Parkinson's disease associated Î±-synuclein familial mutants promote

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Parkinson’s disease associated #-synuclein familial mutants promote dopaminergic neuronal death in Drosophila melanogaster Ganesh M Mohite, Saumya Dwivedi, Subhadeep Das, Rakesh Kumar, sravya paluri, Surabhi Mehra, Neha Ruhela, Arunima S, Narendra Nath Jha, and Samir K. Maji ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00107 • Publication Date (Web): 15 Jun 2018 Downloaded from http://pubs.acs.org on June 16, 2018

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ACS Chemical Neuroscience

Parkinson’s disease associated α-synuclein familial mutants promote dopaminergic neuronal death in Drosophila melanogaster

Ganesh M. Mohitea, Saumya Dwivedia, Subhadeep Dasa,b, Rakesh Kumara, Sravya Paluria, Surabhi Mehraa, Neha Ruhelaa, Arunima Sa, Narendra Nath Jhaa, Samir K. Maji*,a a

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay,

Mumbai, India b

IITB-Monash Research Academy, Indian Institute of Technology Bombay, Mumbai, India

* Correspondence should be addressed to Prof. Samir K Maji Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, India 400076. Telephone: +91-22-2576-7774. Fax: +91-22-2572 3480. E-mail: [email protected]

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Abstract α-Synuclein (α-Syn) aggregation and amyloid formation are associated with loss of dopaminergic neurons in Parkinson’s disease (PD). In addition, familial mutations in α-Syn are shown to be one of the definite causes of PD. Here we have extensively studied familial PD associated α-Syn G51D, H50Q and E46K mutations using Drosophila model system. Our data showed that flies expressing α-Syn familial mutants have a shorter lifespan and exhibit more climbing defects compared to wild-type (WT) flies in an age-dependent manner. The immunofluorescence studies of the brain from the old flies showed more dopaminergic neuronal cell death in all mutants compared to WT. This adverse effect of α-Syn familial mutations highly correlated with the sustained population of oligomer production/ retention in mutant flies. Furthermore, this was supported by our in vitro studies, where significantly higher amount of oligomer was observed in mutants compared to WT. The data suggest that the sustained population of oligomer formation/ retention could be a major cause of cell death by α-Syn familial mutants. Keywords: Parkinson’s disease, α-synuclein mutants, Drosophila melanogaster, oligomers, amyloid

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Introduction Parkinson’s disease (PD) is a chronic neurodegenerative disorder, which affects ~1 % of the population at the age of 65. It is characterized by motor (bradykinesia, resting tremor, rigidity, and postural instability) and non-motor (sleep behavior disorder, hyposmia, rapid eye movements and depression) symptoms, which develop slowly and continue to worsen if not treated1. The major pathological hallmark of PD includes degeneration of dopaminergic neurons in substantia nigra pars compacta of the patient’s brain, which leads to reduce level of dopamine, a neurotransmitter responsible for motor functions. Most of the PD cases are sporadic, however, ~ 10% of the cases have been shown to involve genetic factors where gene multiplication/ mutations lead to an aggressive form of PD2. In this regard, point mutations in the SNCA gene encoding α-Syn, LRRK2 encoding Leucine-rich repeat kinase 2 and GBA encoding glucocerebrosidase are known to cause autosomal dominant PD; whereas mutations in parkin gene family, PINK1 and DJ-1 are associated with autosomal recessive PD3. However, immunostaining and biophysical characterization of intracellular inclusion bodies, i.e. Lewy bodies (LB) and Lewy neurites (LN) found in PD patients brain revealed that α-Syn is the major protein present in these aggregates indicating the involvement of α-Syn as the culprit protein in disease pathology4. α-Syn is 140 amino acid protein majorly expressed in neurons and in red blood cells (RBCs). Although the exact physiological function of α-Syn is largely unknown, it has been suggested that α-Syn is involved in maintaining synaptic vesicle pools at synaptic junction, apoptosis, membrane trafficking and mitochondrial dynamics in normal neurons5. Previous studies have shown that familial disease-associated α-Syn mutations lead to altered oligomerization and aggregation of the protein in vitro6-8. Six familial α-Syn mutations (A30P, A53T, E46K, H50Q, G51D and A53E) and the newly discovered A53V mutation have shown to be associated with early/late-onset PD9-15. Amongst these familial mutations A53T, E46K and H50Q accelerate, whereas A30P, G51D and A53E delay the rate of fibril formation of α-Syn16-20. Enhanced oligomer formation is considered to be the shared property amongst A30P and A53T mutants, which explains their association with early-onset PD16. However, this is not true for G51D and A53E mutations, which showed overall slow aggregation19, 21. Therefore, there is no unifying mechanism underlying the correlation of aggregation/oligomerization propensity of these mutants and with disease severity and onset. Despite the complexity in the field, several 3



attempts have been made to model PD in vivo using various vertebrate and invertebrate model organisms22, 23. Drosophila melanogaster (fruit fly) is one of the most studied model organism in PD due to its advantageous features that include a small and well-defined genome, ease of genetic manipulation, short life span, ease of handling, multiplication and targeted expression of the gene of interest and RNAi mediated gene silencing. Strikingly, Drosophila melanogaster was also found to recapitulate many phenotypes associated with PD24. It has been shown that α-Syn and its familial mutations (A30P and A53T) recapitulates features of locomotory and other phenotypes associated with PD25. Moreover, a study by Karpinar et al. demonstrated that α-Syn mutants [A56P and triple (A30P/A56P/A76P)], which are more prone to oligomerization and/or stay longer in the oligomeric state are more neurotoxic in Drosophila26. In the current work, to establish the phenotypic relationship of aggregation and PD, we developed Drosophila model of PD overexpressing disease associated familial E46K, H50Q and G51D point mutations. These three α-Syn familial mutations H50Q, G51D and E46K have not been studied in Drosophila model system so far. Therefore, studying these mutations in Drosophila model system will be helpful in understanding the molecular mechanism associated with familial PD. In the present work, transgenic Drosophila flies were generated for H50Q, G51D and E46K αSyn mutations. These mutant proteins were expressed in the brain of Drosophila and flies were examined for locomotory defects, survival and dopaminergic neuron degeneration as well as oligomer formation with age. Drosophila expressing familial α-Syn mutants showed enhanced climbing defects, reduced survival, more dopaminergic cell degeneration and sustained formation/retention of oligomers with age compared to WT α-Syn. This was further validated by in vitro aggregation kinetics, where familial mutant proteins showed sustained oligomer formation even after fibrilization compared to WT α-Syn. The study suggests that sustained production of oligomers could be the shared property amongst α-Syn mutations associated with familial PD. Results Transgenic flies show comparable expression of familial α-Syn mutants and WT protein. Previously, α-Syn overexpression has been used to delineate its role in PD pathology using Drosophila as a model system25. Although among previously discovered α-Syn mutants, A30P 4


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and A53T have been studied in Drosophila model for PD 25, E46K, H50Q and G51D have not been investigated yet. In the present study, we compared the phenotypic differences between WT α-Syn and its two faster aggregating familial mutants E46K, H50Q18, 20and a slow aggregating G51D mutant17. Firstly, WT α-Syn and its three familial mutants (E46K, H50Q and G51D) were cloned in the pUAST vector (Figure S1). Gene sequences were confirmed by DNA sequencing of the cloned plasmids. α-Syn mutations were observed after translation of obtained gene sequence using ExPASy translation software. Mutated residues are highlighted with red boxes in the aligned protein sequence of mutants and WT α-Syn (Figure 1A). Thereafter, sequenced pUAST–α-Syn (mutant) plasmids were then used for generation of transgenic flies. Transgenic flies were further examined for the presence of SNCA gene and its mutants. To do so, genomic DNA from transgenic flies were extracted and used as a template to perform PCR with SNCA specific primers. DNA product obtained from PCR showed band of ~430 bp corresponding to the expected size SNCA gene (Figure S2A). PCR product was sequenced to confirm the presence of mutations (Figure S2B). The cross between pUAST α-Syn (WT, E46K, H50Q and G51D) and brain-specific elav-Gal4 was set up and its F1 progeny was analyzed for α-Syn expression in the brain. The F1 generation flies of this cross were used for all the experiments. The elav/+ flies expressing elav-Gal4 (with one copy of elav Gal4) but not α-Syn were used as negative control. The F1 progeny of these control flies (elav/+) were obtained by crossing elavGal4 homozygous virgin female flies with Cantos-S male flies (CS flies do not have α-Syn gene in the genome). F1 generation flies from this cross-served as a negative control for α-Syn expression. To check α-Syn protein expression level in F1 progeny of mutants and WT transgenic Drosophila, brain extract from 10 day old F1 generation flies from all the crosses were used and analyzed by Western blot using LB509 α-Syn monoclonal antibody. F1 progeny of WT and disease-associated mutants showed α-Syn band at expected molecular weight of ~17 KDa in the Western blot (Figure 1B). The control fly elav/+ did not show any presence of α-Syn. Densitometry quantification and normalization with β-tubulin data demonstrated a nearly twofold increase in protein expression for WT and familial mutants (Figure 1C). Moreover, comparable expression of α-Syn in the flies expressing WT and mutants was achieved. This enabled transgenic flies to be used for further behavioral studies where changes in behavioral effect may not be biased due to expression level. 5



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Ectopic expression of α-Syn familial mutants in the brain of flies show locomotory defect with age. The PD pathology is associated with several motor disabilities which include locomotory defects, postural instability, tremors and muscle rigidity in PD patients

27

. Previous studies

demonstrated that transgenic Drosophila overexpressing WT α-Syn or its disease-associated familial mutants in the whole brain show locomotory defects with age as a result of dopaminergic cell death

25

. Usually, these locomotory defects in Drosophila are studied by

climbing assay or negative geotactic response assay 28, where a number of flies that climb above a preset height in 10 seconds is recorded. So, aged matched flies expressing WT α-Syn and its familial mutants under elav-Gal4 promoter were used for the climbing assay. We observed that flies of all genotypes were able to climb 90 ±1.5% on day 10, which decreased to ~ 85±1.3% on day 20 suggesting no significant difference in elav/+ and any of α-Syn expressing flies (Figure 2A). Further analysis of day 20 climbing data suggests no significant difference between WT and mutants or among the different mutants. Climbing ability decreased in 30-day-old flies of elav/+ (75±1.3%), WT (62±2.4%) and drastically in E46K (50±2.6%), H50Q (57±1.8%) and G51D (47±2.1%) (Figure 2A). Among the flies expressing α-Syn, E46K and G51D familial mutations showed significantly higher effect on climbing as compared to WT and H50Q. Furthermore, at day 40 elav/+, WT, E46K, H50Q and G51D showed climbing ability of 67±2.7%, 52±2.68%, 41±5.06%, 50±3.58% and 22±5.0%, respectively (Figure 2A). At day 40, H50Q did not show much change in the climbing ability compared to WT, however, E46K exhibited a significant decrease in climbing ability in comparison to both WT and H50Q. Interestingly, G51D flies showed a significant decrease in climbing ability compared to WT and other two mutants (H50Q and E46K). Collectively, the order of decrease in the climbing ability of flies at day 40 follows is G51D> E46K > H50Q ≥ WT (Figure 2A). Overall, the data demonstrated age-dependent decline in the climbing ability of all the genotypes, with a significant decline in flies expressing WT or its mutants as compared to elav/+ (flies not expressing α-Syn) (Figure 2A). Notably, G51D and E46K expressing flies showed more pronounced climbing defects compared to the flies expressing WT and H50Q mutant.

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α-Syn familial mutants show shorter lifespan compared to WT and control flies. Previous reports have shown reduced lifespan of transgenic Drosophila expressing α-Syn and familial mutants (A30P and A53T) as compared to control flies 25. Similarly, here we examined the lifespan of age-matched flies expressing WT, E46K, H50Q, G51D α-Syn and control flies i.e. elav/+. To avoid any infection, flies under study were transferred to fresh food containing vials every third day. Identical food and day/night regime was maintained for all the flies. Fly death was recorded and final data was plotted as percentage (%) survival. The % survival indicates the number of flies that have survived at any given time point out of the total number of flies initially used for the experiment. α-Syn expressing transgenic Drosophila (WT, E46K, H50Q and G51D) and elav/+ showed ~ 95±0.6% survival until day 25. At day 30, a slight decrease in survival rate was observed in familial mutants compared to WT and elav/+, however this difference was not significant (Figure 2B). At day 35, elav/+, WT, E46K, H50Q and G51D showed % survival of 93±2.40, 95±1.91, 86±7.65, 86±4 and 83±1.5, respectively. From day 40 onwards, differences between familial mutants and WT α-Syn and/or elav/+ were apparent. At day 45, % survival was found to be 84±4.16, 87±6.58, 72±2, 65±0.5 and 69±4.5 for elav/+, WT, E46K, H50Q and G51D, respectively (Figure 2B). WT α-Syn and elav/+ also showed a gradual decrease in lifespan (although the effect was less compared to the mutants) with increasing age from day 0 to day 55, possibly due to the effect of aging on the survival. Moreover, the data also showed life span of WT expressing flies is almost similar to the lifespan of elav/+ flies. Overall, the data indicate that familial mutants show a significantly reduced survival rate as compared to WT αSyn and elav/+ (Figure 2B). Since, % survival of α-Syn mutant flies gradually decreases after day 30, thereby suggesting that familial PD mutants shorten the lifespan of the flies. Familial α-Syn mutant expression induces enhanced dopaminergic neurodegeneration in Drosophila. Degeneration of dopaminergic neurons is one the major pathological hallmark in PD pathogenesis29. Various studies have demonstrated that along with several PD phenotypes, neurodegeneration can also be recapitulated using Drosophila model system

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. The study by

Feany et al showed that WT and mutant (A30P and A53T) α-Syn expression under elav-Gal4 promoter results in an age-dependent degeneration of neurons in the dorsomedial cluster in 7



Drosophila brain 25. Studying the dopaminergic neuronal degeneration in familial α-Syn mutants and WT expressing flies is important as each familial mutation may uniquely affect the dopaminergic clusters. In Drosophila, dopaminergic neurons are present in six distinct clusters, which are grouped into anterior side PAL (paired anterior lateral), PAM (paired anterior medial), PPM1/2, PPM3 (paired posterior medial), PPL1 and PPL2 (paired posterior lateral) 30. Dopamine synthesis in these neuron clusters is regulated by the enzyme tyrosine hydroxylase (TH). TH staining of the brain reveals differences in a number of the clusters with aging and also in the diseased condition. In this study, neuronal degeneration was investigated by taking age-matched (day 10 and 30) flies expressing α-Syn (WT, E46K, H50Q and G51D) and elav/+. TH immunostaining of 10 days old flies expressing elav/+ and WT α-Syn revealed the presence of all the six clusters (Figure 3A). Individual neurons were counted from each cluster and quantitative data was plotted by analyzing six hemispheres for each genotype (Figure 3A and B). Immunostaining and quantification data highlighted that no significant difference exists in any of the dopaminergic clusters among the genotypes under study (Figure 3A and B). This suggested that irrespective of α-Syn expression, at day 10, all the genotypes show no sign of neurodegeneration. Since PD associated phenotypes were apparent from climbing and survival assays after day 30, therefore 30 days old flies were also analyzed for changes in dopaminergic neuronal clusters. Data showed that all the three familial mutants (E46K, G51D and H50Q) had reduced number of neurons in PAL and PPM3 clusters compared to elav/+ (Figure 4A and B). On the day 30, significant differences were observed between WT and all the mutants for PAL cluster. However, among the mutants, we did not find any remarkable difference in number of neurons in this cluster. With respect to the PPM3 clusters, among the mutant expressing flies, only in H50Q and G51D showed significantly decreased number of dopaminergic neurons mutant compared to WT. This decrease is more significant in G51D (**) compared to H50Q (*) (Figure 4A and B). PPM1/2 and PPL1 clusters showed a significant decrease in number of neurons only in G51D mutant expressing flies compared to WT, E46K and H50Q. Notably,WT and elav/+ did not show differences in any of the clusters except for PPL2 (Figure 4A and B). Although PPL2 cluster showed a significant decrease in the number of neurons in all the mutants compared to WT, no considerable change was observed in the number of neurons among the mutants (Figure 4A and B). Moreover, this data was in accordance with climbing (Figure 2A) 8


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and survival studies (Figure 2B) that also suggested that G51D is more aggressive mutant compared to E46K and H50Q mutants. Therefore, based on TH immunostaining, it can be concluded that familial α-Syn mutant expression induces enhanced neurodegeneration in Drosophila. Production of amyloid oligomers and amyloid fibrils in Drosophila overexpressing α-Syn. Initially, it was considered that aggregation and amyloid formation by α-Syn is responsible for dopaminergic neuronal death in PD16. The recent data, however, suggest that α-Syn oligomers produced during aggregation are more toxic species compared to fibrils26,

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. Winner et al.

showed that α-Syn mutant that produced more oligomers was highly toxic compared to the fibrils forming variants of α-Syn 31. Moreover, it was suggested that faster oligomerization is the shared property amongst the familial α-Syn mutants causing PD 8. It has been suggested that both G51D and A53E delay the oligomerization and aggregation whereas H50Q accelerates the aggregation17-19, 21. To analyze the amount of fibrillar aggregates and oligomers generated in the brain of Drosophila due to overexpression of α-Syn, we performed dot blot analysis using amyloid fibril specific OC and oligomers specific A11 antibody (Figure 5A and B). Here, we checked fibril and oligomer formation in 10 and 30 days old flies using dot blot assay. Brain homogenate was made from age-matched flies of all the genotypes under study. An equal amount of protein of each genotype was loaded on nitrocellulose membrane and was subsequently probed with OC and A11 antibody. We further analyzed the dot blot data with Image J software for the relative quantification of different species. Quantified data values have been normalized with respect to WT. The dot blot showed that oligomer (detected by A11) level was high in day 10 flies expressing familial α-Syn mutants compared to WT. We observed that flies expressing H50Q and G51D showed significantly more amount of oligomers (A11 antibody) relative to WT on day 10. However, no significant difference was observed in the amount of oligomers among the mutants (Figure 5A and C). Furthermore, 30 days old flies showed a lower amount of oligomers compared to 10 days old flies in α-Syn WT and familial mutants. Although, at day 30, all the flies expressing mutants showed a significantly higher amount of oligomers compared to WT, this difference was negligible amongst the mutants

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(Figure 5A and C). Data with oligomers analysis, thus indicates that familial mutants tend to produce or sustain more oligomers as compared to WT (Figure 5A). No fibril specific OC antibody signal was observed in any of genotypes at day 10 brain homogenate; however, 30 days old flies showed a prominent signal for OC (Figure 5B and C). Further analysis suggests, no significant difference in OC staining (fibrils specific antibody) between all the mutants relative to WT or among the mutants on day 30 (Figure 5A and C). Oligomer and fibril formation was not observed in day 10 and day 30-brain homogenate of elav/+ flies (Figure 5A and B). In vitro aggregation of WT and familial α-Syn mutants associated with PD. Furthermore, we performed in vitro aggregation study to correlate the aggregation propensity and observed toxicity of WT α-Syn and its familial mutants in Drosophila. Although it has been shown that E46K and H50Q accelerate the α-Syn aggregation, while G51D delayed the aggregation, the amount of oligomers produced by each mutant is not known. In this study, we tried to correlate the oligomers formation by each mutant by measuring A11 positive oligomers. For this, 300 μM LMW protein was prepared and incubated at 37°C with slight agitation. During aggregation, the amount of oligomers formed was qualitatively accessed by dot blot analysis using A11 antibody32. Along with dot blot analysis, at the beginning of incubation and at the end of aggregation (270 hrs), Thioflavin T (ThT) binding and circular dichroism (CD) spectroscopy were performed. At the end of aggregation, morphology of fibril produced by each protein was examined by TEM. The dot blot data suggest that the WT and familial mutants showed increasing amounts of oligomers during aggregation kinetics (Figure 6A). However, at the end of kinetics, the oligomer amount was reduced while the relative content of oligomers was high in case of mutants as compared to WT. This data suggests that familial mutants of α-Syn exhibit enhanced and sustained formation of oligomers. The ThT binding data showed that all the proteins exhibit increase in ThT fluorescence after 270 hrs of incubation compared to soluble protein (Figure 6B). To further confirm the secondary structural changes of protein after aggregation, CD spectroscopy also performed. All proteins showed random coil structure at the beginning of incubation with characteristics minima at ~ 198 nm (Figure 6C). After 270 hrs of incubation, all proteins showed the β-sheet structure as observed by characteristic CD spectra 10


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with minima at ~218 nm (Figure 6D). The morphology of the fibrils at the end of the aggregation showed amyloid-like fibrillar morphology with slight differences in the diameter of the fibrils (Figure 6E).

Discussion Parkinson’s disease is pathologically characterized by the presence of Lewy bodies (LB) and Lewy neurites (LN) in the substantia nigra of PD patient’s brain1,

33

. α-Syn amyloid fibrils

represent the major part of LB and LN and therefore it is suggested that α-Syn aggregation and amyloid formation is the central event in PD pathology4. This has been further substantiated by the discovery of seven familial point mutations of α-Syn, which have been shown to cause an aggressive and definite form of PD9-15. Three of PD associated mutations (A53T, E46K and H50Q) have been shown to accelerate the α-Syn aggregation kinetics, whereas other three (A30P, A53E and G51D) delay the aggregation process16-20. Although oligomeric species formed during the initial stages of aggregation are the most toxic species responsible for PD pathology, only the A30P mutant is shown to exhibit enhanced oligomerization and slow fibrillation rate8. However, this is not known in detail for other mutants. Moreover, despite several efforts to understand the disease-causing mechanism of these mutants, no correlation with the disease pathology and aggregation has been established yet. Here, we tried to delineate the effects of αSyn familial mutants using Drosophila melanogaster as Parkinson’s disease model system. Unlike other PD associated genes, α-Syn has no homolog and is not endogenously produced in Drosophila. So, we performed pan-neural expression of α-Syn WT and familial mutants (E46K, H50Q and G51D) and studied manifestation of PD phenotype in Drosophila. It is also known that amount of α-Syn expression in cells is directly correlated with the development of PD34. So, before the behavioral studies, we examined the expression level of all the proteins in Drosophila brain by Western blotting. We found comparable protein expression in all the cases, which enable us to study the effect of mutations compared to WT. The genetic background and environmental factors directly modulate lifespan of Drosophila35, 36. It has been reported that α-Syn expression induces climbing dysfunctions and shortens the lifespan of flies

37

. In our study, we used age-matched flies to compare the differences in the

climbing ability and survival rate of flies due to α-Syn expression. The climbing assay has been 11



routinely used in Drosophila to study neuronal dysfunctions and associated behavioral changes. The climbing assay showed that initially the control (elav/+) and α-Syn expressing flies climbed efficiently, however, with time, a rapid decline in the performance of α-Syn expressing flies was observed (Figure 2A). This decline was more pronounced for α-Syn mutants than that of WT. Our climbing assay clearly suggests that the expression of α-Syn mutants in Drosophila leads to climbing defects in an age-dependent manner. This was further supported by survival assay wherein α-Syn familial mutants (E46K, H50Q and G51D) expressing flies showed a significant reduction in the lifespan after day 40 compared to WT and elav/+ control flies (Figure 2B). Overall, the data implicate that familial α-Syn mutations affect the climbing ability and shortens the lifespan of the flies. Indeed, the results were in accordance with the previous studies by Feany et al with WT α-Syn and other α-Syn familial mutants 25. Investigating integrity of dopaminergic neuronal clusters in Drosophila is a good system to study neurodegeneration under the influence of genetic and the environmental factors 38. Dopaminergic neuronal clusters are responsible for producing dopamine, which is a neurotransmitter that controls motor functions in Drosophila39. The pathway for producing dopamine is conserved in both human and Drosophila, which involves a key enzyme ‘Tyrosine Hydroxylase’ (TH). Previous studies have shown that the expression of α-Syn mutants leads to dopaminergic cell loss in an age-dependent manner25. We also observed a similar decrease in a number of TH positive cells in flies expressing E46K, H50Q and G51D mutants, with age, indicating the manifestation of PD phenotypes in Drosophila. Indeed, the depletion of TH cell bodies in α-Syn mutant flies was more noticeable than that of WT and control flies. Therefore, selective loss of dopaminergic clusters in familial α-Syn expressing flies may be responsible for climbing defects and reduced survival rate. α-Syn aggregation is central to the development and progression of PD40. There are many intermediates broadly regarded as oligomers and protofibrils, which are formed during fibril formation41-43. Interestingly, many studies have reported that oligomers of α-Syn are more cytotoxic as compared to fibrils26, 31. Studies in Drosophila model system have suggested that phosphorylation of α-Syn at Ser129 reduces the amount of inclusion formation (an aggregated form of α-Syn) and enhance neurodegeneration 44. Previous studies have shown that oligomer is more cytotoxic when overexpressed in rat and Drosophila model systems26, 31. We used oligomer 12


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specific A11 and fibril specific OC antibodies to test the amount of oligomer and fibril formation in young (day 10) and old (day 30) flies. A11 and OC antibodies have been traditionally used in several in vitro and in vivo studies to check the relative amount of oligomers and fibrils 32, 45. We found that oligomeric content was higher in young flies expressing WT and mutants as compared to old flies. Oligomer staining was not observed in elav/+ control flies indicating α-Syn could be the primary source of oligomer formation in α-Syn expressing transgenic flies. Interestingly, even after conversion of oligomers into fibrils as seen by OC staining in old flies, high oligomeric content was also observed in the flies expressing α-Syn mutants. This suggests the sustained formation of oligomers in case of disease-associated α-Syn mutants, which was not the case with flies expressing WT α-Syn (Figure 5). We believe that sustained population of oligomers (irrespective of the aggregation kinetics of mutants) would be responsible for dopaminergic cell death observed in familial PD. Various mechanisms have been proposed for cell death caused by oligomers; including pore formation, impairment in the functionality of mitochondria, endoplasmic reticulum and proteasomal degradation machinery in the cells46. Besides the cytotoxic effect of α-Syn, it is known to play an important role in vesicle recycling and proper functionality of SNARE complexes at synaptic terminal47. Membrane association of α-Syn is essential for its function in vesicle cycling and SNARE48. Mutations are known to affect membrane binding of α-Syn49-51; familial mutations might be perturbing the regular function of α-Syn, which could also be one of the contributing factors for mutation-induced neurotoxicity in PD. Although this study clearly supports that sustained population of oligomers could be common for fast and slow aggregating mutant, but the mechanism of toxicity caused by these oligomers still needs to be further explored. Discovery of familial α-Syn mutations suggested the importance of single point mutation associated with PD. Investigating the change in sequence based conformational propensities of α-Syn and its rate of aggregation can provide insights into the mechanistic basis for abnormal folding associated with PD. We monitored the aggregation of WT and mutant α-Syn and checked the A11 positive oligomer formation during in vitro fibrilization. All the proteins showed typical amyloid formation after incubation as revealed by high ThT fluorescence signal, β-sheet structural conversion in CD and fibrillar morphology observed by under TEM. Furthermore, dotblot analysis using A11 antibody suggested that familial mutants retain more oligomers 13



compared to WT at the end of aggregation. Thus, based on in vivo and in vitro data, it can be concluded that sustained formation of oligomeric species by disease-associated familial mutants of α-Syn may be responsible for enhanced behavioral defects in transgenic Drosophila.

Methods Chemicals and Reagents Most of the chemicals were purchased from Himedia and Merck unless stated. NotI, XbaI and rapid ligation kit were purchased from Fermentas (Vilnius, Lithuania). Antibodies used in this study were following: human α-Syn specific LB509 (Invitrogen, USA), tyrosine hydroxylase (TH) (Novus Biologicals, Littleton, Colorado, USA) and β-tubulin (Sigma). Fluorophore labeled secondary antibodies were purchased from Invitrogen. Fibril Specific OC and oligomer specific A11 antibodies were a kind gift from Prof Charle G. Glabe, University of California, Irvine. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and Triton X-100 were procured from Sigma, USA. Water was double distilled and deionized using a Milli-Q system (Heal Force, Shanghai, China). Protein expression and purification WT, E46K, H50Q and G51D α-Syn were expressed in E. coli BL21 (DE3) strain and purified according to previous method52 with slight modification53. Briefly, IPTG induced bacterial cells were pelleted down by centrifugation at 4000 ×g, 4°C for 30 min. It was resuspended in lysis buffer (50 mM Tris, 10 mM EDTA, 150 mM NaCl, pH 8.0). Protease inhibitor cocktail (Roche), was added to prevent any proteolytic cleavage. It was then sonicated using probe sonicator (Sonics & Materials INC, USA) at 40% amplitude and 45 pulse/min for 10 min, followed by heating at 95°C in water bath for 20 min. It was centrifuged at 10000 ×g for 30 min at 4°C and the supernatant was collected. To the supernatant, 10% streptomycin sulphate (136 μl/ml of supernatant) and glacial acetic acid (228 μl/ml of supernatant) were added to precipitate DNA. The resulting solution was centrifuged at 12000 ×g for 30 min at 4°C and to the supernatant, equal volume of saturated ammonium sulphate (prepared at 4°C) was added. The solution was kept at 4°C for 1 hr for complete precipitation of α-Syn protein and then centrifuged at 12000 ×g for 30 min at 4°C. The pellet was resuspended in 50% ammonium sulfate and centrifuged again 14


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at 12000 ×g for 30 min at 4°C. Finally, the pellet obtained was resuspended in 100 mM ammonium acetate (10 ml/lit growth of culture) followed by adding an equal volume of ethanol for protein precipitation. This step was repeated three times. The solution was centrifuged and redissolved in a minimum volume of ammonium acetate, lyophilized and stored at -20°C until further use. Preparation of α-Syn low molecular weight (LMW) and amyloid fibrils formation Solid lyophilized protein was suspended in 20 mM Gly-NaOH, pH 7.4, 0.01% sodium azide. As the protein was partially soluble, a few drops of 2 N NaOH was added, until the protein solution became clear and the pH was adjusted to 7.4 by adding few drops of 2 M HCl. Final pH of the resulting solution was confirmed by micro pH meter (Model S20 Seven easy, Mettler-Toledo, Switzerland). The solution was then dialyzed for overnight using 10 kDa MWCO mini-dialysis units (Millipore) against the same buffer at 4°C. The low molecular weight (LMW) form of the protein was isolated using centricon YM-100 filter (100 kDa MWCO, Millipore) according to previously described method

54

. Previous studies have shown that LMW preparation of α-Syn

mostly contains monomers along with some amount of low-order multimers. The final concentration of LMW was adjusted to 300 µM. The aggregation study was set up in 20 mM Gly-NaOH, pH 7.4 containing 0.01% sodium azide at 37°C with slight agitation (~ 50 rpm). Aggregation of protein was monitored by ThT fluorescence and secondary structural changes were studied by circular dichroism (CD) spectroscopy. Amyloid formation was confirmed by transmission electron microscopy (TEM). Three independent experiments were performed for each sample. Circular dichroism spectroscopy (CD) CD spectroscopy was performed with 15 µM protein concentration in 20 mM Gly-NaOH buffer, pH 7.4 in a 0.1 cm path-length quartz cell (Hellma, Forest Hills, NY). Spectra were acquired using JASCO-1500 instrument (USA) over the wavelength range of 200-260 nm. Parameters used for acquiring spectra were data interval l.0 nm, bandwidth 1.0 nm, scanning speed 100 nm/min and accumulations 3. Three independent experiments were performed with each sample. Raw data were processed by smoothing and subtraction of buffer spectra, according to the manufacturer’s instructions. All measurements were done at 25°C. 15



Thioflavin T (ThT) fluorescence assay 1 mM ThT was prepared in Tris-HCl buffer, pH 8.0, 0.01% sodium azide. 2 µl of 1 mM ThT solution was added to the 10 µM protein solution in 200 μl Gly-NaOH buffer, pH 7.4, 0.01% sodium azide. ThT fluorescence assay was performed using Horiba-Jobin Yvon Fluomax4 (Kyoto, Japan) instrument with excitation at 450 nm and emission in the range of 460-500 nm at excitation and emission slit width of 5 nm. ThT fluorescence thus obtained at 480 nm was plotted for all proteins at the beginning and at the end of the aggregation. The fluorescence data described in this study were obtained from three different experiments. Transmission Electron Microscopy The end products of aggregation were diluted in distilled water to a final concentration of ~ 40 μM and spotted on a carbon/formvar-coated copper grid (Electron Microscopy Sciences, Fort Washington, PA). The sample was decanted gently on a filter paper after 5 min incubation, and the grid was washed with water. After this, negative staining was done wherein 1% (w/v) aqueous uranyl formate solution was applied to the grid and incubated for 2 min. The stain was then gently removed by filter paper. The grids were air dried for 5 min and then subjected to transmission electron microscopy imaging. Electron microscopy of the samples was carried out using a PHILIPS CM200 (Amsterdam, Netherlands) instrument at 200 kV with 6600X magnification. Randomly 10 -12 images were taken for each sample. Construction of pUAST-Syn vectors E46K, H50Q and G51D α-Syn mutations were created in pRK172 α-Syn vector with sitedirected mutagenesis. Mutations were confirmed by sequencing the plasmids. Familial α-Syn gene carrying the mutation was amplified with primers having NotI and XbaI restriction enzymes sites in it. Primer sequences used for PCR were as follows: UAS NotI Syn FP 5’TACTAATGCGGCCGCATGGATGTATTCATGAAAG-3’ and UAS XbaI Syn RP: 5’TTCAGAGTTCTAGATTAGGCTTCAGGTTCGTAGTC-3’. PCR product and empty pUAST vector were digested with fast digest NotI and XbaI restriction enzymes. Cut vector and insert were ligated using rapid ligation kit (Fermentas, Thermo scientific). Colony PCR and insert release assay were performed for confirmation of α-Syn insert. Finally, mutations were 16


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confirmed by plasmid DNA sequencing. Obtained sequence was checked by nucleotide sequence blast and sequence-based translation in ExPASy translate tool (Figure 1A and S1). Generation of transgenic Drosophila All the sequenced plasmid of familial α-Syn mutants were further amplified by doing maxiprep as per protocol (Quiagen). Plasmid DNA (40 µg total for each plasmid) samples were sent to cCAMP, NCBS, Bangalore, for generation of transgenic Drosophila. Transgenic Drosophila having familial α-Syn mutant insertion balanced with appropriate balancer (Cyo) were generated and provided by c-CAMP, NCBS, Bangalore. Drosophila husbandry and maintenance of flies All the flies were fed and propagated on corn-sugar yeast agar media. To avoid infection to flies, antifungal agents propionic acid (4ml/lit), orthophosphoric acid (0.6ml/lit and methyl parabenzoate (0.7gm/lit) were added to media. Apart from familial α-Syn mutant flies, control flies used in experiments were purchased from Bloomington, USA. All the flies and their crosses were maintained at 25°C with 12 h light/dark (LD) cycle. Fly stocks used in the study were elavGal4, UAS-α-Syn WT (BL 8146) and Canton-S (CS). All the fly stocks were transferred every four days to new vials containing media. Pan-neuronal α-Syn expression in Drosophila In the Drosophila model system, ectopic gene expression of any particular cloned gene is achieved by using the UAS-Gal4 system. Gal4 transcription factor binds to the upstream activating sequence (UAS) and mediates expression of the gene of interest downstream to UAS. In our experiments, the cross between pUAS α-Syn (WT, E46K, H50Q and G51D) andbrain specific elav-Gal4 flies was set up so that in F1 progeny α-Syn will be produced throughout the brain. F1 progeny of mentioned crosses were used for all the fly-based experiments. Negative control flies (elav/+) express elav-Gal4 (with one copy of elav-Gal4) but do not express the α-Syn. The F1 progeny of negative control flies (elav/+) were obtained by crossing elav-Gal4 homozygous virgin female with CS male flies (CS flies do not have α-Syn gene in the genome).

17



Western blot analysis F1 progeny generated from the cross of elav-Gal4 with only CS and UAS α-Syn (WT, E46K, H50Q and G51D) were used in the experiment. Day 10 and 30 age-matched flies were collected and snap frozen in liquid nitrogen and stored at -80°C till further use. Heads of 10 flies of each genotype were crushed in SDS-Triton X-100 buffer, centrifuged and the supernatant was used for Western blot analysis. Samples were resolved on 12% SDS-PAGE and transferred to nitrocellulose membrane, followed by treatment with 0.4% paraformaldehyde (PFA) solution in PBS for 50 min at room temperature (RT). The membrane was blocked with 5% blotto (non-fat milk powder) for 1 hr at RT. Primary antibody was added to the membrane (LB509- 1:1000) and incubated at 4°C for overnight. The membrane was then incubated with the appropriate secondary antibody (1:4000) for 2 hrs at RT. The membrane was exposed to the chemiluminescent substrate (West Femto, Pierce, Waltham, Massachusetts, United States) and the signal was captured on X-ray films. The same membrane was then stripped and re-developed for β-tubulin (1:2000). Images were processed with Image J software for quantitative analysis. Normalized value for α-Syn was obtained by dividing intensity of α-Syn by intensity of β-tubulin for the same lane. Immunostaining of dopaminergic neurons in Drosophila F1 progeny generated from the cross of elav-Gal4 with only CS and UAS α-Syn (WT, E46K, H50Q and G51D) were used in the experiment. Flies of day 10, 20 and 30 were used for this experiment. Drosophila brains of mentioned genotypes and of different age were dissected in PBS by observing under Leica EZ24 stereomicroscope (Leica, Germany). After dissection, brains were fixed in ice-cold 4% PFA for 20 min. Brains were then washed in PBS for 20 min (10 min for each wash). Permeabilization was performed with 1% Triton X-100 made in PBS with Tween (0.01%)(PBST) for 20 min. Blocking was done with blocking solution ( PBST+5% BSA +5% NGS) for 30 min. Dopaminergic neuron-specific marker tyrosine hydroxylase (TH) antibody (Novus Biologicals, Littleton, Colorado, USA) diluted at 1:500 dilution in blocking solution was used to probe the dopaminergic cell death. To do that diluted TH antibody was added to processed brain and samples were kept at 4°C for overnight with slow rocking. Brains were then washed with PBST for 3 times, each of 10 min. Samples were then incubated with secondary antibody (anti-rabbit Alexa 488) for 2 hrs. Further, samples were washed for 10 min 18


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with PBST for 3 times. Samples were mounted and imaged using confocal microscope. Day 10 images were acquired in Olympus FV-500 (Shinjuku, Tokyo, Japan) while Day 30 images were captured in Zeiss Observer Z1 (Oberkochen, Germany). TH positive DA neurons were counted from all the clusters. Minimum six brain sections were analyzed for each genotype. Statistical analysis was performed to compare the differences among all the genotypes. Climbing assay Flies expressing α-Syn WT and other familial mutations were assessed for their ability to climb by using a climbing assay as per established protocol 28. F1 progeny generated from the cross of elav-Gal4 with only CS and UAS α-Syn (WT, E46K, H50Q and G51D) were used in the experiment. Age-matched flies of day 10, 20, 30 and 40 were used for this experiment. All flies under experiment were maintained at 25°C and they were transferred regularly to the new vials. In the climbing assay, 25 flies were allowed to climb upward in a falcon tube (negative geotaxis) and to encourage the climbing, banging of falcons was performed. Video shooting was performed during the climbing assay. Each video was later analyzed for the climbing ability for each genotype. At a given instance, maximum five recording of climbing was recorded. For analyzing data, the height of 3.5 cm was arbitrarily set up as a threshold for climbing. Flies climbed above this threshold in a fixed interval of time (8s) were counted. The percentage of climbing ability represents a number of flies climbed above threshold in a given time interval from the total number of flies examined. In each set, minimum 100 flies were analyzed for each genotype. The experiment was repeated thrice with new F1 generation flies obtained from new parents. Statistical analysis was performed to compare difference among all the genotypes. Survival assay For survival assays, flies were maintained at 25 °C under a 12 h light/dark cycle. 10 adult females of the same age were placed in 1 vials containing fresh food. After every 2 days, the flies were transferred into new vials with fresh food and the number of living and dead flies was registered. In each set of survival assay, minimum 100 flies of F1 generation for each genotype were accessed for survival. The assay was repeated three times. Data is plotted as % survival, which indicates the number of flies survived at a given time point out of a total number of flies

19



used for the study at day 1. Statistical analysis was performed to compare difference among all the genotypes. Dot blot analysis Age-matched flies of day 10 and 30 were used for this experiment. Equal number of flies from the F1 generation of each genotype was used to make brain homogenate. Equal amount of whole cell lysate protein was spotted on the nitrocellulose membrane (Immobilon-NC, Millipore) and allowed to air-dry for 10 min. Two washes (2 × 8 min) were performed with PBST and the membrane was blocked with 5% nonfat milk powder (Himedia, Mumbai, India) in PBST for 1 hr at RT. The blots were incubated with fibril specific OC (1:200 dilutions) and oligomer specific A11 antibody (1:200) for overnight at 4°C followed by three washes of PBST. The membrane was then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000 dilutions, cat. no. 401253, Calbiochem). Three washes were performed with Tris-base saline with 0.01% Tween (TBST) and then blot was developed after it was exposed to chemiluminescent substrate (Super Signal West Pico, Pierce). Densitometry analysis was performed for dot blots. Values obtained were normalized with the respect to signal of WT αSyn expressing flies. Statistical analysis was performed to compare difference among all the genotypes. To analyze the oligomer formation during the in vitro aggregation of α-Syn, 30 μL of sample was taken from each aggregation mixture at regular intervals. Samples were then subjected to ultra-centrifugation at 35,000 rpm for 30 min.

2 μL of the supernatant obtained after

centrifugation were spotted on nitrocellulose membrane. Dot blots then were probed with A11 antibody as discussed above and developed with the ECL substrate. Statistical analysis Statistical significance is calculated by one-way ANOVA followed by Student-Newman-Keuls Multiple Comparison post hoc test, *P

Parkinson's disease associated Î±-synuclein familial mutants promote

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