Extracellular vesicles (EVs) are membrane-enclosed vesicles which play important role for cell communication and physiology. EVs are found in many human biological fluids, including blood, breast milk, urine, cerebrospinal fluid (CSF), ejaculate, saliva etc. These nano-sized vesicles contain proteins, mRNAs, microRNAs, non-coding RNAs and lipids that are derived from producing cells. EVs deliver complex sets of biological information to recipient cells thereby modulating their behaviors by their molecular cargo. In this way EVs are involved in the pathological development and progression of many human disorders, including neurodegenerative diseases. In this study EVs purified by ultracentrifugation from CSF of patients with Parkinson’s disease (PD) and individuals of the comparison group were characterized using nanoparticle tracking analysis, flow cytometry and cryo-electron microscopy. Vesicular size and the presence of exosomal marker CD9 on the surface provided evidence that most of the EVs were exosome-like vesicles. Cryo-electron microscopy allowed us to visualize a large spectrum of extracellular vesicles of various size and morphology with lipid bilayers and vesicular internal structures. Thus, we described the diversity and new characteristics of the vesicles from CSF suggesting that subpopulations of EVs with different and specific functions may exist.

Mutations of the GBA gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are the greatest genetic risk factor for Parkinson’s disease (PD) with frequency between 5% and 20% across the world. N370S and L444P are the two most common mutations in the GBA gene. PD carriers of severe mutation L444P in the GBA gene is characterized by the earlier age at onset compared to N370S. Not every carrier of GBA mutations develop PD during one’s lifetime. In the current study we aimed to find common gene expression signatures in PD associated with mutation in the GBA gene (GBA-PD) using RNA-seq. We compared transcriptome of monocyte-derived macrophages of 5 patients with GBA-PD (4 L444P/N, 1 N370S/N) and 4 asymptomatic GBA mutation carriers (GBA-carriers) (3 L444P/N, 1 N370S/N) and 4 controls. We also conducted comparative transcriptome analysis for L444P/N only GBA-PD patients and GBA-carriers. Revealed deregulated genes in GBA-PD independently of GBA mutations (L444P or N370S) were involved in immune response, neuronal function. We found upregulated pathway associated with zinc metabolism in L444P/N GBA-PD patients. The potential important role of DUSP1 in the pathogenesis of GBA-PD was suggested.

Parkinson’s disease, associated with mutations in the GBA1 gene (GBA1-PD), is the most common genetic form of Parkinson’s disease (PD), marked by clinical heterogeneity influenced by mutation type. Extracellular vesicles (EVs), key mediators of intercellular communication, are implicated in PD pathogenesis through the transport of pathological proteins and lipids. In this study, we analyzed blood plasma-derived EVs from GBA1-PD patients carrying p.N370S and p.L444P mutations and from healthy controls using cryo-electron microscopy, lipidomics, and proteomics. EVs from GBA1-PD patients were significantly larger than those from controls, with the largest size and most multilayered vesicles observed in p.N370S carriers. Lipidomic profiling identified 237 lipid species; of these, 186 lipids were altered in p.N370S and 24 in p.L444P versus controls. Mutation carriers showed distinct lipid signatures, with p.L444P samples enriched predominantly in sphingolipids, while p.N370S carriers exhibited more extensive lipid remodeling across multiple classes, including triglycerides, cholesteryl esters, and phospholipids. Notably, Cer 23:0 was elevated across all GBA1-PD groups. Proteomic analysis revealed enrichment in pathways related to lipid transport, immune regulation, and vesicle-mediated processes. Overall, GBA1-PD patients share a distinct lipidomic EV signature, with mutation-specific patterns reflecting differing mechanisms of lysosomal dysfunction. These findings support the potential of EV profiling to unravel disease heterogeneity and identify biomarkers.

To date, we and others have demonstrated that GBA1-associated Parkinson’s disease (GBA1-PD) exhibits hyperactivation of mTOR and impairment of mTOR-regulated autophagy. Our previous study showed that the degree of autophagy impairment depends on the type of GBA1 mutation in peripheral blood mononuclear cell (PBMC)-derived macrophages. Moreover, the type of GBA1 mutation (“mild”—e.g., p.N370S or “severe”—e.g., p.L444P) correlates with PD severity and may influence therapeutic response. Here, we investigated the dose-dependent effects of GCase inhibition by conduritol β-epoxide (CBE) in SH-SY5Y cells on mTOR signaling, as well as the effects of mTOR inhibition by Torin 1 on mTOR-dependent autophagy-related proteins, lysosomal morphology, and lysosomal hydrolase activities in PBMC-derived macrophages from PD patients carrying GBA1-L444P or GBA1-N370S mutations. CBE induced dose-dependent activation of mTOR signaling in SH-SY5Y, as evidenced by dose-dependent accumulation of p-RPS6 (Ser235/236). mTOR inhibition decreased Beclin-1 protein levels while increasing the LC3B-II/LC3B-I ratio, LC3B–lysosome colocalization, and lysosome number regardless of mutation type in PBMC-derived macrophages. However, Torin1 reduced p62 levels in GBA1-N370S-PD, whereas lysosomal size decreased in GBA1-L444P-PD. Interestingly, Torin 1 increased GCase activity in both patient groups. These findings suggest that mTOR inhibition restores GCase function and autophagy and may represent a potential therapeutic strategy for GBA1-PD.

Alpha-synuclein oligomers are believed to be the major neurotoxic agents in neurodegenerative process in Parkinson’s disease (PD). The SNCA gene expression has been shown to be regulated by methylation of the SNCA intron 1. Polymorphisms in the SNCA promoter and 3′-untranslated regions could affect the SNCA gene expression and gene methylation. The study included 46 L-DOPA-naïve sporadic PD (sPD) patients and 64 individuals in the control group. We evaluated methylation of the SNCA intron 1 in the CD45+ blood cells along with the SNCA expression (mRNA, protein) in the sPD patients and controls. Negative correlations were found between the levels of DNA methylation of all of 23 CpG sites and of individual CpG sites in the SNCA intron 1 and the levels of alpha-synuclein expression (mRNA and protein) in the controls. No difference in the methylation of the SNCA intron 1 in the CD45+ blood cells between the PD patients and controls was found. However, hypermethylation of the CpG site at position 22 in the intron 1 of the SNCA gene in the sPD patients was revealed. It was found that the rs3756063 SNCA genotype influence the SNCA intron 1 methylation level in the CD45+ blood cells both in the sPD patients and controls. Influence of the rs3756063 SNCA genotypes on the alpha-synuclein levels in the CD45+ blood cells was found in the group of sPD patients. Therefore, our data demonstrated that methylation of the SNCA intron 1 is associated with the alpha-synuclein expression levels in the CD45+ blood cells in the controls, but methylation status does not differ between the sPD patients and controls. The obtained results confirmed the effect of rs3756063 polymorphism on methylation of the SNCA intron 1 in the CD45+ blood cells.

Mutations in the GBA1 gene, encoding lysosomal β-glucocerebrosidase (GCase), cause Gaucher disease (GD) and represent the most common genetic risk factor for Parkinson’s disease (PD). To target this pathway, therapeutic approaches for both GD and PD are actively being developed, focusing on two main drug classes: pharmacological chaperones (e.g., ambroxol) and allosteric GCase activators. Here, we characterize a novel allosteric GCase activator, N2, a derivative of the non-inhibitory chaperone NCGC607, and evaluate its therapeutic potential using computational, in vitro, and in vivo approaches. Molecular docking and molecular dynamics simulations indicated that N2 binds within the allosteric site of the mutant p.N370S GCase structure. In patient-derived cells (peripheral blood mononuclear cell-derived macrophages and induced pluripotent stem cell-derived dopaminergic neurons from GD and GBA1-PD patients), N2 enhanced GCase lysosomal translocation, modulated its enzymatic activity and protein level, and reduced substrate accumulation. Furthermore, we demonstrate for the first time that N2 exerts robust neuroprotective effects in an in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Specifically, N2 treatment attenuated MPTP-induced hexosylsphingosine accumulation in the prefrontal cortex, and ameliorated both tyrosine hydroxylase-positive neuronal loss in the substantia nigra and dopamine depletion in the striatum. Our findings first demonstrated the neuroprotective properties of a GCase allosteric activator on an MPTP-induced mouse model of PD and supported the emerging perspectives of such compounds for the treatment of GCase-related disorders.