Caspase inhibition restores dopaminergic identity through the PKA–CREB–BDNF axis in Parkinson’s disease neurons

Caspase inhibition restores dopaminergic identity through the PKA–CREB–BDNF axis in Parkinson’s disease neurons | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Caspase inhibition restores dopaminergic identity through the PKA–CREB–BDNF axis in Parkinson’s disease neurons Stefania Scalise, Giorgia Benedetto, Raffaele Covello, Caterina Gabriele, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7289863/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The progressive loss of dopaminergic identity in midbrain neurons is a hallmark of Parkinson’s disease (PD), contributing to synaptic dysfunction and neurodegeneration. While a subset of PD cases is linked to genetic mutations, the majority are sporadic (sPD) and of unknown etiology. Current therapies offer only symptomatic relief and do not prevent neurodegeneration, underscoring the urgent need for disease-modifying strategies targeting actionable molecular pathways. Here, we used human induced pluripotent stem cell (hiPSC)-derived midbrain dopaminergic neurons (mDAs) from sporadic PD patients to investigate early alterations in neuronal identity, plasticity, and survival. We found that PD-derived mDAs exhibit upregulation of phosphorylated α-synuclein, marked reductions in dopaminergic markers (TH, NURR1), deficient dopamine handling, and impaired synaptogenesis. Transcriptomic and protein analyses revealed sustained activation of apoptotic caspases (caspase-3, -7) and downregulation of the PKA–CREB–BDNF signaling axis, which underpins dopaminergic differentiation and synaptic maturation. Pharmacological inhibition of caspases with Q-VD-OPh restored pCREB, BDNF, and downstream dopaminergic markers, leading to morphological recovery and functional synaptic rescue. Inhibition of PKA with H89 abrogated these effects, positioning the caspase–PKA–CREB cascade as a critical regulator of dopaminergic identity in PD neurons. These findings define a novel non-apoptotic role for caspases in disrupting the transcriptional program of mDAs and identify a druggable pathway capable of rescuing key aspects of dopaminergic function in a patient-derived cellular model. This work provides a mechanistic rationale for targeting caspase signaling in early-stage PD. Biological sciences/Molecular biology Biological sciences/Cell biology Biological sciences/Neuroscience Health sciences/Diseases/Neurological disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Midbrain dopaminergic (mDA) neurons play a central role in motor control, motivation, and reward processing [ 1 ]. Their identity is maintained through the expression of lineage-specific transcription factors such as NURR1 and PITX3, which coordinate dopamine biosynthesis, neuronal connectivity, and synaptic plasticity [ 2 ]. In Parkinson’s disease (PD), the progressive loss of mDA neurons in the substantia nigra pars compacta leads to striatal dopamine depletion and the emergence of motor symptoms including bradykinesia, rigidity, and tremor [ 3 ]. Increasing evidence suggests that disruption of dopaminergic identity may precede neuronal death, representing an early and potentially reversible event in disease progression [ 4 ]. Although a subset of PD cases is linked to genetic mutations, approximately 85–90% are classified as sporadic PD (sPD), with unknown etiology [ 5 ]. To investigate early pathological changes in dopaminergic neurons, we employed a novel differentiation strategy using human induced pluripotent stem cell (hiPSC)-derived mDA neurons obtained from patients with sPD. To enhance reproducibility and minimize batch effects inherent to single-line iPSC differentiation, we implemented a pooled culture system in which independently derived iPSC lines were matured in parallel and then combined into a single culture for terminal dopaminergic differentiation [ 6 , 7 ]. This approach increases throughput, reduces inter-individual variability, and facilitates the detection of conserved disease-relevant phenotypes [ 8 , 9 ]. Transcriptomic and proteomic profiling of sPD-derived mDAs revealed downregulation of dopaminergic markers, impaired synaptic protein expression, elevated phosphorylated α-synuclein (pS129 α-syn), and activation of caspases. Treatment with the pan-caspase inhibitor Quinoline-Val-Asp-Difluorophenoxymethylketone (Q-VD-OPh) suppressed caspase activity, restored expression of key dopaminergic and synaptic proteins, and reduced pS129 α-syn accumulation. Mechanistically, we found that Q-VD-OPh treatment reactivates the PKA–CREB signaling axis, a pathway critically involved in neuronal identity maintenance and plasticity [ 10 , 11 ]. These findings indicate that aberrant caspase activity disrupts transcriptional integrity and functional identity in sPD neurons and that its pharmacological inhibition may represent a promising disease-modifying intervention. MATERIAL AND METHODS Generation of midbrain dopaminergic neurons from iPSCs mDAs were generated from iPSC lines derived from three healthy controls (HC) individuals (hiPSC-1, HC_002, HC_003) and three patients with sPD (PD-2M, PD_003, PD-4F) (Supplementary Table S2 ). All lines were previously established and characterized [ 12 – 15 ]. Differentiation into mDAs was performed using a modified version of the protocol by Kriks et al., 2011 [ 16 ]. iPSCs were cultured on hESC-qualified Matrigel (Corning) and maintained in mTeSR Plus medium (Stem Cell Technologies) at 37°C in a 5% CO₂ humidified incubator. Upon reaching 100% confluency, differentiation was initiated by switching to KnockOut Serum Replacement Medium (SRM), composed of KnockOut DMEM supplemented with 15% KnockOut Serum Replacement, GlutaMAX, MEM-NEAA, 0.2% Penicillin/Streptomycin, and 55 µM 2-Mercaptoethanol (all from Thermo Fisher Scientific). From days in vitro (DIV) 0–4, SRM was supplemented with 100 nM LDN193189, 10 µM SB431542, 100 ng/ml SHH (C24II), 100 ng/ml FGF-8 (Miltenyi Biotec), and 2 µM Purmorphamine (Sigma-Aldrich). From DIV3 to DIV11, 3 µM CHIR99021 (Miltenyi Biotec) was added. Starting from DIV5, SRM was gradually replaced with N2 medium (DMEM/F12 with HEPES, GlutaMAX, N2 supplement, and 0.2% Penicillin/Streptomycin), and cultures were maintained on Matrigel-coated plates with continued LDN193189 and CHIR99021 supplementation. At DIV11, midbrain floor-plate progenitors (mFPPs) were transitioned to Neurobasal/B27 (NB/B27) medium, containing Neurobasal medium, GlutaMAX, Penicillin/Streptomycin, and B27 supplement (Thermo Fisher), enriched with 20 ng/ml BDNF (Miltenyi), 20 ng/ml GDNF (R&D Systems), 1 ng/ml TGF-β3 (Peprotech), 200 µM Ascorbic Acid, 0.5 mM dibutyryl-cAMP, and 10 µM DAPT (Tocris). At DIV20, cells were dissociated with StemPro Accutase (Thermo Fisher), pooled, and replated at 75,000 cells/cm² onto poly-L-ornithine/laminin-coated coverslips or multiwell plates (15 µg/mL and 20 µg/mL, respectively; Sigma-Aldrich) in NB/B27 medium. Neurons were cultured up to 60 days with media changes every 2 days. Cells were collected at defined maturation stages for downstream analyses. For a complete list of reagents and small molecules, see Supplementary Table S1 . Immunofluorescence At the desired time point of differentiation, dopaminergic neurons derived from sPD patients and healthy controls were fixed for immunofluorescence analysis. After removing the culture medium, cells were fixed with 4% formaldehyde (FA; Sigma-Aldrich) for 15 minutes at room temperature (RT), then washed three times with PBS (+/+) (Corning). To block non-specific binding and permeabilize the membranes, cells were incubated for 2 hours at RT in PBS (+/+) containing 10% goat serum (Thermo Fisher Scientific) and 0.1% Triton X-100 (Sigma-Aldrich). Primary antibodies were diluted in PBS (+/+) supplemented with 1% goat serum and 0.1% Triton X-100, and incubated overnight at 4°C. The following day, cells were washed three times in PBS (+/+) and incubated for 1 hour at RT with appropriate AlexaFluor-conjugated secondary antibodies (1:500, Thermo Fisher Scientific): anti-rabbit AlexaFluor-594, anti-mouse AlexaFluor-488, anti-rabbit AlexaFluor-488, and anti-chicken AlexaFluor-594. Nuclei were counterstained with DAPI (1:1000, Carl Roth), and coverslips were mounted using DAKO Fluorescent Mounting Medium (Agilent). Fluorescent images were acquired with a Leica DMi8 inverted microscope (LAS X v3.7.423463) and Leica MICA microscope (LAS X v6.2.2.28360; Leica Microsystems CSM GmbH). Quantification was performed using ImageJ software. Colocalization analysis was conducted using the JaCoP plugin for ImageJ. The list of antibodies used in this study is provided in Supplementary Table S1 . Bulk RNA-sequencing At DIV60, total RNA was extracted from pooled mDAs derived from healthy controls (pHC) and sPD patients (pPD) using TRIzol Reagent (Life Technologies), according to the manufacturer’s protocol. RNA quality and concentration were assessed with the Bioanalyzer 2100 system (Agilent Technologies). For library preparation, mRNA was isolated from total RNA using poly-T oligo-coated magnetic beads. After fragmentation, first-strand cDNA synthesis was performed using random hexamer primers, followed by second-strand synthesis incorporating dUTP. Libraries were validated using Qubit for concentration, real-time PCR for quantification, and Bioanalyzer for fragment size distribution. Sequencing was carried out on the Illumina NextSeq 1000 platform. Reads were aligned to the human reference genome using Hisat2 (v2.0.5), and mapped reads were assembled with StringTie (v1.3.3b). Gene expression was quantified with FeatureCounts (v1.5.0-p3) and normalized as FPKM (Fragments Per Kilobase of transcript per Million mapped reads), based on read counts and gene length. Differential expression analysis between pHC and pPD mDA groups was performed using the DESeq2 R package (v1.20.0). p -values were adjusted using the Benjamini-Hochberg method to control for false discovery rate (FDR). Genes with an adjusted p-value ≤ 0.05 and |log₂(fold change)| ≥ 1 were considered significantly differentially expressed. Functional enrichment and Gene Ontology (GO) analyses were conducted using the ClusterProfiler R package (p < 0.05), and Gene Set Enrichment Analysis (GSEA) was performed with a local version of the GSA tool. Short Tandem Repeat (STR) Analysis Retention of all individual cell lines in the pooled mDA cultures was confirmed throughout differentiation, STR profiling was performed on pPD and pHC mDA samples at both DIV0 and DIV60. The analysis was conducted by Eurofins Genomics Europe (Biotech Products & Services GmbH, Ebersberg, Germany). Genomic DNA was extracted from cell pellets, and STR genotyping was carried out using single-locus PCR amplification. Sixteen independent STR loci were analyzed using the AmpFlSTR® Identifiler® Plus PCR Amplification Kit (Thermo Fisher Scientific). The loci included D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, AMEL, D5S818, FGA, D19S433, vWA, TPOX, and D18S51. For detailed STR profiles, refer to Supplementary Table S3 . Western blotting Neurons were harvested in 1X PBS (−/−) (Corning, NY, USA) and lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS (all from Sigma-Aldrich, St. Louis, MO, USA), supplemented with Halt™ protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Waltham, MA, USA). Lysates were sonicated using a Diagenode Bioruptor (3 cycles of 20 s ON / 20 s OFF) and kept on ice for 30 min. Samples were centrifuged at 21,000 × g for 1 h at 4°C. Protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA) using an Eppendorf spectrophotometer (Hamburg, Germany). For each sample, 20 µg of protein was mixed with 1X sample buffer containing Bolt™ LDS Sample Buffer and Bolt™ Reducing Agent (Thermo Fisher), denatured at 70°C for 10 min, and resolved by SDS-PAGE using Bolt™ 4–12% Bis-Tris Plus gels and 1X MES SDS Running Buffer (Thermo Fisher). Proteins were transferred to nitrocellulose membranes (Bio-Rad) using the Trans-Blot Turbo system (Bio-Rad). Membranes were blocked for 1 h at room temperature in TBS-T containing 5% non-fat milk (PanReac AppliChem, Darmstadt, Germany) and incubated overnight at 4°C with primary antibodies. After washing in TBS-T, membranes were incubated for 1 h at RT with HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit IgG, 1:10,000; Jackson ImmunoResearch, West Grove, PA, USA). Signals were developed using Clarity™ Western ECL substrate (Bio-Rad) and imaged with the Alliance™ Q9-Atom system (Uvitec, Cambridge, UK). GAPDH and Histone H3 were used as loading controls. Band intensities were quantified using the “Analyze Gels” tool in ImageJ. A complete list of antibodies is provided in Supplementary Table S1 . Uncropped Western blot images are available in the Supplementary Material under the section entitled Original Western Blots . Mass Spectrometric Analysis All reagents were from Sigma-Aldrich unless otherwise specified. At DIV60, proteomic profiling was performed on four biological replicates each from pHC and pPD mDA neurons, lysed as previously described. Twenty micrograms of protein from each replicate were subjected to reduction and alkylation by sequential incubation with 3.2 µL of 100 mM DTT (1 h, 37°C), 3.8 µL of 200 mM iodoacetamide (1 h, 37°C), and 0.6 µL of 100 mM DTT (20 min, 37°C). Ten micrograms of protein were then processed by Protein Aggregation Capture (PAC), following the protocol by Murfuni et al., 2024 [ 17 ]. Briefly, 5 µL of MagResyn Hydroxyl beads (100 µg) were equilibrated with two washes in 70% acetonitrile (ACN). Protein binding was induced by adding ACN to 70% final concentration, followed by 10 min incubation at 1100 rpm. The beads were washed three times in ACN and once in 70% ethanol, then resuspended in 50 µL of 50 mM triethylammonium bicarbonate (TEAB). Proteins were digested overnight at 37°C with 200 ng of Trypsin-LysC (Pierce, Thermo Fisher) at a 1:50 enzyme-to-substrate ratio. Peptides were collected, and residual material was eluted with 50 µL of 0.1% formic acid. Peptides were analyzed by nanoLC-MS/MS using an Orbitrap Exploris 480 mass spectrometer coupled to an Easy-nLC 1200 system (Thermo Fisher). Peptides were loaded onto a 17 cm in-house packed silica capillary column (75 µm i.d., 3 µm C18; Dr. Maisch). Separation was achieved using a binary gradient of mobile phase A (2% ACN, 0.1% FA) and mobile phase B (80% ACN, 0.1% FA) at 300 nL/min over 140 min: 3–25% B over 90 min, 25–40% over 30 min, 40–100% over 8 min, followed by 10 min at 100% B. The column was re-equilibrated at 0% B for 2 min. Data were acquired using data-independent acquisition (DIA) mode. Full scans were acquired at 60,000 resolution, followed by 32 MS2 scans at 30,000 resolution with 5e5 AGC target, 50 ms maximum injection time, and 25% normalized collision energy. MS2 isolation windows were 15 m/z in the 350–710 m/z range, 30 m/z in the 710–860 m/z range, and 50 m/z in the 860–1010 m/z range with 0.5 m/z windows overlap. Raw files were analyzed with Spectronaut (v18.7) using the Human 1 Protein 1 Gene database (20,577 sequences, March 2022) for protein identification and quantification using default settings except for the following parameters: proteotypicity filter was “protein group specific”, precursor filtering was “percentile”, fraction was “0.4”, imputation strategy was “use background signal”. Statistical analysis was performed using Perseus (v2.0.11.0). Protein abundances were log2-transformed and filtered to include only proteins detected in ≥ 3 replicates per group. Differentially expressed proteins (DEPs) were identified via two-sample t-test with permutation-based FDR correction (FDR 1.5; q < 0.05). Gene Ontology (GO) enrichment was performed using the enrichGO function from the ClusterProfiler R package with gene symbols (keyType = “SYMBOL”) and adjusted using the Benjamini-Hochberg method. GO terms were classified into Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) categories. KEGG pathway enrichment was performed separately for up- and downregulated proteins using the enrichKEGG function, with gene symbols first converted to Entrez IDs via the bitr function and the org.Hs.eg.db annotation database. Only pathways with adjusted p and q-values < 0.05 were considered significant. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium [ 18 ] via the PRIDE [ 19 ] partner repository. Dopamine assay and quantification of pS129 α-syn by ELISA assay Dopamine levels were measured in media collected from neuronal cultures at DIV30, DIV45, and DIV60. Media were centrifuged at 1000 rpm for 20 minutes at 4°C, and stored at − 20°C until analysis. Dopamine concentrations were quantified using a commercial ELISA kit (ENZ-KIT188-0001, Enzo Biochem) according to the manufacturer’s instructions. Phosphorylated α-synuclein at serine 129 (pS129 α-syn) was quantified in total protein lysates from pPD and pHC mDA neurons at DIV60. A total of 50 µg of protein was analyzed using the PSNCA ELISA kit (MyBioSource, #MBS038716, San Diego, CA, USA), following the manufacturer’s instructions. Absorbance readings were acquired using a Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Live-cell imaging with FFN102 mesylate To evaluate dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) activity, we employed a modified version of the FFN102-based live-imaging protocol described by Virdi et al., 2022 [ 20 ]. Cells were incubated with FFN102 mesylate (10 µM; Tocris, #BT−5200) in pre-warmed (37°C) calcium- and magnesium-free HBSS without phenol red (Thermo Fisher Scientific). After 30 minutes at 37°C, intracellular dye uptake was confirmed. Membrane depolarization was induced by applying 50 mM KCl in HBSS to stimulate FFN102 release. Representative images were acquired using a 405 nm laser on a Leica DMi8 inverted microscope with LAS X software (v3.7.423463; Leica Microsystems). Fluorescence intensity changes were quantified over time using ImageJ (NIH, USA). Pharmacological inhibition of caspase and PKA To inhibit caspase activity, mDA neurons were treated with the pan-caspase inhibitor Q-VD-OPh (1 µM; MedChemExpress, #HY-12305) for 15 consecutive days, from day 45 to day 60 of differentiation. Culture medium was refreshed every other day to maintain constant inhibitor levels. To block protein kinase A (PKA) activity, both pPD and control mDA neurons were treated with H-89 dihydrochloride (10 µM; Cell Signaling, #9844) for 5 days, from day 55 to day 60. In both conditions, neurons treated with vehicle (DMSO) served as controls. At the end of each treatment, cells were harvested for downstream analyses. Statistical analysis Statistical analysis was performed with GraphPad PRISM software (v.10.5.0, GraphPad Software Inc.), and data are represented as mean ± s.e.m. p-value was presented with the following levels of significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and # p ≤ 0.0001. Statistical details for each experiment are provided in the respective figure legends and, for the transcriptomic and proteomic analyses, in the corresponding sections of the Materials and Methods. RESULTS Pooled differentiation of sPD and control iPSCs into midbrain dopaminergic neurons reveals early transcriptional divergence The iPSCs lines from 3 sPD patients and 3 HC (Supplementary Table S2 ) were differentiated into mDAs using the protocols of Kriks et al., 2011 [ 21 ], based on dual SMAD inhibition (LDN193189 and SB431542) for neural identity induction [ 22 ] with SHH and WNT pathway activation (recombinant SHH-C24II, purmorphamine, CHIR99021) to specify ventral midbrain floor plate (mFP) fate (Supplementary Figure S1 A). By day 11, the resulting mFP progenitors (mFPPs) showed robust expression of the neuroepithelial marker NESTIN and the mFP transcription factors FOXA2 (~ 80%) and LMX1A (~ 70%) (Supplementary Figure S1 B–D), while lacking PAX6, confirming ventral identity. Terminal differentiation into mDAs was achieved using a maturation cocktail containing BDNF, GDNF, ascorbic acid, db-cAMP, TGFβ−3, and DAPT. By DIV 2 0, cultures consistently expressed both MAP2 and tyrosine hydroxylase (TH), with comparable TH+/MAP2 + proportions across all lines (Supplementary Figure S1 E–F). To enhance throughput and eliminate the impact of line-to-line variation, immature mDA neurons (imDAs) from the three sPD and three HC lines were combined and plated to yield pooled PD (pPD) and pooled HC (pHC) cultures ( Fig. 1 A ). STR profiling at 16 loci confirmed stable contribution from all donor lines at both DIV0 and DIV60, with unique alleles at four loci in pHC and seven in pPD (Supplementary Table S3 ). By DIV30, both groups exhibited mature neuronal morphology with extended neurites, and by DIV45, brain nucleus-like rosettes with radial cell organization emerged ( Fig. 1 B ). Full differentiation proceeded to DIV60, with sequential marker acquisition: NR4A2 (NURR1) was expressed in ~ 80% of pHC and ~ 75% of pPD neurons at DIV30 ( Fig. 1 C–D ); co-expression of TH and DDC was evident at DIV45 ( Fig. 1 E–F ); and robust GIRK2 expression, marking A9-type mature mDAs, was detected by DIV60 ( Fig. 1 G–H ), confirming efficient and comparable differentiation across groups. To identify disease-associated transcriptional signatures, we performed bulk RNA-sequencing of pPD and pHC cultures at DIV60. A total of 35 804 genes were detected (Supplementary Table S4 ). Principal Component Analysis (PCA) revealed a clear segregation by disease status (PC1, 90% variance) and biological reproducibility (PC2, 7%) ( Fig. 1 I ), supported by Pearson correlation among replicates (Supplementary Figure S1 G). Differential expression analysis (|log2FC| ≥ 1; adjusted p ≤ 0.05) identified 1 191 upregulated and 1 467 downregulated genes in pPD relative to pHC ( Fig. 1 J ). GO term enrichment analysis revealed that downregulated genes were significantly associated with catecholamine biosynthesis, neurotransmitter metabolism, and GPCR signaling ( Fig. 1 K ), while upregulated genes were enriched in pathways related to calcium signaling, synaptic inhibition, and postsynaptic receptor activity ( Fig. 1 L ). These findings indicate that, while sPD-derived mDAs acquire appropriate regional identity and mature comparably to controls, they display robust, disease-specific transcriptomic changes reflecting dopaminergic dysfunction and early synaptic dysregulation. pPD mDA neurons exhibit progressive loss of identity and function To elucidate the phenotypic trajectory of dopaminergic neurons in sPD, we systematically compared dopaminergic identity and function between pPD and pHC mDA neurons. PD is marked by a progressive loss of nigral dopaminergic neurons; we therefore investigated molecular and functional signatures indicative of such degeneration in our human iPSC-derived model. Transcriptomic profiling of DIV60 pPD neurons revealed robust downregulation of genes essential for dopaminergic identity, including transcription factors ( FOXA2 , NR4A2/NURR1 ), biosynthetic enzymes ( DDC , TH ), and key transporters ( SLC6A3/DAT , SLC18A2/VMAT2 ) ( Fig. 2 A ). These transcriptomic defects were confirmed at the protein level: western blotting showed a significant reduction in TH, the rate-limiting enzyme in dopamine synthesis, in pPD versus pHC neurons at DIV60 ( Figs. 2 B–C ). Immunofluorescence analysis corroborated this reduction, demonstrating a marked decrease in TH + neurons in pPD cultures ( Fig. 2 D ). Temporal quantification revealed that while TH+/MAP2 + neuron ratios remained stable in pHC cultures, a progressive decline was evident in pPD neurons from DIV30 to DIV60 ( Fig. 2 E ), suggesting a disease-driven erosion of dopaminergic identity over time. To assess whether molecular defects translated into functional impairment, we quantified dopamine release in the culture medium at DIV30, DIV45, and DIV60. Dopamine secretion progressively declined in pPD neurons, whereas it increased in pHC cultures across the same timepoints ( Fig. 2 F ), indicating a temporal deterioration in dopaminergic output in the disease condition. To investigate synaptic dopamine dynamics, we employed FFN102, a fluorescent analog of dopamine that enables live imaging of vesicular uptake and release [ 23 ]. Both pPD and pHC neurons successfully internalized FFN102, indicating functional uptake mechanisms. However, upon depolarization with KCl, pPD neurons exhibited a significantly blunted and delayed release response compared to pHC ( Figs. 2 G–H ), reflecting compromised vesicular dopamine mobilization or exocytosis. This phenotype is consistent with impaired VMAT2-mediated loading and supports the notion of disrupted synaptic dopamine handling. Collectively, these data demonstrate that sPD-derived mDA neurons undergo progressive dopaminergic degeneration, characterized by transcriptional repression of core identity genes, reduced protein expression, declining dopamine release, and impaired synaptic exocytosis. These early and intrinsic functional deficits recapitulate key aspects of PD pathology. Pathological accumulation of phosphorylated α-synuclein in pPD mDA neurons recapitulates early-stage Lewy pathology To determine whether patient-derived dopaminergic neurons faithfully model key pathogenic features of sPD, we investigated the presence and distribution of pathological α-synuclein species in pPD mDA neurons. Disease Ontology (DO) enrichment analysis of bulk RNA-seq data revealed a significant over-representation of gene signatures associated with neurodegenerative conditions, particularly synucleinopathies and Lewy body dementia (Fig. 3 A). Among the 16 synucleinopathy-associated genes enriched in pPD neurons, several known contributors to oxidative stress and protein misfolding, such as ceruloplasmin ( CP ), monoamine oxidase B ( MAOB ), NAD(P)H quinone dehydrogenase 1 ( NQO1 ), tropomyosin receptor kinase B ( NTRK2 ), and neuronal pentraxin-2 ( NPTX2 ), were significantly upregulated compared to pHC controls (Fig. 3 B), suggesting a molecular landscape permissive to α-synuclein misprocessing. Despite total α-synuclein protein levels did not significantly differ between pPD and pHC neurons (Figs. 3 C–D), immunofluorescence analysis revealed a striking accumulation of serine-129 phosphorylated α-synuclein (pS129 α-syn) in pPD mDA neurons (Figs. 3 E–F), a post-translational modification tightly associated with pathological aggregation and Lewy body formation. This observation was independently validated by western blotting (Figs. 3 G–H) and ELISA quantification (Fig. 3 I), confirming the selective accumulation of the phosphorylated, pathogenic α-synuclein species. To further assess whether pS129 α-syn formed aggregation-prone structures, we analyzed its spatial colocalization with key components of Lewy bodies. In pPD neurons, pS129 α-syn displayed enhanced colocalization with ubiquitin (Figs. 3 J–K), a classical marker of protein aggregates [ 24 ], supporting the presence of insoluble α-synuclein inclusions. Moreover, discrete cytoplasmic puncta showed strong overlap between pS129 α-syn and the autophagy adaptor SQSTM1/p62 (Fig. 3 L), consistent with sequestration of misfolded protein aggregates into autophagosomal structures. Quantitative image analysis confirmed significantly increased pS129 α-syn/p62 colocalization in pPD versus pHC neurons (Fig. 3 M), indicating impaired proteostasis and the formation of early-stage Lewy body–like inclusions [ 25 ]. Collectively, these findings indicate that mDAs derived from pPD exhibit key pathological hallmarks of the α-synuclein pathology. Proteomic profiling reveals early disruption of the PKA–CREB axis in sPD dopaminergic neurons To dissect the molecular mechanisms underlying early dopaminergic vulnerability in sPD, we performed untargeted proteomic profiling on mDA neurons derived from pPD and pHC mDAs at DIV60. Total protein extracts were analyzed via nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). PCA revealed clear clustering of samples according to disease status, indicating robust and reproducible proteomic divergence between pPD and pHC neurons (Supplementary Figure S2 A). A total of 6 414 proteins were identified across all samples, of which 443 met the thresholds for differential expression (|log₂FC| ≥ 1.5; q ≤ 0.05; Supplementary Table S5 ). Among these, 197 proteins were significantly upregulated and 246 were downregulated in pPD neurons compared to pHC (Fig. 4 A). Strikingly, the proteomic alterations mirrored transcriptomic changes observed in bulk RNA-seq, including reduced expression of core dopaminergic proteins such as TH, VMAT2, and DAT (Fig. 4 B), alongside upregulation of molecules involved in PD pathology (Fig. 4 C). GO analysis of differentially expressed proteins (DEPs) highlighted disease-relevant pathways. Upregulated DEPs were enriched in biological processes (BP) such as apoptosis, regionalization, and cytoskeletal organization; associated cellular components (CC) included extracellular matrix, integrin complexes, and adhesion junctions; and molecular functions (MF) encompassed calcium ion binding and integrin signaling (Fig. 4 D). Conversely, downregulated DEPs were associated with synaptic signaling, dopamine biosynthesis, and axon function (BP); presynaptic and postsynaptic compartments (CC); and neurotransmitter receptor interactions (MF) (Fig. 4 E). Pathway analysis (KEGG) further confirmed dysregulation of synaptic architecture and signaling homeostasis in pPD neurons (Supplementary Figures S2 B–C). Notably, multiple nodes within the cAMP–PKA–CREB axis, a pathway critically involved in dopaminergic neuron survival and transcriptional identity [ 10 , 26 , 27 ], were markedly downregulated in pPD neurons (Supplementary Figure S2 D). In particular, levels of ADCY8 and UCN, two upstream regulators of adenylate cyclase activity, were significantly diminished (Fig. 4 F). Likewise, the catalytic subunits of PKA (PRKACA and PRKACB) were downregulated in pPD neurons (Fig. 4 G), suggesting a widespread attenuation of cAMP-mediated signal transduction. These perturbations converged on reduced activation of CREB, as evidenced by lower levels of Ser133-phosphorylated CREB (pCREB) in pPD neurons (Figs. 4 H–I). Moreover, canonical CREB-dependent genes [ 28 ], including those linked to synaptic plasticity and dopaminergic differentiation, exhibited consistent transcriptional and proteomic downregulation (Fig. 4 J; Supplementary Figure S2 E). Together, these proteomic data reveal widespread deficits in dopaminergic identity, synaptic function, and intracellular signaling in sPD-derived neurons. In particular, the downregulation of PKA-CREB pathway components suggests that impaired activation of this neuroprotective axis may play a key role in disease pathogenesis. Loss of synaptic integrity and density in pPD mDA neurons reflects disruption of the PKA–CREB signaling axis The PKA–CREB pathway plays a central role in coordinating synaptic development, plasticity, and neurotransmission [ 29 ]. Building on our proteomic evidence of PKA–CREB pathway repression in pPD neurons, we examined synaptic protein expression and structural integrity in these cells. Differential proteomic analysis revealed significant downregulation of key synaptic components in pPD mDA neurons. Notably, the vesicular monoamine transporter SLC18A2 (VMAT2), sodium/calcium exchanger SLC8A2, neuropeptide precursor VGF, and two canonical synaptic scaffolding proteins, synaptophysin (SYP) and DLG4 (encoding PSD95), were among the significantly downregulated DEPs (Fig. 5 A). To validate these changes at the protein level, we performed western blotting for SYP and PSD95. Both synaptic proteins were significantly reduced in pPD neurons at DIV60 relative to pHC cultures (Figs. 5 B–E), consistent with proteomic findings. To assess whether these reductions reflected impaired synaptogenesis or synapse destabilization, we conducted immunofluorescence staining for SYP and PSD95 in mDA cultures. Quantification revealed a pronounced decrease in the number of PSD95 + puncta in pPD neurons (Figs. 5 F–G), indicative of reduced postsynaptic site density. Fluorescence intensity measurements confirmed a substantial decrease in SYP and PSD95 signal at synaptic compartments in pPD neurons, suggesting impaired accumulation or retention of synaptic proteins (Fig. 5 H). Morphometric analysis further demonstrated a significant reduction in the area occupied by SYP- and PSD95-positive structures in pPD cultures (Fig. 5 I), supporting the hypothesis of synaptic loss rather than redistribution. Interestingly, despite the overall reduction in synaptic marker expression and synapse number, the degree of colocalization between SYP and PSD95 puncta remained comparable between pPD and pHC neurons (Fig. 5 J). This suggests that synapse formation, where it occurs, remains structurally coordinated in pPD neurons, but that the overall number of synapses is markedly diminished. Together, these findings indicate that mDA neurons derived from pPD patients exhibit a substantial loss of synaptic components and reduced synaptic density, likely reflecting early structural and functional synaptic deficits. These alterations are consistent with impaired PKA-CREB signaling and may contribute to the progressive synaptic dysfunction observed in PD. Convergent upregulation of apoptotic effectors and BDNF depletion underlie the intrinsic vulnerability of pPD mDA neurons Apoptotic dysregulation is a well-established contributor to dopaminergic neurodegeneration in PD, and accumulating evidence suggests that early synaptic dysfunction can mechanistically initiate pro-apoptotic cascades [ 30 – 32 ]; PMID: 9006329). Proteomic profiling of pPD mDA neurons at DIV60 revealed robust upregulation of several core apoptotic effectors. Notably, caspase-3 and caspase-7, key executioner caspases, were significantly elevated in pPD neurons, alongside FAS and BAX, critical mediators of receptor-mediated and mitochondrial apoptosis, respectively (Fig. 6 A). These proteomic findings were independently validated by immunoblotting, which demonstrated increased levels of FAS ligand (FASL) and BAX in pPD neurons relative to pHC (Figs. 6 B–C). Western blot analysis further revealed elevated expression of both full-length and cleaved forms of caspase-3 and caspase-7 in pPD neurons (Figs. 6 D–E), confirming activation of the apoptotic cascade at a post-translational level. To functionally assess apoptotic engagement, we performed TUNEL staining, which revealed a significantly greater proportion of TUNEL-positive cells in pPD mDA cultures compared to controls (Figs. 6 F–G). Given the neuroprotective role of brain-derived neurotrophic factor (BDNF) in dopaminergic survival and synaptic maintenance [ 33 , 34 ], we next examined its expression in pPD neurons. Immunoblotting and densitometric analysis revealed a marked reduction in BDNF protein levels in pPD cultures (Figs. 6 H–I), consistent with deficient neurotrophic support. The concurrent elevation of pro-apoptotic signaling and depletion of BDNF suggests a permissive environment for degeneration in pPD neurons. Altogether, these results highlight an intrinsic molecular vulnerability in sPD-derived mDA neurons, characterized by convergent activation of apoptotic machinery and reduced availability of trophic support. Caspase inhibition restores dopaminergic and synaptic identity in pPD mDA neurons and reduces pathological α-synuclein burden To mechanistically test whether excessive caspase activity contributes to the dopaminergic and synaptic deficits observed in pPD mDA neurons, we performed pharmacological rescue experiments using the pan-caspase inhibitor Q-VD-OPh. Chronic administration of Q-VD-OPh (15 days, DIV45–DIV60, Supplementary Figures S3 A) at the optimal concentration of 1 µM, previously defined by effective suppression of cleaved caspase-3 and − 7 levels (Supplementary Figures S3 B), significantly restored key phenotypic features of dopaminergic neurons. Western blotting revealed that Q-VD-OPh treatment led to marked upregulation of TH and NURR1 (Figs. 7 A–B), indicative of transcriptional reactivation of the dopaminergic lineage. In functional terms, treated neurons exhibited significantly enhanced depolarization-induced release of the dopamine surrogate FFN102, pointing to improved vesicular storage and exocytosis (Fig. 7 C; Supplementary Figure S3 C). At the synaptic level, Q-VD-OPh robustly increased the expression of pre- and postsynaptic markers SYP and PSD95 (Figs. 7 D–E). Morphometric analysis revealed a parallel expansion in the synaptic area occupied by SYP and PSD95 (Figs. 7 F–G), as well as a significant rise in the number of discrete PSD95-positive structures (Figs. 7 H), consistent with recovery of synaptic density and architecture. Q-VD-OPh treatment also induced a substantial increase in BDNF protein levels (Figs. 7 I–J), suggesting that caspase inhibition relieves repression of neurotrophic signaling cascades. Furthermore, western blot analysis revealed a significant reduction in pS129 α-syn (Figs. 7 K–L). Collectively, these findings identify caspases as upstream regulators of both dopaminergic transcriptional fidelity and synaptic maintenance. Their pharmacological inhibition not only prevents further molecular degeneration but actively re-engages neurotrophic and functional pathways in mDAs neurons. PKA–CREB signaling mediates the restorative effects of caspase inhibition in PD mDA neurons Having observed that Q-VD-OPh treatment restores the expression of key dopaminergic and neurotrophic genes, many of which are known transcriptional targets of CREB, we hypothesized that activation of the PKA–CREB axis underpins the neuroprotective response to caspase inhibition. Western blot analyses confirmed that Q-VD-OPh selectively enhanced CREB phosphorylation at Ser133 (pCREB) ( Figs. 8 A–B ). To determine whether CREB phosphorylation is PKA-dependent, we co-treated pPD neurons with Q-VD-OPh and the selective PKA inhibitor H89. Co-inhibition of PKA completely abrogated Q-VD-OPh-induced increases in pCREB ( Figs. 8 C–D ), and notably prevented the restoration of BDNF protein expression ( Figs. 8 E–F ). Similarly, H89 blocked Q-VD-OPh-mediated upregulation of TH ( Figs. 8 G–H ), establishing that PKA activity is required for dopaminergic marker reactivation upon caspase inhibition. To further dissect the role of this pathway under basal conditions, we treated healthy pHC neurons with H89 alone. PKA blockade in control neurons resulted in reduced levels of total and phosphorylated CREB ( Fig. 8 I ), and downregulation of downstream targets including TH, NURR1 ( Fig. 8 J ), and the presynaptic protein SYP ( Fig. 8 K ). Importantly, PKA inhibition triggered an increase in pS129 α-syn accumulation ( Fig. 8 L ). Furthermore, H89 led to upregulation of both full-length and cleaved caspase−7 ( Fig. 8 M ), suggesting that disruption of PKA–CREB signaling is sufficient to initiate a pro-apoptotic cascade. Taken together, these findings position the PKA–CREB axis as a pivotal integrator of dopaminergic maintenance, synaptic integrity, and neuronal survival. Caspase inhibition restores PKA–CREB activity, thereby re-engaging neuroprotective transcriptional programs and suppressing PD-related pathomechanisms. Conversely, its pharmacological disruption induces transcriptional silencing, α-synuclein pathology, and apoptosis, even in non-diseased neurons. Overall, the data suggest that caspases act upstream of PKA–CREB and that this signaling axis is essential for caspase-mediated neuroprotection in PD. DISCUSSION In this study, we leveraged an iPSC-based system to model early pathological and molecular features of sPD. mDAs derived from patient-specific iPSCs offer a powerful human cellular platform to investigate disease-initiating mechanisms [ 35 ]. While previous sPD iPSC-derived models have captured aspects of pathology [ 36 – 38 ], the molecular drivers of disease onset remain elusive, and no disease-modifying therapies are currently available. Our findings demonstrate that long-term cultured sPD mDAs recapitulate key pathological features, including progressive loss of dopaminergic identity, pS129 α-syn accumulation, synaptic dysfunction, and caspase activation. Notably, pharmacological inhibition of caspases restored dopaminergic and synaptic markers and unveiled the PKA-CREB signaling axis as a critical mediator of this rescue effect. Caspase activation is a hallmark of apoptotic cell death [ 39 ], but emerging studies have revealed that caspases also exert non-canonical roles in the central nervous system, including the regulation of synaptic remodeling, axonal guidance, and transcriptional programs. In particular, caspase-3 and caspase-9 have been implicated in dendritic pruning and activity-dependent plasticity, even in the absence of overt cell death, suggesting a more nuanced role in neuronal homeostasis [ 40 ]. Accumulation of active caspases has been reported in sPD brains [ 41 , 42 ], and our data confirm upregulation of caspases 3 and 7 in sPD mDAs. Th ese observations align with prior studies implicating caspase-mediated apoptosis in dopaminergic neuron degeneration, particularly via caspase−3 activation [ 43 , 44 ]. A defining pathological feature of PD is α-synuclein misfolding and aggregation into Lewy bodies [ 45 ]. Caspase activity promotes α-synuclein aggregation by generating toxic C-terminal fragments [ 46 , 47 ] and impairs autophagic clearance by cleaving core ATG proteins [ 48 , 49 ]. In our model, pS129 α-syn co-localized with ubiquitin and p62/SQSTM1, recapitulating key features of like-Lewy body composition [ 24 , 25 ]. This pathological accumulation may drive the observed downregulation of dopaminergic regulators TH and NURR1, consistent with studies linking α-syn overexpression to dopaminergic loss [ 50 ]. Beyond their role in apoptosis, caspases regulate synaptic structure and function. Caspase−3, for instance, is essential for long-term depression (LTD) via AMPA receptor internalization [ 51 ]. Caspases also degrade essential synaptic proteins such as PSD95 and synaptophysin [ 52 ] ), which were downregulated in our sPD mDAs. These proteins are critical for synapse formation and plasticity [ 53 , 54 ]. Since α-synuclein also regulates synaptic vesicle cycling and SNARE complex formation [ 55 ], its pathological accumulation likely contributes to the reduced dopamine reuptake observed in sPD mDAs. Given the youthful state of iPSC-derived neurons [ 56 ], these changes likely reflect early, intrinsic disease mechanisms rather than age-related effects [ 57 ]. Therapeutically, our data show that Q-VD-OPh, a broad-spectrum caspase inhibitor with improved bioavailability and reduced toxicity compared to earlier compounds such as zVAD-fmk [ 58 ], effectively restores dopaminergic identity and synaptic markers while reducing pS129 α-syn levels. Previous work confirmed Q-VD-OPh’s neuroprotective effects in MPTP mouse models and human neurons overexpressing α-synuclein [ 59 , 60 ]. Mechanistically, we identified the PKA-CREB signaling axis as the key downstream mediator of this rescue. Proteomic analysis revealed marked downregulation of PKA-CREB signaling in sPD neurons, which was reversed upon Q-VD-OPh treatment. Co-treatment with the PKA inhibitor H89 abolished the beneficial effects of Q-VD-OPh, confirming the pathway’s central role. This signaling cascade has been previously linked to PD pathology and neurotrophic support [ 10 , 11 , 61 ]. Importantly, BDNF, a direct CREB target and key TrkB receptor ligand, was also restored following Q-VD-OPh treatment, suggesting a positive feedback loop that amplifies neuroprotective signaling [ 62 ]. Caspase inhibition may enhance PKA-CREB activity by suppressing phosphatase-mediated dephosphorylation of CREB [ 10 , 63 ]. Strikingly, PKA-CREB inhibition in healthy control neurons via H89 recapitulated pathological phenotypes: decreased dopaminergic and synaptic markers, elevated α-synuclein phosphorylation, and caspase activation. This indicates a reciprocal regulatory loop between caspases and PKA-CREB, positioning this axis as a critical safeguard of dopaminergic neuronal integrity. We propose that caspase activation functions upstream of the PKA–CREB–BDNF axis, repressing the transcriptional program required for dopaminergic identity and synaptic maturation in patient-derived mDA neurons. In this context, caspases contribute to dopaminergic degeneration in sporadic PD not only by promoting cell death but also by disrupting homeostatic signaling essential for neuronal identity. Early pharmacological inhibition with compounds like Q-VD-OPh restored TH and NURR1 expression, improved synaptic markers, and reduced pathogenic pS129 α-synuclein, suggesting potential disease-modifying effects if applied during prodromal or early symptomatic stages. However, limitations of the iPSC model, such as immature neuronal profiles and absence of age-related or glial interactions, must be considered. Additionally, pooled cultures may mask patient-specific traits. Future studies should combine this in vitro approach with in vivo and organoid models to validate the therapeutic value of targeting caspase signaling. Overall, these findings highlight a novel role for caspases in PD pathology and identify the caspase–PKA–CREB–BDNF axis as a promising target for neuroprotection. Declarations Conflict of Interest: The authors declare no competing financial interests. Funding This work was supported by the following funding: PNRR Project #CN00000041: National Center for Gene Therapy and Drugs Based on RNA Technology (CN RNA & Gene Therapy) CUP J33C22001130001 to G.C.; PNRR Project: A multiscale integrated approach to the study of the nervous system in health and disease (MNESYS) CUP D33C22001340002 to G.C co-awarded to Al.Q. Author contributions: G.C. supervised the study. G.C. and S.S. conceived and designed the study and the experiments. G.L.B. and R.C. conducted the experiments. G.L.B., R.C., and S.S. analyzed the data and prepared the figures. G.L.B., R.C., S.S., and E.I.P interpreted the results. C.G. and M.G. performed the proteomic experiment and analyzed the data. B.P., P.H.G., and P.V. conducted the bioinformatic analysis. D.V. and C.Z. assisted with the laboratory experiments. An.Q. and Al.Q. recruited the patients and provided clinical samples and data. V.T. provided critical discussions and suggestions regarding the differentiation process. G.C. and Al.Q. provided financial support. S.S. drafted the manuscript. E.I.P. and G.C. wrote the final version of the manuscript. Acknowledgements: The authors gratefully acknowledge the donors whose contributions made this work possible and thank Dr. Annamaria Aloisio for her technical support. B.P. acknowledges Relatech S.p.A. for the partial funding of her doctoral fellowship. The figures of this manuscript were created using BioRender. Availability of Data and Materials: The raw RNAseq dataset generated and analysed in the current study has been deposited in the Gene Expression Omnibus (GEO) database, under accession number GSE304324. The proteomics data are available via ProteomeXchange with identifier PXD066765. The other datasets are available from the corresponding author on reasonable request. References Bromberg-Martin ES, Matsumoto M, Hikosaka O. Dopamine in Motivational Control: Rewarding, Aversive, and Alerting. Neuron. 2010;68(5):815–34. Weibo Shi, Yaxing Zhang, Guoting Zhao, Songjun Wang, Guozhong Zhang, Chunling Ma, et al. Dysregulation of Dopaminergic Regulatory Factors TH, Nurr1, and Pitx3 in the Ventral Tegmental Area Associated with Neuronal Injury Induced by Chronic Morphine Dependence [Internet]. [cited 2025 July 31]. Available from: https://www.mdpi.com/ 1422-0067/20/2/250 Poewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, Volkmann J, et al. Parkinson disease. Nat Rev Dis Primer. 2017;3(1):17013. Janezic S, Threlfell S, Dodson PD, Dowie MJ, Taylor TN, Potgieter D, et al. Deficits in dopaminergic transmission precede neuron loss and dysfunction in a new Parkinson model. Proc Natl Acad Sci U S A. 2013;110(42):E4016-4025. Tanner CM, Ross GW, Jewell SA, Hauser RA, Jankovic J, Factor SA, et al. Occupation and Risk of Parkinsonism: A Multicenter Case-Control Study. Arch Neurol. 2009 Sept 1;66(9):1106–13. Cederquist GY, Tchieu J, Callahan SJ, Ramnarine K, Ryan S, Zhang C, et al. A Multiplex Human Pluripotent Stem Cell Platform Defines Molecular and Functional Subclasses of Autism-Related Genes. Cell Stem Cell. 2020 July 2;27(1):35–49.e6. Cuomo ASE, Seaton DD, McCarthy DJ, Martinez I, Bonder MJ, Garcia-Bernardo J, et al. Single-cell RNA-sequencing of differentiating iPS cells reveals dynamic genetic effects on gene expression. Nat Commun. 2020;11(1):810. Jerber J, Seaton DD, Cuomo ASE, Kumasaka N, Haldane J, Steer J, et al. Population-scale single-cell RNA-seq profiling across dopaminergic neuron differentiation. Nat Genet. 2021;53(3):304–12. Limone P, Ragni B, Toto GA. The epidemiology and effects of video game addiction: A systematic review and meta-analysis. Acta Psychol (Amst). 2023;241:104047. Xu X, He X, Zhang Z, Chen Y, Li J, Ma S, et al. CREB Inactivation by HDAC1/PP1γ Contributes to Dopaminergic Neurodegeneration in Parkinson’s Disease. J Neurosci. 2022 June 1;42(22):4594–604. Essam RM, Kandil EA. p-CREB and p-DARPP-32 orchestrating the modulatory role of cAMP/PKA signaling pathway enhanced by Roflumilast in rotenone-induced Parkinson’s disease in rats. Chem Biol Interact. 2023;372:110366. Parrotta EI, Scalise S, Taverna D, De Angelis MT, Sarro G, Gaspari M, et al. Comprehensive proteogenomic analysis of human embryonic and induced pluripotent stem cells. J Cell Mol Med. 2019;23(8):5440–53. Parrotta EI, Lucchino V, Zannino C, Valente D, Scalise S, Bressan D, et al. Modeling Sporadic Progressive Supranuclear Palsy in 3D Midbrain Organoids: Recapitulating Disease Features for In Vitro Diagnosis and Drug Discovery. Ann Neurol. 2025;97(5):845–59. Benedetto G, Zannino C, Valente D, Covello R, Scalise S, Lucchino V, et al. Generation of hiPSCs lines from three sporadic Parkinson’s disease patients. Stem Cell Res. 2025;82:103632. Valente D, Zannino C, Scalise S, Bressan D, Lucchino V, Filomena E, et al. Distinct microRNA signatures define sporadic PSP-RS and PD in patient-derived midbrain organoids. iScience. 2025 July 18;113162. Kriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature. 2011;480(7378):547–51. Murfuni MS, Prestagiacomo LE, Giuliano A, Gabriele C, Signoretti S, Cuda G, et al. Evaluation of PAC and FASP Performance: DIA-Based Quantitative Proteomic Analysis. Int J Mol Sci. 2024;25(10):5141. Deutsch EW, Bandeira N, Perez-Riverol Y, Sharma V, Carver JJ, Mendoza L, et al. The ProteomeXchange consortium at 10 years: 2023 update. Nucleic Acids Res. 2023;51(D1):D1539–48. Perez-Riverol Y, Bandla C, Kundu DJ, Kamatchinathan S, Bai J, Hewapathirana S, et al. The PRIDE database at 20 years: 2025 update. Nucleic Acids Res. 2025;53(D1):D543–53. Virdi GS, Choi ML, Evans JR, Yao Z, Athauda D, Strohbuecker S, et al. Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson’s disease in midbrain dopaminergic neurons. Npj Park Dis. 2022;8(1):162. Kriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature. 2011;480(7378):547–51. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27(3):275–80. Rodriguez PC, Pereira DB, Borgkvist A, Wong MY, Barnard C, Sonders MS, et al. Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain. Proc Natl Acad Sci U S A. 2013;110(3):870–5. Spillantini MG, Schmidt ML, Lee VMY, Trojanowski JQ, Jakes R, Goedert M. α-Synuclein in Lewy bodies. Nature. 1997;388(6645):839–40. Masato A, Andolfo A, Favetta G, Bellini EN, Cogo S, Dalla Valle L, et al. Sequestosome-1 (SQSTM1/p62) as a target in dopamine catabolite-mediated cellular dyshomeostasis. Cell Death Dis. 2024 June 18;15(6):424. Niccolini F, Wilson H, Pagano G, Coello C, Mehta MA, Searle GE, et al. Loss of phosphodiesterase 4 in Parkinson disease: Relevance to cognitive deficits. Neurology. 2017;89(6):586–93. Mucignat C, Caretta A. Drug-induced Parkinson’s disease modulates protein kinase A and Olfactory Marker Protein in the mouse olfactory bulb. Behav Brain Funct. 2017;13(1):1. Zhang X, Odom DT, Koo SH, Conkright MD, Canettieri G, Best J, et al. Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues. Proc Natl Acad Sci. 2005;102(12):4459–64. Sakamoto K, Karelina K, Obrietan K. CREB: a multifaceted regulator of neuronal plasticity and protection. J Neurochem. 2011;116(1):1–9. Dauer W, Przedborski S. Parkinson’s Disease: Mechanisms and Models. Neuron. 2003 Sept 11;39(6):889–909. Singh S, Dikshit M. Apoptotic neuronal death in Parkinson’s disease: Involvement of nitric oxide. Brain Res Rev. 2007 June 1;54(2):233–50. Tompkins MM, Basgall EJ, Zamrini E, Hill WD. Apoptotic-like changes in Lewy-body-associated disorders and normal aging in substantia nigral neurons. Am J Pathol. 1997;150(1):119–31. Wu CL, Hwang CS, Chen SD, Yin JH, Yang DI. Neuroprotective mechanisms of brain-derived neurotrophic factor against 3-nitropropionic acid toxicity: therapeutic implications for Huntington’s disease. Ann N Y Acad Sci. 2010 July;1201:8–12. Howells DW, Porritt MJ, Wong JYF, Batchelor PE, Kalnins R, Hughes AJ, et al. Reduced BDNF mRNA Expression in the Parkinson’s Disease Substantia Nigra. Exp Neurol. 2000;166(1):127–35. Bose A, Petsko GA, Studer L. Induced pluripotent stem cells: a tool for modeling Parkinson’s disease. Trends Neurosci. 2022;45(8):608–20. Sánchez-Danés A, Richaud‐Patin Y, Carballo‐Carbajal I, Jiménez‐Delgado S, Caig C, Mora S, et al. Disease‐specific phenotypes in dopamine neurons from human iPS‐based models of genetic and sporadic Parkinson’s disease. EMBO Mol Med. 2012;4(5):380–95. Stern S, Lau S, Manole A, Rosh I, Percia MM, Ben Ezer R, et al. Reduced synaptic activity and dysregulated extracellular matrix pathways in midbrain neurons from Parkinson’s disease patients. Npj Park Dis. 2022;8(1):103. Lin L, Göke J, Cukuroglu E, Dranias MR, VanDongen AMJ, Stanton LW. Molecular Features Underlying Neurodegeneration Identified through In Vitro Modeling of Genetically Diverse Parkinson’s Disease Patients. Cell Rep. 2016 June 14;15(11):2411–26. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 2018;25(3):486–541. Snigdha S, Smith ED, Prieto GA, Cotman CW. Caspase-3 activation as a bifurcation point between plasticity and cell death. Neurosci Bull. 2012;28(1):14–24. Tatton NA. Increased Caspase 3 and Bax Immunoreactivity Accompany Nuclear GAPDH Translocation and Neuronal Apoptosis in Parkinson’s Disease. Exp Neurol. 2000;166(1):29–43. Hartmann A, Troadec JD, Hunot S, Kikly K, Faucheux BA, Mouatt-Prigent A, et al. Caspase-8 Is an Effector in Apoptotic Death of Dopaminergic Neurons in Parkinson’s Disease, But Pathway Inhibition Results in Neuronal Necrosis. J Neurosci. 2001;21(7):2247–55. Hartmann A, Hunot S, Michel PP, Muriel MP, Vyas S, Faucheux BA, et al. Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. Proc Natl Acad Sci. 2000;97(6):2875–80. Hernandez-Baltazar D, Mendoza-Garrido ME, Martinez-Fong D. Activation of GSK-3β and Caspase-3 Occurs in Nigral Dopamine Neurons during the Development of Apoptosis Activated by a Striatal Injection of 6-Hydroxydopamine. PLOS ONE. 2013;8(8):e70951. Xu L, Pu J. Alpha-Synuclein in Parkinson’s Disease: From Pathogenetic Dysfunction to Potential Clinical Application. Park Dis. 2016;2016:1720621. Ma L, Yang C, Zhang X, Li Y, Wang S, Zheng L, et al. C-terminal truncation exacerbates the aggregation and cytotoxicity of α-Synuclein: A vicious cycle in Parkinson’s disease. Biochim Biophys Acta BBA - Mol Basis Dis. 2018;1864(12):3714–25. Wang W, Nguyen LTT, Burlak C, Chegini F, Guo F, Chataway T, et al. Caspase-1 causes truncation and aggregation of the Parkinson’s disease-associated protein α-synuclein. Proc Natl Acad Sci U S A. 2016;113(34):9587–92. Norman JM, Cohen GM, Bampton ETW. The in vitro cleavage of the hAtg proteins by cell death proteases. Autophagy [Internet]. 2010 Nov 16 [cited 2025 July 31]; Available from: https://www.tandfonline.com/doi/abs/ 10.4161/auto.6.8.13337 Pagliarini V, Wirawan E, Romagnoli A, Ciccosanti F, Lisi G, Lippens S, et al. Proteolysis of Ambra1 during apoptosis has a role in the inhibition of the autophagic pro-survival response. Cell Death Differ. 2012 Sept;19(9):1495–504. Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, et al. Dopaminergic Loss and Inclusion Body Formation in α-Synuclein Mice: Implications for Neurodegenerative Disorders. Science. 2000;287(5456):1265–9. Li Z, Jo J, Jia JM, Lo SC, Whitcomb DJ, Jiao S, et al. Caspase-3 Activation via Mitochondria Is Required for Long-Term Depression and AMPA Receptor Internalization. Cell. 2010;141(5):859–71. Liu J, Chang L, Roselli F, Almeida OFX, Gao X, Wang X, et al. Amyloid-β induces caspase-dependent loss of PSD-95 and synaptophysin through NMDA receptors. J Alzheimers Dis JAD. 2010;22(2):541–56. El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS. PSD-95 involvement in maturation of excitatory synapses. Science. 2000;290(5495):1364–8. Janz R, Südhof TC, Hammer RE, Unni V, Siegelbaum SA, Bolshakov VY. Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I. Neuron. 1999;24(3):687–700. Burré J, Sharma M, Tsetsenis T, Buchman V, Etherton MR, Südhof TC. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science. 2010 Sept 24;329(5999):1663–7. Rando TA, Chang HY. Aging, rejuvenation, and epigenetic reprogramming: resetting the aging clock. Cell. 2012;148(1–2):46–57. Milber JM, Noorigian JV, Morley JF, Petrovitch H, White L, Ross GW, et al. Lewy pathology is not the first sign of degeneration in vulnerable neurons in Parkinson disease. Neurology. 2012;79(24):2307–14. Caserta TM, Smith AN, Gultice AD, Reedy MA, Brown TL. Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties. Apoptosis. 2003;8(4):345–52. Yang L, Sugama S, Mischak RP, Kiaei M, Bizat N, Brouillet E, et al. A novel systemically active caspase inhibitor attenuates the toxicities of MPTP, malonate, and 3NP in vivo. Neurobiol Dis. 2004;17(2):250–9. Ganjam GK, Bolte K, Matschke LA, Neitemeier S, Dolga AM, Höllerhage M, et al. Mitochondrial damage by α-synuclein causes cell death in human dopaminergic neurons. Cell Death Dis. 2019;10(11):865. Zhong J, Dong W, Qin Y, Xie J, Xiao J, Xu J, et al. Roflupram exerts neuroprotection via activation of CREB/PGC-1α signalling in experimental models of Parkinson’s disease. Br J Pharmacol. 2020;177(10):2333–50. Gupta AK, Gupta S, Mehan S, Khan Z, Das Gupta G, Narula AS. Exploring the Connection Between BDNF/TrkB and AC/cAMP/PKA/CREB Signaling Pathways: Potential for Neuroprotection and Therapeutic Targets for Neurological Disorders. Mol Neurobiol [Internet]. 2025 May 9 [cited 2025 July 31]; Available from: https://doi.org/10.1007/s12035-025-05001-5 Huang HS, Lee EYC. Protein phosphatase-1 inhibitor-3 is an in vivo target of caspase-3 and participates in the apoptotic response. J Biol Chem. 2008 June 27;283(26):18135–46. Additional Declarations There is no duality of interest Supplementary Files SupplementarytableS1Reagents.xlsx Supplementary table S1 SupplementarytableS2Patients.xlsx Supplementary table S2 SupplementarytableS3STR.xlsx Supplementary table S3 SupplementaryTableS4PDvsHCDEGs.xls Supplementary table S4 SupplementarytableS5PDvsHCDEPs.xlsx Supplementary table S5 SupplementaryFigureandLegends.docx Supplementary Figure and Legends FulllenghtuncroppedoriginalWesternBlots.pdf Full lenght, uncropped, original western blot Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Basel","correspondingAuthor":false,"prefix":"","firstName":"Verdon","middleName":"","lastName":"Taylor","suffix":""},{"id":497064810,"identity":"d51637de-0d90-42ea-95d0-cb2628af6bc7","order_by":11,"name":"Marco Gaspari","email":"","orcid":"https://orcid.org/0000-0002-5411-8800","institution":"Magna Graecia University","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Gaspari","suffix":""},{"id":497064811,"identity":"cafe6e60-f566-43e6-ab1f-d3dc3f661d0b","order_by":12,"name":"Aldo Quattrone","email":"","orcid":"","institution":"University Magna Graecia of Catanzaro","correspondingAuthor":false,"prefix":"","firstName":"Aldo","middleName":"","lastName":"Quattrone","suffix":""},{"id":497064812,"identity":"b0cb24ce-8461-4a20-93a2-2f335f969c6e","order_by":13,"name":"Elvira Parrotta","email":"","orcid":"","institution":"University of Catanzaro","correspondingAuthor":false,"prefix":"","firstName":"Elvira","middleName":"","lastName":"Parrotta","suffix":""},{"id":497064813,"identity":"3dc6b51b-4364-4636-b82b-9412c9012a8e","order_by":14,"name":"Giovanni Cuda","email":"","orcid":"https://orcid.org/0000-0001-6313-1866","institution":"University Magna Graecia","correspondingAuthor":false,"prefix":"","firstName":"Giovanni","middleName":"","lastName":"Cuda","suffix":""}],"badges":[],"createdAt":"2025-08-04 10:11:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7289863/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7289863/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90544466,"identity":"00f7faba-b6c8-45db-ac5a-0905181b9c6a","added_by":"auto","created_at":"2025-09-04 00:19:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3188030,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration, characterization, and transcriptomic profiling of mDAs derived from pooled iPSC lines of sPD patients and HC. A\u003c/strong\u003e Schematic representation of the experimental workflow.\u003cstrong\u003e \u003c/strong\u003eAn equal number of mFPPs from three sPD and three HC were pooled at DIV20 and differentiated together into mature mDAs. \u003cstrong\u003eB \u003c/strong\u003eRepresentative phase-contrast images of differentiating mDA neurons, showing neuron-like morphology at DIV30 and formation of brain nucleus-like structures at DIV45. Magnification: 20X. \u003cstrong\u003eC \u003c/strong\u003eImmunofluorescence staining of TH (green) and early post-mitotic mDA marker NURR1 (red) at DIV30. \u003cstrong\u003eD\u003c/strong\u003e Quantification of NURR1\u003csup\u003e+ \u003c/sup\u003ecells in pHC and pPD cultures at DIV30. \u003cstrong\u003eE\u003c/strong\u003e Expression of TH (red) and mDA marker Dopa Decarboxylase (DDC, green) at DIV45. \u003cstrong\u003eF\u003c/strong\u003e Quantification of DDC signal co-localized with TH. \u003cstrong\u003eG \u003c/strong\u003eExpression of TH (red) and the late maturation marker GIRK2 (green) in mDA neurons at DIV60. \u003cstrong\u003eH\u003c/strong\u003e Quantification of GIRK2 signal co-localized with TH. For (\u003cstrong\u003eC\u003c/strong\u003e), (\u003cstrong\u003eE\u003c/strong\u003e), and (\u003cstrong\u003eG\u003c/strong\u003e), magnification was 63X. For (\u003cstrong\u003eD\u003c/strong\u003e), (\u003cstrong\u003eF\u003c/strong\u003e), and (\u003cstrong\u003eH\u003c/strong\u003e), each dot represents a single image analyzed, derived from two independent differentiation experiments; data are represented as mean ± s.e.m. of N=15, N=10, and N=15, for (\u003cstrong\u003eD\u003c/strong\u003e), (\u003cstrong\u003eF\u003c/strong\u003e), and (\u003cstrong\u003eH\u003c/strong\u003e), respectively; data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test.\u0026nbsp; \u003cstrong\u003eI\u003c/strong\u003e Principal Component Analysis (PCA) of transcriptomic profiles of pooled pHC and pPD mDAs at DIV60, showing group-specific clustering. \u003cstrong\u003eJ\u003c/strong\u003e Volcano plot displaying differentially expressed genes (DEGs) between pPD and pHC mDAs. Threshold: |log₂FC|≥1 and adjusted \u003cem\u003ep-\u003c/em\u003evalue ≤ 0.05. Data were analyzed by DESeq2 R package. \u003cstrong\u003eK\u003c/strong\u003e Gene Ontology (GO) enrichment analysis of downregulated DEGs in pPD vs pHC mDAs. \u003cstrong\u003eL\u003c/strong\u003e GO enrichment analysis of upregulated DEGs in pPD mDAs. For (\u003cstrong\u003eK\u003c/strong\u003e) and (\u003cstrong\u003eL\u003c/strong\u003e), data were analyzed by ClusterProfiler R package, with a significance threshold set at \u003cem\u003ep\u003c/em\u003e-value ≤ 0.05. DIV, days in vitro.\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/4e2912a94acd1ce54d2511fe.png"},{"id":90545068,"identity":"f1301097-8dba-444a-ba18-0657542c81b4","added_by":"auto","created_at":"2025-09-04 00:27:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1590243,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProgressive loss of dopaminergic identity and function in pPD mDA neurons. A \u003c/strong\u003eRNA-seq analysis of key genes involved in dopaminergic neuron differentiation and maintenance in pPD compared to pHC mDA neurons at DIV60. Values are expressed as Normalized Read Counts (NRC), adjusted for sequencing depth across\u003cstrong\u003e \u003c/strong\u003esamples. Data are shown as mean ± s.e.m. of three independent differentiation experiments, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e ≤ 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.001, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e ≤ 0.0001, and were analyzed by DESeq2 R package. \u003cstrong\u003eB \u003c/strong\u003eRepresentative bands of TH in total protein lysates from pPD and pHC mDA neurons at DIV60, as determined by immunoblot analysis. \u003cstrong\u003eC\u003c/strong\u003e Densitometric quantification of TH bands normalized to GAPDH (housekeeping protein) from three independent differentiation experiments. Data are presented as mean ± s.e.m., *\u003cem\u003ep\u003c/em\u003e ≤ 0.05. Data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test. \u003cstrong\u003eD \u003c/strong\u003eRepresentative immunofluorescence images of TH (green) and MAP2 (red) in mDA neurons at DIV60. Magnification: 63X. \u003cstrong\u003eE\u003c/strong\u003e Quantification of the percentage of TH⁺/MAP2⁺ cells performed at DIV30, DIV45, and DIV60. Quantification was based on N=15 images per group for each time point, derived from three independent differentiation experiments. Data are presented as mean ± s.e.m.,\u0026nbsp; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.01, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e ≤ 0.0001. Data were analyzed using two-way ANOVA. \u003cstrong\u003eF\u003c/strong\u003e Dopamine release into the culture medium, measured by ELISA assay at DIV30, DIV45, and DIV60. \u003cstrong\u003eG \u003c/strong\u003eRepresentative images of the dopamine analog FFN 102 uptake (0 min) and KCl-induced release captured at 2, 4, and 6 minutes after KCl administration in pPD and pHC mDAs. \u003cstrong\u003eH \u003c/strong\u003eQuantification of FFN 102 release after KCl stimulation. Values are expressed as Relative Fluorescence Intensity Change: (F\u003csub\u003et\u003c/sub\u003e - F\u003csub\u003e0\u003c/sub\u003e)/F\u003csub\u003e0\u003c/sub\u003e =\u0026nbsp; ΔF/F\u003csub\u003e0\u003c/sub\u003e, where t = time at which the image was captured, and F\u003csub\u003e0\u003c/sub\u003e = baseline fluorescence. Quantification was performed on N = 10 images per group for each time point, derived from two independent differentiation experiments. Data are presented as mean ± s.e.m. DIV, days in vitro; OD, optical density.\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/c5396080aae800c98be12ee2.png"},{"id":90544481,"identity":"f659418a-3f66-4a50-964c-626a3e17e56d","added_by":"auto","created_at":"2025-09-04 00:19:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2075083,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAccumulation of phosphorylated α-synuclein and Lewy body-like pathology in pPD mDA neurons. A\u003c/strong\u003e Significant enrichment of genes associated with synucleinopathies in pPD neurons, identified through Disease Ontology (DO) enrichment analysis. The number next to each bar indicates the number of upregulated genes in our dataset associated with the specific DO term. Data were analyzed by clusterProfiler R package. \u003cstrong\u003eB\u003c/strong\u003e Expression of synucleinopathy-associated genes based on normal read counts (NRC). Each column represents the values corresponding to a single biological replicate (independent differentiation experiment) for pHC and pPD. \u003cstrong\u003eC \u003c/strong\u003eRepresentative bands of total α-synuclein in protein lysates from pPD and pHC mDA neurons at DIV60, as determined by immunoblot analysis. \u003cstrong\u003eD\u003c/strong\u003e Densitometric quantification of α-synuclein bands normalized to GAPDH (housekeeping protein) from three independent differentiation experiments. Data are presented as mean ± s.e.m., \u003csup\u003ens\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u003csup\u003e\u0026nbsp; \u003c/sup\u003e\u0026gt; 0.05. \u003cstrong\u003eE \u003c/strong\u003eRepresentative immunofluorescence images showing phosphorylated α-synuclein at serine 129 (pS129 α-syn, green) in pPD and pHC mDA neurons. Magnification: 20X \u003cstrong\u003eF\u003c/strong\u003e Quantification of the fluorescence intensity for pS129 α-syn, based on N=30 images per group, derived from three independent differentiation experiments. Data are presented as mean ± s.e.m., \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.01. \u003cstrong\u003eG \u003c/strong\u003eRepresentative bands of pS129 α-syn in pPD and pHC mDA neurons at DIV60, as determined by immunoblot analysis. \u003cstrong\u003eH \u003c/strong\u003eDensitometric quantification of pS129 α-syn bands normalized to GAPDH (housekeeping protein) from three independent differentiation experiments. Data are presented as mean ± s.e.m., \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.05. \u003cstrong\u003eI \u003c/strong\u003eLevels of pS129 α-syn in pPD and pHC lysates measured by ELISA assay. Data are presented as mean ± s.e.m. of two independent experiments, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.05. \u003cstrong\u003eJ\u003c/strong\u003e Representative immunofluorescence images of pS129 α-syn (green) and ubiquitin (red) proteins in healthy and disease neurons. Magnification: 63X. Insets show a magnification of the co-localization between the two proteins. \u003cstrong\u003eK\u003c/strong\u003e Quantification of colocalization between pS129 α-syn and ubiquitin, based on N=10 images per group, derived from two independent differentiation experiments. Data are presented as mean ± s.e.m., \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.001. \u003cstrong\u003eL\u003c/strong\u003e Representative immunofluorescence images of pS129 α-syn (green) and SQSTM1/p62 (red) proteins in healthy and disease neurons. Magnification: 63X. Insets show a magnification of the co-localization between the two proteins. \u003cstrong\u003eM\u003c/strong\u003e Quantification of colocalization between pS129 α-syn and SQSTM1/p62, based on N=10 images per group, derived from two independent differentiation experiments. Data are presented as mean ± s.e.m., \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.0001. For (\u003cstrong\u003eD\u003c/strong\u003e), (\u003cstrong\u003eF\u003c/strong\u003e), (\u003cstrong\u003eH\u003c/strong\u003e), (\u003cstrong\u003eI\u003c/strong\u003e), (\u003cstrong\u003eK\u003c/strong\u003e), and (\u003cstrong\u003eM\u003c/strong\u003e), data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test. DIV, days in vitro. OD, optical density.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/c9e6e607029d1c00bfc0c706.png"},{"id":90544471,"identity":"b7bdbcf9-5bf9-4597-a016-395c995e53ea","added_by":"auto","created_at":"2025-09-04 00:19:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":613482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic profiling of dopaminergic neurons in pPD. A\u003c/strong\u003e Volcano plot of differentially expressed proteins (DEPs) in mDA neurons at DIV60, pPD versus pHC. \u003cstrong\u003eB\u003c/strong\u003e Heatmap of proteins associated with dopaminergic neuron identity and function in mDAs. \u003cstrong\u003eC\u003c/strong\u003e Heatmap of proteins associated with PD pathology in mDAs. For (\u003cstrong\u003eB\u003c/strong\u003e) and (\u003cstrong\u003eC\u003c/strong\u003e), each column represents the Z-Score value corresponding to a single biological replicate (independent differentiation experiment). \u003cstrong\u003eD\u003c/strong\u003e GO enrichment analysis for upregulated DEPs. \u003cstrong\u003eE\u003c/strong\u003e GO enrichment analysis for downregulated DEPs. For (\u003cstrong\u003eD\u003c/strong\u003e) and (\u003cstrong\u003eE\u003c/strong\u003e), the fraction next to each bar indicates the Gene Ratio associated in our dataset with the specific GO term. \u003cstrong\u003eF\u003c/strong\u003e Expression of the PKA activation protein urocortin (UCN) and Adenylate Cyclase 8 in mDAs. \u003cstrong\u003eG\u003c/strong\u003e Expression of the PKA cAMP-activated catalytic subunits alpha (PRKACA) and beta (PRKACB) in mDAs. For (\u003cstrong\u003eF\u003c/strong\u003e) and (\u003cstrong\u003eG\u003c/strong\u003e), data are presented as mean ± s.e.m. of four independent differentiation experiments,\u0026nbsp; \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.001, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.0001. Data were analyzed using a two-sample test with a permutation-based FDR. \u003cstrong\u003eH \u003c/strong\u003eRepresentative bands of CREB and phosphorylated CREB at serine 133 (pCREB (Ser133)) in pPD and pHC mDA neurons at DIV60, as determined by immunoblot analysis. \u003cstrong\u003eI \u003c/strong\u003eDensitometric quantification of pCREB (Ser133) bands normalized to total CREB levels from two independent differentiation experiments. Data are presented as mean ± s.e.m., \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.05. Data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test.\u003cstrong\u003e J \u003c/strong\u003eExpression levels of CREB target genes in mDA neurons, as determined by RNA-seq analysis. Values are expressed as log₁₀ of Normalized Read Counts (NRC). DIV, days in vitro. OD, optical density.\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/524eb607a2453b7860c4a07b.png"},{"id":90544475,"identity":"c6233f36-1176-4641-88a8-0951c6861cc0","added_by":"auto","created_at":"2025-09-04 00:19:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1231917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced synaptic marker expression and density in mDAs pPD. A\u003c/strong\u003e Heatmap of proteins involved in synaptic function in mDAs. Each column represents the Z-Score value corresponding to a single biological replicate (independent differentiation experiment). \u003cstrong\u003eB\u003c/strong\u003e Representative bands of Synaptophysin (SYP) in neurons at DIV60, as determined by immunoblot analysis. \u003cstrong\u003eC \u003c/strong\u003eDensitometric quantification of SYP bands normalized to histone H3 (housekeeping protein). \u003cstrong\u003eD\u003c/strong\u003e Representative bands of Post-Synaptic Density Protein 95 (PSD95 or DLG4) in neurons at DIV60, as determined by immunoblot analysis. \u003cstrong\u003eE \u003c/strong\u003eDensitometric quantification PSD95 bands normalized to GAPDH (housekeeping protein).\u0026nbsp; For (\u003cstrong\u003eC\u003c/strong\u003e) and (\u003cstrong\u003eE\u003c/strong\u003e), data are presented as mean ± s.e.m. of three independent differentiation experiments, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.01. data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test. \u003cstrong\u003eF\u003c/strong\u003e Representative immunofluorescence images of SYP (red) and PSD95 (green) in mDA neurons from pPD and pHC at DIV60. Magnification: 20X. \u003cstrong\u003eG\u003c/strong\u003e Quantification of PSD95 puncta. \u003cstrong\u003eH\u003c/strong\u003e Quantification of fluorescence intensity for SYP and PSD95.\u003cstrong\u003e I\u003c/strong\u003e Quantification of the area occupied by SYP and PSD95 immunoreactivity. \u003cstrong\u003eJ\u003c/strong\u003e Quantification of SYP and PSD95 colocalization. For (\u003cstrong\u003eG-J\u003c/strong\u003e), each dot represents a single image analyzed, derived from two independent differentiation experiments; data are represented as mean ± s.e.m.,\u0026nbsp; \u003csup\u003ens\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u003csup\u003e\u0026nbsp; \u003c/sup\u003e\u0026gt; 0.05, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.0001. Data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/34b7e04444ba9983d7839c7e.png"},{"id":90544472,"identity":"559de4c7-b19f-423c-b71c-2f182f1c5d38","added_by":"auto","created_at":"2025-09-04 00:19:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1114183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of apoptotic pathways and reduced BDNF levels in mDAs pPD. A\u003c/strong\u003e Heatmap of pro-apoptotic proteins in pPD and pPHC mDA neurons compared to pHC. Each column represents the Z-Score value corresponding to a single biological replicate (independent differentiation experiment). \u003cstrong\u003eB \u003c/strong\u003eRepresentative bands of pro-apoptotic protein FASL and BAX in neurons at DIV60, as determined by immunoblot analysis. \u003cstrong\u003eC\u003c/strong\u003e Densitometric quantification of FASL and BAX bands normalized to GAPDH (housekeeping protein). \u003cstrong\u003eD \u003c/strong\u003eRepresentative bands of caspase 7 (CASP7, full length and cleaved forms) and caspase 3 (CASP3, full length and cleaved forms) in neurons at DIV60, as determined by immunoblot analysis. \u003cstrong\u003eE\u003c/strong\u003e Densitometric quantification of cleaved CASP7 and CASP3 bands normalized to histone H3 (housekeeping protein). \u003cstrong\u003eF\u003c/strong\u003e Representative immunofluorescence images of TUNEL-positive cells (red) in mDA neurons from pPD and pHC at DIV60. Neurons were marked by Tubulin beta-3 (TUBB3) staining in green. Magnification: 63X. \u003cstrong\u003eG\u003c/strong\u003e Quantification of TUNEL-positive cells. Each dot represents a single image analyzed, derived from two independent differentiation experiments; data are represented as mean ± s.e.m., \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.0001. Data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test. \u003cstrong\u003eH \u003c/strong\u003eRepresentative bands of BDNF in neurons at DIV60, as determined by immunoblot analysis. \u003cstrong\u003eI\u003c/strong\u003e Densitometric quantification of BDNF bands normalized to GAPDH (housekeeping protein). For (\u003cstrong\u003eC\u003c/strong\u003e), (\u003cstrong\u003eE\u003c/strong\u003e), and (\u003cstrong\u003eI\u003c/strong\u003e), data are presented as mean ± s.e.m. of three independent differentiation experiments, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.01. Data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test. DIV, days in vitro; OD, optical density.\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/d553f7f0aeb02b4070da165c.png"},{"id":90544478,"identity":"891fad88-e09f-449d-8b1c-0b72082c5ae8","added_by":"auto","created_at":"2025-09-04 00:19:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1048874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaspase inhibition by Q-VD-OPh restores dopaminergic, synaptic, and neuroprotective markers in mDAs PD. A \u003c/strong\u003eRepresentative immunoblot bands of TH and NURR1 in neurons at DIV60 after 15 days of Q-VD-OPh treatment. \u003cstrong\u003eB\u003c/strong\u003e Densitometric quantification of TH and NURR1 bands normalized to GAPDH (housekeeping protein). \u003cstrong\u003eC\u003c/strong\u003e Quantification of FFN 102 release following KCl stimulation in neurons treated with Q-VD-OPh for 15 days. Values are expressed as Relative Fluorescence Intensity Change: (F\u003csub\u003et\u003c/sub\u003e - F\u003csub\u003e0\u003c/sub\u003e)/F\u003csub\u003e0\u003c/sub\u003e =\u0026nbsp; ΔF/F\u003csub\u003e0\u003c/sub\u003e, where t = time at which the image was captured, and F\u003csub\u003e0\u003c/sub\u003e = baseline fluorescence. Quantification was performed on N =10 images per group for each time point, derived from two independent differentiation experiments. Data are presented as mean ± s.e.m. \u003cstrong\u003eD\u003c/strong\u003e Representative immunoblot bands of SYP and PSD95 in neurons at DIV60 after 15 days of Q-VD-OPh treatment. \u003cstrong\u003eE\u003c/strong\u003e Densitometric quantification of SYP and PSD95 bands normalized to histone H3 and GAPDH (housekeeping proteins), respectively. \u003cstrong\u003eF \u003c/strong\u003eRepresentative immunofluorescence images of SYP (red) and PSD95 (green) in pPD mDAs treated or untreated with Q-VD-OPh. \u003cstrong\u003eG\u003c/strong\u003e Quantification of the area occupied by SYP and PSD95 immunoreactivity in pPD mDAs treated or untreated with Q-VD-OPh. \u003cstrong\u003eH\u003c/strong\u003e Quantification of PSD95 puncta in pPD mDAs treated or untreated with Q-VD-OPh. For (\u003cstrong\u003eG \u003c/strong\u003eand\u003cstrong\u003e H\u003c/strong\u003e), each dot represents a single image analyzed, derived from two independent differentiation experiments; data are represented as mean ± s.e.m., \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.0001. data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test. \u003cstrong\u003eI\u003c/strong\u003e Representative immunoblot bands of BDNF in neurons at DIV60 after 15 days of Q-VD-OPh treatment. \u003cstrong\u003eJ\u003c/strong\u003e Densitometric quantification of BDNF bands normalized to GAPDH (housekeeping protein). \u003cstrong\u003eK\u003c/strong\u003e Representative immunoblot bands of pS129 α-syn in neurons at DIV60 after 15 days of Q-VD-OPh treatment. \u003cstrong\u003eL\u003c/strong\u003e Densitometric quantification of pS129 α-syn bands normalized to GAPDH (housekeeping protein). For (\u003cstrong\u003eB\u003c/strong\u003e), (\u003cstrong\u003eE\u003c/strong\u003e), (\u003cstrong\u003eJ\u003c/strong\u003e), and (\u003cstrong\u003eL\u003c/strong\u003e), data are presented as mean ± s.e.m. of two independent differentiation experiments, \u003csup\u003ens\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u003csup\u003e\u0026nbsp; \u003c/sup\u003e\u0026gt; 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.01. Data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test. DIV, days in vitro; OD, optical density.\u003c/p\u003e","description":"","filename":"FIGURE7.png","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/6b0fb7db31c71f39f7989299.png"},{"id":90545070,"identity":"6f3ea2ec-e538-47d9-9361-cc29dfb23f94","added_by":"auto","created_at":"2025-09-04 00:27:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":564098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQ-VD-OPh promotes recovery of dopaminergic and synaptic markers in PD mDA neurons via PKA-CREB pathway activation. A \u003c/strong\u003eRepresentative immunoblot bands of CREB and pCREB (Ser133) in neurons at DIV60 after 15 days of Q-VD-OPh treatment. \u003cstrong\u003eB\u003c/strong\u003e Densitometric quantification of CREB and pCREB bands normalized to GAPDH (housekeeping protein). \u003cstrong\u003eC\u003c/strong\u003e Representative immunoblot bands and (\u003cstrong\u003eD\u003c/strong\u003e) densitometric quantification of pCREB (Ser133) in pPD neurons at DIV60 after Q-VD-OPh and H89 co-treatment. \u003cstrong\u003eE\u003c/strong\u003e Representative immunoblot bands and (\u003cstrong\u003eF\u003c/strong\u003e) densitometric quantification of BDNF in pPD neurons at DIV60 after Q-VD-OPh and H89 co-treatment. \u003cstrong\u003eG\u003c/strong\u003e Representative immunoblot bands and (\u003cstrong\u003eH\u003c/strong\u003e) densitometric quantification of TH in pPD neurons at DIV60 after Q-VD-OPh and H89 co-treatment. \u003cstrong\u003eI\u003c/strong\u003e Densitometric quantification of CREB and pCREB (Ser133), (\u003cstrong\u003eJ\u003c/strong\u003e) TH and NURR1, (\u003cstrong\u003eK\u003c/strong\u003e) SYP, (\u003cstrong\u003eL\u003c/strong\u003e) pS129 α-syn, and (\u003cstrong\u003eM\u003c/strong\u003e) CASP7 and cleaved CASP7 in pHC neurons at DIV60 after H89 co-treatment. For all densitometric quantification in this figure, data are presented as mean ± s.e.m. of two independent differentiation experiments, \u003csup\u003ens\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u003csup\u003e\u0026nbsp; \u003c/sup\u003e\u0026gt; 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e ≤ 0.01. Data were analyzed using a two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test. DIV, days in vitro; OD, optical density.\u003c/p\u003e","description":"","filename":"FIGURE8.png","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/68f22428a57cc8292d6221c8.png"},{"id":90545612,"identity":"bc4a27c1-8c3f-435b-bd4d-be1f4625cfbf","added_by":"auto","created_at":"2025-09-04 00:51:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11561378,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/17754f9e-49fb-425c-a672-adf4cb1aac03.pdf"},{"id":90545511,"identity":"a31be8d9-bee0-487b-8201-925493f540b2","added_by":"auto","created_at":"2025-09-04 00:43:09","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12402,"visible":true,"origin":"","legend":"Supplementary table S1","description":"","filename":"SupplementarytableS1Reagents.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/7d89226f42f681defe9eee20.xlsx"},{"id":90544461,"identity":"7affe654-6bd9-429d-ac17-7390928b74bc","added_by":"auto","created_at":"2025-09-04 00:19:09","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5759,"visible":true,"origin":"","legend":"Supplementary table S2","description":"","filename":"SupplementarytableS2Patients.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/11e7ac5662b1443bb769307b.xlsx"},{"id":90545069,"identity":"9a60646d-a09d-4d2b-829a-d284cbcd7405","added_by":"auto","created_at":"2025-09-04 00:27:09","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":52894,"visible":true,"origin":"","legend":"Supplementary table S3","description":"","filename":"SupplementarytableS3STR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/c1af24b6cd9df26c390d5d38.xlsx"},{"id":90544476,"identity":"6a997b27-51ba-45bf-96b5-a4fac61c65fb","added_by":"auto","created_at":"2025-09-04 00:19:09","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14311901,"visible":true,"origin":"","legend":"Supplementary table S4","description":"","filename":"SupplementaryTableS4PDvsHCDEGs.xls","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/1e7559653f8192aa67670ff3.xls"},{"id":90544486,"identity":"ef0d9175-e1f8-485f-b669-9ef449d0fcd1","added_by":"auto","created_at":"2025-09-04 00:19:09","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4675183,"visible":true,"origin":"","legend":"Supplementary table S5","description":"","filename":"SupplementarytableS5PDvsHCDEPs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/c23e9cb108591689288d6671.xlsx"},{"id":90545513,"identity":"2b726267-02cc-49fc-9ccf-48e9b4aaf239","added_by":"auto","created_at":"2025-09-04 00:43:09","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2156521,"visible":true,"origin":"","legend":"Supplementary Figure and Legends","description":"","filename":"SupplementaryFigureandLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/ece70b412e8af4a7aaf05cec.docx"},{"id":90544491,"identity":"98980e43-f916-46d4-a654-9c8094fa93d0","added_by":"auto","created_at":"2025-09-04 00:19:10","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":5313204,"visible":true,"origin":"","legend":"Full lenght, uncropped, original western blot","description":"","filename":"FulllenghtuncroppedoriginalWesternBlots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7289863/v1/4a45ca121988239dd161a95e.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Caspase inhibition restores dopaminergic identity through the PKA–CREB–BDNF axis in Parkinson’s disease neurons","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMidbrain dopaminergic (mDA) neurons play a central role in motor control, motivation, and reward processing [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Their identity is maintained through the expression of lineage-specific transcription factors such as NURR1 and PITX3, which coordinate dopamine biosynthesis, neuronal connectivity, and synaptic plasticity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In Parkinson\u0026rsquo;s disease (PD), the progressive loss of mDA neurons in the substantia nigra pars compacta leads to striatal dopamine depletion and the emergence of motor symptoms including bradykinesia, rigidity, and tremor [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Increasing evidence suggests that disruption of dopaminergic identity may precede neuronal death, representing an early and potentially reversible event in disease progression [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although a subset of PD cases is linked to genetic mutations, approximately 85\u0026ndash;90% are classified as sporadic PD (sPD), with unknown etiology [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To investigate early pathological changes in dopaminergic neurons, we employed a novel differentiation strategy using human induced pluripotent stem cell (hiPSC)-derived mDA neurons obtained from patients with sPD. To enhance reproducibility and minimize batch effects inherent to single-line iPSC differentiation, we implemented a pooled culture system in which independently derived iPSC lines were matured in parallel and then combined into a single culture for terminal dopaminergic differentiation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This approach increases throughput, reduces inter-individual variability, and facilitates the detection of conserved disease-relevant phenotypes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Transcriptomic and proteomic profiling of sPD-derived mDAs revealed downregulation of dopaminergic markers, impaired synaptic protein expression, elevated phosphorylated α-synuclein (pS129 α-syn), and activation of caspases. Treatment with the pan-caspase inhibitor Quinoline-Val-Asp-Difluorophenoxymethylketone (Q-VD-OPh) suppressed caspase activity, restored expression of key dopaminergic and synaptic proteins, and reduced pS129 α-syn accumulation. Mechanistically, we found that Q-VD-OPh treatment reactivates the PKA\u0026ndash;CREB signaling axis, a pathway critically involved in neuronal identity maintenance and plasticity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These findings indicate that aberrant caspase activity disrupts transcriptional integrity and functional identity in sPD neurons and that its pharmacological inhibition may represent a promising disease-modifying intervention.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eGeneration of midbrain dopaminergic neurons from iPSCs\u003c/h2\u003e\u003cp\u003emDAs were generated from iPSC lines derived from three healthy controls (HC) individuals (hiPSC-1, HC_002, HC_003) and three patients with sPD (PD-2M, PD_003, PD-4F) (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). All lines were previously established and characterized [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Differentiation into mDAs was performed using a modified version of the protocol by Kriks et al., 2011 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. iPSCs were cultured on hESC-qualified Matrigel (Corning) and maintained in mTeSR Plus medium (Stem Cell Technologies) at 37\u0026deg;C in a 5% CO₂ humidified incubator. Upon reaching 100% confluency, differentiation was initiated by switching to KnockOut Serum Replacement Medium (SRM), composed of KnockOut DMEM supplemented with 15% KnockOut Serum Replacement, GlutaMAX, MEM-NEAA, 0.2% Penicillin/Streptomycin, and 55 \u0026micro;M 2-Mercaptoethanol (all from Thermo Fisher Scientific). From days in vitro (DIV) 0\u0026ndash;4, SRM was supplemented with 100 nM LDN193189, 10 \u0026micro;M SB431542, 100 ng/ml SHH (C24II), 100 ng/ml FGF-8 (Miltenyi Biotec), and 2 \u0026micro;M Purmorphamine (Sigma-Aldrich). From DIV3 to DIV11, 3 \u0026micro;M CHIR99021 (Miltenyi Biotec) was added. Starting from DIV5, SRM was gradually replaced with N2 medium (DMEM/F12 with HEPES, GlutaMAX, N2 supplement, and 0.2% Penicillin/Streptomycin), and cultures were maintained on Matrigel-coated plates with continued LDN193189 and CHIR99021 supplementation. At DIV11, midbrain floor-plate progenitors (mFPPs) were transitioned to Neurobasal/B27 (NB/B27) medium, containing Neurobasal medium, GlutaMAX, Penicillin/Streptomycin, and B27 supplement (Thermo Fisher), enriched with 20 ng/ml BDNF (Miltenyi), 20 ng/ml GDNF (R\u0026amp;D Systems), 1 ng/ml TGF-β3 (Peprotech), 200 \u0026micro;M Ascorbic Acid, 0.5 mM dibutyryl-cAMP, and 10 \u0026micro;M DAPT (Tocris). At DIV20, cells were dissociated with StemPro Accutase (Thermo Fisher), pooled, and replated at 75,000 cells/cm\u0026sup2; onto poly-L-ornithine/laminin-coated coverslips or multiwell plates (15 \u0026micro;g/mL and 20 \u0026micro;g/mL, respectively; Sigma-Aldrich) in NB/B27 medium. Neurons were cultured up to 60 days with media changes every 2 days. Cells were collected at defined maturation stages for downstream analyses. For a complete list of reagents and small molecules, see Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eAt the desired time point of differentiation, dopaminergic neurons derived from sPD patients and healthy controls were fixed for immunofluorescence analysis. After removing the culture medium, cells were fixed with 4% formaldehyde (FA; Sigma-Aldrich) for 15 minutes at room temperature (RT), then washed three times with PBS (+/+) (Corning). To block non-specific binding and permeabilize the membranes, cells were incubated for 2 hours at RT in PBS (+/+) containing 10% goat serum (Thermo Fisher Scientific) and 0.1% Triton X-100 (Sigma-Aldrich). Primary antibodies were diluted in PBS (+/+) supplemented with 1% goat serum and 0.1% Triton X-100, and incubated overnight at 4\u0026deg;C. The following day, cells were washed three times in PBS (+/+) and incubated for 1 hour at RT with appropriate AlexaFluor-conjugated secondary antibodies (1:500, Thermo Fisher Scientific): anti-rabbit AlexaFluor-594, anti-mouse AlexaFluor-488, anti-rabbit AlexaFluor-488, and anti-chicken AlexaFluor-594. Nuclei were counterstained with DAPI (1:1000, Carl Roth), and coverslips were mounted using DAKO Fluorescent Mounting Medium (Agilent). Fluorescent images were acquired with a Leica DMi8 inverted microscope (LAS X v3.7.423463) and Leica MICA microscope (LAS X v6.2.2.28360; Leica Microsystems CSM GmbH). Quantification was performed using ImageJ software. Colocalization analysis was conducted using the JaCoP plugin for ImageJ. The list of antibodies used in this study is provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eBulk RNA-sequencing\u003c/h3\u003e\n\u003cp\u003eAt DIV60, total RNA was extracted from pooled mDAs derived from healthy controls (pHC) and sPD patients (pPD) using TRIzol Reagent (Life Technologies), according to the manufacturer\u0026rsquo;s protocol. RNA quality and concentration were assessed with the Bioanalyzer 2100 system (Agilent Technologies). For library preparation, mRNA was isolated from total RNA using poly-T oligo-coated magnetic beads. After fragmentation, first-strand cDNA synthesis was performed using random hexamer primers, followed by second-strand synthesis incorporating dUTP. Libraries were validated using Qubit for concentration, real-time PCR for quantification, and Bioanalyzer for fragment size distribution. Sequencing was carried out on the Illumina NextSeq 1000 platform. Reads were aligned to the human reference genome using Hisat2 (v2.0.5), and mapped reads were assembled with StringTie (v1.3.3b). Gene expression was quantified with FeatureCounts (v1.5.0-p3) and normalized as FPKM (Fragments Per Kilobase of transcript per Million mapped reads), based on read counts and gene length. Differential expression analysis between pHC and pPD mDA groups was performed using the DESeq2 R package (v1.20.0). \u003cem\u003ep\u003c/em\u003e-values were adjusted using the Benjamini-Hochberg method to control for false discovery rate (FDR). Genes with an adjusted p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 and |log₂(fold change)| \u0026ge; 1 were considered significantly differentially expressed. Functional enrichment and Gene Ontology (GO) analyses were conducted using the ClusterProfiler R package (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Gene Set Enrichment Analysis (GSEA) was performed with a local version of the GSA tool.\u003c/p\u003e\n\u003ch3\u003eShort Tandem Repeat (STR) Analysis\u003c/h3\u003e\n\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eRetention of all individual cell lines in the pooled mDA cultures was\u003c/span\u003e confirmed \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethroughout differentiation, STR profiling was performed on pPD and pHC mDA samples at both DIV0 and DIV60. The analysis was conducted by Eurofins Genomics Europe (Biotech Products \u0026amp; Services GmbH, Ebersberg, Germany). Genomic DNA was extracted from cell pellets, and STR genotyping was carried out using single-locus PCR amplification. Sixteen independent STR loci were analyzed using the AmpFlSTR\u0026reg; Identifiler\u0026reg; Plus PCR Amplification Kit (Thermo Fisher Scientific). The loci included D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, AMEL, D5S818, FGA, D19S433, vWA, TPOX, and D18S51. For detailed STR profiles, refer to Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\u003cp\u003eNeurons were harvested in 1X PBS (\u0026minus;/\u0026minus;) (Corning, NY, USA) and lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS (all from Sigma-Aldrich, St. Louis, MO, USA), supplemented with Halt\u0026trade; protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Waltham, MA, USA). Lysates were sonicated using a Diagenode Bioruptor (3 cycles of 20 s ON / 20 s OFF) and kept on ice for 30 min. Samples were centrifuged at 21,000 \u0026times; g for 1 h at 4\u0026deg;C. Protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA) using an Eppendorf spectrophotometer (Hamburg, Germany). For each sample, 20 \u0026micro;g of protein was mixed with 1X sample buffer containing Bolt\u0026trade; LDS Sample Buffer and Bolt\u0026trade; Reducing Agent (Thermo Fisher), denatured at 70\u0026deg;C for 10 min, and resolved by SDS-PAGE using Bolt\u0026trade; 4\u0026ndash;12% Bis-Tris Plus gels and 1X MES SDS Running Buffer (Thermo Fisher). Proteins were transferred to nitrocellulose membranes (Bio-Rad) using the Trans-Blot Turbo system (Bio-Rad). Membranes were blocked for 1 h at room temperature in TBS-T containing 5% non-fat milk (PanReac AppliChem, Darmstadt, Germany) and incubated overnight at 4\u0026deg;C with primary antibodies. After washing in TBS-T, membranes were incubated for 1 h at RT with HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit IgG, 1:10,000; Jackson ImmunoResearch, West Grove, PA, USA). Signals were developed using Clarity\u0026trade; Western ECL substrate (Bio-Rad) and imaged with the Alliance\u0026trade; Q9-Atom system (Uvitec, Cambridge, UK). GAPDH and Histone H3 were used as loading controls. Band intensities were quantified using the \u0026ldquo;Analyze Gels\u0026rdquo; tool in ImageJ. A complete list of antibodies is provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Uncropped Western blot images are available in the Supplementary Material under the section entitled \u003cem\u003eOriginal Western Blots\u003c/em\u003e.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMass Spectrometric Analysis\u003c/h2\u003e\u003cp\u003eAll reagents were from Sigma-Aldrich unless otherwise specified. At DIV60, proteomic profiling was performed on four biological replicates each from pHC and pPD mDA neurons, lysed as previously described. Twenty micrograms of protein from each replicate were subjected to reduction and alkylation by sequential incubation with 3.2 \u0026micro;L of 100 mM DTT (1 h, 37\u0026deg;C), 3.8 \u0026micro;L of 200 mM iodoacetamide (1 h, 37\u0026deg;C), and 0.6 \u0026micro;L of 100 mM DTT (20 min, 37\u0026deg;C). Ten micrograms of protein were then processed by Protein Aggregation Capture (PAC), following the protocol by Murfuni et al., 2024 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, 5 \u0026micro;L of MagResyn Hydroxyl beads (100 \u0026micro;g) were equilibrated with two washes in 70% acetonitrile (ACN). Protein binding was induced by adding ACN to 70% final concentration, followed by 10 min incubation at 1100 rpm. The beads were washed three times in ACN and once in 70% ethanol, then resuspended in 50 \u0026micro;L of 50 mM triethylammonium bicarbonate (TEAB). Proteins were digested overnight at 37\u0026deg;C with 200 ng of Trypsin-LysC (Pierce, Thermo Fisher) at a 1:50 enzyme-to-substrate ratio. Peptides were collected, and residual material was eluted with 50 \u0026micro;L of 0.1% formic acid. Peptides were analyzed by nanoLC-MS/MS using an Orbitrap Exploris 480 mass spectrometer coupled to an Easy-nLC 1200 system (Thermo Fisher). Peptides were loaded onto a 17 cm in-house packed silica capillary column (75 \u0026micro;m i.d., 3 \u0026micro;m C18; Dr. Maisch). Separation was achieved using a binary gradient of mobile phase A (2% ACN, 0.1% FA) and mobile phase B (80% ACN, 0.1% FA) at 300 nL/min over 140 min: 3\u0026ndash;25% B over 90 min, 25\u0026ndash;40% over 30 min, 40\u0026ndash;100% over 8 min, followed by 10 min at 100% B. The column was re-equilibrated at 0% B for 2 min. Data were acquired using data-independent acquisition (DIA) mode. Full scans were acquired at 60,000 resolution, followed by 32 MS2 scans at 30,000 resolution with 5e5 AGC target, 50 ms maximum injection time, and 25% normalized collision energy. MS2 isolation windows were 15 \u003cem\u003em/z\u003c/em\u003e in the 350\u0026ndash;710 \u003cem\u003em/z\u003c/em\u003e range, 30 m/z in the 710\u0026ndash;860 \u003cem\u003em/z\u003c/em\u003e range, and 50 \u003cem\u003em/z\u003c/em\u003e in the 860\u0026ndash;1010 \u003cem\u003em/z\u003c/em\u003e range with 0.5 \u003cem\u003em/z\u003c/em\u003e windows overlap. Raw files were analyzed with Spectronaut (v18.7) using the Human 1 Protein 1 Gene database (20,577 sequences, March 2022) for protein identification and quantification using default settings except for the following parameters: proteotypicity filter was \u0026ldquo;protein group specific\u0026rdquo;, precursor filtering was \u0026ldquo;percentile\u0026rdquo;, fraction was \u0026ldquo;0.4\u0026rdquo;, imputation strategy was \u0026ldquo;use background signal\u0026rdquo;. Statistical analysis was performed using Perseus (v2.0.11.0). Protein abundances were log2-transformed and filtered to include only proteins detected in \u0026ge;\u0026thinsp;3 replicates per group. Differentially expressed proteins (DEPs) were identified via two-sample t-test with permutation-based FDR correction (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05; S0\u0026thinsp;=\u0026thinsp;0.2). A total of 6,414 proteins were identified, of which 443 were differentially expressed between pPD and pHC mDAs (|log2FC| \u0026gt;1.5; q\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Gene Ontology (GO) enrichment was performed using the enrichGO function from the ClusterProfiler R package with gene symbols (keyType = \u0026ldquo;SYMBOL\u0026rdquo;) and adjusted using the Benjamini-Hochberg method. GO terms were classified into Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) categories. KEGG pathway enrichment was performed separately for up- and downregulated proteins using the enrichKEGG function, with gene symbols first converted to Entrez IDs via the bitr function and the org.Hs.eg.db annotation database. Only pathways with adjusted p and q-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] via the PRIDE [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] partner repository.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDopamine assay and quantification of pS129 α-syn by ELISA assay\u003c/h3\u003e\n\u003cp\u003eDopamine levels were measured in media collected from neuronal cultures at DIV30, DIV45, and DIV60. Media were centrifuged at 1000 rpm for 20 minutes at 4\u0026deg;C, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis. Dopamine concentrations were quantified using a commercial ELISA kit (ENZ-KIT188-0001, Enzo Biochem) according to the manufacturer\u0026rsquo;s instructions. Phosphorylated α-synuclein at serine 129 (pS129 α-syn) was quantified in total protein lysates from pPD and pHC mDA neurons at DIV60. A total of 50 \u0026micro;g of protein was analyzed using the PSNCA ELISA kit (MyBioSource, #MBS038716, San Diego, CA, USA), following the manufacturer\u0026rsquo;s instructions. Absorbance readings were acquired using a Varioskan\u0026trade; LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\n\u003ch3\u003eLive-cell imaging with FFN102 mesylate\u003c/h3\u003e\n\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eTo evaluate dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) activity, we employed a modified version of the FFN102-based live-imaging protocol described by Virdi et al., 2022\u003c/span\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eCells were incubated with FFN102 mesylate (10 \u0026micro;M; Tocris, #BT\u0026minus;5200) in pre-warmed (37\u0026deg;C) calcium- and magnesium-free HBSS without phenol red (Thermo Fisher Scientific). After 30 minutes at 37\u0026deg;C, intracellular dye uptake was confirmed. Membrane depolarization was induced by applying 50 mM KCl in HBSS to stimulate FFN102 release.\u003c/span\u003e Representative images were acquired \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eusing a 405 nm laser on a Leica DMi8 inverted microscope with LAS X software (v3.7.423463; Leica Microsystems). Fluorescence intensity changes were quantified over time using ImageJ (NIH, USA).\u003c/span\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePharmacological inhibition of caspase and PKA\u003c/h2\u003e\u003cp\u003eTo inhibit caspase activity, mDA neurons were treated with the pan-caspase inhibitor Q-VD-OPh (1 \u0026micro;M; MedChemExpress, #HY-12305) for 15 consecutive days, from day 45 to day 60 of differentiation. Culture medium was refreshed every other day to maintain constant inhibitor levels. To block protein kinase A (PKA) activity, both pPD and control mDA neurons were treated with H-89 dihydrochloride (10 \u0026micro;M; Cell Signaling, #9844) for 5 days, from day 55 to day 60. In both conditions, neurons treated with vehicle (DMSO) served as controls. At the end of each treatment, cells were harvested for downstream analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed with GraphPad PRISM software (v.10.5.0, GraphPad Software Inc.), and data are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.e.m. p-value was presented with the following levels of significance: * \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001, and # \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001. Statistical details for each experiment are provided in the respective figure legends and, for the transcriptomic and proteomic analyses, in the corresponding sections of the Materials and Methods.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003ePooled differentiation of sPD and control iPSCs into midbrain dopaminergic neurons reveals early transcriptional divergence\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe iPSCs lines from 3 sPD patients and 3 HC (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) were differentiated into mDAs using the protocols of Kriks et al., 2011\u003c/span\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], based on \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003edual SMAD inhibition (LDN193189 and SB431542) for neural identity induction\u003c/span\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewith SHH and WNT pathway activation (recombinant SHH-C24II, purmorphamine, CHIR99021) to specify ventral midbrain floor plate (mFP) fate (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). By day 11, the resulting mFP progenitors (mFPPs) showed robust expression of the neuroepithelial marker NESTIN and the mFP transcription factors FOXA2 (~ 80%) and LMX1A (~ 70%) (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB–D), while lacking PAX6, confirming ventral identity. Terminal differentiation into mDAs was achieved using a maturation cocktail containing BDNF, GDNF, ascorbic acid, db-cAMP, TGFβ−3, and DAPT. By DIV\u003c/span\u003e2\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e0, cultures consistently expressed both MAP2 and tyrosine hydroxylase (TH), with comparable TH+/MAP2 + proportions across all lines (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE–F). To enhance throughput and eliminate the impact of line-to-line variation, immature mDA neurons (imDAs) from the three sPD and three HC lines were combined and plated to yield pooled PD (pPD) and pooled HC (pHC) cultures (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). STR profiling at 16 loci confirmed stable contribution from all donor lines at both DIV0 and DIV60, with unique alleles at four loci in pHC and seven in pPD (Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). By DIV30, both groups exhibited mature neuronal morphology with extended neurites, and by DIV45, brain nucleus-like rosettes with radial cell organization emerged (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). Full differentiation proceeded to DIV60, with sequential marker acquisition: NR4A2 (NURR1) was expressed in ~ 80% of pHC and ~ 75% of pPD neurons at DIV30 (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC–D\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e); co-expression of TH and DDC was evident at DIV45 (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE–F\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e); and robust GIRK2 expression, marking A9-type mature mDAs, was detected by DIV60 (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG–H\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), confirming efficient and comparable differentiation across groups. To identify disease-associated transcriptional signatures, we performed bulk RNA-sequencing of pPD and pHC cultures at DIV60. A total of 35 804 genes were detected (Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Principal Component Analysis (PCA) revealed a clear segregation by disease status (PC1, 90% variance) and biological reproducibility (PC2, 7%) (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), supported by Pearson correlation among replicates (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG). Differential expression analysis (|log2FC| ≥ 1; adjusted p ≤ 0.05) identified 1 191 upregulated and 1 467 downregulated genes in pPD relative to pHC (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). GO term enrichment analysis revealed that downregulated genes were significantly associated with catecholamine biosynthesis, neurotransmitter metabolism, and GPCR signaling (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), while upregulated genes were enriched in pathways related to calcium signaling, synaptic inhibition, and postsynaptic receptor activity (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). These findings indicate that, while sPD-derived mDAs acquire appropriate regional identity and mature comparably to controls, they display robust, disease-specific transcriptomic changes reflecting dopaminergic dysfunction and early synaptic dysregulation.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003epPD mDA neurons exhibit progressive loss of identity and function\u003c/h2\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eTo elucidate the phenotypic trajectory of dopaminergic neurons in sPD, we systematically compared dopaminergic identity and function between pPD and pHC mDA neurons. PD is marked by a progressive loss of nigral dopaminergic neurons; we therefore investigated molecular and functional signatures indicative of such degeneration in our human iPSC-derived model. Transcriptomic profiling of DIV60 pPD neurons revealed robust downregulation of genes essential for dopaminergic identity, including transcription factors (\u003c/span\u003e\u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eFOXA2\u003c/span\u003e, \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eNR4A2/NURR1\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), biosynthetic enzymes (\u003c/span\u003e\u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eDDC\u003c/span\u003e, \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eTH\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), and key transporters (\u003c/span\u003e\u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eSLC6A3/DAT\u003c/span\u003e, \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eSLC18A2/VMAT2\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e) (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). These transcriptomic defects were confirmed at the protein level: western blotting showed a significant reduction in TH, the rate-limiting enzyme in dopamine synthesis, in pPD versus pHC neurons at DIV60 (\u003c/span\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB–C\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). Immunofluorescence analysis corroborated this reduction, demonstrating a marked decrease in TH + neurons in pPD cultures (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). Temporal quantification revealed that while TH+/MAP2 + neuron ratios remained stable in pHC cultures, a progressive decline was evident in pPD neurons from DIV30 to DIV60 (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), suggesting a disease-driven erosion of dopaminergic identity over time. To assess whether molecular defects translated into functional impairment, we quantified dopamine release in the culture medium at DIV30, DIV45, and DIV60. Dopamine secretion progressively declined in pPD neurons, whereas it increased in pHC cultures across the same timepoints (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), indicating a temporal deterioration in dopaminergic output in the disease condition. To investigate synaptic dopamine dynamics, we employed FFN102, a fluorescent analog of dopamine that enables live imaging of vesicular uptake and release\u003c/span\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eBoth pPD and pHC neurons successfully internalized FFN102, indicating functional uptake mechanisms. However, upon depolarization with KCl, pPD neurons exhibited a significantly blunted and delayed release response compared to pHC (\u003c/span\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG–H\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), reflecting compromised vesicular dopamine mobilization or exocytosis. This phenotype is consistent with impaired VMAT2-mediated loading and supports the notion of disrupted synaptic dopamine handling. Collectively, these data demonstrate that sPD-derived mDA neurons undergo progressive dopaminergic degeneration, characterized by transcriptional repression of core identity genes, reduced protein expression, declining dopamine release, and impaired synaptic exocytosis. These early and intrinsic functional deficits recapitulate key aspects of PD pathology.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePathological accumulation of phosphorylated α-synuclein in pPD mDA neurons recapitulates early-stage Lewy pathology\u003c/h2\u003e\u003cp\u003eTo determine whether patient-derived dopaminergic neurons faithfully model key pathogenic features of sPD, we investigated the presence and distribution of pathological α-synuclein species in pPD mDA neurons. Disease Ontology (DO) enrichment analysis of bulk RNA-seq data revealed a significant over-representation of gene signatures associated with neurodegenerative conditions, particularly synucleinopathies and Lewy body dementia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Among the 16 synucleinopathy-associated genes enriched in pPD neurons, several known contributors to oxidative stress and protein misfolding, such as ceruloplasmin (\u003cem\u003eCP\u003c/em\u003e), monoamine oxidase B (\u003cem\u003eMAOB\u003c/em\u003e), NAD(P)H quinone dehydrogenase 1 (\u003cem\u003eNQO1\u003c/em\u003e), tropomyosin receptor kinase B (\u003cem\u003eNTRK2\u003c/em\u003e), and neuronal pentraxin-2 (\u003cem\u003eNPTX2\u003c/em\u003e), were significantly upregulated compared to pHC controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting a molecular landscape permissive to α-synuclein misprocessing. Despite total α-synuclein protein levels did not significantly differ between pPD and pHC neurons (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC–D), immunofluorescence analysis revealed a striking accumulation of serine-129 phosphorylated α-synuclein (pS129 α-syn) in pPD mDA neurons (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE–F), a post-translational modification tightly associated with pathological aggregation and Lewy body formation. This observation was independently validated by western blotting (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG–H) and ELISA quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), confirming the selective accumulation of the phosphorylated, pathogenic α-synuclein species. To further assess whether pS129 α-syn formed aggregation-prone structures, we analyzed its spatial colocalization with key components of Lewy bodies. In pPD neurons, pS129 α-syn displayed enhanced colocalization with ubiquitin (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ–K), a classical marker of protein aggregates [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], supporting the presence of insoluble α-synuclein inclusions. Moreover, discrete cytoplasmic puncta showed strong overlap between pS129 α-syn and the autophagy adaptor SQSTM1/p62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL), consistent with sequestration of misfolded protein aggregates into autophagosomal structures. Quantitative image analysis confirmed significantly increased pS129 α-syn/p62 colocalization in pPD versus pHC neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM), indicating impaired proteostasis and the formation of early-stage Lewy body–like inclusions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Collectively, these findings indicate that mDAs derived from pPD exhibit key pathological hallmarks of the α-synuclein pathology.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eProteomic profiling reveals early disruption of the PKA–CREB axis in sPD dopaminergic neurons\u003c/h2\u003e\u003cp\u003eTo dissect the molecular mechanisms underlying early dopaminergic vulnerability in sPD, we performed untargeted proteomic profiling on mDA neurons derived from pPD and pHC mDAs at DIV60. Total protein extracts were analyzed via nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). PCA revealed clear clustering of samples according to disease status, indicating robust and reproducible proteomic divergence between pPD and pHC neurons (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). A total of 6 414 proteins were identified across all samples, of which 443 met the thresholds for differential expression (|log₂FC| ≥ 1.5; q ≤ 0.05; Supplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Among these, 197 proteins were significantly upregulated and 246 were downregulated in pPD neurons compared to pHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Strikingly, the proteomic alterations mirrored transcriptomic changes observed in bulk RNA-seq, including reduced expression of core dopaminergic proteins such as TH, VMAT2, and DAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), alongside upregulation of molecules involved in PD pathology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). GO analysis of differentially expressed proteins (DEPs) highlighted disease-relevant pathways. Upregulated DEPs were enriched in biological processes (BP) such as apoptosis, regionalization, and cytoskeletal organization; associated cellular components (CC) included extracellular matrix, integrin complexes, and adhesion junctions; and molecular functions (MF) encompassed calcium ion binding and integrin signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Conversely, downregulated DEPs were associated with synaptic signaling, dopamine biosynthesis, and axon function (BP); presynaptic and postsynaptic compartments (CC); and neurotransmitter receptor interactions (MF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Pathway analysis (KEGG) further confirmed dysregulation of synaptic architecture and signaling homeostasis in pPD neurons (Supplementary Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB–C). Notably, multiple nodes within the cAMP–PKA–CREB axis, a pathway critically involved in dopaminergic neuron survival and transcriptional identity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], were markedly downregulated in pPD neurons (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD). In particular, levels of ADCY8 and UCN, two upstream regulators of adenylate cyclase activity, were significantly diminished (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Likewise, the catalytic subunits of PKA (PRKACA and PRKACB) were downregulated in pPD neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), suggesting a widespread attenuation of cAMP-mediated signal transduction. These perturbations converged on reduced activation of CREB, as evidenced by lower levels of Ser133-phosphorylated CREB (pCREB) in pPD neurons (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH–I). Moreover, canonical CREB-dependent genes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], including those linked to synaptic plasticity and dopaminergic differentiation, exhibited consistent transcriptional and proteomic downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ; Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE). Together, these proteomic data reveal widespread deficits in dopaminergic identity, synaptic function, and intracellular signaling in sPD-derived neurons. In particular, the downregulation of PKA-CREB pathway components suggests that impaired activation of this neuroprotective axis may play a key role in disease pathogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLoss of synaptic integrity and density in pPD mDA neurons reflects disruption of the PKA–CREB signaling axis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe PKA–CREB pathway plays a central role in coordinating synaptic development, plasticity, and neurotransmission [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Building on our proteomic evidence of PKA–CREB pathway repression in pPD neurons, we examined synaptic protein expression and structural integrity in these cells. Differential proteomic analysis revealed significant downregulation of key synaptic components in pPD mDA neurons. Notably, the vesicular monoamine transporter SLC18A2 (VMAT2), sodium/calcium exchanger SLC8A2, neuropeptide precursor VGF, and two canonical synaptic scaffolding proteins, synaptophysin (SYP) and DLG4 (encoding PSD95), were among the significantly downregulated DEPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To validate these changes at the protein level, we performed western blotting for SYP and PSD95. Both synaptic proteins were significantly reduced in pPD neurons at DIV60 relative to pHC cultures (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB–E), consistent with proteomic findings. To assess whether these reductions reflected impaired synaptogenesis or synapse destabilization, we conducted immunofluorescence staining for SYP and PSD95 in mDA cultures. Quantification revealed a pronounced decrease in the number of PSD95 + puncta in pPD neurons (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF–G), indicative of reduced postsynaptic site density. Fluorescence intensity measurements confirmed a substantial decrease in SYP and PSD95 signal at synaptic compartments in pPD neurons, suggesting impaired accumulation or retention of synaptic proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Morphometric analysis further demonstrated a significant reduction in the area occupied by SYP- and PSD95-positive structures in pPD cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), supporting the hypothesis of synaptic loss rather than redistribution. Interestingly, despite the overall reduction in synaptic marker expression and synapse number, the degree of colocalization between SYP and PSD95 puncta remained comparable between pPD and pHC neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). This suggests that synapse formation, where it occurs, remains structurally coordinated in pPD neurons, but that the overall number of synapses is markedly diminished. Together, these findings indicate that mDA neurons derived from pPD patients exhibit a substantial loss of synaptic components and reduced synaptic density, likely reflecting early structural and functional synaptic deficits. These alterations are consistent with impaired PKA-CREB signaling and may contribute to the progressive synaptic dysfunction observed in PD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eConvergent upregulation of apoptotic effectors and BDNF depletion underlie the intrinsic vulnerability of pPD mDA neurons\u003c/b\u003e\u003c/p\u003e\u003cp\u003eApoptotic dysregulation is a well-established contributor to dopaminergic neurodegeneration in PD, and accumulating evidence suggests that early synaptic dysfunction can mechanistically initiate pro-apoptotic cascades [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e–\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; PMID: 9006329). Proteomic profiling of pPD mDA neurons at DIV60 revealed robust upregulation of several core apoptotic effectors. Notably, caspase-3 and caspase-7, key executioner caspases, were significantly elevated in pPD neurons, alongside FAS and BAX, critical mediators of receptor-mediated and mitochondrial apoptosis, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). These proteomic findings were independently validated by immunoblotting, which demonstrated increased levels of FAS ligand (FASL) and BAX in pPD neurons relative to pHC (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB–C). Western blot analysis further revealed elevated expression of both full-length and cleaved forms of caspase-3 and caspase-7 in pPD neurons (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD–E), confirming activation of the apoptotic cascade at a post-translational level. To functionally assess apoptotic engagement, we performed TUNEL staining, which revealed a significantly greater proportion of TUNEL-positive cells in pPD mDA cultures compared to controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF–G). Given the neuroprotective role of brain-derived neurotrophic factor (BDNF) in dopaminergic survival and synaptic maintenance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we next examined its expression in pPD neurons. Immunoblotting and densitometric analysis revealed a marked reduction in BDNF protein levels in pPD cultures (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH–I), consistent with deficient neurotrophic support. The concurrent elevation of pro-apoptotic signaling and depletion of BDNF suggests a permissive environment for degeneration in pPD neurons. Altogether, these results highlight an intrinsic molecular vulnerability in sPD-derived mDA neurons, characterized by convergent activation of apoptotic machinery and reduced availability of trophic support.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCaspase inhibition restores dopaminergic and synaptic identity in pPD mDA neurons and reduces pathological α-synuclein burden\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo mechanistically test whether excessive caspase activity contributes to the dopaminergic and synaptic deficits observed in pPD mDA neurons, we performed pharmacological rescue experiments using the pan-caspase inhibitor Q-VD-OPh. Chronic administration of Q-VD-OPh (15 days, DIV45–DIV60, Supplementary Figures \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA) at the optimal concentration of 1 µM, previously defined by effective suppression of cleaved caspase-3 and − 7 levels (Supplementary Figures \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB), significantly restored key phenotypic features of dopaminergic neurons. Western blotting revealed that Q-VD-OPh treatment led to marked upregulation of TH and NURR1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA–B), indicative of transcriptional reactivation of the dopaminergic lineage. In functional terms, treated neurons exhibited significantly enhanced depolarization-induced release of the dopamine surrogate FFN102, pointing to improved vesicular storage and exocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC; Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC). At the synaptic level, Q-VD-OPh robustly increased the expression of pre- and postsynaptic markers SYP and PSD95 (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD–E). Morphometric analysis revealed a parallel expansion in the synaptic area occupied by SYP and PSD95 (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF–G), as well as a significant rise in the number of discrete PSD95-positive structures (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH), consistent with recovery of synaptic density and architecture. Q-VD-OPh treatment also induced a substantial increase in BDNF protein levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI–J), suggesting that caspase inhibition relieves repression of neurotrophic signaling cascades. Furthermore, western blot analysis revealed a significant reduction in pS129 α-syn (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK–L). Collectively, these findings identify caspases as upstream regulators of both dopaminergic transcriptional fidelity and synaptic maintenance. Their pharmacological inhibition not only prevents further molecular degeneration but actively re-engages neurotrophic and functional pathways in mDAs neurons.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"BoldSmallCaps\" class=\"BoldSmallCaps\" name=\"Emphasis\"\u003ePKA–CREB signaling mediates the restorative effects of caspase inhibition in PD mDA neurons\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eHaving observed that Q-VD-OPh treatment restores the expression of key dopaminergic and neurotrophic genes, many of which are known transcriptional targets of CREB, we hypothesized that activation of the PKA–CREB axis underpins the neuroprotective response to caspase inhibition. Western blot analyses confirmed that Q-VD-OPh selectively enhanced CREB phosphorylation at Ser133 (pCREB) (\u003c/span\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA–B\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). To determine whether CREB phosphorylation is PKA-dependent, we co-treated pPD neurons with Q-VD-OPh and the selective PKA inhibitor H89. Co-inhibition of PKA completely abrogated Q-VD-OPh-induced increases in pCREB (\u003c/span\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC–D\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), and notably prevented the restoration of BDNF protein expression (\u003c/span\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE–F\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). Similarly, H89 blocked Q-VD-OPh-mediated upregulation of TH (\u003c/span\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG–H\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), establishing that PKA activity is required for dopaminergic\u003c/span\u003e marker \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ereactivation upon caspase inhibition. To further dissect the role of this pathway under basal conditions, we treated healthy pHC neurons with H89 alone. PKA blockade in control neurons resulted in reduced levels of total and phosphorylated CREB (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), and downregulation of downstream targets including TH, NURR1 (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), and the presynaptic protein SYP (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). Importantly, PKA inhibition triggered an increase in pS129 α-syn accumulation (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e). Furthermore, H89 led to upregulation of both full-length and cleaved caspase−7 (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eM\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), suggesting that disruption of PKA–CREB signaling is sufficient to initiate a pro-apoptotic cascade. Taken together, these findings position the PKA–CREB axis as a pivotal integrator of dopaminergic maintenance, synaptic integrity, and neuronal survival. Caspase inhibition restores PKA–CREB activity, thereby re-engaging neuroprotective transcriptional programs and suppressing PD-related pathomechanisms. Conversely, its pharmacological disruption induces transcriptional silencing, α-synuclein pathology, and apoptosis, even in non-diseased neurons. Overall, the data suggest that caspases act upstream of PKA–CREB and that this signaling axis is essential for caspase-mediated neuroprotection in PD.\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eIn this study, we leveraged an iPSC-based system to model early pathological and molecular features of sPD. mDAs derived from patient-specific iPSCs offer a powerful human cellular platform to investigate disease-initiating mechanisms\u003c/span\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eWhile previous sPD iPSC-derived models have captured aspects of pathology\u003c/span\u003e [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e–\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethe molecular drivers of disease onset remain elusive, and no disease-modifying therapies are currently available. Our findings demonstrate that long-term cultured sPD mDAs recapitulate key pathological features, including progressive loss of dopaminergic identity, pS129 α-syn accumulation, synaptic dysfunction, and caspase activation. Notably, pharmacological inhibition of caspases restored dopaminergic and synaptic markers and unveiled the PKA-CREB signaling axis as a critical mediator of this rescue effect. Caspase activation is a hallmark of apoptotic cell death\u003c/span\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], but emerging studies have revealed that caspases also exert non-canonical roles in the central nervous system, including the regulation of synaptic remodeling, axonal guidance, and transcriptional programs. In particular, caspase-3 and caspase-9 have been implicated in dendritic pruning and activity-dependent plasticity, even in the absence of overt cell death, suggesting a more nuanced role in neuronal homeostasis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAccumulation of active caspases has been reported in sPD brains\u003c/span\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand our data confirm upregulation of caspases 3 and 7 in sPD mDAs. Th\u003c/span\u003eese observations \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ealign with prior studies implicating caspase-mediated apoptosis in dopaminergic neuron degeneration, particularly via caspase−3 activation\u003c/span\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eA defining pathological feature of PD is α-synuclein misfolding and aggregation into Lewy bodies\u003c/span\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eCaspase activity promotes α-synuclein aggregation by generating toxic C-terminal fragments\u003c/span\u003e [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand impairs autophagic clearance by cleaving core ATG proteins\u003c/span\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eIn our model, pS129 α-syn co-localized with ubiquitin and p62/SQSTM1, recapitulating key features of like-Lewy body composition\u003c/span\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThis pathological accumulation may drive the observed downregulation of dopaminergic regulators TH and NURR1, consistent with studies linking α-syn overexpression to dopaminergic loss\u003c/span\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eBeyond their role in apoptosis, caspases regulate synaptic structure and function. Caspase−3, for instance, is essential for long-term depression (LTD) via AMPA receptor internalization\u003c/span\u003e [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eCaspases also degrade essential synaptic proteins such as PSD95 and synaptophysin\u003c/span\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), which were downregulated in our sPD mDAs. These proteins are critical for synapse formation and plasticity\u003c/span\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eSince α-synuclein also regulates synaptic vesicle cycling and SNARE complex formation\u003c/span\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eits pathological accumulation likely contributes to the reduced dopamine reuptake observed in sPD mDAs. Given the youthful state of iPSC-derived neurons\u003c/span\u003e [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ethese changes likely reflect early, intrinsic disease mechanisms rather than age-related effects\u003c/span\u003e [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eTherapeutically, our data show that Q-VD-OPh, a broad-spectrum caspase inhibitor with improved bioavailability and reduced toxicity compared to earlier compounds such as zVAD-fmk\u003c/span\u003e [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eeffectively restores dopaminergic identity and synaptic markers while reducing pS129 α-syn levels. Previous work confirmed Q-VD-OPh’s neuroprotective effects in MPTP mouse models and human neurons overexpressing α-synuclein\u003c/span\u003e [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eMechanistically, we identified the PKA-CREB signaling axis as the key downstream mediator of this rescue. Proteomic analysis revealed marked downregulation of PKA-CREB signaling in sPD neurons, which was reversed upon Q-VD-OPh treatment. Co-treatment with the PKA inhibitor H89 abolished the beneficial effects of Q-VD-OPh, confirming the pathway’s central role. This signaling cascade has been previously linked to PD pathology and neurotrophic support\u003c/span\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eImportantly, BDNF, a direct CREB target and key TrkB receptor ligand, was also restored following Q-VD-OPh treatment, suggesting a positive feedback loop that amplifies neuroprotective signaling\u003c/span\u003e [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eCaspase inhibition may enhance PKA-CREB activity by suppressing phosphatase-mediated dephosphorylation of CREB\u003c/span\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eStrikingly, PKA-CREB inhibition in healthy control neurons via H89 recapitulated pathological phenotypes: decreased dopaminergic and synaptic markers, elevated α-synuclein phosphorylation, and caspase activation. This indicates a reciprocal regulatory loop between caspases and PKA-CREB, positioning this axis as a critical safeguard of dopaminergic neuronal integrity. We propose that caspase activation functions upstream of the PKA–CREB–BDNF axis, repressing the transcriptional program required for dopaminergic identity and synaptic maturation in patient-derived mDA neurons.\u003c/span\u003e\u003c/p\u003e\u003cp\u003eIn this context, caspases contribute to dopaminergic degeneration in sporadic PD not only by promoting cell death but also by disrupting homeostatic signaling essential for neuronal identity. Early pharmacological inhibition with compounds like Q-VD-OPh restored TH and NURR1 expression, improved synaptic markers, and reduced pathogenic pS129 α-synuclein, suggesting potential disease-modifying effects if applied during prodromal or early symptomatic stages. However, limitations of the iPSC model, such as immature neuronal profiles and absence of age-related or glial interactions, must be considered. Additionally, pooled cultures may mask patient-specific traits. Future studies should combine this in vitro approach with in vivo and organoid models to validate the therapeutic value of targeting caspase signaling. Overall, these findings highlight a novel role for caspases in PD pathology and identify the caspase–PKA–CREB–BDNF axis as a promising target for neuroprotection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the following funding: PNRR Project #CN00000041: National Center for Gene Therapy and Drugs Based on RNA Technology (CN RNA \u0026amp; Gene Therapy) CUP J33C22001130001 to G.C.; PNRR Project: A multiscale integrated approach to the study of the nervous system in health and disease (MNESYS) CUP D33C22001340002 to G.C co-awarded to Al.Q.\u003c/p\u003e\u003ch2\u003eAuthor contributions:\u003c/h2\u003e\u003cp\u003eG.C. supervised the study. G.C. and S.S. conceived and designed the study and the experiments. G.L.B. and R.C. conducted the experiments. G.L.B., R.C., and S.S. analyzed the data and prepared the figures. G.L.B., R.C., S.S., and E.I.P interpreted the results. C.G. and M.G. performed the proteomic experiment and analyzed the data. B.P., P.H.G., and P.V. conducted the bioinformatic analysis. D.V. and C.Z. assisted with the laboratory experiments. An.Q. and Al.Q. recruited the patients and provided clinical samples and data. V.T. provided critical discussions and suggestions regarding the differentiation process. G.C. and Al.Q. provided financial support. S.S. drafted the manuscript. E.I.P. and G.C. wrote the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge the donors whose contributions made this work possible and thank Dr. Annamaria Aloisio for her technical support. B.P. acknowledges Relatech S.p.A. for the partial funding of her doctoral fellowship. The figures of this manuscript were created using BioRender.\u003c/p\u003e\u003ch2\u003eAvailability of Data and Materials:\u003c/h2\u003e\u003cp\u003eThe raw RNAseq dataset generated and analysed in the current study has been deposited in the Gene Expression Omnibus (GEO) database, under accession number GSE304324. The proteomics data are available via ProteomeXchange with identifier PXD066765. The other datasets are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBromberg-Martin ES, Matsumoto M, Hikosaka O. Dopamine in Motivational Control: Rewarding, Aversive, and Alerting. Neuron. 2010;68(5):815\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeibo Shi, Yaxing Zhang, Guoting Zhao, Songjun Wang, Guozhong Zhang, Chunling Ma, et al. Dysregulation of Dopaminergic Regulatory Factors TH, Nurr1, and Pitx3 in the Ventral Tegmental Area Associated with Neuronal Injury Induced by Chronic Morphine Dependence [Internet]. [cited 2025 July 31]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mdpi.com/\u003c/span\u003e\u003cspan address=\"https://www.mdpi.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e1422-0067/20/2/250\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePoewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, Volkmann J, et al. Parkinson disease. Nat Rev Dis Primer. 2017;3(1):17013.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJanezic S, Threlfell S, Dodson PD, Dowie MJ, Taylor TN, Potgieter D, et al. Deficits in dopaminergic transmission precede neuron loss and dysfunction in a new Parkinson model. Proc Natl Acad Sci U S A. 2013;110(42):E4016-4025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTanner CM, Ross GW, Jewell SA, Hauser RA, Jankovic J, Factor SA, et al. Occupation and Risk of Parkinsonism: A Multicenter Case-Control Study. Arch Neurol. 2009 Sept 1;66(9):1106\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCederquist GY, Tchieu J, Callahan SJ, Ramnarine K, Ryan S, Zhang C, et al. A Multiplex Human Pluripotent Stem Cell Platform Defines Molecular and Functional Subclasses of Autism-Related Genes. Cell Stem Cell. 2020 July 2;27(1):35\u0026ndash;49.e6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCuomo ASE, Seaton DD, McCarthy DJ, Martinez I, Bonder MJ, Garcia-Bernardo J, et al. Single-cell RNA-sequencing of differentiating iPS cells reveals dynamic genetic effects on gene expression. Nat Commun. 2020;11(1):810.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJerber J, Seaton DD, Cuomo ASE, Kumasaka N, Haldane J, Steer J, et al. Population-scale single-cell RNA-seq profiling across dopaminergic neuron differentiation. Nat Genet. 2021;53(3):304\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLimone P, Ragni B, Toto GA. The epidemiology and effects of video game addiction: A systematic review and meta-analysis. Acta Psychol (Amst). 2023;241:104047.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu X, He X, Zhang Z, Chen Y, Li J, Ma S, et al. CREB Inactivation by HDAC1/PP1γ Contributes to Dopaminergic Neurodegeneration in Parkinson\u0026rsquo;s Disease. J Neurosci. 2022 June 1;42(22):4594\u0026ndash;604.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEssam RM, Kandil EA. p-CREB and p-DARPP-32 orchestrating the modulatory role of cAMP/PKA signaling pathway enhanced by Roflumilast in rotenone-induced Parkinson\u0026rsquo;s disease in rats. Chem Biol Interact. 2023;372:110366.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParrotta EI, Scalise S, Taverna D, De Angelis MT, Sarro G, Gaspari M, et al. Comprehensive proteogenomic analysis of human embryonic and induced pluripotent stem cells. J Cell Mol Med. 2019;23(8):5440\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParrotta EI, Lucchino V, Zannino C, Valente D, Scalise S, Bressan D, et al. Modeling Sporadic Progressive Supranuclear Palsy in 3D Midbrain Organoids: Recapitulating Disease Features for In Vitro Diagnosis and Drug Discovery. Ann Neurol. 2025;97(5):845\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBenedetto G, Zannino C, Valente D, Covello R, Scalise S, Lucchino V, et al. Generation of hiPSCs lines from three sporadic Parkinson\u0026rsquo;s disease patients. Stem Cell Res. 2025;82:103632.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eValente D, Zannino C, Scalise S, Bressan D, Lucchino V, Filomena E, et al. Distinct microRNA signatures define sporadic PSP-RS and PD in patient-derived midbrain organoids. iScience. 2025 July 18;113162.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson\u0026rsquo;s disease. Nature. 2011;480(7378):547\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMurfuni MS, Prestagiacomo LE, Giuliano A, Gabriele C, Signoretti S, Cuda G, et al. Evaluation of PAC and FASP Performance: DIA-Based Quantitative Proteomic Analysis. Int J Mol Sci. 2024;25(10):5141.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeutsch EW, Bandeira N, Perez-Riverol Y, Sharma V, Carver JJ, Mendoza L, et al. The ProteomeXchange consortium at 10 years: 2023 update. Nucleic Acids Res. 2023;51(D1):D1539\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerez-Riverol Y, Bandla C, Kundu DJ, Kamatchinathan S, Bai J, Hewapathirana S, et al. The PRIDE database at 20 years: 2025 update. Nucleic Acids Res. 2025;53(D1):D543\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVirdi GS, Choi ML, Evans JR, Yao Z, Athauda D, Strohbuecker S, et al. Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson\u0026rsquo;s disease in midbrain dopaminergic neurons. Npj Park Dis. 2022;8(1):162.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson\u0026rsquo;s disease. Nature. 2011;480(7378):547\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27(3):275\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRodriguez PC, Pereira DB, Borgkvist A, Wong MY, Barnard C, Sonders MS, et al. Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain. Proc Natl Acad Sci U S A. 2013;110(3):870\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpillantini MG, Schmidt ML, Lee VMY, Trojanowski JQ, Jakes R, Goedert M. α-Synuclein in Lewy bodies. Nature. 1997;388(6645):839\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMasato A, Andolfo A, Favetta G, Bellini EN, Cogo S, Dalla Valle L, et al. Sequestosome-1 (SQSTM1/p62) as a target in dopamine catabolite-mediated cellular dyshomeostasis. Cell Death Dis. 2024 June 18;15(6):424.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNiccolini F, Wilson H, Pagano G, Coello C, Mehta MA, Searle GE, et al. Loss of phosphodiesterase 4 in Parkinson disease: Relevance to cognitive deficits. Neurology. 2017;89(6):586\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMucignat C, Caretta A. Drug-induced Parkinson\u0026rsquo;s disease modulates protein kinase A and Olfactory Marker Protein in the mouse olfactory bulb. Behav Brain Funct. 2017;13(1):1.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang X, Odom DT, Koo SH, Conkright MD, Canettieri G, Best J, et al. Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues. Proc Natl Acad Sci. 2005;102(12):4459\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSakamoto K, Karelina K, Obrietan K. CREB: a multifaceted regulator of neuronal plasticity and protection. J Neurochem. 2011;116(1):1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDauer W, Przedborski S. Parkinson\u0026rsquo;s Disease: Mechanisms and Models. Neuron. 2003 Sept 11;39(6):889\u0026ndash;909.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh S, Dikshit M. Apoptotic neuronal death in Parkinson\u0026rsquo;s disease: Involvement of nitric oxide. Brain Res Rev. 2007 June 1;54(2):233\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTompkins MM, Basgall EJ, Zamrini E, Hill WD. Apoptotic-like changes in Lewy-body-associated disorders and normal aging in substantia nigral neurons. Am J Pathol. 1997;150(1):119\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu CL, Hwang CS, Chen SD, Yin JH, Yang DI. Neuroprotective mechanisms of brain-derived neurotrophic factor against 3-nitropropionic acid toxicity: therapeutic implications for Huntington\u0026rsquo;s disease. Ann N Y Acad Sci. 2010 July;1201:8\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHowells DW, Porritt MJ, Wong JYF, Batchelor PE, Kalnins R, Hughes AJ, et al. Reduced BDNF mRNA Expression in the Parkinson\u0026rsquo;s Disease Substantia Nigra. Exp Neurol. 2000;166(1):127\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBose A, Petsko GA, Studer L. Induced pluripotent stem cells: a tool for modeling Parkinson\u0026rsquo;s disease. Trends Neurosci. 2022;45(8):608\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eS\u0026aacute;nchez-Dan\u0026eacute;s A, Richaud‐Patin Y, Carballo‐Carbajal I, Jim\u0026eacute;nez‐Delgado S, Caig C, Mora S, et al. Disease‐specific phenotypes in dopamine neurons from human iPS‐based models of genetic and sporadic Parkinson\u0026rsquo;s disease. EMBO Mol Med. 2012;4(5):380\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStern S, Lau S, Manole A, Rosh I, Percia MM, Ben Ezer R, et al. Reduced synaptic activity and dysregulated extracellular matrix pathways in midbrain neurons from Parkinson\u0026rsquo;s disease patients. Npj Park Dis. 2022;8(1):103.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin L, G\u0026ouml;ke J, Cukuroglu E, Dranias MR, VanDongen AMJ, Stanton LW. Molecular Features Underlying Neurodegeneration Identified through In Vitro Modeling of Genetically Diverse Parkinson\u0026rsquo;s Disease Patients. Cell Rep. 2016 June 14;15(11):2411\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGalluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 2018;25(3):486\u0026ndash;541.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSnigdha S, Smith ED, Prieto GA, Cotman CW. Caspase-3 activation as a bifurcation point between plasticity and cell death. Neurosci Bull. 2012;28(1):14\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTatton NA. Increased Caspase 3 and Bax Immunoreactivity Accompany Nuclear GAPDH Translocation and Neuronal Apoptosis in Parkinson\u0026rsquo;s Disease. Exp Neurol. 2000;166(1):29\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHartmann A, Troadec JD, Hunot S, Kikly K, Faucheux BA, Mouatt-Prigent A, et al. Caspase-8 Is an Effector in Apoptotic Death of Dopaminergic Neurons in Parkinson\u0026rsquo;s Disease, But Pathway Inhibition Results in Neuronal Necrosis. J Neurosci. 2001;21(7):2247\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHartmann A, Hunot S, Michel PP, Muriel MP, Vyas S, Faucheux BA, et al. Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson\u0026rsquo;s disease. Proc Natl Acad Sci. 2000;97(6):2875\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHernandez-Baltazar D, Mendoza-Garrido ME, Martinez-Fong D. Activation of GSK-3β and Caspase-3 Occurs in Nigral Dopamine Neurons during the Development of Apoptosis Activated by a Striatal Injection of 6-Hydroxydopamine. PLOS ONE. 2013;8(8):e70951.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu L, Pu J. Alpha-Synuclein in Parkinson\u0026rsquo;s Disease: From Pathogenetic Dysfunction to Potential Clinical Application. Park Dis. 2016;2016:1720621.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa L, Yang C, Zhang X, Li Y, Wang S, Zheng L, et al. C-terminal truncation exacerbates the aggregation and cytotoxicity of α-Synuclein: A vicious cycle in Parkinson\u0026rsquo;s disease. Biochim Biophys Acta BBA - Mol Basis Dis. 2018;1864(12):3714\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang W, Nguyen LTT, Burlak C, Chegini F, Guo F, Chataway T, et al. Caspase-1 causes truncation and aggregation of the Parkinson\u0026rsquo;s disease-associated protein α-synuclein. Proc Natl Acad Sci U S A. 2016;113(34):9587\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNorman JM, Cohen GM, Bampton ETW. The in vitro cleavage of the hAtg proteins by cell death proteases. Autophagy [Internet]. 2010 Nov 16 [cited 2025 July 31]; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.tandfonline.com/doi/abs/\u003c/span\u003e\u003cspan address=\"https://www.tandfonline.com/doi/abs/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4161/auto.6.8.13337\u003c/span\u003e\u003cspan address=\"10.4161/auto.6.8.13337\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePagliarini V, Wirawan E, Romagnoli A, Ciccosanti F, Lisi G, Lippens S, et al. Proteolysis of Ambra1 during apoptosis has a role in the inhibition of the autophagic pro-survival response. Cell Death Differ. 2012 Sept;19(9):1495\u0026ndash;504.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMasliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, et al. Dopaminergic Loss and Inclusion Body Formation in α-Synuclein Mice: Implications for Neurodegenerative Disorders. Science. 2000;287(5456):1265\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Z, Jo J, Jia JM, Lo SC, Whitcomb DJ, Jiao S, et al. Caspase-3 Activation via Mitochondria Is Required for Long-Term Depression and AMPA Receptor Internalization. Cell. 2010;141(5):859\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu J, Chang L, Roselli F, Almeida OFX, Gao X, Wang X, et al. Amyloid-β induces caspase-dependent loss of PSD-95 and synaptophysin through NMDA receptors. J Alzheimers Dis JAD. 2010;22(2):541\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS. PSD-95 involvement in maturation of excitatory synapses. Science. 2000;290(5495):1364\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJanz R, S\u0026uuml;dhof TC, Hammer RE, Unni V, Siegelbaum SA, Bolshakov VY. Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I. Neuron. 1999;24(3):687\u0026ndash;700.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBurr\u0026eacute; J, Sharma M, Tsetsenis T, Buchman V, Etherton MR, S\u0026uuml;dhof TC. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science. 2010 Sept 24;329(5999):1663\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRando TA, Chang HY. Aging, rejuvenation, and epigenetic reprogramming: resetting the aging clock. Cell. 2012;148(1\u0026ndash;2):46\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMilber JM, Noorigian JV, Morley JF, Petrovitch H, White L, Ross GW, et al. Lewy pathology is not the first sign of degeneration in vulnerable neurons in Parkinson disease. Neurology. 2012;79(24):2307\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCaserta TM, Smith AN, Gultice AD, Reedy MA, Brown TL. Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties. Apoptosis. 2003;8(4):345\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang L, Sugama S, Mischak RP, Kiaei M, Bizat N, Brouillet E, et al. A novel systemically active caspase inhibitor attenuates the toxicities of MPTP, malonate, and 3NP in vivo. Neurobiol Dis. 2004;17(2):250\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGanjam GK, Bolte K, Matschke LA, Neitemeier S, Dolga AM, H\u0026ouml;llerhage M, et al. Mitochondrial damage by α-synuclein causes cell death in human dopaminergic neurons. Cell Death Dis. 2019;10(11):865.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhong J, Dong W, Qin Y, Xie J, Xiao J, Xu J, et al. Roflupram exerts neuroprotection via activation of CREB/PGC-1α signalling in experimental models of Parkinson\u0026rsquo;s disease. Br J Pharmacol. 2020;177(10):2333\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGupta AK, Gupta S, Mehan S, Khan Z, Das Gupta G, Narula AS. Exploring the Connection Between BDNF/TrkB and AC/cAMP/PKA/CREB Signaling Pathways: Potential for Neuroprotection and Therapeutic Targets for Neurological Disorders. Mol Neurobiol [Internet]. 2025 May 9 [cited 2025 July 31]; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12035-025-05001-5\u003c/span\u003e\u003cspan address=\"10.1007/s12035-025-05001-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang HS, Lee EYC. Protein phosphatase-1 inhibitor-3 is an in vivo target of caspase-3 and participates in the apoptotic response. J Biol Chem. 2008 June 27;283(26):18135\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7289863/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7289863/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe progressive loss of dopaminergic identity in midbrain neurons is a hallmark of Parkinson\u0026rsquo;s disease (PD), contributing to synaptic dysfunction and neurodegeneration. While a subset of PD cases is linked to genetic mutations, the majority are sporadic (sPD) and of unknown etiology. Current therapies offer only symptomatic relief and do not prevent neurodegeneration, underscoring the urgent need for disease-modifying strategies targeting actionable molecular pathways. Here, we used human induced pluripotent stem cell (hiPSC)-derived midbrain dopaminergic neurons (mDAs) from sporadic PD patients to investigate early alterations in neuronal identity, plasticity, and survival. We found that PD-derived mDAs exhibit upregulation of phosphorylated α-synuclein, marked reductions in dopaminergic markers (TH, NURR1), deficient dopamine handling, and impaired synaptogenesis. Transcriptomic and protein analyses revealed sustained activation of apoptotic caspases (caspase-3, -7) and downregulation of the PKA\u0026ndash;CREB\u0026ndash;BDNF signaling axis, which underpins dopaminergic differentiation and synaptic maturation. Pharmacological inhibition of caspases with Q-VD-OPh restored pCREB, BDNF, and downstream dopaminergic markers, leading to morphological recovery and functional synaptic rescue. Inhibition of PKA with H89 abrogated these effects, positioning the caspase\u0026ndash;PKA\u0026ndash;CREB cascade as a critical regulator of dopaminergic identity in PD neurons. These findings define a novel non-apoptotic role for caspases in disrupting the transcriptional program of mDAs and identify a druggable pathway capable of rescuing key aspects of dopaminergic function in a patient-derived cellular model. This work provides a mechanistic rationale for targeting caspase signaling in early-stage PD.\u003c/p\u003e","manuscriptTitle":"Caspase inhibition restores dopaminergic identity through the PKA–CREB–BDNF axis in Parkinson’s disease neurons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-04 00:19:04","doi":"10.21203/rs.3.rs-7289863/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de0fa0b8-94ac-4c31-89f1-99a9598a5507","owner":[],"postedDate":"September 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52802039,"name":"Biological sciences/Molecular biology"},{"id":52802040,"name":"Biological sciences/Cell biology"},{"id":52802041,"name":"Biological sciences/Neuroscience"},{"id":52802042,"name":"Health sciences/Diseases/Neurological disorders"}],"tags":[],"updatedAt":"2025-09-15T14:50:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-04 00:19:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7289863","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7289863","identity":"rs-7289863","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}