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Identification of an alternatively-spliced #-synuclein isoform that generates a 41-amino acid N-terminus truncated peptide, 41-syn: Role in dopamine homeostasis Ravali Vinnakota, DEEPTHI YEDLAPUDI, Krishna Madhuri Manda, Keerti Bhamidipati, Kirthi Theja Bommakanti, G Sree RangaLakshmi, and Shasi Vardhan Kalivendi ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00140 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

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ACS Chemical Neuroscience

Identification of an alternatively-spliced α-synuclein isoform that generates a 41-amino acid Nterminus truncated peptide, 41-syn: Role in dopamine homeostasis Ravali L Vinnakota#1, Deepthi Yedlapudi#2, Krishna Madhuri Manda3, Keerti Bhamidipati1, Kirthi Theja Bommakanti4, G Sree RangaLakshmi4 and Shasi V Kalivendi1* From The 1Centre for Chemical Biology and Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad 500 007, T.S, India and 4 Department of Neurology, Osmania General Hospital, Afzal Gunj, Hyderabad 500012, T.S., India. Running title: Identification of a 41aa isoform of α-synuclein # Both the authors contributed equally to the work * To whom correspondence should be addressed: Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology (Council of Scientific and Industrial Research), Uppal Road, Tarnaka, Hyderabad 500 007, T.S, India. Tel: 91-040-2719 1865; Fax: 91-040-2716 0387; Email: [email protected]

Abstract The presynaptic protein, α-synuclein (α-syn), has been shown to play a crucial role in multiple neurodegenerative diseases, such as, Parkinson’s (PD), Alzheimer’s (AD) and Dementia with Lewy bodies (DLB). The three major domains of α-syn protein were shown to govern its membrane interaction, protein fibrillation and chaperone activity. So far four different alternatively spliced isoforms of α-syn were identified which lack either exon 3 (syn-126) or 5 (syn-112) or both (syn-98) resulting in altered function of the proteins. In the present study, we have identified the smallest isoform of α-syn due to the skipping of exons 3 and 4 generating a 238bp transcript. Due to the presence of premature stop codon, the 238bp transcript generated a 41aa N-terminus peptide instead of 78aa protein which is secreted into the extracellular medium when overexpressed in cells. The presence of 41-syn was initially noticed in the substantia nigra of PD autopsy tissues as well as in cells undergoing oxidative stress. In vitro studies inferred that 41-syn neither aggregates nor alters the aggregation propensity of either WT or 112-syn. Overexpression of 41-syn or treatment of cells with 41-syn peptide did not affect cell viability. However, PC-12 cells treated with 41-syn exhibited a time and dose dependent enhancement in the cellular uptake of dopamine. Based on physiological role of the N-terminus region of α-syn in modulating membrane trafficking events, we believe that the identification of 41-syn may provide novel impetus in unraveling the physiological basis of alternative splicing events in governing PD pathophysiology. Key words: Parkinson’s disease, Oxidative stress, alternative splicing, 41-synuclein, dopamine homeostasis.

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Introduction α-Synuclein (α-syn) is a 140aa natively unfolded protein and was found to play a pivotal role in the pathophysiology of PD (1-4). The ability of missense mutations of α-syn as well as its increased gene dosage (duplication and triplication of α-syn locus) clearly established the role of α-syn in PD (5,6). Also, the lack of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced PD biochemical manifestations in α-syn null mice highlights the crucial role of this protein in both familial and sporadic cases of the disease (7). In fact, the discovery of α-syn as non-amyloid component precursor (NACP) in AD patients itself indicates that this protein plays an important role in multiple neurodegenerative diseases and could form a basis for identifying any common denominator in addressing multiple neurodegenerative disease processes (8). In vitro, α-syn aggregates and forms fibrils with similar morphology and properties to that of the amyloid fibrils extracted from LB (9,10). The integral region, namely non-amyloid component (NAC), of α-syn which is encoded by exon 4 was found to be responsible for its aggregation propensity (11). The N-terminus region of α-syn contains several repeat regions of the consensus sequence, KTKEGV, which are putative PDZ binding domains and were shown to interact with membranes at the endoplasmic reticulum and modulate the trafficking of vesicles out of plasma membrane (12). Further, the N-terminal repeat domain of α-syn has been shown to inhibit beta-sheet and amyloid fibril formation (13). The unstructured acidic C-terminus of the protein possesses chaperone activity and is involved in protein-protein interactions (14,15). The above findings highlight the physiological importance of different domains of α-syn in governing cellular processes and any alteration in the domain structure by way of exon skipping might result in the generation of α-syn isoforms with altered function. The role of alternative splicing events is becoming more evident in several neurodegenerative disorders in recent years (16-19). Increased levels of WT-syn, 112-syn with a concomitant decrease in 126-syn was observed in multiple system atropy (MSA) patients at both transcriptional and translational levels (20). It has been shown that the expression of all the four isoforms of α-syn were up-regulated in the frontal cortex of PD patients, whereas, in the Substantia nigra pars compacta (SNPc), only three shorter isoforms were overexpressed (21). In PD, increased levels of 126-syn were found in SNPc when compared to controls (22). Also, 112syn was found to be up-regulated in dementia with lewy bodies (DLB) (23). In-frame skipping of exon 3 and exon 5 generates 98-syn and mRNA expression analysis revealed significant increase in 98-syn in frontal cortices of DLB, AD and PD patients (24). Studies aimed at understanding the aggregation propensities of spliced isoforms of α-syn demonstrated that while the WT-syn forms straight fibrils, 126-syn forms shorter fibrils in parallel bundles and 98-syn forms annular structures (25). However, their exact relevance in disease pathology needs to be established. Earlier we have reported that oxidants and various Parkinsonian mimetics induced the generation of 112-syn and the expression of 112-syn was found to be deleterious to dopamine neurons possibly due to the enhanced aggregation propensity of 112-syn (26,33). The induced-alternative


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splicing events along with the observed alteration in the transcript levels of α-syn isoforms clearly indicate that they may have a specific role in disease pathophysiology which is still far from clear. In the present study, we report the identification of a novel alternatively spliced isoform of α-syn resulting in a 238 bp mRNA transcript lacking exons 3 and 4 in the SNPc of human PD autopsy samples. The presence of premature stop codon due to the excision of out-of-frame exon i.e., exon 3 and 4, generates a 41aa truncated peptide form of α-syn from this transcript (referred as 41-syn). The ability of 41-syn on some of the crucial events underlying PD pathophysiology, such as protein aggregation and dopamine homeostasis has been examined. Results Identification of 41-syn in human PD brain samples: RT-PCR analysis of the autopsy brain tissue (SNPc and cerebellum of human PD patients) employing full length α-syn primers revealed the expression of a shorter isoform of αsyn of 238 bp (human 41-syn GeneBank accession numbers: BankIt2051233 Seq1 MG016711), apart from WT and 112-syn isoforms corresponding to 423 and 339 bp in both SNPc as well as cerebellum regions (Figure 1A). The expressions of these three transcripts were found to be nearly in equal proportions in SNPc (lane 1 and 2). In cerebellum, WT-syn transcript was found to be prominent as compared to the two shorter transcripts [Figure 1A]. Next, in order to identify 41-syn peptide expression, CSF proteins from age matched healthy controls and PD patients were analyzed. Results indicate that the expression of peptide corresponding to 41-syn is clearly evident in three out of four PD samples analyzed, however, no corresponding band was evident in CSF from control subjects (Figure 1B). In order to examine the role of oxidative stress in the generation of 238 bp transcript of α-syn, human SK-N-SH cells were treated with Parkinsonian mimetic MPP+ (500 µM), doxorubicin (DOX) (0.5 µM), paraquat (PQ) (100-500 µM), rotenone (0.5-1 µM) or 6hydroxydopamine (6-OHDA) (50-100 µM) for 24 h. Results indicate that while control cells express predominantly WT-syn, treatment with MPP+, paraquat, rotenone and 6hydroxydopamine induced alternative splicing resulting in the generation of 112-syn and 41-syn transcripts [Figure 1C and D]. Treatment of cells with the known oxidative stress inducer, doxorubicin (DOX) also resulted in the generation of 112 and 41-syn (Figure 1C and D). The transcript levels of 41-syn when normalized with 18S rRNA displayed nearly 2-4 fold increase over controls under all the treatment conditions, whereas rotenone at 1µM exhibited >6-fold enhancement (Figure 1C and D). Analysis of 41-syn peptide expression by Western blotting also indicated an increase in the expression of 41-syn by ~ 3-4-fold in cells overexpressing pcDNA encoding 238 bp fragment as well as in rotenone treated cells (Figure 1E and F). 41-Syn represents the N-terminus of α-syn and possesses putative PDZ binding domains which are known to interact with membranes (12,15). Possibly, due to the presence of membrane


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interaction domains in addition to its smaller size, 41-syn appears to be a secretory peptide as it was detected in the extracellular medium, but not in the cell lysates (Figure 1E). Treatment of primary cultures of rat mesencephalic neurons isolated from 14-day old embryos with either 0.5µM DOX or 50µM MPP+ or 100µM H2O2 for a period of 12 h also induced the alternative splicing of rSyn1 (human homolog of rat α-syn) leading to the generation of 41-rSyn1 [Figure 1G and H] (rat 41-syn GeneBank accession number:BankIt2051233 Seq2 MG016712). The expression of 41-rSyn1 was increased to ~2.5-fold in DOX treated cells, whereas, treatment with MPP+ or H2O2 resulted in ~1.5-2.0-fold increase. On contrary, 112-syn transcript was found to be more abundant in H2O2 treated cells. The overall transcript abundance of all the isoforms was found to be very low in primary neuronal cultures as compared to SK-NSH cells, probably due to the mixed lineage of cells and relatively lower number of dopamine neurons in primary cultures. The obtained smaller transcript of α-syn was excised from agarose gel and cloned into a TA vector (Thermo). Sequence analysis of the spliced transcript demonstrated the deletion of exon-3 and exon-4 (corresponding to 185 bases, encoding 62 aa) resulting in the generation of a 238 bp transcript, supposedly coding for 78 aa [Figure 2A]. However, due to emergence of a premature stop codon by alternative splicing, this isoform expresses only a 41 aa N-terminus peptide [Figure 2B], hence we referred it as 41-syn. DNA sequence analysis confirmed the deletion of exon-3 and 4 even in rat α-syn (rSyn1) gene which results in the generation of 41rSyn1 peptide [Figure 2A & B]. Though the first 40 amino acids of human and rat sequence are identical, the 41st amino acid in human sequence is glutamic acid, whereas, in rat gene it is encoded by glycine due to the change in codon sequence (Figure 2B) (Gene Bank accession numbers for human and rat 41-syn are BankIt2051233 Seq1 MG016711and BankIt2051233 Seq2 MG016712). 41-syn neither aggregates nor affects the fibrillation of WT or 112-syn in vitro To investigate whether 41-syn is prone for aggregation in vitro, we have incubated 41syn (0.5 mg/mL) in 20 mM Tris buffer, pH-7.5 for 48h and found no increase in ThT fluorescence indicating that it is not prone for self-fibrillation [Figure 3A]. Under similar conditions, Aβ (1-42) peptide exhibited a ~ 4-fold enhancement in ThT fluorescence, whereas the values for WT and 112-syn were found to be enhanced by ~3 and ~9-fold respectively [Figure 3A]. Also, 41-syn did not inhibit the heat induced aggregation of aldolase at 1:2 and 1:4 molar concentrations indicating that 41-syn lacks chaperone activity unlike WT-syn [data not shown]. To examine the effect of 41-syn on the fibrillation of either WT or 112-syn, we have incubated both WT and 112-syn individually with varying concentrations of 41-syn at a ratio of 1:1 and 1:2 for a period of 24-72h and protein fibrillation was analyzed by ThT staining. However, no apparent changes in ThT emission spectrum of either WT or 112-syn were evident in the presence or absence of varying concentrations of 41-syn, indicating that 41-syn neither seeds nor inhibits the fibrillation of either WT or 112-syn in vitro [Figure 3 B-E].


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41-syn is not deleterious to dopaminergic cells in vitro To examine the effect of α-syn isoforms on cell viability, we have overexpressed the plasmids encoding either WT-syn, 112-syn or 41-syn in N27 cells (rat dopaminergic neuronal cell line) and the expression of α-syn mRNA was analyzed employing gene specific primers for WT, 112 and 41-syn [Figure 4A]. Following 48h of incubation, data from cell viability assay revealed that overexpression of 112-syn reduced the cell viability by ~25% as compared to its wild-type counterpart, wherein, the viability was reduced by only ~12% [Figure 4B], corroborating with the earlier studies that 112-syn overexpression was deleterious to dopaminergic cells (26). However, overexpression of 41-syn did not affect the viability of cells significantly (Figure 4B). Further, cells treated with 41-syn peptide (5-40 µM) for 48 h also did not exhibit any decrease in cell viability. Under similar incubation conditions, aggregated amyloid beta peptide (Aβ 1-42, 5-20 µM) exhibited a dose-dependent decrease in cell viability as well as the potent oxidative stress inducer, DOX [Figure 4C]. Altered dopamine homeostasis by 41-syn peptide Incubation of PC-12 cells with [3H]DA(5 to 60µM) exhibited a dose and time dependent enhancement in the cellular uptake of dopamine (Figure 5A). Cells were pretreated with 10, 25 and 50 µM 41-syn peptide for 30 min prior to the addition of [3H]DA (60µM). Following 15 min of incubation, cellular uptake of [3H]DA was analyzed. Control cells were incubated with equivalent volume of buffer in place of [3H]DA. In some wells, cells were incubated with GBR12909 (GBR) (5µM), a DA reuptake inhibitor for 15 min prior to the addition of [3H]DA. Results indicate that as compared to controls, there was a significant increase (~30-40%) in cellular uptake of [3H]DA in cells pre-treated with 41-syn peptide (Figure 5B). The observed increase in DA uptake was nearly the same in cells that were pre-exposed to 41-syn for 15, 30 or 60 min (data not shown). Under similar conditions, GBR inhibited DA uptake to a greater extent (Figure 5B). 41-syn induced DA uptake was inhibited in presence of GBR, indicating the involvement of dopamine transporter (DAT) in uptake of DA (Figure 5B). Under similar experimental conditions 112-syn overexpression resulted in a significant increase in cellular dopamine uptake but not by WT-syn (Figure 5C). In order to examine the effect of 41-syn on DA release, PC12 cells were pre-loaded with [ H]DA (60µM) for 15, 30 and 60 min, cells were washed and replaced with fresh media and the time dependent release of [3H]DA into extracellular medium was analyzed by measuring the radioactivity at 15, 30 and 60 min. Results indicate that there was a time-dependent increase in the release of [3H]DA under all the conditions examined (Figure 6A). Though there was a significantly increased release of [3H]DA in cells preloaded for 30 min as compared to 15 min, the release of [3H]DA in cells preloaded for 30 and 60 min were nearly the same at the end of 30 and 60 min (Figure 6A). Next, to examine the effect of 41-syn on DA release, PC-12 cells were preloaded with [3H]DA for 30 min, following replacement of medium with fresh medium, cells were incubated with different concentrations of 41-syn (10, 25 and 50 µM) and control cells 3


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were treated with equal volume of buffer. It was noticed that treatment of cells with 41-syn did not show any significant changes in the release of [3H]DA as compared to controls at all the concentrations examined (Figure 6B). Discussion The genetic link of α-syn to PD established in 1997 prompted several studies in understanding the molecular mechanisms governing the regulation of α-syn and its plausible down-stream effects (27). Four different protein forms of α-syn were shown to be the products of alternative splicing till date with altered structure and function (25). Of the different α-syn isoforms described, α-syn 140 is the major transcript of the protein and alternative splicing of exons 3 and 5 gives rise to α-syn 126 and 112 respectively, whereas, splicing of exons 3 and 5 results in α-syn 98. Emerging literature indicates that apart from WT-syn, the abundance of its transcript variants, such as, 112-syn, 126 syn and 98-syn were found to be altered in some of the neurodegenerative disorders (21-24). Reported abnormalities in the alternative splicing events leading to disease pathologies were ascribed to the presence of mutations either in the cis- or trans-acting regions (28). Earlier, we have reported the induced-alternative splicing of α-syn by various oxidants resulting in the generation of 112-syn, which is more prone for fibrillation (26). The above findings clearly indicate that there are highly regulated physiological processes that control the generation of protein isoforms with altered function by way of alternative splicing. However, in the context of neurodegenerative diseases the role of alternative splicing events in the mechanism mediating the disease is still elusive. A random analysis of α-syn gene expression pattern in the postmortem autopsy samples of PD patients revealed the presence of three different transcripts of α-syn gene in both the SNPc and cerebellum. However, the relative ratios of the smaller genes in SNPc are more abundant as compared to the cerebellum (Figure 1). Sequence analysis identified a novel isoform of 238 bp in addition to WT and 112-syn genes (Gene Bank accession numbers are BankIt2051233 Seq1 MG016711 and BankIt2051233 Seq2 MG016712). Though the number of samples employed in the present study is not enough to arrive at the statistical significance, nevertheless, it will not negate that fact that the present study identified a novel and the smallest isoform of α-syn which lacks both exons 3 and 4 due to the skipping of 185 bp fragment. As exons 3 and 4 are out of frame as compared to the rest of the sequence, deletion of this sequence introduced a premature stop codon after 41 aa which resulted in the generation of 41-syn. Except for the 41st amino acid (E in human sequence and G in rat sequence) the rest of 40 aa are identical in both human and rat sequence. Under in vitro conditions, Parkinsonian mimetics could induce the generation of 41syn albeit to different extents in addition to 112-syn. Similar findings were obtained even from the rat mesencephalic neurons treated with DOX and other oxidants (Figure 1). To examine the plausible biological effects of 41-syn, we have examined its in vitro aggregation propensity and its ability to inhibit the aggregation of WT or 112-syn in vitro.


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However, it was found that 41-syn is neither prone for aggregation nor inhibited the fibrillation of either WT or 112-syn in vitro (Figure 3). We have also examined the ability of 41-syn in possessing chaperone activity against the heat-induced aggregation of aldolase, effects on in vitro tubulin polymerization as well as on 20s proteasome activity, however, none of these events were found to be inhibited by 41-syn (data not shown). Overexpression of 41-syn (as a 238 bp transcript in pcDNA3) in N27 cells did not reduce the viability of cells following 48h post transfection, however, 112-syn overexpression resulted in a significant reduction in cell viability (Figure 4B). As 41-syn was found to be secreted into the medium, we have examined the effects of extracellular addition of 41-syn peptide (5-40 µM) in N27 cells for 48h and did not find any significant reduction in the viability of cells unlike aggregated Aβ peptide indicating that 41-syn is not cytotoxic to cells (Figure 4C). As there are three repeat sequences with the consensus KTKEGV in the 41-syn peptide which are predicted to be involved in the membrane interaction, we next analyzed whether this peptide is involved in the modulation of dopamine homeostasis in vitro. It was interesting to notice that cells incubated with 41-syn exhibited a significant increase in the cellular uptake of [3H]DA in a dose-dependent manner (Figure 5). The ability of GBR in inhibiting 41-syn induced cellular uptake of DA indicates the plausible role of DAT in 41-syn induced DA uptake (Figure 5B). Though dopamine has been reported to directly interact with α-syn, however, the interaction involves five residues at the C-terminus (YEMPS) and E83 residue of α-syn (29). Hence, it is unlikely that 41-syn induced dopamine uptake involves any direct binding to dopamine per se. A survey of literature indicates that α-syn interacts with DAT through its NAC region and modulate DAT activity (30). It was also shown that CAMKII phosphorylates DAT by binding to its C-terminus region (30). α-Syn and CAMKII were shown to bind to similar region on DAT and thereby modulate DAT activity (30). Later studies identified that calcium bound calmodulin interacts with α-syn at its N-terminus (31). In a recent study, it was shown that calcium binds to the C terminus of α-syn and thereby increases its lipid-binding capacity (32). Overall, there appears to be a stringent regulation of DAT by α-syn which involves both its N and C-terminus regions along with other mediators, such as calmodulin which needs further investigation to understand their precise role in disease pathogenesis. Nevertheless, based on the available literature, it appears that any alterations in the structure of α-syn by way of alternative splicing may result in the altered homeostasis of dopamine. Furthermore, recent studies demonstrated the ability of N-terminus peptide of α-syn in activating microglial mediated superoxide production and subsequent neuronal damage (33). Also, α-syn N-terminus peptide was shown to act as antigenic epitope that drives helper and cytotoxic T-cells in PD patients (34). All the above observations clearly suggest a crucial role for the N-terminus region of α-syn in PD pathophysiology. Hence, the findings of our present study on the identification of a novel alternatively spliced isoform of α-syn in generating a truncated form of 41aa N-terminus peptide


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may have direct implications in the pathogenesis of PD and warrants further investigation. Materials & methods

Materials Cell culture media, 1- Methyl-4-phenylpyridinium iodide (MPP+ iodide), rotenone, paraquat, 6-hydroxy dopamine, hydrogen peroxide, aldolase (ALD) from rabbit muscle, thioflavinT (ThT) and all other chemicals were purchased from Sigma, St. Louis, MO, USA. Restriction enzymes were obtained from Fermentas Inc, Fisher Scientific, PA, USA. All other reagents were of analytical grade. Peptides of 41-syn, Aβ (1-42) were from Kric, India. Dopamine ([3H]DA) was purchased from Perkin Elmer, USA.

Methods Cell culture and treatments SK-N-SH cells (ATCC, USA) were grown in T75 flasks in either Eagle’s minimal essential medium supplemented with 10% fetal bovine serum until 80% confluence. Cells were plated in 6-well plates one day before the start of the experiment. Twelve hours prior to the treatments, the medium was replaced with minimum essential medium containing 2% serum and the cells were treated with either MPP+ (500 µM), doxorubicin hydrochloride (DOX) (0.5 µM), paraquat (100-500 µM), rotenone (0.5-1 µM) or 6-OHDA (50-100 µM) for a period of 24 h. PC12 cells (NCCS, Pune, India) were maintained in Rosewell Park Memorial Institute (RPMI) medium supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum in poly-D-lysine (0.1mg/mL) coated dishes at 37°C with 5% CO2. N27 cells (Millipore, USA) were grown in RPMI medium supplemented with 10% FBS. Primary cultures of mesencephalic dopamine neurons isolated from embryonic day 14 pups were cultured as described previously (24). The animal experimental protocols used were approved by Institutional Animal Ethical Committee (IAEC) and were in accordance with the NIH guidelines for the care and use of laboratory animals (IICT/09/2015). RT-PCR analysis Following the termination of experiment, the medium was aspirated and 1 ml of TRIzol (Invitrogen Inc, USA) was added to cells in 6-well plates and total RNA was extracted using the manufacturer’s protocol. 5 µg of RNA was used for the first strand cDNA synthesis using a First strand cDNA synthesis kit (Thermo Scientific, USA) according to the manufacturer’s protocol. In the experiments involving human tissue, brain samples of PD patients were obtained from Alzheimer’s disease core facility, Pennsylvania. Brain tissues from SNPc and cerebellum were directly homogenized in Trizol and RT-PCR was performed by employing full length α-syn primers. The obtained transcripts were cloned into TA vector (Thermo) for DNA sequencing (the sequence of human and rat 41-syn was submitted to Gene Bank with accession numbers, BankIt2051233 Seq1 MG016711 and BankIt2051233 Seq2 MG016712).


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Cloning of 41-syn SK-N-SH cells were treated with 500 µM MPP+or1 µM DOX for a period of 24 h. Following the termination of incubation, the medium was aspirated and 1 ml of Trizol reagent (Invitrogen Inc, USA) was added to the cells in 6-well plates and total RNA was extracted using the manufacturer’s protocol (Invitrogen). RT-PCR was performed employing full length α-syn primers: (F: 5’-AAC TCG AGA TGG ATG TAT TCA TGA AAG GAC-3’) and (R: 5’- AAA AGC TTG GCT TCA GGT TCG TAG T -3’). Following PCR, the amplified 238 bp product was digested with Hind III and XhoI and ligated into pcDNA3 plasmid that was predigested with the same restriction enzymes to obtain the plasmid expressing 41-syn. Cell viability assay: To assess the effect of 41-syn overexpression on cell viability, N27 cells were transfected with WT-syn, 112-syn and 41-syn plasmids using Lipofectamine 2000 as per manufacturer protocol (Invitrogen). Following the termination of incubation, cells were fixed with 10% icecold trichloroacetic acid and incubated at 4°C for 1h and then washed with deionized water and air dried. Sulforhodamine B (SRB) (0.04%, w/v) was added to the cells and incubated further for 30 min at room temperature. The residual dye was removed by washing thrice in 1% acetic acid and the plates were air dried. SRB bound to the cells was subsequently eluted with 10mM Trisbase and absorbance was measured at 565 nm using EnSpire multimode plate reader (Perkin Elmer) (35). Western blot analysis: Cells in 90 mm plates were transfected with 41-syn in pcDNA3 using Lipofectamine 2000 as per manufacturer’s protocol (Millipore, USA) and 48h post transfection, the extracellular medium was replaced with 6 mL of serum-free medium and incubated further for 8h. In parallel experiments, cells were treated with either paraquat (100 µM) or rotenone (1 µM) for 24h. Following the termination of incubation, the culture media was collected, centrifuged at 3000rpm for 5-6 min at 4°C and further at 8000rpm for 5-6 min to remove any floating cells or debris. Later, the supernatant was concentrated to ~150µL using Amicon Ultra 15mL centrifugal devices by centrifugation at 4000 rpm for 120 min. 30 µL of the final concentrate was run on a 16% Tris-tricine peptide gel and blotted on to 0.1µm PVDF membrane and fixed with 0.4% paraformaldehyde for 30 min. The blot was probed with N-terminal α-syn antibody (custommade, Genscript) and anti-goat IgG conjugated to HRP was used as a secondary antibody. Bands were developed using ECL Prime reagent (Pierce, Thermo). CSF sample collection and analysis: The participants in the study include patients suffering from PD and healthy controls subjects who did not have Parkinson’s disease and the cerebrospinal fluid (CSF) analysis was done for other investigative purpose. CSF was collected following informed consent from


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control and PD subjects using standard lumbar puncture procedures [Approval No. SS15071008]. Immediately after collecting the CSF, aliquots of 0.5mL were flash frozen and stored for further analysis. 40 µL of each CSF sample was denatured by heating at 95°C for 5min in presence of SDS-PAGE loading dye (1x final concentration). The proteins were resolved on 16% Tris-Tricine peptide gel and Western blot was performed for the detection of 41-syn. Fibril formation Solutions of WT-syn, 112syn, 41-syn and Aβ (1-42) at a concentration of 0.5 mg/mL in 20 mM Tris buffer, pH 7.5 were incubated under shaking conditions (1000 rpm) at 37°C in a thermo mixer (Eppendorf) as described previously (36). Aliquots from the incubation mixtures were withdrawn at 48h for fluorescence measurements. In experiments where interdependency of isoforms was analyzed, 41-syn (0.5 and 1.0 mg/mL) and WT or 112-syn at a concentration of 1:1 and 1:2 were incubated at different ratios and kinetics of fibrillation was analyzed by Thioflavin (ThT) staining at every 24h for up to 72h. Analysis of fibril formation by ThT binding assay Fibril formation was monitored with ThT fluorescence as described previously with slight modifications (36). Briefly, aliquots of 5 µL from the incubation mixtures were withdrawn at various time points, diluted to 100 µL with 25 mM ThT in 50 mM Tris buffer (pH 8.0). The fluorescence emission spectrum (470–600 nm) of the samples was recorded following the excitation of samples at 450 nm in an Enspire 800 model Perkin-Elmer multimode reader. The blank measurement recorded prior to the addition of proteins was subtracted from the signal obtained from each sample. Dopamine uptake studies PC-12 cells were grown in poly-D-lysine coated 48-well plates for 24h. Media was aspirated from the wells and cells were washed twice with Krebs-Ringer HEPES buffer (KRH buffer - pH 7.4, 25 mM HEPES, 125 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.3 mM CaCl2, 1.2 mM MgSO4, 5.6 mM glucose, 50 µM pargyline, and 50 µM ascorbic acid) before the treatments as described previously (37). GBR12909 was used as negative control for dopamine uptake. After pre-incubation with 41-syn peptide for the indicated time points, [3H]DA (60 µM) uptake was performed for a period of 15 min. In parallel experiments, PC-12 cells overexpressing either WT or 112-syn were also employed to examine their effect on dopamine uptake. [3H]DA uptake was terminated by aspiration of the solution followed by two rapid washes with KRH buffer. Cells in each well were lysed with 100µL of 0.1%SDS and transferred to scintillation vials with 2 mL of Optiphase Supermix Scintillation Cocktail (Perkin Elmer) and counted on a Microbeta Scintillation Counter (Perkin Elmer).


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Dopamine release assay Plating of cells was performed as described in the uptake assay. 24h later, media was aspirated from wells and washed three times with KRH buffer. The cells were pre-loaded with 60µM [3H]DA in KRH buffer at 37°C for 30 min, washed three times with KRH buffer and thereafter incubated with peptides/inhibitors as described (37). At 0, 15, 30 and 60 min time points, 25 µL buffer was collected from each well and replaced with fresh buffer. The collected fractions were added to 200 µL scintillation liquid and analyzed for [3H]DA content using liquid scintillator. Following removal of the final fraction, cells were lysed with 0.1% SDS to quantify the remaining dopamine content. Experiments were carried out in triplicate.

Statistical analysis Statistical analysis was done using Prism 7 software (Graph Pad). Data are expressed as means ± S.D. Data with single variable were analyzed by Student’s t-test. One-way ANOVA was employed to assess statistical differences among the experimental groups. Grant support: This work was supported by the XII FYP projects, MiND (BSC-115) from CSIR, India and the Research project from DST, India (EMR/2016/001119).

Research

Fellowship to RLV, KMM and DY (from CSIR, India) are gratefully acknowledged. Abbreviations used: PD, Parkinson’s disease; α-syn, α-synuclein; MPP+, 1-Methyl-4phenylpyridinium ion; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NACP, Nonamyloid component precursor; DAT, dopamine transporter; DA, dopamine; SNPc, Substantia nigra pars compacta; NAC, non amyloid component; CAMK, calcium calmodulin kinase; PQ, paraquat; DOX, doxorubicin; Rot, rotenone; 6-OHDA, 6-hydroxydopamine. Conflict of interest No conflict of interest to disclose Author Contributions: RLV performed in vitro neurotransmitter transport and viability experiments, wrote the manuscript; KMM performed protein purification and in vitro protein fibrillation assays; DY performed gene expression studies; KB performed protein purification; KTB and GSR coordinated with patients for CSF sample collection; SVK planned experiments, coordinated the work elements and wrote the manuscript. Acknowledgements: We greatly acknowledge the Alzheimer’s disease core facility, Pennsylvania for providing us with post-mortem samples of PD patients. Footnotes: 1: Present address- Department of Pharmaceutical Sciences, Wayne State University, Michigan, USA. 2: Excelra knowledge solutions, IDA Uppal, Hyderabad 500039, T.S., India.


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Figure legends: Figure 1: Identification of a novel isoform of α-syn: (A) Total RNA was isolated from the human post mortem brain tissue of PD patients using Trizol and RT-PCR was performed by employing full length α-syn primers and α-syn mRNA levels were resolved on a 2% agarose gel. (B) 41-syn peptide levels in the CSF of control and PD patients (C) RT-PCR analysis of α-syn isoforms in SK-N-SH cells treated with either MPP+ (500 µM ), DOX (1µM), paraquat (PQ) (100-500 µM), rotenone (Rot) (0.5-1 µM) or 6-hydroxydopamine (6-OHDA) (50-100 µM) for 24 h. (D) Densitometric analysis of 41-syn levels from (C). (E) Immunoblot demonstrating the peptide expression of 41-syn in the extracellular medium. (F) Densitometry analysis of 41-syn staining in the extracellular medium from (E). (G) RT-PCR analysis of α-syn isoforms of rat mesencephalic dopamine neurons treated with MPP+ (50 µM), DOX (0.5 µM) or H2O2 (100 µM) for 24h. (H) Densitometric analysis of 41-syn from (G). From A, C, E and G data shown are the pooled samples from three separate wells and is a representative of two independent experiments. For (D and F), *p

Identification of an Alternatively Spliced Î± ... - ACS Publications

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