Search Results (16)

Parkinson’s disease (PD) is defined by the progressive degeneration of dopaminergic neurons within the substantia nigra and the pathological accumulation of α-synuclein (α-syn) aggregates. Beyond these intracellular hallmarks, the extracellular matrix (ECM) has emerged as an active regulator of synaptic dysfunction, neuroinflammation, and disease progression. Recent multi-omics evidence, including transcriptomic and proteomic profiling of post-mortem tissue and iPSC-derived neurons, demonstrates consistent dysregulation of ECM components across both sporadic and genetic PD subtypes—typified by downregulation of basement membrane collagens (COL4A) and integrin signaling (ITGB1), alongside upregulation of matrix metalloproteinases (MMPs). These alterations destabilize perineuronal nets (PNNs), allow prion-like α-syn propagation, and promote glial activation through TLR-mediated signaling. Circulating ECM-derived neoepitopes (C1M, C4M) may complement neurofilament light chain as prognostic biomarkers of disease progression, although prospective validation remains necessary. Pharmacological strategies targeting MMP activity (e.g., doxycycline), integrin–FAK signaling (ATN-161), and MMP–TIMP balance (mesenchymal stem cells) represent emerging therapeutic avenues, though clinical evidence in PD remains limited. This review synthesizes current evidence on ECM dysregulation in PD and discusses its implications for biomarker development and disease-modifying intervention. Full article

Progressive supranuclear palsy-Richardson’s syndrome (PSP-RS) is a primary 4R tauopathy in which early axonal dysfunction may precede overt neurodegeneration; however, the mechanisms linking Tau dysregulation to cytoskeletal vulnerability remain poorly defined. Here, we generated induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons from individuals with sporadic PSP-RS and matched healthy controls and performed integrated transcriptomic and proteomic analyses. PSP-RS neurons exhibited coordinated suppression of dopaminergic and synaptic programs alongside activation of cytoskeletal remodeling and stress-related pathways. These changes were accompanied by increased Tau phosphorylation, neurofilament accumulation, and structural alterations of the axonal compartment, consistent with an early axonopathic phenotype. Notably, mechanistic target of rapamycin (mTOR) signaling significantly increased. Pharmacological inhibition of mTOR reduced Tau phosphorylation and neurofilament levels, indicating that mTOR activity contributes to the maintenance of cytoskeletal imbalance. In conclusion, our findings support a model in which early cytoskeletal dysfunction in PSP-RS arises from the convergence of Tau dysregulation, impaired structural homeostasis, and altered signaling pathways. Rather than acting as a primary driver, mTOR appears to function as a pathogenic amplifier that sustains axonal stress. This study provides a human cellular framework to investigate early axonopathic mechanisms in sporadic PSP-RS. Full article

With emerging genetic association studies, new genes and pathways are revealed as causative factors in the development of Parkinson’s disease (PD). However, many of these PD genes are poorly characterized in terms of their function, subcellular localization, and interaction with other components in cellular pathways. This represents a major obstacle towards a better understanding of the molecular causes of PD, with deeper molecular studies often hindered by a lack of high-quality, validated antibodies for detecting the corresponding proteins of interest. In this study, we leveraged the nanoluciferase-derived LgBiT-HiBiT system by generating a cohort of tagged PD genes in both induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells. To promote luminescence signals within cells, a master iPSC line was generated, in which LgBiT expression is under the control of a doxycycline-inducible promoter. LgBiT could bind to HiBiT when present either alone or when tagged onto different PD-associated proteins encoded by the genes GBA1, GPNMB, LRRK2, PINK1, PRKN, SNCA, VPS13C, and VPS35. Several HiBiT-tagged proteins could already generate luminescence in iPSCs in response to the doxycycline induction of LgBiT, with the enzyme glucosylceramidase beta 1 (GCase), encoded by GBA1, being one such example. Moreover, the GCase chaperone ambroxol elicited an increase in the luminescence signal in HiBiT-tagged GBA1 cells, correlating with an increase in the levels of GCase in dopaminergic cells. Taken together, we have developed and validated a Doxycycline-inducible luminescence system to serve as a sensitive assay for the quantification, localization, and activity of HiBiT-tagged PD-associated proteins with reliable sensitivity and efficiency. Full article

Cell death involving oxidative stress and mitochondrial dysfunction is a major cause of dopaminergic neuronal loss in the substantia nigra (SN) of Parkinson’s disease patients. Ergothioneine (ET), a natural dietary compound, has been shown to have cytoprotective functions, but neuroprotective actions against PD have not been well established. 6-Hydroxydopamine (6-OHDA) is a widely used neurotoxin to simulate the degeneration of dopaminergic (DA) neurons in Parkinson’s disease. In this study, we investigated the protective effect of ET on 6-OHDA treated iPSC-derived dopaminergic neurons (iDAs) and further confirmed the protective effects in 6-OHDA-treated human neuroblastoma SH-SY5Y cells. In 6-OHDA-treated cells, decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial reactive oxygen species (mROS), reduced cellular ATP levels, and increased total protein carbonylation levels were observed. 6-OHDA treatment also significantly decreased tyrosine hydroxylase levels. These effects were significantly decreased when ET was present. Verapamil hydrochloride (VHCL), a non-specific inhibitor of the ET transporter OCTN1 abrogated ET’s cytoprotective effects, indicative of an intracellular action. These results suggest that ET could be a potential therapeutic for Parkinson’s disease. Full article

Mutations in the GBA1 gene increase the risk of developing Parkinson’s disease (PD). However, most carriers of GBA1 mutations do not develop PD throughout their lives. The mechanisms of how GBA1 mutations contribute to PD pathogenesis remain unclear. Cerebrospinal fluid (CSF) is used for detecting pathological conditions of diseases, providing insights into the molecular mechanisms underlying neurodegenerative disorders. In this study, we utilized the proximity extension assay to examine the levels of metabolism-linked protein in the CSF from 17 PD patients carrying GBA1 mutations (GBA1-PD) and 17 idiopathic PD (iPD). The analysis of CSF secretome in GBA1-PD identified 11 significantly altered proteins, namely FKBP4, THOP1, GLRX, TXNDC5, GAL, SEMA3F, CRKL, APLP1, LRP11, CD164, and NPTXR. To investigate GBA1-associated CSF changes attributed to specific neuronal subtypes responsible for PD, we analyzed the cell culture supernatant from GBA1-PD-induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic (mDA) neurons. The secretome analysis of GBA1-PD iPSC-derived mDA neurons revealed that five differently regulated proteins overlapped with those identified in the CSF analysis: FKBP4, THOP1, GLRX, GAL, and CRKL. Reduced intracellular level of the top hit, FKPB4, was confirmed via Western Blot. In conclusion, our findings identify significantly altered CSF GBA1-PD-associated proteins with FKPB4 being firmly attributed to mDA neurons. Full article

Advances in stem cell technologies open up new avenues for modelling development and diseases. The success of these pursuits, however, relies on the use of cells most relevant to those targeted by the disease of interest, for example, midbrain dopaminergic neurons for Parkinson’s disease. In the present study, we report the generation of a human induced pluripotent stem cell (iPSC) line capable of purifying and tracing nascent midbrain dopaminergic progenitors and their differentiated progeny via the expression of a Blue Fluorescent Protein (BFP). This was achieved by CRISPR/Cas9-assisted knock-in of BFP and Cre into the safe harbour locus AAVS1 and an early midbrain dopaminergic lineage marker gene LMX1A, respectively. Immunocytochemical analysis and single-cell RNA sequencing of iPSC-derived neural cultures confirm developmental recapitulation of the human fetal midbrain and high-quality midbrain cells. By modelling Parkinson’s disease-related drug toxicity using 1-Methyl-4-phenylpyridinium (MPP⁺), we showed a preferential reduction of BFP⁺ cells, a finding demonstrated independently by cell death assays and single-cell transcriptomic analysis of MPP⁺ treated neural cultures. Together, these results highlight the importance of disease-relevant cell types in stem cell modelling. Full article

Aside from the degeneration of dopaminergic neurons, inflammation is a key component in the movement disorder Parkinson’s disease (PD). Microglia activation as well as elevated cytokine levels were observed in the brains of PD patients, but the specific role of microglia in the disease process is unknown. Here, we generate human cellular models by differentiating iPSCs into dopaminergic neurons and microglia. We combine these cells in co-culture to perform cytokine profiling, representing the final functional outcome of various signaling pathways. For this, we used unstimulated conditions and treatment with inflammatory stressors. Importantly, only co-cultures but not the monocultures responded to IL-1β treatment suggesting co-culture-related crosstalk. Moreover, we identified the main types of released cytokines and chemokines in this model system and found a preference for the activation of the chemotaxis pathway in response to all treatments, which informs future studies on the cell-type-specific reaction to inflammatory stimulation. Finally, we detected protein level changes in PD risk factor GPNMB upon stress in microglia, further confirming the link between PD-associated genes and inflammation in human-derived cellular models. Full article

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Some cases of PD may be caused by genetic factors, among which mutations in the LRRK2 and SNCA genes play an important role. To develop effective neuroprotective strategies for PD, it is important to diagnose the disease at the earliest stages of the neurodegenerative process. Therefore, the detection of diagnostic and prognostic markers of Parkinson’s disease (PD) is an urgent medical need. Advances in induced pluripotent stem cell (iPSC) culture technology provide new opportunities for the search for new biomarkers of PD and its modeling in vitro. In our work, we used a new technology for multiplex profiling of gene expression using barcoding on the Nanostring platform to assess the activity of mitochondrial genes on iPSC-derived cultures of dopaminergic neurons obtained from patients with LRRK2- and SNCA-associated genetic forms PD and a healthy donor. Electron microscopy revealed ultrastructural changes in mitochondria in both LRRK2 and SNCA mutant cells, whereas mitochondria in cells from a healthy donor were normal. In a culture with the SNCA gene mutation, the ratio of the area occupied by mitochondria to the total area of the cytoplasm was significantly lower than in the control and in the line with the LRRK2 gene mutation. Transcriptome analysis of 105 mitochondria proteome genes using the Nanostring platform revealed differences between the diseased and normal cells in the activity of genes involved in respiratory complex function, the tricarboxylic acid cycle, ATP production, mitochondria–endoplasmic reticulum interaction, mitophagy, regulation of calcium concentration, and mitochondrial DNA replication. Full article

Mutations in the GBA1 gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), cause Gaucher disease (GD) and are the most common genetic risk factor for Parkinson’s disease (PD). Pharmacological chaperones (PCs) are being developed as an alternative treatment approach for GD and PD. To date, NCGC00241607 (NCGC607) is one of the most promising PCs. Using molecular docking and molecular dynamics simulation we identified and characterized six allosteric binding sites on the GCase surface suitable for PCs. Two sites were energetically more preferable for NCGC607 and located nearby to the active site of the enzyme. We evaluated the effects of NCGC607 treatment on GCase activity and protein levels, glycolipids concentration in cultured macrophages from GD (n = 9) and GBA-PD (n = 5) patients as well as in induced human pluripotent stem cells (iPSC)—derived dopaminergic (DA) neurons from GBA-PD patient. The results showed that NCGC607 treatment increased GCase activity (by 1.3-fold) and protein levels (by 1.5-fold), decreased glycolipids concentration (by 4.0-fold) in cultured macrophages derived from GD patients and also enhanced GCase activity (by 1.5-fold) in cultured macrophages derived from GBA-PD patients with N370S mutation (p < 0.05). In iPSC-derived DA neurons from GBA-PD patients with N370S mutation NCGC607 treatment increased GCase activity and protein levels by 1.1-fold and 1.7-fold (p < 0.05). Thus, our results showed that NCGC607 could bind to allosteric sites on the GCase surface and confirmed its efficacy on cultured macrophages from GD and GBA-PD patients as well as on iPSC-derived DA neurons from GBA-PD patients. Full article

GBA variants increase the risk of Parkinson’s disease (PD) by 10 times. The GBA gene encodes the lysosomal enzyme glucocerebrosidase (GCase). The p.N370S substitution causes a violation of the enzyme conformation, which affects its stability in the cell. We studied the biochemical characteristics of dopaminergic (DA) neurons generated from induced pluripotent stem cells (iPSCs) from a PD patient with the GBA p.N370S mutation (GBA-PD), an asymptomatic GBA p.N370S carrier (GBA-carrier), and two healthy donors (control). Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), we measured the activity of six lysosomal enzymes (GCase, galactocerebrosidase (GALC), alpha-glucosidase (GAA), alpha-galactosidase (GLA), sphingomyelinase (ASM), and alpha-iduronidase (IDUA)) in iPSC-derived DA neurons from the GBA-PD and GBA-carrier. DA neurons from the GBA mutation carrier demonstrated decreased GCase activity compared to the control. The decrease was not associated with any changes in GBA expression levels in DA neurons. GCase activity was more markedly decreased in the DA neurons of GBA-PD patient compared to the GBA-carrier. The amount of GCase protein was decreased only in GBA-PD neurons. Additionally, alterations in the activity of the other lysosomal enzymes (GLA and IDUA) were found in GBA-PD neurons compared to GBA-carrier and control neurons. Further study of the molecular differences between the GBA-PD and the GBA-carrier is essential to investigate whether genetic factors or external conditions are the causes of the penetrance of the p.N370S GBA variant. Full article

Parkinson’s disease (PD) is the second most common neurodegenerative diseases characterized by progressive loss of midbrain dopaminergic neurons in the substantia nigra. Mutations in the PARK2 gene are a frequent cause of familial forms of PD. Sustained chronic neuroinflammation in the central nervous system makes a significant contribution to neurodegeneration events. In response to inflammatory factors produced by activated microglia, astrocytes change their transcriptional programs and secretion profiles, thus acting as immunocompetent cells. Here, we investigated iPSC-derived glial cell cultures obtained from healthy donors (HD) and from PD patients with PARK2 mutations in resting state and upon stimulation by TNFα. The non-stimulated glia of PD patients demonstrated higher IL1B and IL6 expression levels and increased IL6 protein synthesis, while BDNF and GDNF expression was down-regulated when compared to that of the glial cells of HDs. In the presence of TNFα, all of the glial cultures displayed a multiplied expression of genes encoding inflammatory cytokines: TNFA, IL1B, and IL6, as well as IL6 protein synthesis, although PD glia responded to TNFα stimulation less strongly than HD glia. Our results demonstrated a pro-inflammatory shift, a suppression of the neuroprotective gene program, and some depletion of reactivity to TNFα in PARK2-deficient glia compared to glial cells of HDs. Full article

Established disease models have helped unravel the mechanistic underpinnings of pathological phenotypes in Parkinson’s disease (PD), the second most common neurodegenerative disorder. However, these discoveries have been limited to relatively simple cellular systems and animal models, which typically manifest with incomplete or imperfect recapitulation of disease phenotypes. The advent of induced pluripotent stem cells (iPSCs) has provided a powerful scientific tool for investigating the underlying molecular mechanisms of both familial and sporadic PD within disease-relevant cell types and patient-specific genetic backgrounds. Overwhelming evidence supports mitochondrial dysfunction as a central feature in PD pathophysiology, and iPSC-based neuronal models have expanded our understanding of mitochondrial dynamics in the development and progression of this devastating disorder. The present review provides a comprehensive assessment of mitochondrial phenotypes reported in iPSC-derived neurons generated from PD patients’ somatic cells, with an emphasis on the role of mitochondrial respiration, morphology, and trafficking, as well as mitophagy and calcium handling in health and disease. Furthermore, we summarize the distinguishing characteristics of vulnerable midbrain dopaminergic neurons in PD and report the unique advantages and challenges of iPSC disease modeling at present, and for future mechanistic and therapeutic applications. Full article

Accumulation of α-synuclein (α-syn) into Lewy bodies (LBs) and mitochondrial abnormalities are the two cardinal pathobiological features of Parkinson’s disease (PD), which are associated with the loss of dopaminergic neurons. Although α-syn accumulates in many different cellular and mouse models, these models generally lack LB features. Here, we generated midbrain dopaminergic (mDA) neuronal cultures from induced pluripotent stem cells (iPSCs) derived from familial PD (fPD) patients and healthy controls. We show that mDA neuronal cultures from fPD patients with A53T mutation and α-syn gene (SNCA) triplication display pathological α-syn deposits, which spatially and morphologically resemble LBs. Importantly, we did not find any apparent accumulation of pathological α-syn in mDA neuronal culture derived from a healthy donor. Furthermore, we show that there are morphological abnormalities in the mitochondrial network in mDA neuronal cultures from fPD patients. Consequently, these cells were more susceptible to mitochondrial damage compared with healthy donor-derived mDA neuronal cultures. Our results indicate that the iPSC-derived mDA neuronal culture platform can be used to investigate the spatiotemporal appearance of LBs, as well as their composition, architecture, and relationship with mitochondrial abnormalities. Full article

Parkinson’s Disease (PD) is a chronic neurodegenerative disorder characterized by motor and non-motor symptoms, among which are bradykinesia, rigidity, tremor as well as mental symptoms such as dementia. The underlying cause of Parkinson disease is degeneration of dopaminergic neurons. It has been challenging to develop an efficient animal model to accurately represent the complex phenotypes found with PD. However, it has become possible to recapitulate the myriad of phenotypes underlying the PD pathology by using human induced pluripotent stem cell (iPSC) technology. Patient-specific iPSC-derived dopaminergic neurons are available and present an opportunity to study many aspects of the PD phenotypes in a dish. In this review, we report the available data on iPSC-derived neurons derived from PD patients with identified gene mutations. Specifically, we will report on the key phenotypes of the generated iPSC-derived neurons from PD patients with different genetic background. Furthermore, we discuss the relationship these cellular phenotypes have to PD pathology and future challenges and prospects for iPSC modelling and understanding of the pathogenesis of PD. Full article

Cardiotonic steroids (CTS) are specific inhibitors and endogenous ligands of a key enzyme in the CNS—the Na⁺, K⁺-ATPase, which maintains and creates an ion gradient on the plasma membrane of neurons. CTS cause the activation of various signaling cascades and changes in gene expression in neurons and other cell types. It is known that intracerebroventricular injection of cardiotonic steroid ouabain causes mania-like behavior in rodents, in part due to activation of dopamine-related signaling cascades in the dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) expressing medium spiny neurons in the striatum. Dopaminergic projections in the striatum innervate these GABAergic medium spiny neurons. The objective of this study was to assess changes in the expression of all genes in human iPSC-derived expressing DARPP-32 and GABA receptors neurons under the influence of ouabain. We noted a large number of statistically significant upregulated and downregulated genes after a 16-h incubation with non-toxic concentration (30 nM) of ouabain. These changes in the transcriptional activity were accomplished with activation of MAP-kinase ERK1/2 and transcriptional factor cAMP response element-binding protein (CREB). Thus, it can be concluded that 30 nM ouabain incubated for 16 h with human iPSC-derived expressing DARPP-32 and GABA receptors neurons activates genes associated with neuronal maturation and synapse formation, by increasing the expression of genes associated with translation, vesicular transport, and increased electron transport chain function. At the same time, the expression of genes associated with proliferation, migration, and early development of neurons decreases. These data indicate that non-toxic concentrations of ouabain may induce neuronal maturation, neurite growth, and increased synaptogenesis in dopamine-receptive GABAergic neurons, suggesting formation of plasticity and the establishment of new neuronal junctions. Full article