Analysis of α-syn and parkin interaction in mediating neuronal death in Drosophila model of Parkinson's disease

One of the hallmarks of Parkinson's Disease (PD) is aggregation of incorrectly folded α-synuclein (SNCA) protein resulting in selective death of dopaminergic neurons. Another form of PD is characterized by the loss-of-function of an E3-ubiquitin ligase, parkin. Mutations in SNCA and parkin result in impaired mitochondrial morphology, causing loss of dopaminergic neurons. Despite extensive research on the individual effects of SNCA and parkin, their interactions in dopaminergic neurons remain understudied. Here we employ Drosophila model to study the effect of collective overexpression of SNCA along with the downregulation of parkin in the dopaminergic neurons of the posterior brain. We found that overexpression of SNCA along with downregulation of parkin causes a reduction in the number of dopaminergic neuronal clusters in the posterior region of the adult brain, which is manifested as progressive locomotor dysfunction. Overexpression of SNCA and downregulation of parkin collectively results in altered mitochondrial morphology in a cluster-specific manner, only in a subset of dopaminergic neurons of the brain. Further, we found that SNCA overexpression causes transcriptional downregulation of parkin. However, this downregulation is not further enhanced upon collective SNCA overexpression and parkin downregulation. This suggests that the interactions of SNCA and parkin may not be additive. Our study thus provides insights into a potential link between α-synuclein and parkin interactions. These interactions result in altered mitochondrial morphology in a cluster-specific manner for dopaminergic neurons over a time, thus unraveling the molecular interactions involved in the etiology of Parkinson's Disease.


Introduction
Parkinson's Disease (PD), first characterized by James Parkinson in 1817 (Jost and Reichmann, 2017), is the second most common persistent movement neurodegenerative disorder, after Alzheimer's disease, with no cure to date.PD affects 0.8% of the worldwide population among other neurological disorders (Ray Dorsey et al., 2018).It is characterized by the specific loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) region of the midbrain which leads to motor symptoms including bradykinesia, muscular rigidity, resting tremor, postural instability (Wood-Kaczmar et al., 2006;Chartier and Duyckaerts, 2018).Progressive loss of DA neurons and the presence of intra-neuronal Lewy bodies (mainly aggregation of α-synuclein protein) are majorly considered neuropathological features of PD (Chartier and Duyckaerts, 2018;Shahmoradian et al., 2019).
SNCA and parkin are the two major genes involved in both sporadic as well as genetic forms of PD (Corti et al., 2011).SNCA encodes an α-synuclein protein and mutations in SNCA are associated with the Autosomal-Dominant form of PD (Polymeropoulos et al., 1997;Kachergus et al., 2004;Appel-Cresswell et al., 2013).Different in-vitro and in-vivo studies have shown that misfolded α-synuclein aggregation causes neurotoxicity by influencing the neurotransmission, synaptic vesicle exocytosis, recycling as well as endocytosis in the substantia nigra region.Migration of α-synuclein between neurons in a prion-like manner to propagate the formation of Lewy bodies throughout the substantia nigra has also been suggested (Li et al., 2008;Burré et al., 2010;Olanow and Brundin, 2013).Parkin encodes an E3 ubiquitin ligase and is the second most common cause of autosomal recessive early-onset PD (Kitada et al., 1998).Parkin loss-offunction causes neurodegeneration with or without forming Lewy bodies in PD patients (Yokochi, 1997;Pramstaller et al., 2005;Johansen et al., 2018).Studies have reported that overexpression of parkin results in reduced neurotoxicity caused by α-synuclein in different models (Oluwatosin-Chigbu et al., 2003;Haywood and Staveley, 2004;Khandelwal et al., 2010).Studies have also reported that parkin mutation results in no aggregation of α-synuclein in mice (Goldberg et al., 2003;Van Rompuy et al., 2015).However, very limited in-vivo studies have been done to explore the link between SNCA and parkin.
At the cellular level, mitochondria dysfunction has been considered a major hallmark of PD (Nicoletti et al., 2021).Studies in different model systems have shown that α-synuclein causes mitochondria fragmentation, disturbed membrane potential, complex I deficits, and reduced ATP production.In Drosophila, elongated as well as fragmented mitochondria have been reported due to α-synuclein overexpression (Nakamura, 2013;Ordonez et al., 2018;Krzystek et al., 2021).Loss-of-function mutation in parkin has also shown the mitochondrial pathology demonstrated as mitochondrial elongation, swelling, and cristae disruption in in-vitro and in-vivo models (Greene et al., 2003;Pesah et al., 2004;Deng et al., 2008;Yu et al., 2011;Noda et al., 2020).However, fused and fragmented mitochondria have also been reported in DA neurons of parkin mutant Drosophila (Cackovic et al., 2018).Moreover, it has been found that mitochondrial fragmentation caused by α-synuclein overexpression can be rescued by coexpression of parkin, PINK1, or DJ-1, indicating that α-synuclein and parkin may function in the same pathway (Kamp et al., 2010;reviewed in Jeśko et al., 2019).However, limited in-vivo studies have been done to test the effect of the interaction of SNCA and parkin on mitochondrial morphology in Parkinson's disease.
In this report, we have tested the interaction of parkin and SNCA and their effect on mitochondria in DA neurons using a humanized Drosophila melanogaster model of PD.Drosophila provides a simple, yet powerful in-vivo system to study neurodegenerative diseases including Parkinson's disease.It has a compact genome size (1/30th of the human genome), limited genetic redundancy, and a shorter generation time (12-15 days) and life span (60-80 days) (Bier, 2005).Almost 75% of all human disease genes are well conserved in the Drosophila genome sequence (Dawson et al., 2010;Aryal and Lee, 2019).Although Drosophila does not have an SNCA homolog, it mimics major PD symptoms i.e., locomotor dysfunction, age-dependent DA neuronal loss, and Lewy bodies aggregation upon overexpression of human SNCA using the GAL4/UAS system (Feany and Bender, 2000).Drosophila adult fly brain contains approximately 100 DA neurons, which are grouped into different clusters according to their anatomical position: PAL (paired anterior lateral), PAM (paired anterior medial), PPM1/2 and PPM3 (paired posterior medial), and PPL1 and PPL2 (paired posterior lateral) (Monastirioti, 1999;Mao and Davis, 2009).It has been suggested that each clusterspecific DA neuron projects to distinct functional areas of the brain (White et al., 2010), although the function of each of the DA clusters is not completely explored.PPL1 and PPM3 are more explored clusters in the case of PD using animal models because these are functionally homologous to the mammalian substantia nigra pars compacta region (Strausfeld and Hirth, 2013).It has been shown that PPL1 is associated largely with memory formation (Heisenberg, 2003) whereas, PPM3 is reported to be a center for the control of locomotor behavior (Strauss, 2002).In PD models of Drosophila, different clusters have been reported to be affected in a context-dependent manner.
In this study, we have used GAL4/UAS system specific for DA neurons to understand DA neuronal loss because of accumulation of α-synuclein as well as downregulation of parkin in a timedependent manner in adult fly brain, mimicking the PD in humans.We found DA neuronal loss upon genetic alterations is clusterspecific, exhibiting altered mitochondrial morphology.Our studies have implications in providing leads for cellular and molecular interactions on DA neuronal degeneration during the onset and progression of PD.

Climbing assays
To determine locomotor activity, climbing assays were performed (Pendleton et al., 2002).Ten flies per genotype were transferred into a cylindrical glass tube after anesthetization and left for 5-10 min for revival and acclimatization at room temperature.Tubes were marked up to 8 cm above the bottom of the vial.After acclimatization, flies were gently tapped down to the bottom of the vial and the number of flies that crossed the 8 cm mark was recorded after 10 s.Three trials were performed, and numbers were then averaged, and the resulting mean was used as the overall value for each single group of flies.For all genotypes, three replicates were carried out.

Quantitative real-time PCR
Total RNA was extracted from 25 to 30 fly heads using the TRIzol method (Invitrogen) specified in the Jove protocol (URL: https://www.jove.com/video/50245)(Mehta et al., 2019).RNA concentrations were measured with a Nanodrop ND-1000 Spectrophotometer and equal amounts of RNA were reverse transcribed using Verso cDNA Synthesis Kit (AB1453A).qPCR was performed using 5x HOT FIREPol R EvaGreen R qPCR Mix Plus (Solis BioDyne).RP49 was used as the internal control gene.Primer sequences were as follows:

Adult brain immunohistochemistry
Brain preparation for confocal microscopy imaging was done as described by Tito et al. (2016).Briefly, Adult brains of the desired genotype were dissected in cold 1X PBS and incubated with fixative solution (4% formaldehyde) in 1XPBST (0.1%TritonX-100) for 20 min at room temperature.After three washes with 1XPBST for 10 min each wash, blocking was done using 1% BSA for 1hr at room temperature.Brains were probed with rabbit anti-TH (#AB152) at 1:1,000, overnight (12-16) at 4 • C. Following three washes for 10 min, brains were incubated with Goat anti-Rabbit, Alexa-Fluor Plus 555 (#A32732) (1:4,000) secondary antibodies at room temperature for 2 h.Brains were washed three times for 15 min, then they were mounted between two glass coverslips by using an antifade medium on microscope slides.A confocal microscope was used to acquire z-stacks at 1 µm intervals with 20×/N.A.0.60 Plan-Apochromat objective.The number of THpositive neurons was counted manually within each cluster of posterior regions of the brains.

Mitochondrial morphology measurement
To assess the mitochondrial morphology in DA neurons, the UAS-MitoGFP fly strain was used to tag the mitochondria, and DA neurons were stained using anti TH antibody.Z-stack of one PPL1 and one PPM3 DA neuronal cluster per brain was imaged using confocal microscope at 63×/N.A.1.30oil with 1.5 zoom.A total of four brains per genotype were scanned.We used publicly available ImageJ Mito-Morphology Macro created by Dagda et al. (2009), to quantify the mitochondria.Average area and circularity were calculated representing elongation and fragmentation of mitochondria respectively.

Statistical analysis
GraphPad Prism 8.0.1 was used for statistical analysis and graphical display of the data.Significance is expressed as p values which were determined with one-way ANOVA and Two-way ANOVA followed by Tukey's multiple comparison tests as indicated in the figure legends.
Each result is representative of at least three biological and three technical repeats.
7-day and 21-day respectively).We observed that flies that expressed SNCA with parkin knockdown (UAS-parkin IR ; UAS-SNCA) displayed loss of climbing ability as compared to control (Figure 1); however, this was to a lesser extent with respect to parkin downregulation alone in age an age-dependent manner.Though, both SNCA overexpression and parkin downregulation independently displayed a loss of climbing ability with age (3-21 days) as compared to control (TH>GFP) (Figure 1), supporting the existing studies (Feany and Bender, 2000;Wang et al., 2007;Paricio and Muñoz-Soriano, 2011;Zhang et al., 2013;Mohite et al., 2018).We also confirmed the phenotype of parkin downregulation by overexpressing wild type-parkin with SNCA (UAS-SNCA;UASparkin). Notably, wild-type parkin overexpression with SNCA was able to restore the climbing ability (Supplementary Figure 2).We also confirmed the overexpression of α-synuclein using H3C antibody in UAS-SNCA; UAS-parkin transgene through fluorescence microscopy (Supplementary Figure 1C).These observations suggest that SNCA and parkin alteration together do not worsen the locomotor defects.

SNCA and parkin alterations cause dopaminergic neurodegeneration
In PD, cardinal motor symptoms are caused by the death of DA neurons in the substantia nigra pars compacta (SNpc).Thus, we examined the DA neuronal clusters in adult fly brains using antibodies against tyrosine hydroxylase to investigate whether locomotor defects observed in SNCA and parkin alteration are due to loss of DA neurons.We have explored posterior protocerebrum DA neuronal clusters: PPM1/2, PPM3, PPL1, and PPL2 (Figure 2A).To visualize the DA neurons in the adult brain, we stained the DA neurons of the TH-Gal4 driving GFP flies with TH antibody (Figure 2B), hence confirming the activity of TH-Gal4>UAS-GFP and TH antibody.In our study, only TH-positive neurons were monitored for DA neuron quantification.We have found a substantial reduction in specific DA neuronal clusters in UAS-SNCA, UAS-parkin IR individually, and the UAS-parkin IR ; UAS-SNCA transgene adult brain with respect to age-matched controls (Figure 2).In 7-day-old adult fly brains, quantification of PPL1, PPM1&2, PPM3, and PPL2 DA neuron clusters, SNCA, and parkin IR showed significant reduction in number of DA neurons only in PPL1 and PPM1&2 clusters (Figures 2C-E', G).UASparkin IR ; UAS-SNCA also showed a significant reduction in the number of DA neurons in PPL1 and PPM1&2 as compared to control (Figures 2F, F', G).However, the number of DA neuronal losses in UAS-parkin IR ; UAS-SNCA adult fly brains were less pronounced as compared to SNCA and parkin IR alone (Figures 2D-F', G).In 21-day-old fly brains, DA neurons numbers were further decreased in PPL1 and PPM1 & 2 clusters in a similar manner to 7-day-old adult fly brains of SNCA, parkin IR, and in UAS-parkin IR ; UAS-SNCA (Figures 2H-K', L).In PPM3 DA neuron clusters, we have observed that the numbers of DA neurons were reduced only in parkin IR adult fly brains (Figures 2G, L).In the PPL2 DA cluster, we have observed no change in the number of DA neurons in UAS-SNCA, UAS-parkin IR , and UAS-parkin IR ; UAS-SNCA of 7-day-old and 21-day-old adult fly brains.These observations suggest that SNCA and parkin cause DA clusters specific neuronal loss in the posterior region of adult fly brain.

SNCA a ects the expression of parkin at the transcriptional level but not at the translational level
Several in-vitro and in-vivo studies have shown the reduced neurotoxicity caused by SNCA upon parkin overexpression and suggested that parkin plays a vital role in the molecular pathway of PD pathogenesis.It has been reported that mutations in SNCA and parkin affect DA neuronal loss as well as the formation of Lewy bodies (Madsen et al., 2021).However, the effect of wildtype SNCA on parkin is not much explored.Therefore, we tested the parkin mRNA and protein levels of flies with UAS-parkin IR ; UAS-SNCA, SNCA overexpression, and parkin downregulation ), ** ( .), ns-not significant ( .).
independently.We found decreased parkin mRNA levels in UASparkin IR ; UAS-SNCA flies as compared to control flies; however, this decreased transcript was at a lesser extent with respect to parkin downregulation and SNCA overexpression independently in 7-day adult fly brains (Figure 3A).In 21-day-old adult fly brains with UAS-parkin IR ; UAS-SNCA has not shown a further decrease in parkin mRNA level as compared to control and as well as with SNCA overexpression and parkin downregulation independently (Figure 3B).Wilkaniec et al., 2019 have reported a decreased parkin protein level upon α-synuclein oligomerization which induces cell death in an in-vitro model.In contrast, in our study, we have found no significant change in parkin protein level upon SNCA overexpression in 7 (Figures 3C, E) and 21-day-old (Figures 3D, F) adult fly brains.However, we observed decreased parkin protein levels in flies expressing UAS-parkin IR ; UAS-SNCA as compared to control but it was to a lesser extent to parkin downregulation independently (Figures 3E, F).Altogether, these data suggest that SNCA affects the parkin level.

SNCA and parkin alteration a ect mitochondrial morphology independently in PPL and PPM clusters of the adult fly brain
Mitochondria are highly dynamic organelles and maintenance of mitochondrial morphology is essential for the survival of the neurons.Therefore, we investigated whether α-synuclein and parkin alteration-induced PD phenotypes have any relation ), ns-not significant ( .).
Frontiers in Cellular Neuroscience frontiersin.orgwith mitochondrial morphology.We have considered only PPL1 and PPM3 DA neuronal clusters for mitochondrial morphology assessment.This is because we have found a decrease in the number of TH-positive DA neurons in PPL1 clusters of UASparkin IR ; UAS-SNCA (Figure 2), SNCA overexpression, and parkin downregulation independently and in PPM3 due to parkin downregulation only.We assessed the mitochondrial morphology using UAS-mitoGFP in TH-positive neurons.We have found that in PPL1 neuronal clusters, UAS-parkin IR ; UAS-SNCA (Figures 4D, D', E; Supplementary Figure 4D) has shown swollen mitochondria as compared to control (TH>mito-GFP) (Figures 4A, A'; Supplementary Figure 4A) in 7-day adult fly brains.Whereas we observed fragmented mitochondria in 21-day-old adult fly brains (Figures 4I, I', J; Supplementary Figure 4H) as compared to control (Figures 4F, F', J; Supplementary Figure 4E).SNCA overexpression (Figures 4B,  B', E; Supplementary Figure 4B) and parkin downregulation (Figures 4C, C', E; Supplementary Figure 4C) independently have shown swollen and/or enlarged mitochondria, which degenerate, in 7-day-old adult fly brains, as compared to control (Figures 4A, A',  E; Supplementary Figure 4A).SNCA overexpression (Figures 4G,  G', J; Supplementary Figure 4F) and parkin downregulation (Figures 4H, H', J; Supplementary Figure 4G) independently have shown further enhanced mitochondrial morphology in 21-day-old adult fly brains as compared to control (Figures 4F, F', J; Supplementary Figure 4E).In PPM3 neuronal clusters, UAS-parkin IR ; UAS-SNCA (Figures 5D, D', E) and SNCA overexpression (Figures 5B, B',  E) have shown fragmented mitochondria in 7-day-old adult fly brains as compared to control (Figures 5A, A', E).In 21-dayold adult brains of UAS-parkin IR ; UAS-SNCA (Figures 5I, I', J) and SNCA overexpression (Figures 5G, G', J) have also shown fragmented mitochondria as compared to control (Figures 5F, F',  J) which do not degenerate.The parkin downregulation only has shown enlarged and/or swollen mitochondria in PPM3 clusters which degenerate, in 7-day (Figures 5C, C', 5E) as well as in 21-day old adult fly brains (Figures 5H, H', J) as compared to control (Figures 5A, A', E).To confirm the role of parkin in SNCA-induced mitochondrial morphology defects, we performed mitochondrial fractionation.We observed non-statistically different reductions in mitochondrial localization of parkin protein (Supplementary Figure 3).Hence, these data confirm the role of parkin being independent of α-synuclein to cause altered mitochondrial morphology in PD progression.

Discussion
Here, we characterize the cluster-specific DA neuronal loss associated with the interaction of SNCA and parkin.We have highlighted the effect of SNCA overexpression and parkin knockdown together in terms of DA neuronal loss in PPL1 and PPM3 clusters of adult fly brains over a time period of 21 days.
SNCA and parkin mutations have been found to be involved in motor (postural instability, tremor, bradykinesia) and non-motor symptoms (sleep disorders, depression, anxiety, hallucinations) in PD patients.SNCA overexpression and parkin mutation have also been shown to cause age-dependent locomotor dysfunction and neurodegeneration in in-vivo models (Feany and Bender, 2000;Wang et al., 2007;Ordonez et al., 2018;Yan et al., 2019).In our study, SNCA overexpression and parkin knockdown in DA neurons have also shown progressive locomotor dysfunction.Interaction of SNCA and parkin in PPL1 clusters results in progressive locomotor dysfunctions.However, the effect of genetic alterations is not as pronounced to indicate an additive effect.This is further supported by observed mitochondrial morphology in UAS-parkin IR ; UAS-SNCA.With respect to the reports published previously, our work uncovers the relationship between SNCA and parkin in an in-vivo system in a novel way by bringing the gene alterations together.This enables us to understand the relationship, between SNCA and parkin at both transcriptional and translational levels.
Studies have reported that wild-type SNCA causes a reduction in the number of TH-positive neurons in PPM1&2 and PPL1 clusters but not in PPM3 clusters (Trinh et al., 2008(Trinh et al., , 2010;;Barone et al., 2011;Agostini et al., 2023).We have also found that SNCA overexpression has reduced the number of TH-positive neurons in PPM1&2 and PPL1 and not in PPM3, which are consistent with aforementioned reports.Flies with parkin mutation have also been shown to cause progressive loss of DA neurons in the PPL1 cluster but not in PPM3 (Whitworth et al., 2005;Wang et al., 2007) after 20 days post-eclosion.No loss of neurons has been reported in the dorsomedial clusters (DMC) (also known as PPM) in parkin loss of function mutation in the adult fly brain after 21 days post-eclosion (Pesah et al., 2004).However, in our study, parkin knockdown alone shows loss of DA neurons in PPM3 along with PPM1&2 and PPL1 clusters in 7 and 21-day-old adult fly brains.SNCA with parkin knockdown (UAS-parkin IR ; UAS-SNCA) showed a decreased number of DA neurons in PPM1&2, PPL1 clusters as compared to control, though at a lesser extent with SNCA and parkin knockdown independently.Numbers of DA neurons in PPL2 clusters were unaltered in SNCA and parkin knockdown independently and together, in 7-day as well as in 21-day-old fly brains.Hence, these observations suggest that DA neuronal loss was correlated with locomotor dysfunctions.Since we did not observe aggravated phenotype in SNCA overexpression and parkin knockdown together (UAS-parkin IR ; UAS-SNCA); this may suggest that SNCA doesn't affect parkin directly.Alternatively, this could also mean that parkin downregulation is not the only mechanism involved in SNCA-induced locomotor dysfunction and neurodegeneration.
Neurons have highly dynamic energy requirements, and hence intact mitochondrial morphology is an important aspect to preserve neuronal health.In post-mortem brains of Parkinson's patients, it has been shown that α-synuclein localizes to mitochondria and affects mitochondrial homeostasis (Devi et al., 2008;Nakamura et al., 2011;Wang et al., 2019;Choi et al., 2022).Although, α-synuclein does not have an exact mitochondrial targeting sequence, studies suggest that α-synuclein contains a cryptic mitochondrial targeting sequence in the N-terminus region (Devi et al., 2008).Recently, it has been reported that N-terminus of α-synuclein plays a role in mitochondrial fragmentation via a DRP1-dependent pathway in Drosophila (Krzystek et al., 2021).In Drosophila, C. elegans, dorsal root ganglia of Danio rerio (zebra fish) and in cellular models as well, SNCA overexpression causes ), ns-not significant ( .).
Frontiers in Cellular Neuroscience frontiersin.orgmitochondrial fragmentation (Kamp et al., 2010;Butler et al., 2012;O'Donnell et al., 2014).In our study, SNCA overexpression has caused more elongated/or swollen mitochondria in PPL1 clusters (Supplementary Figures 4B, F), while in PPM3 clusters, it results in more fragmented mitochondria in a progressive manner.These results are thus align with the DA neuronal loss in PPL1 clusters but not in PPM3 clusters.This may also be an indication of some other mechanisms involved in rescuing the effect of SNCA overexpression and parkin knockdown together.Loss-of-function mutations in parkin are the most prevalent cause of recessive form PD (Corti et al., 2011).Upon mitochondria depolarization, parkin is activated by PINK1 and promotes degradation of Mitofusin 1 and 2 (Poole et al., 2008;Gegg et al., 2010;Sarraf et al., 2013) and recruits Drp1 to mitochondria which leads to fission (Buhlman et al., 2014).Parkin is also involved in the selective degradation of damaged mitochondria through the mitophagy process (Pickrell and Youle, 2015).In tissues of parkin-null Drosophila mutants, swollen mitochondria have been observed and this suggests that parkin may either promote fission or inhibit fusion (Greene et al., 2003;Pesah et al., 2004).Conversely, in DA neurons of parkin knockout mice, more fragmented mitochondria have been shown to cause neuronal loss (Noda et al., 2020).The presence of parkin mutation in causing accumulation of dysfunctional mitochondria in PD patients has also been established.In our study, parkin downregulation caused the enlargement of mitochondria in PPL1 (Supplementary Figures 4C,  G) and PPM3 clusters in age age-dependent manner.These results were correlating with the DA neuronal loss in PPL1 and PPM3 clusters.
Several studies have reported that overexpression of parkin restores mitochondrial morphology and function caused by SNCA, but it is still not clear whether this is through a direct link between parkin and SNCA, or the neuroprotective role of parkin in maintaining mitochondrial dynamics (Kamp et al., 2010;Lonskaya et al., 2013;Krzystek et al., 2021).In the in-vitro model, exogenous α-synuclein oligomers or fibrils caused a reduction in parkin expression and wild-type parkin overexpression rescues αsynuclein-induced mitochondrial fragmentation (Wilkaniec et al., 2021).However, they have shown that the toxic effects of αsynuclein on mitochondria were higher as compared to parkin silencing-induced mitochondrial dysfunction and suggested that α-synuclein-induced parkin downregulation is not the only mechanism for mitochondrial dysfunction (Wilkaniec et al., 2021).Similarly, in our study, overexpression of SNCA with parkin downregulation (UAS-parkin IR ; UAS-SNCA) shows more fragmented mitochondria in PPL1 (Supplementary Figure 4E) as well in PPM3 clusters in age-dependent manner, which is just opposite to SNCA overexpression and parkin downregulation individually.We have found no significant changes in parkin expression at the protein level in SNCA overexpressed flies; however, parkin transcript was significantly reduced.In our fractionation studies as well, we did not see statistically significant differences in the localization of Parkin protein.This warrants that further studies need to be carried out to validate the transcriptional correlation.Also, critical differences in gene expression related to mitochondrial morphology can be explored further.It is important to note that our observations of mitochondrial morphology in specific clusters pertain to the TH-positive neurons only, based on the limited number of trials performed.Thus, further comprehension of mitochondrial morphology within these particular clusters can be attained by exploring other aspects of mitochondrial homeostasis, such as mitochondrial transport and mitophagy, in the progression of Parkinson's Disease.
The current study provides insights into cellular and molecular etiology in the case of PD in a time-dependent manner specifically in DA neurons, using an overexpression system.Since neuronal mitochondria are highly dynamic, depending on ever-changing metabolic requirements, the morphology changes driving degeneration are limited to TH-positive neurons (Figure 6).Using cell isolation techniques from individual DA neuronal clusters of brains will be able to provide more insights at the individual neuronal and organelle level since the number of these neurons is limited.Furthermore, to understand the mechanisms involved in regulation at the organelle level, more research will be required in other animals to understand if these interactions are conserved at the cellular and molecular level affecting the pathogenesis of Parkinson's disease.In addition, it would be crucial to understand if mitochondria are affected by other direct or indirect genetic and molecular factors affecting the progression of Parkinson's disease.

FIGURE
FIGURESNCA overexpression and parkin downregulation (parkin IR ) independently, and together (parkin IR ; SNCA) in dopaminergic neurons (DA), exhibit locomotor dysfunctions: Climbing assay indicates loss of locomotor function with age in flies.A total of (N = ) flies were used per genotype and flies (n = ) were used for the climbing assay.Data is represented as mean with SEM.Statistical analysis was performed using Two-way ANOVA followed by Tukey's multiple comparison test.P value: * ( .), ** ( .), *** (< .),ns-not significant ( .).

FIGURE
FIGURE SNCA a ects the expression of parkin at the transcriptional level but not the translational level.(A) -day old parkin mRNA level (B) -day-old parkin mRNA level.(C) Parkin immunoblot and (E) quantification of -day-old adult brains normalized to beta-Tubulin.(D) Parkin immunoblot and (F) quantification of -day old adult brains normalized to beta-Tubulin.Data is represented as mean with SEM.Statistical analysis was performed using One-way ANOVA followed by Tukey's multiple comparison test.P value: *( .),** ( .), ns-not significant ( .).

FIGURE
FIGURE SNCA overexpression results in swollen mitochondria, parkin IR expression has shown elongated whereas together (parkin IR ; SNCA) shows fragmented mitochondria in PPL DA clusters.Adult brains of the desired genotype expressing the mitochondria-targeted green fluorescent protein (mitoGFP) in TH-TH-positive (red) cells.(A-J) -day & -day old adult fly brains showing mitoGFP in PPL cluster.Control brains showing mitoGFP at (A, A') -day and (F, F') -day.SNCA overexpressing flies show mitoGFP (B, B') in -day and in (G, G') -day.Parkin IR flies show mitoGFP in (C, C') -day and in (H, H') -day.Parkin IR ; SNCA expressing flies show mitoGFP in (D, D') -day and further enhanced in (I, I') -day.(E) Quantification of mitochondria morphology (area and circularity) was done using ImageJ Mito-Morphology Macro.Scale bar µm.A total of four adult brains were used (n = ) per genotype.Data is represented as mean with SEM.Statistical analysis was performed using Two-way ANOVA followed by Tukey's multiple comparison test.P value: *( .),ns-not significant ( .).

FIGURE
FIGURE SNCA overexpression and parkin IR ; SNCA have shown fragmented mitochondria, whereas parkin IR expression has shown elongated mitochondria in PPM DA clusters.Adult brains of the desired genotype expressing the mitochondria-targeted green fluorescent protein (mitoGFP) in TH-TH-positive (red) cells.(A-J) -day & -day old adult fly brains showing mitoGFP in PPM cluster.Control brains showing mitoGFP at (A, A') -day and (F, F') -day.SNCA overexpressing flies show mitoGFP (B, B') in -day and in (G, G') -day.Parkin IR expressing flies show mitoGFP in (C, C') -day and in (H, H') -day.Parkin IR ; SNCA expressing flies show mitoGFP in (D, D') -day and in (I, I') -day also.(E) Quantification of mitochondria morphology (area and circularity) was done using ImageJ Mito-Morphology Macro.Scale bar µm.A total of four adult brains were used (n = ) per genotype.Data is represented as mean with SEM.Statistical analysis was performed using Two-way ANOVA followed by Tukey's multiple comparison test.P value: *( .), **( .), ***(< .),ns-not significant ( .).

FIGURE
FIGURESchematic representation of the e ect of α-syn and parkin on specific DA neuronal clusters in the adult fly brain.