Dose-Dependent Alterations of Lysosomal Activity and Alpha-Synuclein in Peripheral Blood Monocyte-Derived Macrophages and SH-SY5Y Neuroblastoma Cell Line by upon Inhibition of MTOR Protein Kinase - Assessment of the Prospects of Parkinson's Disease Therapy.

To date, the molecular mechanisms of the common neurodegenerative disorder Parkinson's disease (PD) are unknown and, as a result, there is no neuroprotective therapy that may stop or slow down the process of neuronal cell death. The aim of the current study was to evaluate the prospects of using the mTOR molecule as a potential target for PD therapy due to the dose-dependent effect of mTOR kinase activity inhibition on cellular parameters associated with, PD pathogenesis. The study used peripheral blood monocyte-derived macrophages and SH-SY5Y neuroblastoma cell line. As a result, we have for the first time showed that inhibition of mTOR by Torin1 only at a concentration of 100 nM affects the level of the lysosomal enzyme glucocerebrosidase (GCase), encoded by the GBA1 gene. Mutations in GBA1 are considered a high-risk factor for PD development. This concentration led a decrease in pathological phosphorylated alpha-synuclein (Ser129), an increase in its stable tetrameric form with no changes in the lysosomal enzyme activities and concentrations of lysosphingolipids. Our findings suggest that inhibition of the mTOR protein kinase could be a promising approach for developing therapies for PD, particularly for GBA1-associated PD.

The Effect of p.G2019S Mutation in the LRRK2 Gene on the Activity of Lysosomal Hydrolases and the Clinical Features of Parkinson's Disease Associated with p.N370S Mutation in the GBA1 Gene.

BACKGROUND: Mutations in the glucocerebrosidase (GBA1) and leucine-rich repeat kinase 2 (LRRK2) genes, encoding lysosomal enzyme glucocerebrosidase (GCase) and leucine-rich repeat kinase 2 (LRRK2), respectively, are the most common related to Parkinson's disease (PD). Recent data suggest a possible functional interaction between GCase and LRRK2 and their involvement in sphingolipid metabolism. The aim of the present study was to describe the clinical course and evaluate the lysosomal enzyme activities and sphingolipid concentrations in blood of patients with PD associated with dual mutations p.N370S GBA1 and p.G2019S LRRK2 (p.N370S/GBA-p.G2019S/LRRK2-PD) as well as in blood of asymptomatic mutation carriers (p.N370S/GBA1-p.G2019S/LRRK2-carrier). METHODS: One patient with p.N370S/GBA1-p.G2019S/LRRK2-PD and one p.N370S/GBA1-p.G2019S/LRRK2-carrier were enrolled. GBA1-associated PD (GBA1-PD), LRRK2-associated PD (LRRK2-PD), sporadic PD (sPD) patients were described earlier by our research group. A neuropsychiatric examination of the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was carried out using scales (Montreal Cognitive Assessment scale (MoCA), Mini-mental State Examination scale (MMSE), Frontal Assessment Batter scale (FAB), Hospital Anxiety, and Depression Scale (HADS), etc). Lysosomal enzyme activity (GCase, alpha-galactosidase [GLA], acid sphingomyelinase [ASMase], galactosylcerebrosidase [GALC]) and sphingolipid concentrations (hexasylsphingosine [HexSph], lysoglobotriaosylsphingosine [LysoGb3], lysosphingomyelin [LysoSM]) were assessed with high-performance liquid chromatography-tandem mass spectrometry in blood. The following comparison with the previously described groups of GBA1-PD and sPD patients were conducted. RESULTS: Clinical features of p.N370S/GBA1-p.G2019S/LRRK2-PD included an early age of onset of the disease (46 years) and mild cognitive and affective disorders (MMSE = 29, MoCA = 23), despite a long (24 years) course of the disease. Interestingly, no differences were found in hydrolase activity and lysosphingolipid concentrations between the p.N370S/GBA1-p.G2019S/LRRK2-PD patient and GBA1-PD patients. However, GCase activity was lower in these groups than in LRRK2-PD, sPD, and controls. Additionally, the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was characterized by a pronounced decreased in ASMase activity and increased LysoSM concentration compared to the p.N370S/GBA1-p.G2019S/LRRK2-carrier (p = 0.023, p = 0.027, respectively). CONCLUSIONS: Based on one patient, our results indicate a protective effect of the p.G2019S mutation in the LRRK2 gene on clinical course of p.N370S/GBA1-PD. The identified pronounced alteration of ASMase activity and LysoSM concentration in p.N370S/GBA1-p.G2019S/LRRK2-PD provide the basis for the further research.

Extracellular Vesicles Secreted by Adipose Tissue during Obesity and Type 2 Diabetes Mellitus Influence Reverse Cholesterol Transport-Related Gene Expression in Human Macrophages.

Obesity is a risk factor for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). Adipose tissue (AT) extracellular vesicles (EVs) could play a role in obesity and T2DM associated CVD progression via the influence of their specific cargo on gene expression in recipient cells. The aim of this work was to evaluate the effects of AT EVs of patients with obesity with/without T2DM on reverse cholesterol transport (RCT)-related gene expression in human monocyte-derived macrophages (MDMs) from healthy donors. AT EVs were obtained after ex vivo cultivation of visceral and subcutaneous AT (VAT and SAT, respectively). ABCA1, ABCG1, PPARG, LXRß (NR1H2), and LXRα (NR1H3) mRNA levels in MDMs as well as in origine AT were determined by a real-time PCR. T2DM VAT and SAT EVs induced ABCG1 gene expression whereas LXRα and PPARG mRNA levels were simultaneously downregulated. PPARG mRNA levels also decreased in the presence of VAT EVs of obese patients without T2DM. In contrast ABCA1 and LXRß mRNA levels tended to increase with the addition of obese AT EVs. Thus, AT EVs can influence RCT gene expression in MDMs during obesity, and the effects are dependent on T2DM status.

Association of Rare Variants in ARSA with Parkinson's Disease.

BACKGROUND: Several lysosomal genes are associated with Parkinson's disease (PD), yet the association between PD and ARSA remains unclear. OBJECTIVES: To study rare ARSA variants in PD. METHODS: To study rare ARSA variants (minor allele frequency < 0.01) in PD, we performed burden analyses in six independent cohorts with 5801 PD patients and 20,475 controls, followed by a meta-analysis. RESULTS: We found evidence for associations between functional ARSA variants and PD in four cohorts (P ≤ 0.05 in each) and in the meta-analysis (P = 0.042). We also found an association between loss-of-function variants and PD in the United Kingdom Biobank cohort (P = 0.005) and in the meta-analysis (P = 0.049). These results should be interpreted with caution as no association survived multiple comparisons correction. Additionally, we describe two families with potential co-segregation of ARSA p.E382K and PD. CONCLUSIONS: Rare functional and loss-of-function ARSA variants may be associated with PD. Further replications in large case-control/familial cohorts are required. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Neurofilament light levels predict clinical progression and death in multiple system atrophy.

Disease-modifying treatments are currently being trialled in multiple system atrophy. Approaches based solely on clinical measures are challenged by heterogeneity of phenotype and pathogenic complexity. Neurofilament light chain protein has been explored as a reliable biomarker in several neurodegenerative disorders but data on multiple system atrophy have been limited. Therefore, neurofilament light chain is not yet routinely used as an outcome measure in multiple system atrophy. We aimed to comprehensively investigate the role and dynamics of neurofilament light chain in multiple system atrophy combined with cross-sectional and longitudinal clinical and imaging scales and for subject trial selection. In this cohort study, we recruited cross-sectional and longitudinal cases in a multicentre European set-up. Plasma and CSF neurofilament light chain concentrations were measured at baseline from 212 multiple system atrophy cases, annually for a mean period of 2 years in 44 multiple system atrophy patients in conjunction with clinical, neuropsychological and MRI brain assessments. Baseline neurofilament light chain characteristics were compared between groups. Cox regression was used to assess survival; receiver operating characteristic analysis to assess the ability of neurofilament light chain to distinguish between multiple system atrophy patients and healthy controls. Multivariate linear mixed-effects models were used to analyse longitudinal neurofilament light chain changes and correlated with clinical and imaging parameters. Polynomial models were used to determine the differential trajectories of neurofilament light chain in multiple system atrophy. We estimated sample sizes for trials aiming to decrease neurofilament light chain levels. We show that in multiple system atrophy, baseline plasma neurofilament light chain levels were better predictors of clinical progression, survival and degree of brain atrophy than the neurofilament light chain rate of change. Comparative analysis of multiple system atrophy progression over the course of disease, using plasma neurofilament light chain and clinical rating scales, indicated that neurofilament light chain levels rise as the motor symptoms progress, followed by deceleration in advanced stages. Sample size prediction suggested that significantly lower trial participant numbers would be needed to demonstrate treatment effects when incorporating plasma neurofilament light chain values into multiple system atrophy clinical trials in comparison to clinical measures alone. In conclusion, neurofilament light chain correlates with clinical disease severity, progression and prognosis in multiple system atrophy. Combined with clinical and imaging analysis, neurofilament light chain can inform patient stratification and serve as a reliable biomarker of treatment response in future multiple system atrophy trials of putative disease-modifying agents.

Whole Transcriptome Analysis of Substantia Nigra in Mice with MPTP-Induced Parkinsonism Bearing Defective Glucocerebrosidase Activity.

Mutations in the GBA1 gene represent the major genetic risk factor for Parkinson's disease (PD). The lysosomal enzyme beta-glucocerebrosidase (GCase) encoded by the GBA1 gene participates in both the endolysosomal pathway and the immune response. Disruption of these mechanisms is involved in PD pathogenesis. However, molecular mechanisms of PD associated with GBA1 mutations (GBA-PD) are unknown today in particular due to the partial penetrance of GBA1 variants in PD. The modifiers of GBA1 penetrance have not been elucidated. We characterized the transcriptomic profiles of cells from the substantia nigra (SN) of mice with co-injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and selective inhibitor of GCase activity (conduritol-ß-epoxide, (CBE)) to mimic PD bearing GCase dysfunction (MPTP+CBE), mice treated with MPTP, mice treated with CBE and control mice treated with injection of sodium chloride (NaCl) (vehicle). Differential expression analysis, pathway enrichment analysis, and outlier detection were performed. Functional clustering of differentially represented transcripts revealed more processes associated with the functioning of neurogenesis, inflammation, apoptosis and autophagy in MPTP+CBE and MPTP mice than in vehicle mice, with a more pronounced alteration of autophagy processes in MPTP+CBE mice than in MPTP mice. The PI3K-Akt-mTOR signaling pathway may be considered a potential target for therapy in PD with GCase dysfunction.

Potential Binding Sites of Pharmacological Chaperone NCGC00241607 on Mutant ß-Glucocerebrosidase and Its Efficacy on Patient-Derived Cell Cultures in Gaucher and Parkinson's Disease.

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.

ABCA1 and ABCG1 DNA methylation in epicardial adipose tissue of patients with coronary artery disease.

BACKGROUND: Recent studies have focused on the potential role of epicardial adipose tissue (EAT) in the development of coronary artery disease (CAD). ABCA1 and ABCG1 transporters regulate cell cholesterol content and reverse cholesterol transport. We aimed to determine whether DNA methylation and mRNA levels of the ABCA1 and ABCG1 genes in EAT and subcutaneous adipose tissue (SAT) were associated with CAD. METHODS: Paired EAT and SAT samples were collected from 82 patients undergoing elective cardiac surgery either for coronary artery bypass grafting (CAD group, N = 66) or valve surgery (NCAD group, N = 16). ABCA1 and ABCG1 mRNA levels in EAT and SAT samples were analyzed using real time polymerase chain reaction, ABCA1 protein levels in EAT samples were assessed by western blotting. ABCA1 and ABCG1 DNA methylation analysis was performed in 24 samples from the CAD group and 9 samples from the NCAD group via pyrosequencing. RESULTS: DNA methylation levels in the ABCA1 promoter and ABCG1 cg27243685 and cg06500161 CpG sites were higher in EAT samples from patients with CAD compared with NCAD (21.92% vs 10.81%, p = 0.003; 71.51% vs 68.42%, p = 0.024; 46.11% vs 37.79%, p = 0.016, respectively). In patients with CAD, ABCA1 and ABCG1 DNA methylation levels were higher in EAT than in SAT samples (p < 0.05). ABCA1 mRNA levels in EAT samples were reduced in the subgroup of patients with CAD and concomitant carotid artery disease or peripheral artery disease compared with the NCAD group (p = 0.024). ABCA1 protein levels in EAT samples tended to be lower in CAD patients than in the NCAD group (p = 0.053). DNA methylation levels at the ABCG1 cg27243685 site positively correlated with plasma triglyceride concentration (r = 0.510, p = 0.008), body mass index (r = 0.556, p = 0.013) and waist-to-hip ratio (r = 0.504, p = 0.012) in SAT samples. CONCLUSION: CAD is associated with ABCA1 and ABCG1 DNA hypermethylation in EAT. CAD with concomitant carotid artery disease or peripheral artery disease is accompanied by decreased ABCA1 gene expression in EAT. DNA methylation levels at the ABCG1 cg27243685 locus in SAT are associated with hypertriglyceridemia and obesity.

FABP4 and omentin-1 gene expression in epicardial adipose tissue from coronary artery disease patients.

Omentin-1 and fatty acid-binding protein 4 (FABP4) are adipose tissue adipokines linked to obesity-associated cardiovascular complications. The aim of this study was to investigate epicardial adipose tissue (EAT) omentin-1 and FABP4 gene expression in obese and non-obese patients with coronary artery disease (CAD). Omentin-1 and FABP4 mRNA levels in EAT and paired subcutaneous adipose tissue (SAT) as well as adipokine serum concentrations were assessed in 77 individuals (61 with CAD; 16 without CAD (NCAD)). EAT FABP4 mRNA level was decreased in obese CAD patients when compared to obese NCAD individuals (p=0.001). SAT FABP4 mRNA level was decreased in CAD patients compared to NCAD individuals without respect to their obesity status (p=0.001). Omentin-1 mRNA level in EAT and SAT did not differ between the CAD and NCAD groups. These findings suggest that omentin-1 gene expression in adipose tissue is not changed during CAD; downregulated FABP4 gene expression in SAT is associated with CAD while EAT FABP4 gene expression is decreased only in obesity-related CAD.

Blood lysosphingolipids accumulation in patients with parkinson's disease with glucocerebrosidase 1 mutations.

INTRODUCTION: Glucocerebrosidase 1 mutations, the most common genetic contributor to Parkinson's disease (PD), have been associated with decreased glucocerebrosidase enzymatic activity in PD patients with glucocerebrosidase 1 mutations (glucocerebrosidase 1-PD). However, it is unknown whether this decrease in enzymatic activity leads to lysosphingolipid accumulations. METHODS: The levels of hexosylsphingosines, globotriaosylsphingosine, sphingomyelin, and sphingomyelin-509 were measured in dried blood spots from glucocerebrosidase 1-PD patients (n = 23), sporadic PD patients (n = 105), Gaucher disease patients (n = 32), and controls (n = 88) by liquid chromatography-tandem mass spectrometry. RESULTS: Glucocerebrosidase 1-PD patients had increased hexosylsphingosine levels when compared with sporadic PD patients (P < .001) and controls (P < .0001). Hexosylsphingosine levels were increased in glucocerebrosidase 1 mutation carriers of glucocerebrosidase 1 (L444P; N370S; n = 11, P = .001) and glucocerebrosidase 1 polymorphic variants (E326K, T369M) associated with PD (n = 12, P = .04) when compared with controls. CONCLUSIONS: Lysosphingolipid accumulations in PD patients who bear glucocerebrosidase 1 mutations suggest that substrate reduction therapy might be viewed as a possible strategy for glucocerebrosidase 1-PD treatment. © 2018 International Parkinson and Movement Disorder Society.

Plasma oligomeric alpha-synuclein is associated with glucocerebrosidase activity in Gaucher disease.

BACKGROUND: The link between Parkinson's disease (PD) and Gaucher disease (GD), the most common lysosomal storage disease associated with loss of glucocerebrosidase (GBA) activity, can be explained by abnormal accumulation of oligomeric alpha-synuclein (α-Syn) species resulting from mutations in the GBA gene. However, in GD, the relationship between GBA activity and α-Syn accumulation in biological fluids has not been investigated. METHODS: We analyzed plasma oligomeric α-Syn levels, leucocyte GBA activity, and plasma chitotriosidase activity in 21 patients with GD. RESULTS: Negative correlation between plasma oligomeric α-Syn levels, and leucocyte GBA activity was observed in patients with GD (R(2) = 0.487; P < 0.001). CONCLUSION: The decrease in GBA activity may influence α-Syn oligomerization, explaining the high risk of PD development in GD patients. © 2015 International Parkinson and Movement Disorder Society.

Are rare heterozygous SYNJ1 variants associated with Parkinson's disease?

Previous studies have established that rare biallelic SYNJ1 mutations cause autosomal recessive parkinsonism and Parkinson's disease (PD). We analyzed 8165 PD cases, 818 early-onset-PD (EOPD, < 50 years) and 70,363 controls. Burden meta-analysis revealed an association between rare nonsynonymous variants and variants with high Combined Annotation-Dependent Depletion score (> 20) in the Sac1 SYNJ1 domain and PD (Pfdr = 0.040). A meta-analysis of EOPD patients demonstrated an association between all rare heterozygous SYNJ1 variants and PD (Pfdr = 0.029).

Are rare heterozygous SYNJ1 variants associated with Parkinson's disease?

Previous studies have suggested that rare biallelic SYNJ1 mutations may cause autosomal recessive parkinsonism and Parkinson's disease (PD). Our study explored the impact of rare SYNJ1 variants in non-familial settings, including 8,165 PD cases, 818 early-onset PD (EOPD, <50 years) and 70,363 controls. Burden meta-analysis using optimized sequence Kernel association test (SKAT-O) revealed an association between rare nonsynonymous variants in the Sac1 SYNJ1 domain and PD (Pfdr=0.040). Additionally, a meta-analysis focusing on patients with EOPD demonstrated an association between all rare SYNJ1 variants and PD (Pfdr=0.029). Rare SYNJ1 variants may be associated with sporadic PD, and more specifically with EOPD.

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons.

Background: Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Methods: Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. Results: We first identified specific non-coding variants in CTSB that drive the association with PD and are linked to changes in brain CTSB expression levels. Using iPSC-derived dopaminergic neurons we then find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. Moreover, in cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. Similarly, in midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition or knockout potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. Conclusions: The results of our genetic and functional studies indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

Fraction of plasma exomeres and low-density lipoprotein cholesterol as a predictor of fatal outcome of COVID-19.

Transcriptomic analysis conducted by us previously revealed upregulation of genes involved in low-density lipoprotein particle receptor (LDLR) activity pathway in lethal COVID-19 caused by SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2). Last data suggested the possible role of extracellular vesicles in COVID-19 pathogenesis. The aim of the present study was to retrospectively evaluate parameters of cholesterol metabolism and newly identified EVs, exomeres, as possible predictors of fatal outcome of COVID-19 patients infected by the Alpha and the Delta variants of SARS-CoV-2 virus. Blood from 67 patients with severe COVID-19 were collected at the time of admission to the intensive care unit (ICU) and 7 days after admission to the ICU. After 30 days patients were divided into two subgroups according to outcome-34 non-survivors and 33 survivors. This study demonstrated that plasma low- and high-density lipoprotein cholesterol levels (LDL-C and HDL-C) were decreased in non-survivors compared to controls at the time of admission to the ICU. The conjoint fraction of exomeres and LDL particles measured by dynamic light scattering (DLS) was decreased in non-survivors infected by the Alpha and the Delta variants compared to survivors at the time of admission to the ICU. We first showed that reduction of exomeres fraction may be critical in fatal outcome of COVID-19.

Downregulation of Exosomal hsa-miR-551b-3p in Obesity and Its Link to Type 2 Diabetes Mellitus.

Obesity is a significant risk factor for the development of type 2 diabetes mellitus (T2DM). Adipose tissue dysfunction can affect the pool of circulating exosomal miRNAs, driving concomitant disease in obesity. These exosomal miRNAs can reflect adipose tissue functionality, thus serving as prognostic biomarkers for disease monitoring in case of T2DM. In the present study, we conducted NanoString microRNA profiling of extracellular vesicles (EVs) secreted by adipose tissue of obese patients (body mass index (BMI) > 35) without T2DM and nonobese individuals (BMI < 30) as a control group. Functional and pathway enrichment analysis showed that miRNAs associated with obesity in this study were implicated in insulin signaling and insulin resistance biological pathways. Further, these microRNAs were screened in serum EVs in the following groups: (1) obese patients with T2DM, (2) obese patients without T2DM, and (3) nonobese individuals as a control group. has-miR-551b-3p was shown to be downregulated in adipose tissue EVs, as well as in serum EVs, of patients with obesity without T2DM. At the same time, the serum exosomal hsa-miR-551b-3p content was significantly higher in obese patients with T2DM when compared with obese patients without T2DM and may be a potential biomarker of T2DM development in obesity.

Cryo-electron microscopy of adipose tissue extracellular vesicles in obesity and type 2 diabetes mellitus.

Extracellular vesicles (EVs) are cell-derived membrane vesicles which play an important role in cell-to-cell communication and physiology. EVs deliver biological information from producing to recipient cells by transport of different cargo such as proteins, mRNAs, microRNAs, non-coding RNAs and lipids. Adipose tissue EVs could regulate metabolic and inflammatory interactions inside adipose tissue depots as well as distal tissues. Thus, adipose tissue EVs are assumed to be implicated in obesity-associated pathologies, notably in insulin resistance and type 2 diabetes mellitus (T2DM). In this study we for the first time characterize EVs secreted by visceral (VAT) and subcutaneous adipose tissue (SAT) of patients with obesity and T2DM with standard methods as well as analyze their morphology with cryo-electron microscopy. Cryo-electron microscopy allowed us to visualize heterogeneous population of EVs of various size and morphology including single EVs and EVs with internal membrane structures in samples from obese patients as well from the control group. Single vesicles prevailed (up to 85% for SAT, up to 75% for VAT) and higher proportion of EVs with internal membrane structures compared to SAT was typical for VAT. Decreased size of single and double SAT EVs compared to VAT EVs, large proportion of multilayered EVs and all EVs with internal membrane structures secreted by VAT distinguished obese patients with/without T2DM from the control group. These findings could support the idea of modified biogenesis of EVs during obesity and T2DM.