Abstract

   Duplication of the SNCA gene (SNCA^Dupl), linked to elevated levels of
   α-synuclein (aSyn), is a genetic cause of Parkinson’s disease (PD). Our
   prior work with human-induced pluripotent stem cell (hiPSC)–derived
   midbrain neurons generated from patients with PD SNCA^Dupl identified
   neuritic deficits, accompanied by decreased levels of cytoskeletal
   element β-tubulin-III (bTubIII). To explore mechanisms underlying these
   effects in SNCA^Dupl neurons, we used CRISPR-Cas9 to generate isogenic
   control hiPSCs. Isogenic correction of SNCA dosage restored
   SNCA^Dupl-induced neurite defects and bTubIII levels. Multi-omics
   analyses revealed SNCA^Dupl-induced alterations in neuronal
   differentiation, with a notable down-regulation of PAX6. Moreover,
   SNCA^Dupl induced an up-regulation of vimentin. Further
   characterization revealed heightened vimentin truncation associated
   with altered distribution and organization. Similar changes in vimentin
   levels and truncation were observed in postmortem putamen tissue from
   patients with sporadic PD. Notably, targeting vimentin with okadaic
   acid and withaferin A restored bTubIII- and neurite-associated defects,
   suggesting its potential to prevent aSyn-mediated neuritic
   degeneration.
     __________________________________________________________________

   Find the effect of SNCA duplication on Parkinson’s neurons and how
   targeting SNCA and vimentin reverses neuronal deficits.

INTRODUCTION

   Parkinson’s disease (PD) is the most common neurodegenerative movement
   disorder, clinically characterized by motor symptoms, including
   bradykinesia, rigidity, and resting tremor. These symptoms are
   attributed to the progressive degeneration of midbrain dopaminergic
   neurons primarily located in the substantia nigra pars compacta.
   Neuropathologically, PD is defined by the presence of intracellular
   inclusions within neuronal bodies and neurites, so-called Lewy bodies
   (LB) and Lewy neurites (LN), respectively. Comprehensive investigations
   have identified α-synuclein (aSyn), especially in aggregated forms, as
   the major protein component in Lewy inclusions ([46]1, [47]2).
   Moreover, point mutations and the multiplication of the SNCA gene,
   which encodes aSyn, are associated with autosomal dominant forms of PD
   ([48]3). Hence, both neuropathological and genetic evidence underscores
   the pivotal role of aSyn in the pathogenesis of PD.

   Initially found in the electric ray Torpedo californica, aSyn was
   detected in the nucleus and nerve terminals of neurons ([49]4). Because
   of its predominant localization in neurites, extensive research has
   been dedicated to understanding its role at the neuronal terminal. aSyn
   has been shown to modulate soluble N-ethylmaleimide–sensitive factor
   attachment protein receptor complex assembly ([50]5), synaptic vesicle
   recycling ([51]6, [52]7), and neurotransmission ([53]8, [54]9).
   Coincidently, it has been implicated in the regulation of neurite
   development and function. Specifically, pathological forms of aSyn have
   been shown to impair axonal transport by interfering with cytoskeletal
   elements ([55]10, [56]11). These findings on the interplay between
   aSyn, neuritic, and synaptic functions are particularly intriguing, as
   a growing body of evidence suggests that neurodegeneration originates
   at the distal portion of neurites ([57]12). However, the precise
   biological mechanisms underlying this interplay remain elusive.

   Human-induced pluripotent stem cell (hiPSC)–derived neurons from
   patients with PD offer a unique opportunity to investigate PD-related
   aSyn pathology. Since an elevated level of aSyn is an established risk
   factor for PD pathology, the use of hiPSCs derived from patients with
   PD carrying SNCA gene multiplications has emerged as an important tool
   for modeling PD. Numerous studies, often using SNCA triplication hiPSC
   models, have revealed PD-associated cellular phenotypes such as
   mitochondrial dysfunction ([58]13) and alterations in protein
   degradation pathways ([59]14, [60]15). Moreover, several studies on
   neurons derived from patients with PD carrying SNCA triplication have
   demonstrated impaired neurite morphology, vesicle transport deficits,
   and axonal degeneration compared to neurons from healthy individuals
   ([61]16, [62]17). Consistent with these findings, our own studies
   comparing neurons derived from patients with SNCA duplication
   (SNCA^Dupl) PD with those from control donors, revealed altered neurite
   morphology ([63]18) and axonal transport activity ([64]19), along with
   disruption of microtubule organization ([65]18). These alterations
   appeared to be linked to aSyn pathology: We observed increased aSyn
   accumulation along neurites ([66]20) and interaction of aSyn with
   microtubule elements in SNCA^Dupl neurons ([67]18).

   Patients with PD harboring SNCA^Dupl or triplication have varying sizes
   of polygenic loci multiplicated in the genomic region of chromosome
   4q22 ([68]21). The rapid development of the CRISPR-Cas9–based genome
   editing tool provides a valuable tool for specifically modifying SNCA
   loci in cells derived from patients carrying SNCA multiplications.
   CRISPR-Cas9–mediated SNCA gene correction has been used in hiPSCs with
   SNCA triplication ([69]14, [70]22, [71]23). This enables investigation
   into the specific effects of additional SNCA alleles without the
   influence of other multiplicated genes in the affected genomic regions.

   In this study, we investigated whether the observed SNCA^Dupl-mediated
   neurite impairments and cytoskeletal alterations in midbrain neurons
   are specific effects resulting from increased SNCA gene dosage. In
   addition, we explored the mechanisms underlying these SNCA^Dupl-induced
   alterations. We reprogrammed fibroblasts from a female patient with PD
   carrying an SNCA^Dupl to hiPSCs. In addition, we generated an isogenic
   control from this SNCA^Dupl hiPSC line by restoring the SNCA gene
   expression. By comparing patient-derived SNCA^Dupl midbrain neurons
   with isogenic neurons, we revealed specific effects of elevated SNCA
   dosage. These effects include dynamic alterations in aSyn homeostasis
   during neuronal differentiation and impairments in neurite morphology.
   Using RNA sequencing (RNA-seq), assay for transposase-accessible
   chromatin sequencing (ATAC-seq), and proteomics, we identified that
   cellular pathways related to extracellular matrix (ECM) organization,
   neuronal differentiation, and synaptic function are significantly
   affected by increased SNCA dosage. In particular, SNCA^Dupl induced a
   remarkable up-regulation of vimentin. Further characterization revealed
   increased vimentin truncation, alongside its network disorganization
   and altered distribution along neurites in response to elevated SNCA
   dosage. Notably, by small molecule–mediated interference with the
   vimentin network, we were able to restore cytoskeletal and neuritic
   phenotypes related to SNCA gene duplication. Our findings shed light on
   an intrinsic interplay between aSyn and vimentin organization in
   neurons, thereby influencing cytoskeletal organization and neurite
   integrity.

RESULTS

SNCA duplication alters aSyn dynamics and distribution during neuronal
differentiation

   We obtained a skin biopsy from a 44-year-old female patient with PD
   with a genetically confirmed heterozygous duplication spanning an
   estimated region of 3.59 to 4.32 Mb on chromosome 4q22.1, which
   includes the SNCA gene. The patient began to develop PD-associated
   motor symptoms at age 39. In our previous study, we analyzed aSyn
   levels in neural precursor cells (NPCs) and midbrain neurons
   differentiated from hiPSCs, which were generated by reprogramming
   fibroblasts obtained from skin biopsies of this patient and healthy
   control donors ([72]18). We observed significantly higher levels of
   aSyn in SNCA^Dupl NPCs and midbrain neurons at an early differentiation
   stage compared to their respective controls. These elevations were
   accompanied by neurite deficits and microtubule disorganization
   ([73]18). To determine whether these phenotypes observed in SNCA^Dupl
   neurons are primarily attributable to SNCA^Dupl and whether correcting
   SNCA dosage can alleviate these effects, we used CRISPR-Cas9 technology
   to generate an isogenic hiPSC line (hereafter termed SNCA^Iso) from the
   patient with SNCA^Dupl by inducing a heterozygous out-of-frame deletion
   of 118 base pair (bp) within exon 2 of SNCA [fig. S1, A to E,
   CRISPR-Cas9 procedure was described in more detail in ([74]24)].

   Next, we investigated aSyn levels at different stages of neuronal
   differentiation, ranging from hiPSCs to midbrain neurons by comparing
   cells derived from SNCA^Dupl, SNCA^Iso, and control donors ([75]Fig.
   1A). We induced midbrain neuronal differentiation from hiPSCs via NPCs
   using a small molecule–based protocol ([76]18, [77]25). Following this
   protocol, neuronal differentiation yielded 80 to 90% neurons, as
   confirmed by flow cytometric analysis of the neuronal marker NeuN (fig.
   S1F). In addition, 30 to 50% of these neurons exhibited
   immunopositivity for both NeuN and tyrosine hydroxylase, indicative of
   dopaminergic neuron identity (fig. S1F). Immunocytochemistry (ICC)
   analysis using neuronal subtype–specific markers [tyrosine hydroxylase
   (TH) for dopaminergic neurons, glutamate decarboxylase 67 (GAD67) for
   GABAergic neurons, vesicular glutamate transporter 1 (vGlut1) for
   glutamatergic neurons, and choline acetyltransferase (ChAT) for
   cholinergic neurons] revealed that our midbrain cell model developed a
   small proportion of neurons positive for GAD67, vGlut1, and ChAT, while
   a larger proportion was positive for TH (fig. S1G).

Fig. 1. aSyn levels in SNCA^Dupl hiPSCs and NPCs.

   [78]Fig. 1.
   [79]Open in a new tab

   (A) Midbrain neuronal differentiation from hiPSC lines via NPCs.
   Created in BioRender. Xiang, W. (2025)
   [80]https://BioRender.com/i9n38rh. (B) Agarose gel electrophoresis of
   PCR products. L, DNA ladder. The SNCA^Iso line displays a modified SNCA
   DNA fragment (~300 bp), alongside the unmodified fragment (~420 bp).
   (C) RT-qPCR analysis of SNCA mRNA, showing a higher level in the
   SNCA^Dupl compared to SNCA^Iso and control hiPSCs; N = 3
   differentiation rounds. (D to F) RT-qPCR, WB, and ELISA analyses of
   SNCA mRNA (D) and aSyn protein [(E) and (F)] at the NPC stage. SNCA
   mRNA and aSyn levels are significantly higher in SNCA^Dupl NPCs than
   those in control [dashed line in (D)] and SNCA^Iso NPCs; N = 3
   differentiation rounds. For (E), LC, Coomassie brilliant-blue staining.
   For (D), an additional unpaired t test was performed to compare
   SNCA^Iso or SNCA^Dupl with control NPCs, with ##P < 0.01.

   At the hiPSC stage, polymerase chain reaction (PCR) amplification of
   the CRISPR-Cas9 targeted region followed by gel electrophoresis
   confirmed the presence of a modified SNCA DNA fragment with a deletion
   of approximately 100 bp in the isogenic hiPSC line ([81]Fig. 1B). In
   addition, reverse transcription quantitative PCR (RT-qPCR) analysis
   revealed a 1.5-fold higher level of SNCA mRNA in the SNCA^Dupl line
   compared to the SNCA^Iso line and hiPSCs from a healthy control
   individual ([82]Fig. 1C). Moreover, SNCA mRNA levels in the SNCA^Iso
   line were comparable to those of the control hiPSC line ([83]Fig. 1C).
   These findings suggest heterozygous editing of SNCA and restoration of
   the SNCA dosage in the SNCA^Iso line.

   To further analyze aSyn protein levels, hiPSCs were differentiated into
   NPCs. This step was necessary since aSyn expression is known to be
   induced during neuronal development ([84]26, [85]27), and full-length
   aSyn is absent in hiPSCs in our model (fig. S1D). At the NPC stage, we
   detected a significant increase in SNCA expression in SNCA^Dupl NPCs
   compared to SNCA^Iso or control NPCs, both at transcriptional and
   protein levels as determined by RT-qPCR ([86]Fig. 1D) and Western blot
   (WB) ([87]Fig. 1E), respectively. An additional enzyme-linked
   immunosorbent assay (ELISA) analysis revealed a 1.7-fold increase in
   aSyn levels in SNCA^Dupl compared to SNCA^Iso ([88]Fig. 1F).

   During the midbrain neuron differentiation, we assessed aSyn levels
   using WB analysis across early and late differentiation stages
   ([89]Fig. 2, A to C, and fig. S2, A to B). Specifically, SNCA^Dupl
   neurons exhibited significantly higher aSyn levels during early
   differentiation (days 12 to 16) compared to SNCA^Iso neurons. These
   differences, however, became less pronounced as differentiation
   progressed to 24 days ([90]Fig. 2A and fig. S2A). At day 24, SNCA^Dupl
   neurons exhibited even lower aSyn levels than SNCA^Iso neurons
   ([91]Fig. 2A), as assessed by quantitative ELISA analysis ([92]Fig.
   2B). Similar trends were observed when comparing SNCA^Dupl neurons to
   healthy control neurons: aSyn levels were significantly elevated in
   SNCA^Dupl neurons at 10 days of differentiation compared to controls,
   but the differences declined by day 24 (fig. S2B).

Fig. 2. Dynamic aSyn expression during midbrain neuronal differentiation.

   [93]Fig. 2.
   [94]Open in a new tab

   (A) WB analysis of aSyn in SNCA^Dupl neurons during differentiation (12
   to 24 days) compared to SNCA^Iso counterparts (green dashed line). aSyn
   levels are significantly higher in SNCA^Dupl neurons at days 12 to 16;
   however, the differences between SNCA^Dupl and SNCA^Iso neurons
   diminish over time. LC, revert 520 total protein stain; N = 4–8
   differentiation rounds. An additional unpaired t test was performed to
   compare all SNCA^Dupl groups with their SNCA^Iso counterparts, with
   #P < 0.05. (B) ELISA analysis of total aSyn levels in neurons (24
   days). SNCA^Dupl neurons display a significant lower aSyn levels than
   SNCA^Iso neurons. (C) ELISA analysis of the ratio of aggregated/total
   aSyn in neurons (24 days), demonstrating an increase in aggregated aSyn
   in SNCA^Dupl neurons. (D to F) ICC analysis of aSyn distribution using
   the 15G7 and LB509 antibodies, showing an increased ratio of intensity
   in neurite/soma in SNCA^Dupl compared to SNCA^Iso neurons, with a more
   pronounced effect observed using the LB509 antibody [(D) and (E)]. In
   (D), aSyn in the soma and along the neurite is highlighted with
   arrowheads and arrows, respectively. n = 12 neurons per group from
   N = 3 differentiation rounds. (G) Schematic summary of aSyn dynamics.
   Created in BioRender. Xiang, W. (2025)
   [95]https://BioRender.com/yqichdf.

   To investigate whether SNCA^Dupl leads to a disturbance in aSyn
   homeostasis, we conducted an ELISA measuring the ratio of aggregated
   aSyn to total aSyn ([96]Fig. 2C). This analysis revealed a significant,
   proportional increase of aggregated aSyn in SNCA^Dupl neurons compared
   to SNCA^Iso neurons.

   We previously demonstrated increased aSyn accumulation along neurites
   in neurons from different patients with SNCA^Dupl compared to control
   individuals ([97]20). Building upon this finding, we compared aSyn
   distribution in neurites and soma of SNCA^Dupl and SNCA^Iso midbrain
   neurons using ICC with two antibodies against aSyn: 15G7 and LB509
   ([98]Fig. 2, D to F). LB509 is frequently used to detect aggregated
   forms of aSyn ([99]28), while the 15G7 antibody detects total aSyn.
   Consistent with our previous observations, we found increased
   accumulation of aSyn in neurites of SNCA^Dupl neurons compared to
   SNCA^Iso neurons. This was particularly evident with the LB509
   antibody, indicating enhanced accumulation of aggregated aSyn in
   SNCA^Dupl neurites ([100]Fig. 2, D and E).

   In summary, SNCA^Dupl led to higher aSyn levels in NPCs and early
   differentiated neurons. However, as differentiation progressed, the
   difference in total aSyn levels between SNCA^Dupl and SNCA^Iso or
   control neurons diminished. Nevertheless, increased aSyn aggregation
   was observed in SNCA^Dupl neurons compared to controls, both in this
   study (SNCA^Dupl versus SNCA^Iso) and in our previous study [SNCA^Dupl
   versus controls ([101]18)]. In addition, SNCA^Dupl neurons exhibited
   increased accumulation of aSyn in neurites compared to controls
   [SNCA^Dupl versus SNCA^Iso in this study; SNCA^Dupl versus healthy
   control neurons in ([102]20)]. Together, these collective findings
   suggest a spatially and temporally disturbed aSyn homeostasis in
   SNCA^Dupl neurons (summarized in [103]Fig. 2G).

SNCA duplication impairs neurite morphology and restored by corrected SNCA
dosage

   Our previous study comparing SNCA^Dupl- and control individual–derived
   midbrain neurons demonstrated impaired neuritic morphology in neurons
   carrying SNCA^Dupl ([104]18). This was accompanied by reduced levels
   and reorganization of β-tubulin-III (bTubIII), a crucial structural
   component of neuronal microtubules. To investigate whether these
   observed effects are specifically attributable to SNCA^Dupl, we
   performed ICC analysis of bTubIII and found an overall impaired
   neuritic network characterized by remarkably lower neurite density in
   SNCA^Dupl neurons compared to SNCA^Iso neurons ([105]Fig. 3, A and B)
   and those from control donors [[106]Fig. 3A and also characterized in
   ([107]18)]. Isogenic correction of SNCA^Dupl restored this neurite
   phenotype to a level comparable to that of control neurons ([108]Fig.
   3A). Further analyses revealed that neurites of SNCA^Dupl neurons
   exhibited a significant decrease in diameter and volume compared to
   those of SNCA^Iso neurons ([109]Fig. 3, C and D, and fig. S3, A and B).
   In addition, we assessed cell death in SNCA^Iso and SNCA^Dupl neurons
   using the lactate dehydrogenase cytotoxicity assay without detecting
   significant differences (fig. S3C). These findings suggest that the
   neurite deficits observed in SNCA^Dupl neurons are not due to extensive
   neuronal loss, highlighting neurite degeneration as an early event
   preceding neuronal loss. Coinciding with neurite deficits, bTubIII
   levels were lower in SNCA^Dupl compared to control midbrain neurons,
   and bTubIII levels were restored in SNCA^Iso neurons ([110]Fig. 3E).

Fig. 3. Analysis of neurites and bTubIII levels.

   [111]Fig. 3.
   [112]Open in a new tab

   (A) ICC analysis of the neuronal network using fluorescent staining for
   bTubIII, demonstrating a decrease in the number of neurites developed
   from SNCA^Dupl midbrain neurons compared to control and SNCA^Iso
   neurons. (B) Quantification of neurite density between SNCA^Iso and
   SNCA^Dupl. (C) ICC images of bTubIII^+ primary neurites (white) at
   higher magnification. (D) Analysis of the diameter and volume of
   bTubIII^+ neurites using IMARIS, demonstrating a significant decrease
   in SNCA^Dupl neurons compared to SNCA^Iso neurons. For neurite
   diameter, n = 320–501 bTubIII^+ neurites per group; for neurite volume,
   n = 70–900 bTubIII^+ neurites per group from N = 3 differentiation
   rounds. (E) WB analysis of bTubIII levels. Left, a representative
   image; right, quantification of bTubIII showing lower levels in
   SNCA^Dupl neurons compared to control and SNCA^Iso neurons. LC, Ponceau
   staining. Quantitative data were derived from N = 3 differentiation
   rounds for control neurons and N = 6 differentiation rounds for
   SNCA^Dupl and SNCA^Iso neurons.

   Together, our results indicate that the altered neuronal network,
   including structural changes associated with neurite development and
   bTubIII changes observed in SNCA^Dupl midbrain neurons are specific
   outcomes of SNCA gene duplication during neuronal differentiation. In
   addition, correcting SNCA dosage reverses the neurite phenotypes and
   bTubIII levels in SNCA^Dupl midbrain neurons.

SNCA duplication changes gene expression affecting neuronal differentiation
and function

   On the basis of the observed alterations in aSyn dynamics and neurite
   deficits mediated by SNCA^Dupl, we sought to explore underlying
   mechanisms using multiple omics approaches. Since similar patterns were
   observed in these phenotypes between SNCA^Iso and control cells, and to
   dissect the specific effects of SNCA^Dupl, we focused on comparisons
   between SNCA^Dupl and SNCA^Iso midbrain neurons. We performed
   transcriptomic analysis using RNA-seq and found extensive alterations
   in SNCA^Dupl neurons, evident by the identification of 3011
   up-regulated and 909 down-regulated transcripts (adjusted P value <0.01
   and fold change >2) compared to SNCA^Iso neurons ([113]Fig. 4A). Gene
   ontology analysis of significantly up-regulated transcripts in
   SNCA^Dupl neurons further revealed enrichment in genes associated with
   ECM-related activities, such as ECM organization and cell-cell adhesion
   ([114]Fig. 4B, top). Notably, VIM (encoding for vimentin) was
   identified as one of the most up-regulated genes in SNCA^Dupl midbrain
   neurons. Vimentin is an element of intermediate filaments, a crucial
   component of the cytoskeleton, and is involved in the modulation of
   signaling cascades that regulate ECM modeling ([115]29). Consistent
   with alterations in ECM, genes encoding several collagen isoforms were
   found to be up-regulated in SNCA^Dupl midbrain neurons ([116]Fig. 4A).
   In addition, we observed the down-regulation of genes associated with
   neuronal maintenance and function in SNCA^Dupl neurons, consistent with
   the observed neurite deficits. Enriched pathways included synaptic
   signaling, synapse organization, neuronal projection, and neuronal
   differentiation ([117]Fig. 4B, bottom). Regarding neuronal
   differentiation, PAX6 was identified as one of the most down-regulated
   genes. In addition, LHX5, a member of the LHX family of transcription
   factors, which is involved in brain development, was found to be
   another gene among the most down-regulated genes in SNCA^Dupl neurons
   ([118]Fig. 4A). GPRIN3, a member of the G protein–regulated inducer of
   neurite outgrowth family known for its regulatory role in dopamine
   receptor signaling ([119]30), exhibited significant down-regulation in
   SNCA^Dupl midbrain neurons ([120]Fig. 4A and fig. S4A). GPRIN3 is
   situated adjacent to the SNCA locus and is also present within the
   heterozygous duplicated locus in SNCA^Dupl cells. As expected,
   CRISPR-Cas9–mediated gene editing of SNCA (SNCA^Iso) did not initially
   affect the gene expression of GPRIN3, indicated by consistently
   elevated mRNA levels, determined using RT-qPCR in both SNCA^Iso and
   SNCA^Dupl NPCs compared to control NPCs (fig. S4A). During midbrain
   neuronal differentiation, however, GPRIN3 expression is substantially
   down-regulated in SNCA^Dupl neurons, implying its role in neuronal
   differentiation. This observation also highlights the impact of
   increased SNCA dosage on the dynamic regulation of GPRIN3 expression.

Fig. 4. Transcriptomic and proteomic analyses.

   [121]Fig. 4.
   [122]Open in a new tab

   (A) Volcano plot of differentially expressed genes identified by
   transcriptomics via RNA-seq comparing SNCA^Dupl to SNCA^Iso midbrain
   neurons. Thresholds for log[2]-fold changes were set to 1 and −1 (gray
   lines). Transcript annotation was performed using the ggrepl package in
   R. Significantly up- (right) and down-regulated (left) genes are
   highlighted in red. X axis, log[2]-fold change of the mean expression
   levels; y axis, negative decade logarithm of the P value [−log[10] (P
   value)]. (B) Gene ontology pathway analysis of differentially regulated
   genes. Top 10 pathways for up- (top) and down-regulated genes (bottom)
   in SNCA^Dupl midbrain neurons are displayed. For (A) and (B), N = 2
   differentiation rounds, with experimental duplicates per round. (C)
   Correlation analysis of proteomic and transcriptomic hits based on
   Pearson correlation coefficient. The analysis includes 421
   differentially expressed proteins and mRNAs with at least a twofold
   change (log[2] <−1 or >1) and an adjusted P value <0.05. A significant
   correlation is observed (r^2 = 0.64, P = 2.69 × 10^−49) between the
   transcriptomic and proteomic hits. VIM as one of the most up-regulated
   elements and PAX6 as one of the most down-regulated elements are
   highlighted. (D) Gene ontology pathway analysis of differentially
   expressed features both at protein and mRNA levels in SNCA^Dupl
   compared to SNCA^Iso midbrain neurons. Top 10 pathways for commonly
   up-regulated (top) and down-regulated features (bottom) in SNCA^Dupl
   midbrain neurons are depicted. X axis, adjusted – log[10] P value; y
   axis, top 10 identified pathways for up-regulated (top) or
   down-regulated features (bottom). For (C) and (D), N = 2
   differentiation rounds for SNCA^Dupl midbrain neurons and N = 3
   differentiation rounds for SNCA^Iso midbrain neurons, with experimental
   duplicates per round.

   Next, we conducted a proteomic analysis of SNCA^Dupl and SNCA^Iso
   midbrain neurons. Consistent with the transcriptomic results, we
   observed increased protein levels of vimentin and proteins crucial for
   ECM activity, such as collagen isoforms, alongside decreased protein
   levels of neuronal differentiation–associated transcription factors
   like PAX6, LHX5, and GPRIN3 (fig. S4B). This prompted us to investigate
   whether changes in gene transcription are reflected in protein
   abundance. We compared differentially regulated transcripts and
   proteins, exhibiting changes of log[2] <−1 or >1 fold with an adjusted
   p value <0.05. We identified 425 up-regulated and 60 down-regulated
   features that are changed in SNCA^Dupl neurons at both mRNA and protein
   levels, displaying a robust correlation of Pearson = 0.64 [P = 2.69 ×
   10^−49; 95% confidence interval (0.58. 0.69), [123]Fig. 4C]. Gene
   ontology analysis using these features regulated at both mRNA and
   protein levels confirmed the enrichment of ECM-associated processes
   (such as ECM organization and cell-cell adhesion) specifically through
   SNCA dosage enhancement, while processes associated with the nervous
   system (such as brain development and synapse organization) were
   down-regulated ([124]Fig. 4D). In summary, through a combined
   transcriptomic and proteomic analysis, we established a robust
   correlation between gene and protein expression levels, highlighting
   the impact of SNCA dosage on cellular processes related to neuronal
   differentiation, function, and cellular structural integrity.

SNCA duplication alters the chromatin accessibility landscape

   SNCA duplication resulted in substantial changes in the gene expression
   and proteome of midbrain neurons, but the molecular mechanisms
   underlying these alterations remain incompletely understood. Since gene
   expression is tightly regulated by chromatin accessibility, we
   performed ATAC-seq to investigate changes in the chromatin
   accessibility landscape. To further identify transcriptional regulators
   driving gene expression, we conducted motif enrichment analysis of open
   chromatin regions to infer key transcription factors driving gene
   expression during development mediated by SNCA duplication.

   Analysis of ATAC-seq data indicated that SNCA^Dupl was associated with
   a >2-fold increase in ATAC signal at 3476 and a >2-fold decrease at
   4939 distal sites (>±3 kb from transcription start site; adjusted P
   value <0.05) in midbrain neurons ([125]Fig. 5A). As two examples of
   affected genes, PAX6 demonstrated decreased open chromatin regions,
   while VIM exhibited increased open chromatin regions in SNCA^Dupl
   midbrain neurons ([126]Fig. 5B), consistent with their altered
   expression levels identified through transcriptomic analysis ([127]Fig.
   4A).

Fig. 5. Analysis of chromatin accessibility and transcription factor motif
enrichment using ATAC-seq.

   [128]Fig. 5.
   [129]Open in a new tab

   (A) Scatterplot of ATAC-seq peaks in SNCA^Dupl versus SCNA^Iso neurons.
   Color codes indicate significant changes (orange and green are fold
   change >2 and <−2, respectively, Padj <0.05, calculated from DESeq2
   analysis). (B) Genome browser tracks of ATAC-seq of SNCA^Dupl (orange)
   and SNCA^Iso (green) neurons at the PAX6 and VIM loci. (C) De novo
   motif analysis of ATAC-seq peaks at regions of open chromatin. Target
   (%) is the number of target sequences with motifs over total target
   sequences; %BG (%background) is the number of background sequences with
   motif over total background sequences. Data were derived from N = 2
   differentiation rounds, with experimental duplicates/round. P values
   were calculated using binomial distribution in HOMER.

   Additional gene ontology analysis of genes exhibiting differential
   chromatin accessibility between SNCA^Dupl and SNCA^Iso neurons revealed
   enrichment of genes associated with various aspects of nervous system
   development, such as head development and regulation of nervous system
   development as well as pathways related to neuronal functions like
   synaptic signaling. In addition, genes involved in cellular structural
   elements, including cell morphogenesis and cell-cell adhesion, were
   found to be enriched (fig. S5). Notably, the enriched pathways exhibit
   notable similarities to pathways found in transcriptomics and
   proteomics ([130]Fig. 4, B and D). This implies an interplay between
   expression regulation and chromatin accessibility in those observed
   biological processes.

   To identify key transcription factors underlying the observed changes
   in gene expression between SNCA^Dupl and SNCA^Iso midbrain neurons, we
   performed de novo motif analysis of differentially regulated enhancers
   ([131]Fig. 5C). One common transcription factor–binding motif
   identified was associated with AP-1. This AP-1–binding motif is highly
   enriched in open chromatin regions exhibiting both increased and
   reduced accessibility in SNCA^Dupl compared to SNCA^Iso midbrain
   neurons. AP-1 is known to be involved in various neuronal functions
   including cell growth, differentiation, survival, and synaptic
   plasticity ([132]31). Moreover, the most significantly enriched
   sequences in SNCA^Dupl corresponded to motifs recognized by several
   additional transcriptional factors like FOXA1, RFX5, and SMAD2. These
   transcription factors have been associated with neuronal development
   and survival ([133]32), development and maintenance of specific
   neuronal subtypes ([134]33, [135]34), as well as neuronal function
   ([136]32). In contrast, binding motifs for MEIS1 and CUX2 were enriched
   in peaks reduced or lost in SNCA^Dupl compared to SNCA^Iso midbrain
   neurons. These transcription factors are also implicated in neuronal
   differentiation and brain development ([137]35, [138]36). In summary,
   SNCA duplication in midbrain neurons induced significant alterations in
   chromatin accessibility and usage of transcription factors involved in
   neuronal development and synaptic signaling.

SNCA duplication decreases PAX6 expression during neuronal differentiation

   Our combined multi-omics approaches unveiled alterations in neuronal
   differentiation induced by SNCA^Dupl, characterized particularly by a
   down-regulation of PAX6 expression ([139]Fig. 4). Since PAX6 is a
   well-known NPC marker, we examined its expression at the NPC stage. WB
   analysis using an antibody targeting a 17–amino acid PAX6 peptide
   showed no significant difference in PAX6 levels between SNCA^Dupl and
   SNCA^Iso NPCs (fig. S6A). However, mass spectrometric (MS) analysis for
   detecting various proteoforms across the entire protein sequence,
   demonstrated a significant reduction in PAX6 protein expression
   ([140]Fig. 6A, left). Supporting this finding, ICC analysis showed a
   significant decrease in PAX6 fluorescence intensity ([141]Fig. 6B and
   fig. S6B), and flow cytometry revealed a smaller PAX6-immunoreactive
   population (fig. S6C) in SNCA^Dupl NPCs compared to SNCA^Iso NPCs.
   Consistently, RT-qPCR further confirmed reduced PAX6 expression at the
   mRNA level ([142]Fig. 6A, right). Among crucial transcriptional factors
   involved in neuronal development and differentiation into mature
   neurons, such as Nestin, Sox2, and PAX6 ([143]37–[144]39), which were
   all expressed in both SNCA^Iso and SNCA^Dupl NPCs ([145]Fig. 6B), only
   PAX6 exhibited a significant reduction in SNCA^Dupl NPCs as assessed by
   MS analysis ([146]Fig. 6A, left).

Fig. 6. PAX6 expression.

   [147]Fig. 6.
   [148]Open in a new tab

   (A) Left, MS protein analysis of Nestin, Sox2, and PAX6, demonstrating
   unchanged levels of Nestin and Sox2 between SNCA^Dupl and SNCA^Iso
   NPCs. However, PAX6 levels are significantly reduced in SNCA^Dupl NPCs.
   Right, RT-qPCR analysis of PAX6 mRNA, showing a significant
   down-regulation in SNCA^Dupl NPCs. (B) ICC analysis of NPC markers
   Nestin (red), Sox2 (cyan), and PAX6 (green) in NPCs. (C) Left, MS
   analysis of PAX6 protein levels; right, RNA-seq analysis of PAX6 mRNA
   levels in differentiated midbrain neurons. Both display a significant
   decrease in PAX6 gene and protein levels in SNCA^Dupl neurons. For (A)
   left and (C), N = 2–3 differentiation rounds for SNCA^Dupl midbrain
   neurons and N = 3 differentiation rounds for SNCA^Iso neurons, with
   n = 2 technical replicates. For (A) right, N = 3 independent
   differentiation rounds. Statistics in [(A), left], two-way analysis of
   variance (ANOVA) with Šídák’s multiple comparisons.

   Notably, the down-regulation of PAX6 expression persisted during
   midbrain neuronal differentiation. Both MS and WB demonstrated
   decreased PAX6 protein levels ([149]Fig. 6C, right, and fig. S6A),
   while RNA-seq analysis and RT-qPCR confirmed reduced PAX6 mRNA
   expression ([150]Fig. 6C, left, and fig. S6D). In total, multiple
   complementary approaches revealed a significant reduction in PAX6
   expression in SNCA^Dupl cells, both at the mRNA and protein level, as
   well as in both NPCs and differentiated midbrain neurons.

SNCA duplication disrupts vimentin levels, truncation, organization, and
neurite distribution

   Our unbiased multi-omics analyses identified vimentin as one of the
   most significantly differentially expressed proteins. Vimentin, an
   integral component of the cytoskeleton, is involved in ECM
   organization, regulating collagen expression and release [reviewed in
   ([151]29)], as well as in neuronal differentiation ([152]40) and
   neurite development ([153]39). Since these cellular processes are
   particularly affected in SNCA^Dupl midbrain neurons according to
   pathway enrichment analyses ([154]Fig. 4, B and D), we proceeded to
   validate the effects of SNCA^Dupl on vimentin biology compared to
   SNCA^Iso neurons.

   Firstly, we validated vimentin expression levels via WB analysis, using
   two different antibodies targeting C-terminal and distal N-terminal
   regions of the protein ([155]Fig. 7, A to C). We confirmed markedly
   elevated vimentin levels in SNCA^Dupl midbrain neurons using both
   antibodies, although the determined fold changes varied. These
   differences may result from variations in antibody binding affinity,
   potentially influenced by distinct posttranslational modification
   patterns of vimentin in hiPSC-derived neurons. The significant increase
   in vimentin expression in SNCA^Dupl neurons was already evident at the
   NPC stage (fig. S7A). Intriguingly, we detected a vimentin band below
   55 kDa, which was exclusively recognized by the C-terminal antibody and
   showed a significant increase in intensity ([156]Fig. 7A). Extending
   the SDS–polyacrylamide gel electrophoresis (SDS-PAGE) run time resolved
   several additional vimentin-related bands below the full-length
   vimentin band, which were detected by either N- or C-terminal vimentin
   antibodies, suggesting the presence of various truncated forms
   ([157]Fig. 7D and fig. S7B). Since the truncated forms below 55 kDa
   were barely detected in SNCA^Iso neurons and increased in SNCA^Dupl
   ([158]Fig. 7A), we focused on the characterization of these forms in
   the following analyses. We conducted MS analysis following in-gel
   digestion of full-length vimentin (466 amino acids) and the <55-kDa
   truncated forms (fig. S7C and table S5). Consistent with their absence
   when using the distal N-terminal antibody, MS revealed the missing
   N-terminal fragment ^13RMFGGPGTASRPSSSR^28 in the truncated forms,
   indicating that the N-terminal truncation involves this fragment.
   Furthermore, MS identified the loss of the distal C-terminal fragment
   ^451DGQVINETSQHHDDLE^466. Despite the absence of this most C-terminal
   fragment, the C-terminal antibody can still detect truncated vimentin,
   assuming the binding to a broader region within the C-terminal domain.

Fig. 7. Vimentin expression, truncation, subcellular localization, and
organization.

   [159]Fig. 7.
   [160]Open in a new tab

   (A and B) WB analysis of vimentin using the C-terminal [C-Vim-Ab, (A)]
   and the N-terminal [N-Vim-Ab, (B)] vimentin antibodies. Vimentin levels
   are significantly increased in SNCA^Dupl neurons compared to SNCA^Iso
   neurons. Notably, the levels of truncated vimentin <55 kDa (Tr, red) is
   increased in SNCA^Dupl neurons (A). LC, Ponceau staining. For (A),
   N = 6; for (B) N = 3 differentiation rounds. (C) Vimentin amino acid
   sequence and epitopes targeted by the N- and C-terminal antibodies. (D)
   WB image of a SNCA^Dupl sample, probed with the C- (red) or N-terminal
   (green) vimentin antibody, following SDS-PAGE with extended run time.
   Truncated vimentin forms are detected by C- and N-terminal antibodies
   and highlighted in green and red, respectively. (E and F) WB analysis
   of vimentin in the putamen of patients with sporadic PD and controls
   using C- and N-terminal vimentin antibodies. Truncated forms detected
   by the C-terminal antibody only are highlighted in red. LC, Ponceau
   staining. Both antibodies detected a trend toward increased vimentin
   levels in sporadic PD. Additional quantification of the ratio of
   truncated/full-length vimentin [(E) bottom right] demonstrates a trend
   toward elevated truncation in sporadic PD. N = 5 per group. (G) ICC
   images of vimentin (white) and bTubIII (red). Arrows highlight
   vimentin^+ and bTubIII^+ neurites. (H) Quantification of vimentin^+ and
   bTubIII^+ double-positive neurites among all bTubIII^+ neurite,
   demonstrating a significant decrease in neuritic vimentin distribution
   in SNCA^Dupl neurons compared to SNCA^Iso neurons. n = 6 individual
   cells/group of N = 3 differentiation rounds. (I) Analysis of vimentin
   solubility using a solubility assay followed by WB analysis of vimentin
   using the C-terminal vimentin antibody. SNCA^Dupl neurons show a
   significant increase in the ratio of soluble (S)/insoluble (IS)
   vimentin and an overall higher amount of truncated vimentin forms in
   both soluble and insoluble fractions, compared to SNCA^Iso neurons. LC,
   Ponceau staining. N = 3 differentiation rounds.

   To assess the pathological relevance of these observations on vimentin
   in synucleinopathies, we extended the vimentin analysis to human
   postmortem putamen tissue derived from patients with sporadic PD and
   controls. Despite the limited sample size (n = 5 per group), we noted a
   trend toward increased total vimentin levels in patients with PD using
   both vimentin antibodies ([161]Fig. 7, E to F, left and right), in line
   with our findings in hiPSC-derived SNCA^Dupl midbrain neurons.
   Moreover, WB analysis revealed at least three different vimentin
   truncation bands ([162]Fig. 7E) in human putamen tissue. These forms
   were detected either by both antibodies or only by the C-terminal
   antibody, with higher levels observed in patients with PD ([163]Fig.
   7E, middle quantification). In addition, we analyzed vimentin in mouse
   models of PD and multiple system atrophy (MSA), overexpressing human
   aSyn either under the Thy-1 promoter [Thy1-SNCA, line 61, ([164]41)] or
   the myelin basic protein promoter [MBP-SNCA, line 29, ([165]42)],
   respectively. In both synucleinopathy mouse models, we observed
   significantly elevated vimentin levels in brain tissue compared to
   their respective nontransgenic counterparts (fig. S7, D and E).
   Increased vimentin truncation was also observed in synucleinopathy
   mouse brains, detected by the N-terminal antibody. The discrepancy in
   the sensitivity of vimentin antibodies to detect truncated forms in
   different models, such as hiPSC-derived neurons, postmortem PD brains,
   and synucleinopathy mouse models, may result from variations in
   truncation sites, protease involvement, and the degree of vimentin
   cleavage in each system. These factors likely lead to differential
   exposure of C-terminal or N-terminal epitopes, which are recognized by
   the respective antibodies. Despite these discrepancies in truncation
   detection, our collective data from different models point to an
   interplay between aSyn overexpression and increased vimentin expression
   and truncation.

   Given the pronounced neuritic phenotypes in SNCA^Dupl midbrain neurons,
   we proceeded to analyze vimentin distribution in neuritic processes by
   ICC analysis. We observed a notable decrease in vimentin-positive
   neurites despite increased protein expression, with vimentin
   accumulating in somata ([166]Fig. 7, G to H).

   Further, we explored potential alterations in vimentin organization by
   analyzing its solubility via ultracentrifugation, thereby evaluating
   its integration into cytoskeletal networks. In SNCA^Iso midbrain
   neurons, vimentin was predominantly found in the insoluble fraction,
   whereas a notable shift toward the soluble fraction was observed in
   SNCA^Dupl midbrain neurons accompanied by increased truncated vimentin
   levels in both soluble and insoluble fractions ([167]Fig. 7I). In
   summary, our analysis of vimentin in SNCA^Dupl midbrain neurons
   revealed significant effects of increased SNCA dosage on various
   aspects of vimentin biology, including elevated expression and
   truncation, reorganization from neuronal processes to cell bodies, and
   redistribution toward the soluble fraction.

Vimentin interference reverses SNCA duplication–induced bTubIII and neurite
disruptions

   After identifying vimentin alterations specifically in response to
   SNCA^Dupl, we explored whether changes in vimentin affect key aspects
   of SNCA^Dupl-induced phenotypes. To address this, we interfered with
   vimentin organization using okadaic acid and withaferin A. Okadaic acid
   induces vimentin hyperphosphorylation and reorganization ([168]43),
   while withaferin A directly binds to vimentin, influencing the
   integrity of the vimentin network ([169]44, [170]45).

   Following okadaic acid treatment, we observed a significant reduction
   in vimentin levels and truncation in SNCA^Dupl neurons, effectively
   counteracting the key effects of SNCA^Dupl on vimentin when compared to
   SNCA^Iso neurons ([171]Fig. 8A). In the present and our previous
   studies, SNCA^Dupl has been linked to impaired neurite phenotypes
   ([172]18) and axonal transport dysfunction ([173]19) in neurons derived
   from different patients with SNCA^Dupl compared to neurons of healthy
   donors, accompanied by a characteristic decrease in bTubIII levels
   ([174]18, [175]46). For this reason, we further investigated bTubIII
   levels via WB and observed a significant decrease in bTubIII levels in
   SNCA^Dupl neurons reversed by okadaic acid treatment ([176]Fig. 8B).
   Moreover, okadaic acid significantly enhanced neurite diameter and
   volume in SNCA^Dupl midbrain neurons ([177]Fig. 8, C and D), indicating
   a restoration of SNCA^Dupl neurite phenotypes.

Fig. 8. Effects of vimentin interference on vimentin, bTubIII, and neuritic
phenotype.

   [178]Fig. 8.
   [179]Open in a new tab

   (A) WB analysis of vimentin in okadaic acid–treated neurons using the
   C-terminal vimentin antibody, showing reduced levels of total and
   truncated vimentin in SNCA^Dupl neurons upon treatment. (B) WB analysis
   of bTubIII, demonstrating reversed bTubIII levels in SNCA^Dupl neurons
   treated with okadaic acid. For (A) and (B), LC, Ponceau staining. Blots
   within the black frame were derived from the same membrane. Lanes from
   different parts of the same membrane are separated by dashed lines. (C
   and D) ICC analysis of neurite diameter and volume of bTubIII^+
   neurites. Okadaic acid treatment rescues the decrease in neurite
   diameter and volume observed in SNCA^Dupl neurons compared to SNCA^Iso
   neurons. (E) Solubility analysis of vimentin, showing a dose-dependent
   decrease in soluble vimentin levels in SNCA^Dupl neurons treated with 2
   or 5 μM withaferin A (WFA), reversing the effect of SNCA^Dupl compared
   to SNCA^Iso neurons. (F) WB analysis of bTubIII levels, demonstrating a
   restoration of bTubIII levels in SNCA^Dupl neurons upon withaferin A
   treatment. For (E) and (F), LC, revert 520 Total Protein Stain. (G and
   H) ICC analysis of bTubIII^+ neurites demonstrates that withaferin A
   treatment (2 μM) increases neurite diameter (G) and volume (H) in
   SNCA^Dupl neurons. (I) Schematic overview of the effects of vimentin
   interference on SNCA^Dupl phenotypes. Solid lines indicate findings
   revealed in this study, while dashed lines represent proposed
   interactions based on current data. For (C), (D), (G), and (H), neurite
   diameter was analyzed for 320 to 500 bTubIII^+ neurites per group, and
   neurite volume was measured for 71-1142 bTubIII^+ neurites per group.
   For all quantifications, N = 3 differentiation rounds.

   The effects of targeting vimentin via withaferin A were analyzed by
   assessing vimentin levels, truncation, and solubility. Withaferin A did
   not induce a statistically significant decrease in total vimentin
   levels; however, a slight reduction was observed at the 5 μM
   application dose (fig. S8A). Notably, a decrease in truncated vimentin
   levels was observed, reaching statistical significance at a
   concentration of 5 μM withaferin A (fig. S8A). Intriguingly, withaferin
   A modulated the vimentin network in both SNCA^Iso and SNCA^Dupl
   neurons, as evidenced by a reduction in soluble vimentin ([180]Fig.
   8E). Notably, withaferin A treatment restored the increased soluble
   vimentin levels observed in SNCA^Dupl neurons. Similar to the effect of
   okadaic acid on bTubIII, withaferin A treatment resulted in an increase
   in bTubIII levels in SNCA^Dupl neurons ([181]Fig. 8F). Neurite
   phenotype analysis revealed a significant increase in both neurite
   diameter ([182]Fig. 8G) and neurite volume ([183]Fig. 8H) in SNCA^Dupl
   neurons exposed to withaferin A. In addition, a restoration of neurite
   volume could be observed in treated SNCA^Dupl neurons compared to
   SNCA^Iso neurons ([184]Fig. 8H).

   In SNCA^Iso neurons, the effects of both vimentin-interfering compounds
   were less pronounced for most measured parameters ([185]Fig. 8). In
   certain instances, such as bTubIII levels ([186]Fig. 8, B and F) and
   neurite volume ([187]Fig. 8, D and H), the effects even trended in the
   opposite direction compared to those observed in SNCA^Dupl neurons.
   These findings highlight the critical role of aSyn dosage in preserving
   neuronal integrity.

   Given disturbed aSyn homeostasis observed in SNCA^Dupl neurons, we
   additionally analyzed aSyn following treatment of okadaic acid or
   withaferin A. WB analysis of aSyn levels revealed a significant
   increase in aSyn levels (~2-fold) in SNCA^Dupl neurons after okadaic
   acid treatment (fig. S8B), thus restoring aSyn levels, which tended to
   be reduced at the late neuronal differentiation stages compared to
   SNCA^Iso neurons ([188]Fig. 2, A and B). However, aSyn levels remained
   unchanged following withaferin A treatment (fig. S8D), and neither
   compound restored the increased aSyn accumulation in neurites of
   SNCA^Dupl neurons (fig. S8, C and E). These results suggest that
   okadaic acid and withaferin A have distinct effects on aSyn levels but
   do not affect its spatial distribution within SNCA^Dupl neurons under
   our experimental conditions.

   In summary, our study using two independent vimentin-interfering
   compounds highlights the potential of modulating vimentin network to
   restore SNCA^Dupl-induced alterations in bTubIII levels and neurite
   phenotypes ([189]Fig. 8I). These findings underscore the significance
   of vimentin in modulating the impact of increased SNCA dosage on
   cytoskeletal organization and ultimately neurite integrity.

DISCUSSION

   In this study, we identified specific effects of elevated SNCA gene
   dosage on dynamic aSyn changes during neuronal differentiation combined
   with alterations in the regulatory differentiation machinery and
   modifications in the cytoskeletal network. These molecular events
   resulted in a profound neurite phenotype at the cellular level. In
   particular, the data strongly underscore the critical interplay between
   neuronal aSyn and vimentin, both influencing neurite phenotypes.

   Our findings provide notable insights into the effects of SNCA
   multiplication on aSyn dynamics during neuronal differentiation. Of
   particular interest is the initial rise in aSyn levels in
   differentiating SNCA^Dupl cells, followed by a pronounced decrease
   compared to control or SNCA^Iso neurons. This dynamic change is rarely
   reported in hiPSC-based studies, which predominantly focus on SNCA
   triplication and endpoint comparisons. However, a study by Fukusumi
   et al. ([190]47) similarly observed a dynamic decline in aSyn levels
   during midbrain neuronal differentiation in SNCA triplication neurons,
   albeit using a growth factor–based protocol. The dynamic aSyn changes
   in SNCA^Dupl cells suggest a finely tuned regulation of aSyn
   homeostasis during neuronal differentiation. A plausible explanation
   for the reduced total aSyn levels in SNCA^Dupl neurons at advanced
   differentiation stages may involve the activation of compensatory
   mechanisms, such as the autophagy-lysosomal protein clearance pathway,
   in response to the initially elevated aSyn levels to degrade toxic
   forms of aSyn. Despite the overall reduction in total aSyn levels, we
   detected a significant increase in aSyn aggregation in SNCA^Dupl
   neurons, along with a pronounced neuritic accumulation of aSyn in
   SNCA^Dupl neurons, especially evident when using an aSyn
   aggregate–sensitive antibody. These findings indicate a shift in aSyn
   homeostasis toward aggregation and a spatial redistribution of
   aggregated aSyn to neurites, correlating with the neurite deficits in
   these neurons. Elevated aSyn accumulation within neurites can disrupt
   crucial cytoskeleton-dependent transport processes between the cell
   body and nerve terminals, leading to neurite dysfunction, as evidenced
   in prior research ([191]10, [192]11, [193]48), including our own
   ([194]19). Given that dopaminergic neurons are the predominant neuronal
   subtype in our model, they may likely be the primary contributor to the
   distinct pattern of intraneuronal aSyn dynamics we observed. This
   pattern may contrast with findings in the limited postmortem brain
   studies of SNCA multiplication patients, where increased soluble aSyn
   protein levels have been reported ([195]49). The differences may
   reflect the complexity of cell populations in postmortem tissue, which
   contribute to a more heterogeneous landscape of aSyn regulation.

   Neurite deficits may be a result of disturbed neuronal differentiation
   pathways in SNCA^Dupl, as evidenced by comprehensive transcriptome and
   proteome analyses in our study. Moreover, our ATAC-seq data, showing
   SNCA^Dupl-mediated changes in the chromatin landscape, support a
   regulatory role of aSyn at the transcriptional levels. Particularly
   noteworthy was the pronounced down-regulation of PAX6 expression in
   SNCA^Dupl NPCs and midbrain neurons compared to their respective
   SNCA^Iso counterparts, suggesting an intricate interplay between aSyn
   and PAX6. Tight regulation of PAX6 is crucial for balancing cell
   proliferation, neuronal differentiation, and proper brain patterning
   ([196]50). Specifically, in dopaminergic neurons, PAX6 deficiency has
   been linked to abnormal neuronal localization and impaired axonal
   pathfinding in murine brains ([197]51). These effects may be associated
   with disturbances in the ECM organization, which play a key role in the
   axonal guidance environment. Furthermore, a reduction in PAX6
   immunoreactive cells has been documented in the postmortem brain of
   patients with PD ([198]52). Our findings highlight the impact of
   elevated SNCA dosage, which triggers reduced PAX6 expression, alongside
   alterations in ECM organization and neurite deficits. However, the
   exact regulatory mechanisms through which aSyn influences PAX6 remains
   unclear. Considering the nuclear localization of aSyn ([199]20,
   [200]53) and accumulating evidence for its role as a transcriptional
   modulator ([201]54), nuclear aSyn may participate in the
   transcriptional regulation of PAX6 expression (i) indirectly via
   modification of histones ([202]55) or interaction with collaborating
   transcription factors or (ii) directly via binding to the PAX6 gene,
   thus acting as a transcription factor ([203]56). Exploring whether and
   how aSyn regulates transcription and in particular PAX6 expression
   could be further investigated in future studies using genomics
   approaches, such as transcription factor chromatin immunoprecipitation
   followed by sequencing.

   hiPSC cells generated from individuals with SNCA multiplication exhibit
   variations in the length of the multiplication stretch ([204]57). Some
   phenotypes described in this study, such as disrupted aSyn homeostasis,
   altered bTubIII expression, and neurite abnormalities, were
   consistently observed in SNCA^Dupl neurons. These findings were
   replicated in comparisons with both neurons from healthy donors without
   any SNCA mutations [in this and our previous study ([205]18)] and
   isogenic control neurons (in this study). These results suggest that
   the effects observed are primarily attributable to SNCA^Dupl. However,
   the contribution of other genes flanking the SNCA gene within the
   duplicated genomic region cannot be excluded. One such neighboring gene
   is GPRIN3, directly located at the 3′ site of SNCA. It was unexpectedly
   identified in our study using unbiased multi-omics approaches as one of
   the most down-regulated features in SNCA^Dupl neurons at both the mRNA
   and protein levels. While the specific function of the GPRIN3 protein,
   particularly in the context of PD, remains undefined, a study by
   Karadurmus et al. ([206]30), characterizing a GPRIN3 knockout mouse
   model, demonstrated the role of GPRIN3 protein as a mediator of
   dopamine receptor D2R in the striatum. This finding suggests the GPRIN3
   protein as a functional partner of the midbrain dopaminergic pathway.
   Our finding of reduced GPRIN3 gene expression in SNCA^Dupl midbrain
   neurons could be explained by regulatory interaction of SNCA with
   GPRIN3, both possibly sharing common regulatory genomic features. The
   potential contribution of neighboring genes in the SNCA multiplication
   region is largely unexplored and should be considered in future
   studies. Further functional investigations are necessary to dissect the
   individual effects and elucidate interplay of SNCA and neighboring
   genes affected by multiplication. These studies will provide a more
   comprehensive understanding of the mechanisms underlying SNCA
   multiplication–related phenotypes.

   Our previous studies comparing midbrain neurons from different
   SNCA^Dupl cases and control donors revealed a direct interaction
   between aSyn and microtubule elements ([207]18). The microtubule
   network, composed of α- and β-tubulin subunits, is a key component of
   the cytoskeleton and plays a pivotal role in neurite organization and
   development. Notably, we observed a significant reduction in bTubIII
   across all investigated SNCA^Dupl cases ([208]18, [209]46), alongside
   increased coaggregation and co-redistribution of aSyn and bTubIII
   ([210]18). In our present study comparing SNCA^Dupl and SNCA^Iso
   midbrain neurons, we highlight the specific effect of elevated SNCA
   dosage on bTubIII reduction, linked to a massive decrease in neurite
   network formation and parameters associated with neurite development
   such as neurite diameter and volume.

   An important finding of this study is the identification of the
   interaction between aSyn homeostasis and the vimentin network.
   Vimentin, an essential intermediate filament protein, represents
   another crucial component of the cytoskeleton in addition to
   microtubule. While previous research predominantly focused on its role
   in astrocytes, particularly in astrogliosis ([211]58), recent studies
   have highlighted its significance in neuronal differentiation, axon
   outgrowth, and regenerative processes in neurons and neurites
   ([212]59). Beyond its role as a cellular structural organizer, vimentin
   has been demonstrated to inhibit the long noncoding RNA Gas5, which is
   expressed age-dependently and suppresses axonal regeneration ([213]60).
   In the context of neurodegeneration, neuronal vimentin has been
   observed in the cytoplasm and dendrites of neurons in the cortex,
   hippocampus, and cerebellum of patients with Alzheimer’s disease,
   regions known to exhibit intra- and extracellular amyloid-β (1–42)
   (Abeta42) deposition. Colocalization of neuronal vimentin and Abeta42
   is prevalent in these brain regions ([214]61).

   In this study, we shed light on the multifaceted impact of increased
   SNCA dosage on vimentin biology within neurons. This encompasses a
   notable increase in vimentin levels, enhanced truncation (especially
   evident in differentiated neurons), a shift from insoluble to soluble
   fractions (indicating its release from the cytoskeletal network), and
   its redistribution from neurites to the soma. Although the relevant
   data are limited, several studies have suggested a regulatory role of
   aSyn in vimentin: A significant decrease in vimentin levels upon
   depletion of aSyn expression was demonstrated in SNCA knockout melanoma
   cells; conversely, in SH-SY5Y cells, overexpression of aSyn led to a
   drastic increase in vimentin levels ([215]62). Another study, which
   induced aggresomal structures resembling LB via Parkin overexpression,
   confirmed the presence of both aSyn and vimentin within these
   structures ([216]63). Vimentin was reported to coordinate the transport
   of proteasome to the aggresome, thereby contributing to proteostasis
   ([217]64). Regarding vimentin truncation, limited proteolysis of
   vimentin from both the N and C termini has been documented in several
   studies, demonstrating that vimentin truncation significantly increases
   its solubility, thereby interrupting its assembly ([218]65–[219]67).
   Our data obtained from SNCA^Dupl cell model further support this
   notion. Intriguingly, both vimentin levels and truncation were markedly
   elevated in mouse models of PD and MSA overexpressing human aSyn.
   Similar patterns were also observed in postmortem putamen from patients
   with sporadic PD. Proteases, such as caspases and calpains ([220]65,
   [221]66), have been shown to cleave vimentin, producing various
   truncated vimentin forms. Notably, studies on postmortem brains and PD
   animal models have reported elevated calpain levels and activity, as
   well as the protective effects of calpain inhibition ([222]68–[223]70).
   Further research is needed to identify the precise cleavage sites
   involved in aSyn-mediated vimentin truncation. Overcoming the technical
   challenges in isolating specific truncated forms and dissecting their
   functions in biological models will be crucial. The regulatory
   mechanisms driving aSyn-mediated vimentin truncation, including the
   role of proteases, require further investigation.

   While elucidating the effect of increased SNCA dosage on vimentin
   organization, we also highlighted the impact of vimentin interference
   on key phenotypes observed in SNCA^Dupl midbrain neurons. Both okadaic
   acid and withaferin A, despite different mechanisms of action, reversed
   vimentin-related phenotypes. Okadaic acid reduced vimentin levels,
   withaferin A restored the proportion of soluble vimentin, and both
   vimentin parameters were altered in SNCA^Dupl neurons. In addition,
   both compounds corrected deficits in bTubIII levels and neurite
   structure. These significant findings suggest that SNCA^Dupl-mediated
   vimentin alterations precede the microtubule- and neurite-associated
   phenotypes in midbrain neurons. Considering the emerging evidence in
   regard to the neuroprotective effects of withaferin A in PD-related
   cell and animal models ([224]71, [225]72), our findings highlight the
   intervention potential of targeting the vimentin pathway to protect
   against aSyn-mediated neurite degeneration, a very early cellular event
   in neurodegeneration. To validate this potential, further precise and
   comprehensive studies using more targeted interference of vimentin,
   such as genetic modification, are required. Moreover, further
   validation of compounds like withaferin A is necessary to explore their
   effects on the interplay between aSyn, vimentin, as well as neurite
   structure and function in different neural cell types using both
   cell-based and in vivo models, to assess their clinical potential.

   The vimentin phenotypes demonstrated in our studies are either less
   pronounced or rarely documented in other hiPSC-based neuronal models
   derived from SNCA triplication. This discrepancy may partly be
   influenced by variations in SNCA copy number. Clinical studies have
   provided evidence that different SNCA copy numbers in multiplication
   patients influence the penetrance of symptoms as well as disease onset
   and progression ([226]73–[227]75). In addition, various neighboring
   multiplication loci of SNCA may also contribute to these differences,
   underscoring the need for consideration in future studies.

   In summary, our present study highlights the specific impact of
   SNCA^Dupl on neuronal differentiation and neurite development through
   cytoskeletal disruption. SNCA dosage correction of aSyn to a normal
   level is able to restore these effects. Our findings further emphasize
   the modulatory potential of targeting the vimentin network.

MATERIALS AND METHODS

Experimental design with human-derived materials

Human iPSCs

   hiPSCs were derived from dermal fibroblasts obtained through skin
   biopsies from a patient with PD and healthy donors. Skin biopsies were
   conducted at the Movement Disorder Clinic, Department of Molecular
   Neurology, University Hospital Erlangen, following written informed
   consent obtained from voluntary donors. All experiments involving
   hiPSC-derived cells were conducted in accordance with Institutional
   Review Board approval (Nr. 259_17B) guidelines.

   Skin biopsy specimens were collected from a female patient with PD
   carrying SNCA duplication (SNCA^Dupl) (*3; table S1), as well as from
   two healthy individuals without history of neurological disease (*1 and
   *2; table S1). The patient with PD, who was 44 years old at the time of
   biopsy, began experiencing disease symptoms at the age of 39. The
   presence of SNCA^Dupl was confirmed using next-generation sequencing,
   which revealed a heterozygous duplication of a DNA region on chromosome
   4q22.1. The minimal estimated duplicated region spans from 88,007,635
   to 91,301,913 (3.59 Mb), while the maximal estimated duplicated region
   extends from 87,987,426 to 92,303,966 (4.32 Mb). The healthy donors, a
   male (*1) and a female (*2), were respectively 69 and 42 years old at
   the time of biopsy. Two PD-hiPSC clones [UKERi7GG-S1-004 (hiPSC clone
   SNCA^Dupl#1) and UKERi7GG–S1-005 (hiPSC clone SNCA^Dupl#2], derived
   from the patient with PD SNCA^Dupl (*3) and three control hiPSC lines
   (UKERiG3G-R1-039 from donor *1 (hiPSC clone Ctrl#1); UKERi1JF-R1-011
   (hiPSC clone Ctrl#2) and UKERi1JF-R1-018 (hiPSC clone Ctrl#3) from
   donor *2 were used in this study (table S1).

   CRISPR-Cas9 gene editing was conducted by using the PD SNCA^Dupl#1
   hiPSC line, generating the hiPSC line SNCA^Iso. Details regarding the
   skin biopsy donors and the hiPSC lines generated for this study are
   provided in table S1 and in our previous publication ([228]24). Unless
   specifically indicated in the figure legends, hiPSC lines Ctrl#3,
   SNCA^Dupl#2, and SNCA^Iso were used in analyses.

Human postmortem tissue

   Human postmortem samples of five patients with PD and five age- and
   gender-matched controls (table S2) were obtained from the Netherlands
   Brain Bank, Netherlands Institute for Neuroscience, Amsterdam (open
   access: [229]http://brainbank.nl). PD cases were clinically diagnosed
   by the presence of motor symptoms (bradykinesia, rigidity, and tremor)
   and neuropathologically confirmed by the occurrence of LB pathology. In
   this study, all PD cases exhibited neocortical LB, according to Braak
   Lewy stages 5 and 6 ([230]76).

Generation of an isogenic control hiPSC line through heterozygous knockout of
SNCA in a SNCA^Dupl hiPSC line

   For the design of single guide RNAs (sgRNAs) targeting SNCA gene, the
   web tool CRISPOR was used ([231]http://crispor.tefor.net/). Two sgRNAs
   were selected flanking the whole exon 2 of SNCA (table S3). Synthetic
   sgRNAs and recombinant Cas9 were both purchased from Synthego, CA, USA.
   The subsequent SNCA gene editing in the SNCA^Dupl hiPSC line was
   performed as described previously ([232]24) and summarized in fig. S1A.

   Specifically, hiPSCs were cultivated in mTeSR on a six-well plate
   precoated with GeltrexTM (Thermo Fisher Scientific, concentration
   according to the manufacturer’s recommendation) in mTeSR Plus (STEMCELL
   Technologies) with 1% penicillin-streptomycin at 37°C and 5% CO[2]. For
   CRISPR-Cas9, hiPSCs were dissociated using Accutase (Sigma-Aldrich) and
   nucleofected with the synthetic sgRNAs. Before transfection, 100 μM
   sgRNAs were mixed with 20 μM of recombinant Cas9 in a ratio of 7.5:1
   and incubated for 10 min at room temperature (RT) for in vitro
   assembly. hiPSCs were replated on six-well plates for 48 hours before
   single-cell isolation. Single cells were isolated via flow cytometry on
   96-well plates in mTeSR Plus containing CloneR (STEMCELL Technologies).
   After reaching confluency of 80 to 100%, each clone was split 1:2 into
   two 48-well plates for genotyping and downstream validation analyses.
   DNA of clones was isolated using QuickExtract (Lucigen) according to
   the manufacturer’s protocol, and DNA was amplified with the Q5
   High-Fidelity DNA Polymerase (New England Biolabs) using SNCA-specific
   primers (table S3) and separated by agarose gel electrophoresis
   ([233]Fig. 1B). Modified DNAs of candidate clones were isolated from
   agarose gel using QIAquick PCR Purification Kit (Qiagen) and validated
   by sequencing (LGC Genomics, Berlin; fig. S1B). Obtained sequences were
   prescreened for genetic insertion and deletion (indel) using the
   Inference of CRISPR Edits tool provided by Synthego
   ([234]https://ice.synthego.com/#/) (fig. S1C). The candidate clone
   showing an indel frequency between ~20 and 40% was harvested and
   verified for modification by sequencing (LGC Genomics, Berlin,
   Germany). Ultimately, modifications were confirmed by expression
   analysis via WB and RT-qPCR. In addition, off-target analysis was
   performed amplifying the five most homologous genomic sequences
   provided by the CRISPOR web tool (fig. S1E).

Differentiation of midbrain neurons from hiPSC

   Midbrain neurons were generated by small molecule–based induction of
   hiPSCs according to Reinhardt et al. ([235]25) and previously published
   studies by our group introducing minor modifications ([236]18,
   [237]46). For the generation of NPCs, neural induction medium (Nid0),
   including mTeSR Plus supplemented with 1 μM LDN, 10 μM SB-431542, 3 μM
   Chir, 0.5 μM purmorphamine (PurMA), and 10 μM Y-27632 2HCL (ROCK
   inhibitor) was initially used. HiPSCs were dissociated using
   collagenase IV and resuspended in this Nid0 medium on Ultra-Low
   Adhesion Surface Plates (Corning). After 24 hours, the Nid0 medium was
   replaced by medium without ROCK inhibitor (Nid1). On day 3, Nid1 was
   replaced by N2B27 medium, consisting of 50% Dulbecco’s modified Eagle’s
   medium/F12 Glutamax, 50% Neurobasal medium, 1:200 N2, 1:100 B27, 1:100
   penicillin-streptomycin supplemented with all factors mentioned above
   (Nid2). On day 4, LDN was withdrawn from Nid2 and supplemented with 150
   μM ascorbic acid (smNPC medium). On day 6, the generated embryoid
   bodies were dissociated using a P1000 pipette and transferred to
   12-well plates in a 1:4 ratio in smNPC medium supplemented with 5 μM
   ROCK inhibitor. After 24 hours, ROCK inhibitor was withdrawn and cells
   were cultivated until 80 to 100% confluency. For NPC passaging, the
   cells were detached with Accutase (Sigma-Aldrich) and split in 1:3 to
   1:5 ratios on 12-well plates coated with Geltrex according to the
   manufacturer’s instruction. NPCs were frozen in Knockout SR,
   supplemented with 10% dimethyl sulfoxide (DMSO), and stored at −150°C
   or immediately used for differentiation into midbrain neurons.

   For midbrain differentiation, NPCs of more than eight passages were
   cultivated on 12-well plates in smNPC medium until 80 to 100%
   confluency. Afterward, the smNPC medium was replaced by N2B27 medium
   containing FGF-8 (100 ng/ml), 1 μM PurMA, and 200 μM ascorbic acid
   (differentiation medium). The cells were cultivated in differentiation
   medium until day 8 of differentiation, and medium was renewed every
   other day. On day 8 of neuronal differentiation, differentiation medium
   was replaced by medium containing brain-derived neurotrophic factor (10
   ng/ml), glial cell line–derived neurotrophic factor (10 ng/ml),
   transforming growth factor–β3 (1 ng/ml), 500 μM cyclic adenosine
   monophosphate, 0.5 μM PurMA, and 200 μM ascorbic acid (maturation
   medium). On day 9, 0.75 × 10^6 cells were transferred to 12-well plates
   in maturation medium supplemented with 10 μM ROCK inhibitor and removed
   after additional 24 hours. On day 11, PurMA was withdrawn from
   maturation medium, and the cells were cultivated in this medium until
   day 24 of maturation, replacing maturation medium every other day.
   Unless otherwise specified, midbrain neurons differentiated for 24 days
   were used for the analyses.

Reverse transcription quantitative PCR

   For RT-qPCR, cell pellets were homogenized in RLT buffer containing 1%
   β-mercaptoethanol (74136, Qiagen) and RNA isolated according to the
   manufacturer’s protocol. RNA concentration was determined via Nano-Drop
   (PeqLab). Complementary DNA was generated using the GoScript Reverse
   Transcription System (A5004, Promega). For quantification of transcript
   levels, qPCR was performed on a LightCycler 480 (Roche) using the
   SsoFast EvaGreen Supermix (1725205, Bio-Rad Laboratories) with specific
   primers. The program contained the following five steps: (i) enzyme
   activation for 30 s at 95°C; (ii) denaturation for 5 s at 95°C; (iii)
   annealing for 20 s at 64° to 66°C depending on optimal temperature for
   primers; (iv) acquisition for 20 s at 73°C; (v) melt curve with
   continuous temperature elevation from 65°C to 95°C. While steps (i) and
   (v) were performed only once at the beginning and the end of the
   program, respectively, 55 cycles of steps (ii), (iii), and (iv) were
   conducted. For qPCR analysis, each sample was measured in duplicates.
   Gene expression levels were determined using LightCycler480 Software
   (Roche) and normalized to housekeeping genes 18S and
   glyceraldehyde-3-phosphate dehydrogenase. All primers used are given in
   table S3.

WB analyses

   For WB analysis, the cells were harvested in cold phosphate-buffered
   saline (PBS) and centrifuged at 300g for 5 min at 4°C. Unless stated
   otherwise, the pellet was resuspended in radioimmunoprecipitation assay
   (RIPA) lysis buffer [50 mM tris/HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA,
   1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS], followed by three
   freeze and thaw cycles at −80°C and +37°C and incubation on ice for 30
   min. For the preparation of human brain tissue, the samples were first
   homogenized in homogenization buffer [50 mM tris/HCl and 150 mM NaCl
   (pH 8)] using a Braun Potter S Homogenizer (Sartorius AG), then mixed
   with 4× RIPA in a ratio of 1:3, and incubated on ice for 30 min. After
   centrifugation at 10,000g for 20 min at 4°C, the protein concentration
   of the supernatant was determined using the Pierce bicinchoninic acid
   assay (BCA, 23225, Thermo Fisher Scientific). Afterward, 20 to 30 μg of
   total protein was either mixed with 4× LDS and 10× reducing agent (all
   from Thermo Fisher Scientific) or with 5× Laemmli buffer [0.3 M
   tris/HCl (pH 6.8), 50% glycerol, 1% SDS, 0.05% bromophenol blue, and 5%
   2-mercaptoethanol], followed by incubation at 70°C for 10 min (for
   samples in LDS buffer) or 99°C for 10 min (for samples in Laemmli
   buffer). SDS-PAGE was performed using MES buffer on NuPAGE 4 to 12%
   bis-tris protein gels (Thermo Fisher Scientific) or using tris/glycine
   buffer with selfcast 12% acrylamide gels at 120 V for 1 hour and 30
   min. For detection of vimentin truncation, Laemmli buffer and 12%
   acrylamide gels were used. Protein was transferred to a polyvinylidene
   fluoride (PVDF-FL) membrane in a Wet/Tank Blotting System (Bio-Rad) at
   90 V for 1.5 hours. Membranes were fixed using 4% paraformaldehyde
   (PFA) before blocking in 1% bovine serum albumin (BSA) in TBS-T for
   1 hour at RT. Primary antibody incubation was performed at 4°C
   overnight followed by incubation with fluorophore-coupled secondary
   antibodies for 1.5 hours. All primary and secondary antibodies and
   their working dilutions are listed in table S4. Before imaging, the
   membranes were dried in the dark to improve signal contrast. Protein
   loading control (LC in WB images) was carried out by staining the gel
   with Coomassie Brilliant Blue G 250 (Bio-Rad), or the PVDF-FL membrane
   was stained with either Ponceau S (Sigma-Aldrich) or Revert 520 Total
   Protein Stain (Li-Cor Bioscience) according to the manufacturer’s
   instruction. Membranes were imaged either using a Fusion FX7 system
   (Peqlab) or an Odyssey M (Li-Cor Biosciences) imaging system.

Solubility assay

   For assessment of protein solubility, the cells were homogenized in 50
   mM tris/HCl (pH 7.4), 175 mM NaCl, 5 mM EDTA, and 1% Triton X-100 using
   a Braun Potter S Homogenizer (Sartorius AG). Protein concentration was
   determined using the BCA assay and adjusted to 1 μg/μl, after which 100
   μl of the protein homogenate was processed further. The homogenate was
   centrifuged at 100,000g for 60 min at 4°C using the Sorvall WX+
   ultracentrifuge (Thermo Fisher Scientific). The pellet was resuspended
   in half the volume of the supernatant using a buffer containing 0.5 M
   urea and 5% SDS. Both fractions were mixed with 5× Laemmli buffer.
   After boiling at 99°C for 10 min, the samples were loaded on 12%
   SDS-PAGE gels for electrophoresis. WB and immunostaining were carried
   out as described above.

Enzyme-linked immunosorbent assay

   Levels of total and aggregated aSyn were analyzed using the LEGEND MAX
   Human a-Synuclein ELISA kit (BioLegend, catalog no. 448607) and LEGEND
   MAX a-Synuclein Aggregate ELISA Kit (BioLegend, catalog no. 449407)
   following the manufacturer’s instructions. Specifically, cells were
   harvested in PBS and resuspended in a lysis buffer, containing 20 mM
   Hepes, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl[2], 10% glycerol, and 1%
   Triton X-100. After three freeze-thaw cycles (−80° and 37°C), the cell
   lysate was incubated on ice for 30 min and centrifuged at 10,000g for
   10 min. The resulting supernatant was used to measure total and
   aggregated aSyn using 0.1 and 20 μg of total protein, respectively.

Immunocytochemistry

   For ICC, cells were initially fixed with 2% PFA for 10 min, followed by
   a subsequent fixation step using 4% PFA for an additional 10 min.
   Following cell fixation, PBS was used to rinse the cells three times at
   RT. To minimize nonspecific antibody binding, the cells were treated
   with fish skin gelatin buffer (FSGB: tris-buffered saline with 0.4%
   cold water fish skin gelatin in water, 1% BSA, and 0.1% Triton X-100)
   for 30 min at RT. Primary antibodies, diluted in FSGB, were then
   applied and incubated for 1 hour at RT. After primary antibody
   incubation, the cells were rinsed three times with PBS at RT, followed
   by incubation with the appropriate secondary antibody for 1 hour at RT.
   Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (0.1
   μg/ml) for 5 min at RT. Subsequently, the cells were washed three more
   times with PBS and mounted using ProLong solution before imaging. All
   antibodies used for ICC are listed in table S4. Imaging was performed
   using the fluorescence Observer microscope and a Zeiss LSM 780 confocal
   laser scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

Analysis of neuronal and neurite phenotypes

   The distribution of aSyn in neurites and neuronal somata was assessed
   using Zen 2013 software. Each neuron was examined by randomly selecting
   one or two primary neurites. Cytosolic and neuritic regions were
   delineated, and the intensity of aSyn signals was quantified in both
   compartments.

   For analyzing neurite parameters, each neuron was examined by randomly
   selecting one or two primary neurites. Neurite diameter and volume were
   quantified using IMARIS (Bitplane, Zurich, Switzerland). The filament
   tracing feature of the software was used to automatically trace
   neurites within a randomly chosen image area (fig. S3A). Neurite
   diameter analysis focused solely on primary neurites before the first
   branching point, whereas neurite volume determination involved neurites
   spanning from the first to the fifth branching point.

   Neurite density was assessed using fluorescent images of neurons
   stained for bTubIII, recorded with a 10× objective on a Zeiss LSM 780
   microscope and analyzed using ImageJ software ([238]77). Five regions
   of interest per image were unbiasedly selected and processed with
   consistent parameters, including thresholding (15 pixels) to binarize
   the image and background subtraction (10 pixels). Neurites were
   skeletonized, and the resulting two-dimensional (2D)/3D skeletons were
   analyzed to calculate the number of skeletons per area (in pixels^2) as
   a measure of neurite density.

   Immunofluorescence staining of vimentin and bTubIII was imaged using
   the Zeiss LSM microscope. Quantification of vimentin immunoreactive
   neurites was performed using the Fiji Plugin of the ImageJ software.
   For this, the focus was set on neuronal cell accumulations showing
   neuritic outgrowth. Absolute number of neurites was assessed counting
   the bTubIII^+ neurites using the cell counter plugin of ImageJ. For
   calculation of the proportion of the Vim^+/bTubIII^+ neurites, the
   number of Vim^+ neurites was assessed and normalized to the absolute
   number of bTubIII^+ neurites.

MS-based proteomics

Sample preparation and mass spectrometric measurements

   All samples were prepared in 96-well plate format using the optimized
   SP3 protocol according to Hughes et al. ([239]78). Specifically, cell
   pellets were lysed in 1% NP-40 and 0.2% (w/v) SDS in 20 mM Hepes (pH
   7.5). Protein concentration in lysates was determined using BCA (Thermo
   Fisher Scientific, #23225). Protein amount was adjusted to 10 μg in a
   total volume of 40 μl of lysis buffer. The protein was loaded onto a
   1:1 mixture of hydrophilic and hydrophobic carboxylate–coated magnetic
   beads (Cytiva, #45152105050250 and #65152105050250, 10 μl each)
   prewashed three times with 100 μl of MS-grade H[2]O (Honeywell,
   #15665350). The magnetic beads with protein samples were mixed at 850
   rpm for 1 min at RT. To initiate the binding, 60 μl of absolute EtOH
   was added, and the mixture was incubated at RT for 5 min at 850 rpm.
   Subsequently, the beads were washed three times with 80% (v/v) EtOH,
   with incubation at RT for 1 min and 850 rpm between each wash. After
   the last wash, the beads were resuspended in 50 μl of 100 mM ammonium
   acetate buffer (ABC, Sigma-Aldrich, #09689). The wash steps and ABC
   buffer addition were performed by a liquid handling robot (Hamilton
   Microlab Prep). The on-beads digestion was performed with trypsin
   (Promega, V5113, 1 μg) overnight at 37°C and 850 rpm. The resulting
   peptide mixture was eluted from the magnetic beads into a new 1.5-ml
   tube. The magnetic beads were washed with 50 and 30 μl of 1% (v/v)
   formic acid (TCI, # F0654) and incubated at 40°C at 850 rpm for 5 min.
   The fractions were added to the first elution fraction. The combined
   fractions were further purified from remaining magnetic beads. MS
   measurements were performed on an Orbitrap Eclipse Tribrid Mass
   Spectrometer (Thermo Fisher Scientific) coupled to an UltiMate 3000
   Nano-HPLC (Thermo Fisher Scientific) via a nanospray Flex ion source
   (Thermo Fisher Scientific) equipped with column oven (Sonation) and
   FAIMS interface (Thermo Fisher Scientific). Peptides were loaded on an
   Acclaim PepMap 100 μ-precolumn cartridge (5 μm, 100 Å, 300 μm ID by 5
   mm, Thermo Fisher Scientific) and separated at 40°C on a PicoTip
   emitter (noncoated, 15 cm, 75 μm ID, 8 μm tip, New Objective) that was
   in-house packed with Reprosil-Pur 120 C18-AQ material (1.9 μm, 150 Å,
   Dr. A. Maisch GmbH). Buffer composition: buffer A consists of MS-grade
   H[2]O supplemented with 0.1% formic acid (FA); buffer B consists of
   acetonitrile supplemented with 0.1% FA. The 105-min LC gradient from 4
   to 35.2% buffer B was used. The flow rate was 0.3 μl/min.

Data-independent acquisition

   The DIC duty cycle comprised one MS1 scan followed by 30 MS2 scans. The
   MS2 scans had an isolation window of 4 mass/charge ratio (m/z) range,
   which overlapped with an adjacent window at the 2 m/z range. MS1 scan
   was conducted with Orbitrap at 60,000 resolution power and a scan range
   of 200 to 1800 m/z with an adjusted radio frequency (RF) lens at 30%.
   MS2 scans were conducted with Orbitrap at 30,000 resolution power, and
   RF lens was set to 30%. The precursor mass window was restricted to a
   500 to 740 m/z range. HCD fragmentation was enabled as an activation
   method with a fixed collision energy of 35%. FAIMS was performed with
   one CV at −45 V for both MS1 and MS2 scans during the duty cycle.

Computational evaluation of DIA raw files

   Raw files were converted in the first step with “MSConvertGUI” as a
   part of the “ProteoWizard” software package
   ([240]http://www.proteowizard.org/download.html) to an output mzML
   format applying the “peakPicking” filter with “vendor msLevel = 1,” and
   the “Demultiplex” filter with parameters “Overlap Only” and “mass
   error” set to 10 ppm. Standalone DIA-NN software under version 1.8.1
   was used for protein identification and quantification. First, a
   spectral library was predicted in silico by the software’s deep
   learning-based spectra, RTs, and IMs prediction using UniProt database
   for Homo sapiens (taxon identifier: 9606; containing canonical and
   isoforms, decoys, and common contaminants). DIA-NN search settings are
   as follows: FASTA digest for library-free search/library generation
   option was enabled, together with a match between runs option and
   precursor FDR level set at 1%. Library generation was set to smart
   profiling, Quantification strategy - Robust LC. The mass accuracy and
   the scan window were set to 0 to allow the software to identify optimal
   conditions. The precursor m/z range was changed to 500 to 740 m/z to
   fit the measuring parameters.

Proteome analysis

   Perseus (2.0.9.0) was used to log[2] transform LFQ intensities, replace
   missing values from normal distribution, and construct the volcano
   plots ([241]79).

RNA-seq library preparation

   RNA-seq libraries were prepared as previously described ([242]80).
   Approximately ~200,000 iPSC-derived neurons were collected in TRIzol.
   Total RNA was extracted from cells using the Direct-zol RNA MicroPrep
   Kit (Zymo Research R2052) and stored at −80°C until RNA-seq library
   preparation. mRNAs were enriched by incubation with Oligo d(T) magnetic
   beads (NEB, S1419S) in 1× DTBB buffer [20 mM tris-HCl (pH 7.5), 1 M
   LiCl, 2 mM EDTA, 1% lithium dodecyl sulfate, and 0.1% Triton X-100] at
   65°C for 2 min and were incubated at RT while rotating for 10 min. The
   beads were washed 1× with RNA wash buffer 1 [10 mM tris-HCl (pH 7.5),
   0.15 M LiCl, 1 mM EDTA, 0.1% lithium dodecyl sulfate, and 0.1% Triton
   X-100] and 1× with RNA wash buffer 3 [10 mM tris-HCl (pH 7.5), 0.15 M
   NaCl, and 1 mM EDTA] before elution in RNA elution buffer [10 mM
   tris-HCl (pH 7.5) and 1 mM EDTA] at 80°C for 2 min. PolyA selection was
   performed a second time, and samples were washed 1× with wash buffer 1,
   1× with wash buffer 3, and 1× with 1× SuperScript III first-strand
   buffer. Beads were resuspended in 10 μl 2× SuperScript III buffer plus
   10 mM dithiothreitol (DTT), and RNA was fragmented at 94°C for 9 min
   and immediately chilled on ice before the next step. For first-strand
   synthesis, 10 μl of fragmented mRNA, 0.5 μl of random primers (50 μM)
   (Thermo Fisher Scientific), 0.5 μl of SUPERase-In (Ambion), 1 μl of
   deoxynucleotide triphosphates (dNTPs, 10 mM), and 0.5 μl of oligo(dT)20
   Primer (Thermo Fisher Scientific) were heated for 50°C for 1 min. At
   the end of incubation, 5.8 μl of water, 1 μl of DTT (100 mM), 0.1 μl of
   actinomycin D (2 μg/μl), 0.2 μl of 1% Tween-20 (Sigma-Aldrich), and
   0.5 μl of SuperScript III (Thermo Fisher Scientific) were added and
   incubated in a PCR machine using the following conditions: 25°C for 10
   min and 50°C for 50 min. The product was then immediately purified with
   RNAClean XP beads (Beckman Coulter) according to the manufacturer’s
   instruction and eluted with 10 μl of nuclease-free water. The RNA/cDNA
   double-stranded hybrid was then added to 1.5 μl of Blue Buffer
   (Enzymatics), 1.0 μl dNTP mix with deoxyuridine triphosphate (dUTP) (10
   mM each deoxyadenosine triphosphate, deoxycytidine triphosphate,
   deoxyguanosine triphosphate, and dUTP), 0.1 μl of dUTP (100 mM), 0.2 μl
   ribonuclease H (5 U/μl), 1.05 μl of water, 1 μl of DNA polymerase I
   (Enzymatics), and 0.15 μl of 1% Tween-20. The mixture was incubated at
   16°C overnight. The following day, the dUTP-marked double-stranded DNA
   (dsDNA) was purified using 28 μl of SpeedBeads (GE Healthcare), diluted
   with 20% polyethylene glycol, molecular weight 8000 (PEG8000) and 2.5 M
   NaCl to a final concentration of 13% PEG, and eluted with 40 μl of
   elution buffer (DNA elution buffer from Zymo ChIP Clean and
   Concentrator Kit). The purified dsDNA underwent end repair by blunting,
   A-tailing, and adaptor ligation as previously described ([243]81) using
   barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR
   amplified for 12 cycles, size selected for 200 to 500 bp, quantified
   using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), and
   sequenced on a NovaSeq 6000 for 51 cycles according to the
   manufacturer’s instructions.

ATAC-seq library preparation

   ATAC-seq libraries were prepared as follows: Approximately 1 × 10^6
   iPSC-derived neurons were resuspended in 47.5 μl of lysis buffer [10 mM
   tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl[2], 0.1% IGEPAL, and CA-630 in
   water] at RT. After the addition of 2.5 μl of DNA enzyme I (Illumina),
   the cells were incubated at 37°C for 30 min. DNA was purified with Zymo
   ChIP DNA concentrator columns (Zymo Research), eluted with 11 μl of
   elution buffer, and amplified using NEBNext High-fidelity 2× PCR
   MasterMix (New England BioLabs) with the Nextera primer Ad1 (1.25 μM)
   and a unique Ad2.n barcoding primer (1.25 μM) for 8 to 10 cycles. The
   resulting libraries were size selected by gel excision to 165 to 400
   bp, purified, and single-end sequenced using a NovaSeq 6000 (Illumina)
   sequencer for 51 cycles according to the manufacturer’s instructions.

RNA-seq analysis

   FASTQ files from RNA-seq experiments were mapped to hg38 using STAR
   ([244]82) with default parameters. After mapping, tag directories were
   created using the HOMER command makeTagDirectory. The gene expression
   raw counts were quantified by HOMER’s ([245]81) analyzeRepeats command
   with the option “-condenseGenes -count exons –noadj.” Differential gene
   expression was calculated using the HOMER command
   “getDiffExpression.pl.” TPM (transcript per kilobase million) were
   quantified for all genes matching accession number to raw counts.
   Differentially expressed genes were assessed with DESeq2 ([246]83) at
   Padj (adjusted P value) < 0.05 and fold change >2 where indicated. Gene
   ontology enrichment analyses were performed using Metascape ([247]84).

ATAC-seq analysis

   ATAC-seq FASTQ files were trimmed to 30 bp before mapping to hg38
   genome with Bowtie2 with default parameters. HOMER was used to convert
   aligned reads into “tag directories” for further analysis. To call
   peaks, we used HOMER’s findPeaks with “-style factor -size 200 -minDist
   200 – L 0 – C 0 -fdr 0.9.” Next, we ran irreproducibility discovery
   rate (IDR) pairwise on ATAC-seq for each condition. We applied IDR
   analysis for replicates. We used DESeq2 using HOMER’s getDIffExpression
   to identify differential chromatin accessibility with fold change >2
   and Padj <0.05. The UCSC genome browser was used to visualize ATAC-seq
   data.

Gene ontology pathway enrichment analysis

   Gene ontology pathway enrichment analysis was performed using R.
   Conversion of gene symbols to EntrezID was performed using the
   AnnotationDbi-package ([248]85). Transformation, conversion of P
   values, and ranking of genes were conducted using the tidyverse package
   ([249]86). For enrichment of transcripts, the clusterProfiler- and the
   ReactomePA-package ([250]87, [251]88) were used. Enriched pathways were
   filtered for adjusted P values <0.05 and subsequently organized in
   descending order based on the normalized enrichment score. For
   Graphical visualization, the ggplot2-package was used ([252]89).

Treatment with okadaic acid and withaferin A

   For the treatment of neurons, maturation medium was replaced on
   differentiation day 24 by N2B27 medium containing 25 nM okadaic acid
   and 2 or 5 μM withaferin A for 3 hours. DMSO was used as the vehicle
   control for okadaic acid treatment and ethanol for withaferin A
   treatment. Subsequently, the medium was discarded and cells were washed
   with prechilled PBS twice. Afterward, the cells were detached using
   prewarmed Accutase at 37°C for 5 min. Accutase was rinsed using PBS,
   and cells were centrifuged for 5 min at 300g at RT. Supernatant was
   discarded, and cell pellets were snap-frozen in liquid nitrogen and
   stored at −80°C until further processing.

Statistical analysis

   Statistical analyses were conducted using GraphPad Prism software
   (version 9.5.1), except for omics studies. Unless otherwise stated, all
   graphs depict the mean of various neuronal differentiation experiments
   ± SD. Unless otherwise indicated, a two-tailed unpaired t test was used
   for comparisons between two groups. For comparisons involving three or
   more groups or conditions, one-way or two-way analysis of variance
   (ANOVA) was applied, followed by Tukey’s post hoc multiple comparisons
   test. A P value <0.05 was considered statistically significant, with
   * < 0.05, ** < 0.01, *** < 0.005, and **** < 0.001. If statistical
   significance is not depicted in the figure, or described as such in the
   figure legend, the results are considered statistically not
   significant.

Acknowledgments