro tein, for expression only in specific brain centers and for expression in the motor neurons. Overall Objectiv e and G oal Objective The overall objective of this thesis project was to i dentify the underlying mechanisms that lead to PrP instability For this, two pathogenic mutations that perturb HC of PrP were introduced in mouse Prnp (D201N, Q21 1P; mouse numbering hereinafter) These constructs were expressed in Drosophila and the biochemical, behavioral and immunohistochemical abnormalities were analyzed. Drosophila was chosen as the model organism because it lacks endogenous PrP and hence can be used to engineer PrP without any interference from the internal machinery. Hypothesis T he globular domain plays a central role in stabilizing PrP. Disturbances in any of the stabilizing interactions, specifically to HC in the globular domain can signifi cantly affect the native conformation of the PrP leading to aberrant folding patterns. Molecular dynamics analyses support the hypothesis that point mutations induce thermodynamic instabil ity in the protein. Hence, the underlying hypothesis of this study i s that the mutations (D201N, Q211P) destabi lize the structure of the PrP by altering th e interaction between the conserved stabilizing regions in the globular domain, thereby inducing the conversion of PrP to pathogenic forms

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23 Goal The g oal of this project is to utilize two pathogenic mutations to gain insight into the mechanisms that modulate the aberrant folding/processing of PrP. By identifying thermodynamic instability or aberrant cellular trafficking, we can throw light on similar patte rns in other mut ations associated with facilitate the growth of novel therapeutics to combat this currently incurable disease.

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24 CHAPTER 2 MATERIALS AND METHODS Genetic Manipulation of Drosophila T he positive control MoPrP WT was a gift from Dr. S. Supattapone (42) and the negative control UAS LacZ was obtained from the Bl oomington Drosophila Stock Center at Indiana University ( http://flystocks.bio.ind iana.edu / ) All t he Drosophila stock lines were maintained at 18 or 25 The Jazz mix Drosophila food (Fisher) was used as a standard medium for maintaining the stocks (c onstituents include brown sugar, corn meal, yeast, agar, benzoic acid, methyl paraben and propionic acid ) Wide and narrow vials were used to house smaller population of flies. When larger populations of flies were desired for an e xperimental set up, wide bottles were used. For all the experiments, homozygous virgins from the Gal4 strains were crossed with the UAS males to generate the desired progeny. The crosses were all placed at the assays unless specified otherwise Gal4 Lines To drive the expression of the UAS constructs, the following GAL4 lines were used : OK107 Gal4 expressed in the mushroom bodies, Kenyon cells and the eyes; B g 380 Gal4 expressed in the motor neurons, and da Gal4 expressed ubiquitously. Generation of UAS Lines MoPrnp sequence with the D201N and Q211P were synthesized and cloned into the pUAST vector (containing UAS sequence and mini white + (w + ) by Genscript (41) The plasmids were injected into the germline of the yw flies by Rainbow genetics

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25 following standard proce dures (43) Adults from these embryos were grown on the standard Drosophila medium at 25 and c r o ssed with yw flies. The F1 generation of this cross contains a population of flies with and without the w + gene Since the vectors were cloned with both w + and Prnp gene, flies with red eye s also carry the Prnp gene w + ma les were crossed with w; X a /Cyo; MKRS females From the progeny, m ales and virgins of w + / CyO ; MKRS were crossed to each other to generate stable lines and the chromosomal insertions were identified (Figure 2 1 ) Figure 2 1 Genetic manipulation of Drosophila The white+ and the Prnp gene were introduced into the p UAST plasmid. The plasmid was then injected into yw embryos. The F1 generation was crossed with balancer flies to obtain stable lines expressing PrP. Four independent lines were obtained for the PrP D201N and PrP Q211P mutant flies

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26 Behavioral S tudies Viability For studying viability PrP WT, PrP D201N, PrP Q211P and LacZ males were collected and crossed with da Gal4 virgins. The crosses were kept at 25 and after 2 days shifted to 28 60 females were collected from the F1 generation of each of the c rosses and separated into six groups of ten flies per tube The flies were counted and transferred to new vials every day. The experiment was continued un til all the flies died. % Viability was calculated using ( ). The gr aph of % Viability against the t ime period of survival was plotted on Microsoft Excel. Cl imbing A ssay Locomotor dysfunction in the flies was analyzed and documented. PrP WT, PrP D201N, PrP Q211 P and LacZ males were crossed with the B g 380 Gal4 driver. 50 males were collected from the F1 generation s and were split into two gro ups of 25 each and placed at 27 The flies were rested at room temperature (with occasional tapping) for 30 min every day prior to performing the assay. The flies from each tube were transferred to an empty tube with a 5 cm line marked on it placed on a stand for ease of viewing. The tube was tapped thrice so that all the flies were at the bottom of the tube. T he flies w ere a llowed to climb up for 10 sec The number of flies above the 5 cm line was recorded each time Eight trials were performed for each genotype to average the climbing index. Once all the flies of a genotype did not climb above the 5 cm line the assay was stopped. % climbing was calculated as the ( The graph of % climbing against the number of days was plotted in Excel.

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27 Bioche mical Assays PrP WT, PrP D201N PrP Q211P and Lac Z were expressed under the control of da Gal4 to obtain ubiquitous ex pression of the protein. All the crosses were set at nd day 30 after eclosion and frozen at Tissue Homogenates A single whole fly was homoge nized in 30 l of RIPA buffer ( 1% NP 40, 0.1% SDS +50 mM Tris HCl pH 7 .4+150 mM NaCl+ 0.5% sodium d eoxycholate+ 1 mM EDTA Thermo Scientific) containing 20X complete p rotease inhibitors (Roche) using a sterile motorized pestle (Kontes) The samples were centrifuged (Eppendorf MiniSpin) for 1 min at 13,000 rpm and the supernatant was collected discarding the tissu e debris. The supernatant was mixed with 8 l of LDS Sample Loading buffer (Invitrogen ) and the samples were incuba Western Blot 25 l of the homogenate was loaded in a pre cast 12% Bis Tris Gel (Invitrogen) housed in a chamber (Invitrogen ) carr ying the running buffer along with a protein standard broad r ange (2 200 kDa, Biorad ). The gel was run at 90 V for 3 h and was transferred on to a nitrocellulose membrane housed in a cassette carrying the transfer buffer at 800 mA for 9 0 min (1X Running buffer: 10X NuPAGE MES SDS; 1X Tr ansfer buffer: 10X Tris Glycine + Methanol + dH 2 O) The membrane was blocked with 5% non fat dry milk in TBS 0.3% tween 20 (TBST) for 1 h at room temperature and was ve primary antibody in 5 ml of 5% milk. The next day, the membranes were washed with TBS T three times for 10 min each to remo ve any unbound antibody. The membrane was incubated with the secondary

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28 ant ibody diluted in 5% milk in TBS T at room temperature for 2 h T he membranes were again washed with TBS T 3 times for 10 min each. The signal thus obtained w as visualized using SuperSignal West Pico Chemilumiscent Substrate (Thermo Sc ientific) and developed using a Konica SRX 101A developer. Antibodies The prima Tubulin (1:125,000, Sigma), Anti PrP 6D11 (1:5,000, Covance), Anti PrP 6H4 (1:10,000, Prionics), IPC 2 ( 1:200 a gift of R. Gabizon (29) ) 15B3 (3 g/ml, Prionics). Anti M ouse HRP (1:2,000, Sigma) was used as secondary antibody. Glycosylation A ssay Flies expressing PrP WT PrP D201N and PrP Q211P at day 1 were analyzed to determine their glycosylation pattern. Homogenates were prepared as previously described and div ided into three parts of 9 l each One part was treated as the negative control, and kept at 20 sed for Endoglycosidase H (10X G5 Reaction Buffer + EndoH + dH 2 O) or PNGase F ( 10% NP 40 + 10X G7 Reaction Buffer + PNGase F +dH 2 O) assay (New E ngland Biolabs). First, homogenates were incubated with 1 denaturing buffer for 10 min at 100C. After 10 min EndoH and PNGase protocol. The samples were incubated at 37 for 1 h a nd the reaction was terminated by adding 8 l of LDS Sample Loading buff er. Samples were analyzed by a western b lot. P roteinase K (PK) Assay Homogenates were prepared as previously described, and treated with PK at six different concentrations: 0, 2.5, 5, 7.5, 10 and 20 /ml. The samples were incubated at

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29 25 f or 30 min The digestion was stopped by adding the LDS sample loading buffer and was analyzed by western b lot. Immunoprecipitation with 15B3 Misfolded conformations of PrP in young and old flies were recognized using 15B3. Reagents provided by the manufacturer (Prionics) include the 5X Homogenization buffer, IP (Assay) buffer and the purified 15B3 antibody (2.2 mg/ml). Dynabeads M 450 Rat Anti Mouse IgM (Invitrogen) and DynaMag Magnetic Particle Concentrator (Invitrogen) were used. Coating buffer was prepared with 1X PBS containing 0.1% BSA and was stored at 4 till use. The d ynabeads were mixed and shaken before use to obtain a homogenous s u spension of the beads. 10 l of the antibody coated bea d s for each genotype and unbound beads for the control were taken in a tube. The tube was placed on the magnet a nd the supernatant was decanted Then the tube was removed from the magnet and five v olu mes of the coating buffer were added. The beads were vortexed for 1 min and the process was repeated twice 3 g of the antibody per 100 l of the beads was added and the beads were gently mixed on a shak er at room temperature for 2 h to fac ilitate the cap ture. After 2 h the tube was placed on the magnet and the supernatant was removed and discarded Th e beads were washed twice with five volumes of coating buffer with gentle mixing for 10 min each. The supernatant was removed and one volume of the coating buffer was added to the beads They were stored at 4 until further use. A single whole fly was homogenized in 30 l of the 1X Homogenization buffer. The samples were centrifuged at high speed for 1 min and 20 l of the protein was collected. To this, 470 l of the IP buffer and 10 l of the coated/uncoated beads

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30 (depending on the type of reaction) were added. The samples were then gently mix ed at room temperature for 2 h After this, the samples were placed on the magnet a nd the sup ernatant was decanted The samples were washed twice for 10 min each b y gently mixing the beads with five volumes of 1X PBS. 30 l of 1X Loading buffer was added to the samples and heated at 95 for 5 min and the western blot analysis was performed. Immu nohistochemistry and Microscopy PrP WT PrP D201N PrP Q211P and LacZ were co expressed with CD8 GFP in the brain under the control of the OK107 G al4 driver. The crosses were i nitially C and shifted to 28 C after 2 days. After eclosion, the d ay 1 flies were collected for subsequent analysis. were then used for subsequent analysis. Dissections of L arval and Adult B rains Brains were dissected and collected in cold 1X PBS (10X p hosphate buffered s aline) They were fixed in 4% formaldehyde for 20 min The samples were washed thrice with PBT (PBS 0.3% Triton X 100) for 10 min each and then block ed in PBT BSA (PBT 0.5% b ovine serum a lbumin) for 1 h at room temperature The primary antibody was prepared in PBT BSA and added to the brains and incubated overnight at 4C. On the next day, the samples were washed three times with PBT for 10 min each and the secondary antibody prepared in PBT BSA was added and left at room temperature for 2 h Fin ally, the samples were washed again with PBT and mounted on Superfrost Microscope Slides and Corning cover glass (1.5 inch, 22 mm ) using the Vectashield Hard Set mounting medium (Vector Laboratories) for their visualization.

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31 Antibodies The primary antibod ies used were: Anti PrP 6D11 (1:1,000, Covance), and Anti PrP 6H4 (1:1 000 Prionics ). The secondary antibody used was Anti Mouse Cy3 (1: 600, Molecular Technologies) Stacks of the Mushroom bodies and K enyon cells were imaged and documented. Microscopy A n Axio Observer Z1 microscope (Zeiss) was used to visualize the specimen and the Axio Vision software was used to record the images for further analysis. The samples were excited at 488 nm to see GFP + cells and at 543 nm to see the i mmunostaining of PrP by Cy3. The zeiss ApoTome was used to obtain optical sections from fluorescent samples T he AxioVision software, the ApoTome was loaded and multid imensional Acquisition was selected. The slides were placed on the stage and the oil immersion objectives with 4 0X (numerical aperture of 0.65) and 63X (numerical aperture of 1.4) magnification w ere calibrated for further use. After checkin g the exposure times of each filter (Green and DsRed), the images were taken as Z stacks and later were reconstructed using the ortho projection All the images were processed with Adobe Photoshop CS5 Statistical Analysis GraphPad Prism 6 was used to analyze the statistical significance of the results. The data was collected and entered in the software A two way ANOVA with multip le comparisons was used to obtain the p value and e stimate the significance.

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32 CHAPTER 3 RESULTS PrP D201N had Decreased Survival Rates in C omparison to PrP WT PrP WT, PrP D201N, PrP Q211P and UAS LacZ were expressed ubiquitously in Drosophila under the control of da Gal4 driver The flies were maintained and were counted every day to analyze their longevity (Figure 3 1 ) More than 50% of the negative control ( LacZ) flies were alive at day 45. O n the other hand PrP WT reached 50% survival rate s by day 3 2 Interestingly, PrP D201N flies took 22 days to reach 50% survival indicating stunted longevity. However it took 37 days for PrP Q211P to reach 50% viability. Therefore while PrP D201N induced stunted survival of the flies, PrP Q2 11P did not cause significant differences in the longevity of the flies. Figure 3 1 PrP D201N induced stunted viability: UAS lines were expressed ubiquitously under the control of da Gal4 driver to study their l ongevity Negative control or the LacZ flies (red) reached 50% survival rates by day 55 when the experiment was stopped. PrP WT (blue) flies reached 50% survival rates in 32 days and all the flies died in 51 days. PrP D201N (green) reached

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33 50% survival rates by day 22 and all the flies died by day 45. PrP Q211P (purple) reached 50% survival rates by day 37 and <20% of the flies were alive by day 55 when the assay was stopped. Locomotor D ysfunc tion was I nduced by PrP D201N but not b y PrP Q211P For analyz ing if the mutations created any significant changes in the motor skills of the flies, PrP WT, PrP D201N, PrP Q211P and UAS LacZ were expressed in Drosophila under the control of the Bg 380 Gal4 driver (Figure 3 2 ) Bg 380 Gal4 d rives the expression of the UAS lines in motor neurons Male flie s used for this experi ment were maintained C. PrP WT showed 50% climb by day 6 whereas the negative controls UAS LacZ showed 50% climb by day 14. However, PrP D201N and PrP Q211P showed 50% climb by days 9 an d 15 respectively. This result proves that PrP Q211P flies d o not exhibit any relevant locomotor dysfunction. PrP D201N on the other hand moved from 50% climb on day 9 to 0% climb on day 16. Similarly, in PrP WT the % climb moved from 50% on 7 to 0% climb on day 18. This implies that although it takes PrP WT 11 days to drop from 50% climb to no climb, it takes PrP D201N only 7 days to drop from 50% climb to no climb (Table 3 1 ) This resul t suggests that, although the time of onset of locomotor dysfunction is delayed in PrP D201N as opposed to the PrP WT it progresses rapidly to induce complete dysfunction. Table 3 1 Climbing index of PrP WT and mutant PrP % Climb Number of days LacZ PrP WT PrP D201N PrP Q211P 100 80 7 3 5 5 60 12 6 7 10 40 14 7 9 15 20 22 8 9 16 0 30 18 16 30

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34 Figure 3 2 PrP D201N induces locomotor dysfunction. Locomotor dysfunction was analyzed in PrP WT (yellow), PrP D201N (green), PrP Q211P (blue) and UAS LacZ (red). In PrP WT locomotor dysfunction in the flies was induced by day 7 and it took 18 days for all the flies to stop climbing. However in PrP D201N, locomotor dysfunction was induced in 9 day. However, all the flies stopped climbing in 16 days. It took 3 0 days for the LacZ flies to stop climbing. The PrP Q211P flies mirrored this pattern, all the flies stopped climbing by 30 days. Neuropathology of the Mushroom B odies The mushroom bodies are a collection of ~2500 neurons are referred to as the learning centers in the brain. Axonal projections from the K enyon cells move through the peduncle to the lobes where they terminate (Figure 3 3) PrP is expressed throughout the axon, hence studying the gross anatomy changes in the mushroom bodies will shed light on the neurodegeneration caused by PrP PrP WT, PrP D201N, PrP Q211P and LacZ were co expressed with CD8 : GFP /CyO under the control of the OK107 Gal4 driver Adults were collected on day 1 and day 40 and the brains were % Climb No of days

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35 immunostained to study the m orphological changes in the mushroom bodies. Mushroom bodies of LacZ on day 1 and day 40 did not show any significant differe nces in the anatomy of the lobes (Figure 3 4 : A and PrP WT on day 1 looked similar to the UAS LacZ flies. However in old er fli es, it appeared that there was an onset of membrane blebbing (Figure 3 4 : B and ) Bleb or blob refers to any outward growth of the membrane or the cytoskeleton as a consequence of apoptosis. Thus membrane blebbing was a characteristic indicator of the o nset of neurodegeneration. Interestingly, PrP D201N showed comparable levels of membrane blebbing in the old flies to PrP WT The young flies looked similar to LacZ and PrP W T (Figure 3 4 : C and ) The PrP Q211P mutant flies looked similar to LacZ flies on day 1 and day 40 (Figure 3 4 : D and Figure 3 3 Anatomy of the mushroom body projections in the fly brain. The anterior lobes a bodies. The peduncle connects the mushroom body projections to the Kenyon cells and extends beyond the plane of this paper (Source: http://web.neurobio.arizona.edu/Flybrain/ )

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36 Figure 3 4 Neuropathological anatomy of mushroom body projections in PrP WT and mutant PrP. The structural integrity of the mushroom body projections in the lobes ( was studied using the CD8 GFP/OK107 Gal4 driver. had a homogenous distribution of GFP. PrP membrane blebbing in the older flies in comparison to the youn ger flies. PrP younger flies looked like the LacZ flies. PrP Q211P displays a homogenous distribution of neurons carrying PrP through the lobes and displays no Quan tification of Kenyon C ells Neuropathological changes in the axon terminals suggest a subsequent change in the cell bodies of the Kenyon cells which form a compact triangular cluster. Brains for each genotype ( PrP WT, PrP D201N, PrP Q211P and LacZ ) were imaged The surface area occupied (measured in m 2 ) by the K enyon cells were calculated using Adobe Photoshop CS5 (Figure 3 5, 3 6 ) A 25.45% increase in the size of this cluster in the negative control ( LacZ) was observed in the older flies as opposed to the younger flies. This was attributed to the fact that as the flies age, the Kenyon cells are restructured and hence become enlarged. However, in older flies from the PrP WT and PrP D201N,

PAGE 37

37 there was a 19.54% and a 19.62% decrease in the size of the cluster respectively. This was indicative of the fact that there is a loss in the number of cell bodies leading to neurodegeneration. A n 11.72% increase in the size of the cluster of the PrP Q211P flies was observed similar to t he negative control LacZ suggesting that the mutant PrP either causes no neurodegeneration or it is not significant enough to cause neurodegeneration and the answer to that question is subject to more investigation. Figure 3 5 Anatomy of the Kenyon c ells. Kenyon cells were imaged to look for differences in their anatomy or the distribution of PrP in the cell bodies. The Kenyon cells appear as a triangular shaped compact set of cell bodies (~2000). The Kenyon cells in older LacZ flies looked structural ly similar to the younger flies, with no loss of neurons. The anatomy of PrP WT, PrP D201N and PrP Q211P also looked similar to the LacZ flies. However, it appeared that the surface area occupied by the cells decreased in the older flies of PrP WT and PrP D201N genotype suggesting a possible loss of neurons.

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38 Figure 3 6 Quantification of the surface area occupied by the Kenyon cells. Surface area occupied by the Kenyon cells was quantified. N=8 Kenyon cells were imaged on AxioVision, for the analysis. The surface area occupied by the LacZ flies increased in the older flies in comparison to the younger flies. This could be due to the rearrangement of cell bodies. PrP WT showed a significant decrease in the surface area occupied by the Kenyon cells suggesting neuronal loss owing to neurodegeneration. PrP D201N also displayed a smaller cluster of Kenyon cells in the older f lies in comparison to the younger flies suggesting an ongoing pathogenic process inducing neurotoxicity. Ab normal C ellular D istribution of PrP D201N and PrP Q211P To further the understanding of the distribution of PrP in intact structures, the localization of Pr P in individual neurons at the cellular level was analyzed. PrP was co expressed with mCD8 : GFP under the control of the OK107 Gal4 driver. Third instar larvae were dissected to study the expression profile of PrP in the interneurons of t he ventral ganglion. The immunostained brains were analyzed under high magnification for sub cellular distribution of PrP (Figure 3 7 ) (44) previously reported that PrP WT accumulated in the interneurons as di stinct puncta However, PrP D201N and Pr P Q211P formed smaller puncta and these were present diffusely throughout the cytoplasm To substantiate this further, brains from the young and old adult flies from

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39 the same cross were dissected to study the distrib ution of PrP in the K enyon cells (Figure 3 8 ) Since PrP is a membrane anchored protein, it is found on the cell surface. T he PrP D201N mutants displayed increased accumulation of PrP on the cell surface in aged flies a s opposed to young flies. PrP Q211P mutants did not show any differences. Figure 3 7 Subcellular distribution of PrP in larvae. PrP was coexpressed in the interneurons of the ventral ganglion using mCy3. Subcellular distribution indicates that PrP aggregates in the interneurons of PrP WT indicating that it is stuck in the secretory pathway, whereas in PrP Q211P and PrP D201N, there is a punctate distribution of PrP across the cytoplasm.

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40 Figure 3 8 Aggregation of PrP in the Kenyon cells. The distribu tion of PrP in the Kenyon cells was studied by coexpressing PrP in the cell bodies using mCy3 as the secondary antibody. PrP D201N aggregated in the Kenyon cells more than PrP WT in older flies. Biochemical A nalysis of MoPrnp Revealed D ifferenc es in PrP glycoforms To investigate the aberrant behavior of mutant PrP in the viability assay its consequent behavior on the climbing assay and their patterns of local ization at the cellular level, the biochemistry of mutant and PrP WT was compared ( Figu re 3 9 ) Whole fly protein extracts were immunoblotted with mAb 6D11 6D11 has a linear epitope in the unstructured N terminal chain Typically in a western blot, PrP WT appears in three glycoforms un mono and di glycosylated, with relatively larger quantitie s of un and mono glycosylated forms PrP D201N had abundant di and mono gl ycosylated glycoforms whereas PrP Q211P had all the three glycoforms. Two

PAGE 41

41 PrP WT lines were used for comparison. The stronger line was used for qualitative assays and the w eaker line was used for quantitative comparisons Figure 3 9 Expression of MoPrP WT, MoPrP D201N, MoPrP Q211P and negative control UAS LacZ in flies. PrP was extracted from each of the genotypes and a western blot was performed. PrP was visu alized with the antibody 6D11 and tubulin was used as an endogenous control. UAS LacZ does not carry any PrP. In a western blot, PrP WT typically appears in mono and unglycosylated glycoforms. The mutants however displayed aberrant glycosylation patterns. PrP D201N on the other hand appeared to have di and monoglycosylated glycoforms. PrP Q211P appeared to have all the three glycoforms. Resistance of PrP D201N to C omplete D igestion of N linked glycosylation As previously stated glycosylations are a chara cteristic post translational modification in PrP and it has two N linked glycosylation sites ( N 181 and N 197). E ndoglycosidase H (EndoH) is a recombinant glyc osidase which cleaves high mannose and some hybrid oligosaccharides from N linked glycoproteins whereas Peptide N Glycosidase F (PNGase F ) is an amidase that cleaves the innermost high mannose, hybrid, and complex oligosaccharides from N linked glycoproteins This differential action of both the enzymes removes the immature (EndoH) and complete glyc osylations on PrP (PNGase F), respectively.

PAGE 42

42 Homogenates were obtained from the single flies and were treated with EndoH and PNGase F (Figure 3 10 ). PrP WT when untreated appears on an immunoblot with 6D11 with abundant quantities of un and monoglycosylat ed forms. PrP is not sensitive to EndoH indicating maturation of the protein in the Golgi. PNGase F, on the other hand, digested all the glycoforms in PrP WT appearing as a single band corresponding to the molecular weight of the un glycosylated glycoform ~27 kDa. Untreated PrP D201N has a higher concentration of the protein in its mature conformation, in the mono and di glycosylated forms. Upon treatment with EndoH, there is a remarkable decrease in the intensity of the mono and diglycosylated is o forms of the protein indicating partial digestion of the protein with the enzyme. Interestingly, when treated with PNGase F, there is a shift in the glycosylation pattern with the digested PrP resembling the untreated PrP WT. There was an increase in the un and m onoglycosylated isoforms, however PNGase F was unable to cleave all the N linked glycosylations. These results concur with the hypothesis that the protein is misfolded in an alternate conformation such that it is inaccessible to the cleavage by enzymes. Pr eviously, it was reported that in cell culture, PrP D202N (human numbering) forms detergent insoluble aggregates (35) Based on this evidence, aggregates formed by PrP D201N in Drosophila make it difficult for PNGase F to penetrate and digest the N linked glycosylations. PrP Q211P, on the other hand, displayed resistance to digestion by EndoH and sensitivity to digestion by PNGase F in a fashion similar to PrP WT.

PAGE 43

43 Figure 3 10 PrP D201N displays aberrant glycosylations. PrP WT, PrP D201N and PrP Q211P were digested with EndoH and PNGase F. PrP WT was not sensitive to digestion by EndoH, h owever was completely digested by PNGase F. PrP D201N was also not digested by EndoH, however display ed resistance to digestion by PNGase F. PrP Q211P displays resistance to digestion by EndoH providing important clues towards problems in protein processing and trafficking. Amino Acid Substitutions Introduce C onformational A bnormalities Structural perturbations due to p oint mutations on HC lead to thermodynamic instabilities in PrP disrupting long range i nteractions therefore causing the secondary structure to collapse. The a bnormal glycosylation patterns of PrP D201N and PrP Q211P led to the hypothesis that the key to understanding the abnormal behavior of the was by understanding how the p rotein folds. I used t hree different conformational antibodies to study the protein folding in mutant PrP (Figure 3 11 ). 6D11 has its linear epitope in the N terminal region (93 109) and was used as a control since it recognized PrP WT and mutant PrP (Figu re 3 1 2 ) mAb 6H4 has its epitope on HA (144 152). In PrP WT, HA interacts with HC when the globular fold is formed. Decreased sensitivity to 6H4 has been reported when there is a perturbation to the globular domain. Whole fly protein extracts from PrP WT, PrP D201N and PrP Q211P were resolved on a n SDS/PAGE and immunoblotted wi th 6H4 and compared with 6D11 (Figure 3 12 ) PrP WT and PrP Q211P were sensitive to 6H4. However PrP D201N

PAGE 44

44 was not recognized by 6H4. This suggests that in PrP D201N, the epitope fo r 6H4 is masked and unavailable for the antibody to bind. To confirm this hypothesis, in adult brains PrP WT, PrP D201N, and PrP Q211P were co expressed with mCD8:GFP and immunostained with 6H4. The distribution of PrP was analyzed in the K enyon cells and images were procured K enyon cells in the adult brain of PrP WT and PrP Q211P were recognized by 6H4. However PrP D201N was not recognized by 6H4, consistent with the results from the western blot (Figure 3 13 ) T he disulfide bond between Cys17 8 and Cys2 1 3 is a highly conserved structure and plays a role in holding HB and HC in close proximity to facilitate their stabilizing interactions. IPC 2 identified this disulfide bond because its epitope is composed of Cys178 on HB and Cys213 and Met212 on HC (28) Surprisingly, when PrP WT, PrP D201N and PrP Q21 1P we re immunoblotted with IPC2, PrP D201N and PrP Q211P were not detected by IPC2 (Figure 3 12 ) This suggests that P rP D201N and PrP Q211P do not have HB and HC together leading to alterations in the globular domain. Lack of sensitivity of PrP D201N to 6H4 also indicates that there is a possible local disruption of the disulfide bond which can lead to interruptions in t he global structural stability.

PAGE 45

45 Figure 3 11 Epitopes specific for the conformational antibodies. A. Linear epitope for 6D11 and HA epitope for 6H4. B. Co nformational epitope for IPC2 Cys178, Cys213 and Met212 holding HB and HC together (29) C. Complex epitope for 15B3 encompassing 3 peptide sequences:142 148, 162 170, and 214 226 (45) B A

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46 Figure 3 12 Conformational antibodies reveal aberrant protein fold in PrP D201N and PrP Q211P. PrP WT is detected by all the three antibodies (6D11, 6H4, and IPC2) in an immunoblot. PrP D201N was not detected by the HA recognizing antibody 6H4 and I PC2 specific for the disulfide bond. However PrP Q211P was identified only selectively by 6H4 and not IPC 2.

PAGE 47

47 Figure 3 13 Immunostaining of Kenyon cells with 6H4. The Kenyon cells were immunostained with 6H4 and coexpression of Pr P was analyzed. It was assumed that the denaturing conditions of the western blot may influence the epitope availability. The intact brains of PrP WT, PrP D201N and PrP Q211P were stained with mAb6H4 to detect the presence of PrP. PrP D201N could not be de tected with 6H4. 15B3 is a monoclonal antibody that distinguishes between the native fold and the pathogenic PrP Sc like conformation selectively precipitating on ly the protein with the abnormal fold. Three distinct peptide sequences have been identifie d as the epitope for 15B3 amino acids 142 148, 162 170 and 214 226. On availability of all three sequences to the antibody 15B3 recognizes PrP PrP WT was not recognized by 15B3

PAGE 48

48 in young flies. However in the aged flies, 15B3 recognized th e un and mono g lycosylated forms of PrP. Similarly, PrP D201N in young flies was not recognized by 15B3 but in aged flies, the mono and di glycosylated forms were recognized. In contrast, P rP Q211P was not recognized by 15B3 in younger and older flies ( Figure 3 14 ) The inference drawn from these results is that PrP WT and PrP D201N acquire a more misf olded conformation as the flies age One of the peptide sequences involved in the protein recognition by 15B3 (a.a: 214 226) is believed to be disrupted because of the Q211P mutation. Figure 3 14 Immunoprecipitation of misfolded PrP in old flies of the PrP WT and PrP D201N. 15B3 precipitated PrP Sc like conformers in old flies and not young flies. 15B3 has a complex epitope containing three peptide sequences. Older flies ca rrying PrP WT and PrP D201N were immunoprecipitated with 15B3. PrP Q211P was not recognized by 15B3. However, structural perturbations to HC due to the mutation might make it infeasible for detection of PrP Q211P. PrP D201N A cquires Partial R esistance to PK and an Alternate T opolog y To understand if the misfolding of PrP produce s PK resistant isoforms, PrP D201N and PrP Q211P were digested with PK Tissue homogenates from single flies (day 1 and day 30) w ere treated with a concentration gradient of PK from 0, 2.5, 5, 7.5,

PAGE 49

49 10, and 20 g/ml (Figure 3 15 ) PrP WT was completely digested by the smallest concentration of PK ( 2.5 g/ml ) PrP D201N displayed resistance to PK and the pattern of resistance differed be tween the young and old flies. The young flies were resistant to PK at a concentration of 2.5 g/ml, a higher conc entration completely digested PrP. The mole cular mass of the resistant PrP was the same as the untreated PrP (25 27 kDa). However in aged flie s, PrP was resistant to PK at a highest concentration of 5 g/ml and the molecular mass of the resistant PrP was 20 kDa. This pat tern of PK resistance indicates the presence of a topological isoform of PrP called as the C terminal or the Ctm PrP. This topo example the point mutat ion A117V causing GSS syndrome However previous studies on PrP D201N in in vitro cultures suggested that PrP accumulates in the cell. The appearance of Ctm topology in a ged flies provides important clues to the neuroto xicity of PrP D201N. Like PrP WT PrP Q211P was digested by PK at a concentration of 2.5 g/ml in young and old flies. This result suggests that although PrP Q211P has conformational a berrations during prote in folding, it does not create a PK resistant conformer.

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50 Figure 3 15 Proteinase K resistance. PrP WT, PrP D201N, and PrP Q211P were treated with a concentration gradient of PK. PrP WT was sensitive to the least concentration of PK and was compl etely digested in young and old flies. PrP D201N was resistant to digestion of PK at a concentration of 2.5 g/ml in younger flies and at 5 g/ml in older flies. Also, Ctm PrP resistant to PK is formed in the older flies of the PrP D201N mutant. PrP Q211P mirrored the digestion pattern of PrP WT.

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51 C HAPTER 4 DISCUSSION Global health concerns have driven forward the understanding of PrD and the central role played by PrP in the pathogenesis of these diseases. The s tructural and functional properties of PrP determine the integrity of the protein in vivo Specifically, HC in PrP is a long and rigid structure that is conserved across specie s. HC is structurally autonomous but is also involved in a large number of stabilizing interactions in the globular domain. It can be hypothesized that alterations in conserved domains on HC cause structural perturbations increasing the propensity of HC to misfold. In spite of the vast literature available, pathogenic mutations on HC have only been tested in vitro to establish their ability to cause PrD. Therefore, understanding the mechanisms by which mutations destabilize HC will shed light on the comm on events across several For this, we expressed two pathogenic mutations ( D201N and Q211P ) in Drosophila and studied their processing and trafficking using biochemical, behavioral, and immunohistochemic al approaches. D201 and Q211 stabilize HC through important interactions with other residues on HC or the globular domain hence contributing to the overall integrity of PrP (17) Our aims with this study were two fold: (i) to study the differences in the processing, conformation and structural stability of PrP D201N and PrP Q211P, and (ii) to analyze the ability of PrP D201N and PrP Q211P to induce neurotoxicity. In a first of its kind in vivo study on PrP D201N and PrP Q211P, we observed interesting differences between the protein folding pathways in young and old flies. Previously, these mutations were engineered only in cell culture or cell free systems and results did not indicate significant neurotoxicity or the identification of a

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52 characteristic PrP signature. However the Drosophila model provided useful answers to the pathogenesis of these two mutations. Aspartic A cid to Asparagine: T he S tructural J ourney in PrP WT This loop is attached to HC at T199. D201 on HC forms a conserved capping box with T199, which is the starting point of HC (30) The N capping box formed by D201 with T199 and the hydrophobic staple motif dictate the direction of orientation of HC. D201N and T199A disrupt this capping box; hence the interaction of T199 with HB is altered What structural changes in PrP D201N are likely to play a role in its misfolding? In order to answer the question, we tested PrP D201N against conformational antibodies and we observed that: (i) the disulfide bond between Cys178 and Cys213 was not recog nized by IPC 2, (ii) mAb 6H4 antibody did not identify PrP D201N in an immunoblot and in intact brains, and (iii) immunoprecipitation specifically isolated PrP Sc like conformers in older flies that is assumed to be neurotoxic. The explanation to these dras tic events might be the hypothetical collapse of the conserved disulfide bond, leading to misfolding of the globular domain (29,46) The disruption of the capping box at the beginning of HC is likely to create a thermodynamically unfavorable twist of HC away from HB, therefore introducing aberrations in the pr otein fold. Also, it can be argued that PrP D201N misfolds initially during biogenesis and accumulates as the fly ages leading to the formation of neurotoxic PrP that is 15B3 positive. One of the most striking features of PrP D201N is the improper process ing and trafficking of the protein. PrP D201N was incompletely processed with only the mono and diglycosylated glycoforms, did not leave the cis Golgi, and formed aggregates that could not be digested by PNGase F. Previously, PrP D202N (human numbering) was

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53 reported to accumulate in the ER of the neuroblastoma cells, as curly aggregates (35) In Drosophila we report a shift in the glycoform pattern and partial digestion on treatment with PNGase F. N glycosidase F is a potent enzyme that eliminates all N linked glycosylation. These observations indicate that PrP D201N is stuck in the ER or the cis Golgi forming aggregates that mask the N linked glycans for PNGase F to cleave. However, resistance of PrP D201N to digestion with PNGase F was speculated to be due to the clumping of protein as aggregates, impenetrable for cleavage. To resolve these aggregates, PrP D201N was treated with 2% SDS and 2% Triton X and then digested with PNGase F. However, both the treatments were unable to disaggregate PrP D201N and hence the digestion with PNGase F remained infeasible. Interestingly, digestion of PrP D201N by P K revealed a resistant 20 kDa band corresponding to Ctm PrP in older but not younger flies Based on th is I hypothesize that PrP D201N has the tendency to form Ctm PrP, however the quality control machinery keeps this pathway in check in young flies In older flies, the cell s commit more mistakes in protein folding leading to the accumulation of Ctm PrP. Previously, studies on human PrP determined that the membrane spanning stretch (TMD ) and the stop transfer effector (STE) domain govern the formation of topological isoforms of PrP (47) Until recently, only mutations in the signal peptide and the TMD were associated with the formation of Ctm PrP. Mutations on HB and HC (D178N, H187R, F198S, E200K, D202N, V210I, and Q212P) in cell culture did not produce alternate topologies but induce d protein accumulation (48) Interestingly, substitution of methionine residues with serine on HC of PrP (M et 205 and M et 213) revealed a failure in oxidative folding (28,29) and the formation of Ctm PrP (46) Recent studies with recPrP support the

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54 evidence that the disulfide bond determines the membrane insertion of PrP (49,50) Therefore, apart from mutations in the signal peptide, TMD, and the synthetic M>S substitutions, Ctm PrP can be formed due to perturbations in other conserved regions of PrP. However, the biogenesis of Ctm PrP from PrP D201N follows a unique pathway because the abnormal isoform is acquired only in aged flies Since, PrP WT does not display a similar biochemical process, it is possible that the spontaneously misfolded PrP D201N accelerates its conversio n to other pathogenic isoforms. Epidemiological data from GSS syndrome kindreds linked to P102L, A117V, D202N, Q212P and Q217R have shown an early onset of the disease, short duration and early death (51) The neuropathology revealed widespread deposition of multicentric plaques, but no spongiform degenera tion. To examine neurotoxicity in the in vivo model, we compared: (i) expression of PrP D201N in Drosophila, (ii) PrP D201N in cell culture systems, and (iii) other Ctm PrP mutants and their neuropathology. I n cell culture, PrP D201N induced neurodegenerat ion but i n vitro studies limit the relev ance to progressive neurotoxici t y and are performed more as a screen for potential pathogenic mutants. This could explain why all the newly reported features of PrP D201N were not observed in vitro PrP A117V produced astrogliosis and neurodegeneration in mice. However, sections of the brains revealed no PrP Sc and only Ctm PrP. Also, mouse models were generated with mutations that prevented the formation of Ctm PrP (STE TM1 and G123P) showed no neuro degeneration (25,47) To substantiate this further, mice expressing PrP A117V were exposed to infectious PrP Sc and these developed the disease much faster than PrP Ctm PrP increases susceptibility to PrP Sc (47) PrP D201N decreased the survival rates

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55 of the flies alongside locomotor dysfunction. Also, in adult flies, PrP D201N displayed membrane bleb bing, an indicator of neuronal degeneration The Kenyon cells were smaller in size and displayed accumulation of PrP. Although, Ctm PrP has been established to be neurotoxic, it appears that in PrP D201N, Ctm PrP is not the only conformation driving pathog enesis. The interplay between normal aging processes structural perturbations in the structure of PrP might lead to the abnormal behavior of the protein and accelerated neurotoxicity. Evidence from the available literature and our work sheds light on the f act that PrP D201N is abnormally folded, accumulates in the secretory pathway, acquires an abnormal topology and is neurotoxic is older flies. Helix B reaks due to P roline does not Induce N eurodegeneration Q211 is located centrally on HC of PrP and is in c lose proximity to the disulfide bond forming residue: Cys213. Proline does not have an amide hydrogen and, hence it cannot form a hydrogen bond with the preceding amino acid on the helix. Also, its side chain is large and this sterically hinders any stabilizing hydrogen bonds. T hese interactions force about 30 on the helix axis (38,52) In PrP, this backward bend induces an unfavorable interaction of Pro211 with Gl u210 (36) It has been reported previously, that proline can only cause helical breaks at in the beginning or the end of a helix. However if the helix is more than 13 amino acids long introducing a proline can destabilize the entire helix (37) NMR studies on PrP Q212P (human numbering) reveal characteristic fea tures: a helical break at Ser 22 2 and a change in the long range S2 HB interactions. This abnormal S2 HB interaction in PrP Q211P causes a change in the orientation of Tyr162, Tyr168 and Phe175 (present in the hydrophobic cluster), globally causing an increased flexibility in the C term inal part of HC. In the R220K artificial mutant (human numbering), HC is completely disordered beyond the

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56 mutation, making HC flexible (53) In PrP E200K, another mutation on HC causing fCJD, Tyr168 is exposed and the S2 HB region shows increased flexibility (54) Recent evidence is surfacing regarding the flexibility of conserved portions of the globular domain due to structural perturbations by the mutations. Flexibility of the C terminus of the globular domain makes the structure vulnerab le to misfolding. Studies on PrP Q211P in cell culture revealed that it induces misfolding and perturbations in the secretory pathway (39,48) The authors identi fied a pathway common to several PrD causing mutants, wherein misfolded PrP is alternatively routed to the lysosomes for degradation. Using conformational antibodies, we identified an atypical disulfide bond in Pr P Q211P like in the PrP D201N mutants. How ever, we could not identify any other cues to establish the pathogenicity of these mutants. Immunoprecipitation with 15B3 did not detect PrP Q211P in young and old flies. Three peptide sequences on PrP form the epitope for 15B3. The helical break caused by proline in PrP Q211P might displace one of the peptide sequences away from the other two peptide sequences therefore making PrP Q211P insensitive to the 15B3. Another possible explanation could be that the turnover of PrP Q211P is very high and hence is d egraded quickly, which prevents its build up in the cell and neurotoxicity. PrP Q211P has only been established to cause neuropathology in a patient reported to have GSS syndrome (unpublished). However, there are no in vivo models to study the neurotoxici ty of PrP Q211P. Our study could not establish neurotoxicity by PrP Q211P either However we identified clues that suggest that PrP Q211P has the potential to misfold and in theory should be neurotoxic. The next step would be to

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57 combine PrP Q211P with P rP WT to study if PrP Q211P accelerates neurotoxicity through inducing the misfolding of PrP WT. Conclusion Neurodegenerative diseases are an increasing cause of socio economic burden in the United States. Threat of a silent epidemic like the BSE outbreak in the 1980s necessitates the urgency to understand the pathogenesis of the PrDs better and to develop disea se protective strategies. Our findings establish that two pathogenic mutations located in close proximity to each other and to several conserved stabilizing bonds on HC of PrP produce completely independent and distinct phenotypes in Drosophila This simu ltaneous study of the mutations also helps us to understand that the mechanisms of pathogenesis is distinct amongst these mutations irrespective of position of the mutation. Hence a good therapeutic strategy would be to identify a common link to all these mutations to understand the regulatory pathways where the mistakes occur in protein folding. Owing to ease of genetic manipulations in Drosophila

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63 BIOGRAPHICAL SKETCH Amrutha Pa ttamatta was born in Chennai, India 1989 and spent all her growing up years in Chennai until she finally moved to Gainesville, FL She was always proficient in her studies, whic h has won he r several accolades. Amrutha pursued her undergraduate education in Sri Venkateswara College of Engineering, Chennai, India (2006 2010) majoring in Biotech nology. She has kept herself constantly occupied through four years of undergraduate education by pur suing internships in research wings of several medical hospitals all over the country. Her father, now retired, worked in the private sector of the Indian economy as an Executive Director handling off shore business, for over two decades. S he has thus had the opportunity to grow up among children of other faiths and nationalities. Her mother is a high school teacher in Chemistry. She has always drawn inspiration from her parents and her brother, all three of whom have driven the aspiration of crea ting a successful career path for herself. She fondly states that life as a graduate student in University of Florida was not very easy or rosy. But she ended up learning the most important professional and personal lessons from here. She is very passionat e about her research in the Fernandez Funez and Rincon Limas lab and believes that she could not have asked for a better platform of understanding and knowledge before pursuing a doctoral degree. All set to complete r faith that her family, friends and mentors are instrumental in bringing her this far. Apart from her studies and work, she is an avid reader and enjoys cooking.