LRRK2 exonic variants are associated with lysosomal hydrolase activities and lysosphingolipid alterations in Parkinson's disease.

Last data demonstrated that exonic variants of LRRK2 (p.G2019S, p.M1646T) may affect the catalytic activity of lysosomal enzyme glucocerebrosidase (GCase) probably through the phosphorylation of Rab10 protein. We aimed to evaluate an association of LRRK2 exonic variants previously associated with alteration of phosphorylation levels for Rab10Thr73 with PD risk in Russian population and analyze an impact of p.G2019S mutation and selected LRRK2 variants on lysosomal hydrolase activities. LRRK2 variants were determined by full sequencing of LRRK2 in 508 PD patients and 470 controls from Russian population. Activity of lysosomal enzymes (glucocerebrosidase (GCase), alpha-galactosidase A (GLA), acid sphingomyelinase (ASMase) and concentrations of their corresponded substrates (hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM), respectively) were estimated in 211 PD patients and 179 controls by liquid chromatography with tandem mass spectrometry (LC-MS-MS) in dry blood spots. p.M1646T and p.N2081D were associated with PD (OR = 2.33, CI 95%: 1.1215 to 4.8253, p = 0.023; OR = 1.89, 95%CI: 1.0727 to 3.3313, p = 0.028, respectively) in Russian population. An increased LysoGb3 concentration was found in p.G2019S and p.N2081D LRRK2 carriers among PD patients compared to both PD patients and controls (p.G2019S: p = 0.00086, p = 0.0004, respectively; p.N2081D: p = 0.012, p = 0.0076, respectively). A decreased ASMase activity in p.G2019S LRRK2 carriers among PD patients (p = 0.014) was demonstrated as well. Our study supported possible involvement of LRRK2 dysfunction in an alteration of sphingolipid metabolism in PD.

Impaired Sphingolipid Hydrolase Activities in Dementia with Lewy Bodies and Multiple System Atrophy.

The synucleinopathies are a group of neurodegenerative diseases characterized by the oligomerization of alpha-synuclein protein in neurons or glial cells. Recent studies provide data that ceramide metabolism impairment may play a role in the pathogenesis of synucleinopathies due to its influence on alpha-synuclein accumulation. The aim of the current study was to assess changes in activities of enzymes involved in ceramide metabolism in patients with different synucleinopathies (Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA)). The study enrolled 163 PD, 44 DLB, and 30 MSA patients as well as 159 controls. Glucocerebrosidase, alpha-galactosidase, acid sphingomyelinase enzyme activities, and concentrations of the corresponding substrates (hexosylsphingosine, globotriaosylsphingosine, lysosphingomyelin) were measured by liquid chromatography tandem-mass spectrometry in blood. Expression levels of GBA, GLA, and SMPD1 genes encoding glucoceresobridase, alpha-galactosidase, and acid sphingomyelinase enzymes, correspondently, were analyzed by real-time PCR with TaqMan assay in CD45 + blood cells. Increased hexosylsphingosine concentration was observed in DLB and MSA patients in comparison to PD and controls (p < 0.001) and it was associated with earlier age at onset (AAO) of DLB (p = 0.0008). SMPD1 expression was decreased in MSA compared to controls (p = 0.015). Acid sphingomyelinase activity was decreased in DLB, MSA patients compared to PD patients (p < 0.0001, p < 0.0001, respectively), and in MSA compared to controls (p < 0.0001). Lower acid sphingomyelinase activity was associated with earlier AAO of PD (p = 0.012). Our data support the role of lysosomal dysfunction in the pathogenesis of synucleinopathies, namely, the pronounced alterations of lysosomal activities involved in ceramide metabolism in patients with MSA and DLB.

Parkinson's Disease Associated with GBA Gene Mutations: Molecular Aspects and Potential Treatment Approaches.

Parkinson's disease (PD) is a multifactorial neurodegenerative disease. To date, genome-wide association studies have identified more than 70 loci associated with the risk of PD. Variants in the GBA gene encoding glucocerebrosidase are quite often found in PD patients in all populations across the world, which justifies intensive investigation of this gene. A number of biochemical features have been identified in patients with GBA-associated Parkinson's disease (GBA-PD). In particular, these include decreased activity of glucocerebrosidase and accumulation of the glucosylceramide substrate. These features were the basis for putting forward a hypothesis about treatment of GBA-PD using new strategies aimed at restoring glucocerebrosidase activity and reducing the substrate concentration. This paper discusses the molecular and genetic mechanisms of GBA-PD pathogenesis and potential approaches to the treatment of this form of the disease.

[Plasma Exosomes in Inherited Forms of Parkinson's Disease].

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Alpha-synuclein misfolding and aggregation resulting in neurototoxicity is a hallmark of PD. The prion properties of alpha-synuclein are still under discussion. Exosomes (extrcellular vesicles 40-100 nm in size) can play a key role in the transport of pathogenic forms of alpha-synuclein. The most frequent inherited forms of the disease are PD associated with mutation in the leucine-rich repeat kinase 2 (LRRK2-PD) and glucocerebrosidase (GBA-PD) genes. The aim of our work is to evaluate the concentration and size of exosomes derived from blood plasma of patients with GBA-PD, asymptomatic GBA mutation carriers, and the effect of GBA and LRRK2 mutations on alpha-synuclein level in exosomes derived from peripheral blood plasma. Plasma extracellular vesicles were isolated via chemical precipitation and sequential ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis (NTA), and flow cytometry. Total alpha-synuclein level in plasma exosomes was estimated by enzyme-linked immunosorbent assay. Patients with sporadic PD, PD with dementia, patients with inherited PD (GBA-PD, LRRK2-PD), and GBA mutation carriers were included in the study. The concentration on plasma exosomes was higher in GBA-PD patients that in sporadic PD patients, asymptomatic carriers of mutations on GBA gene, and control (p = 0.004, 0.019 and 0.0001 respectively). The size of plasma exosomes was higher in GBA-PD patients compared to asymptomatic carriers of GBA mutations and control (p = 0.009 and 0.0001, respectively). No significant difference was found for exosomal alpha-synuclein levels in the studied groups. Our results allowed us to suggest that a decrease in GBA activity may affect the pool of plasma exosomes, and mutations in the LRRK2 and GBA genes do not influence the level of plasma exosomal alpha-synuclein.

Ambroxol increases glucocerebrosidase (GCase) activity and restores GCase translocation in primary patient-derived macrophages in Gaucher disease and Parkinsonism.

Mutations in the glucocerebrosidase gene (GBA) encoding the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease (GD) and are the most commonly known genetic risk factor for Parkinson disease (PD). Ambroxol is one of the most effective pharmacological chaperones of GCase. Fourteen GD patients, six PD patients with mutations in the GBA gene (GBA-PD), and thirty controls were enrolled. GCase activity and hexosylsphingosine (HexSph) concentration were measured in dried blood and macrophage spots using liquid chromatography coupled with tandem mass spectrometry. The effect of ambroxol on GCase translocation to lysosomes was assessed using confocal microscopy. The results showed that ambroxol treatment significantly increased GCase activity in cultured macrophages derived from patient blood monocytic cell (PBMC) of GD (by 3.3-fold) and GBA-PD patients (by 3.5-fold) compared to untreated cells (p < 0.0001 and p < 0.0001, respectively) four days after cultivation. Ambroxol treatment significantly reduced HexSph concentration in GD (by 2.1-fold) and GBA-PD patients (by 1.6-fold) (p < 0.0001 and p < 0.0001, respectively). GD macrophage treatment resulted in increased GCase level and increased enzyme colocalization with the lysosomal marker LAMP2. The possible binding modes of ambroxol to mutant GCase carrying N370S amino acid substitution at pH 4.7 were examined using molecular docking and molecular dynamics simulations. The ambroxol position characterized by minimal binding free energy was observed in close vicinity to the residue, at position 370. Taken together, these data showed that PBMC-derived macrophages could be used for assessing ambroxol therapy response for GD patients and also for GBA-PD patients.

Genetics variants and expression of the SCARB2 gene in the pathogenesis of Parkinson's disease in Russia.

Lysosomal integral membrane protein-2 (LIMP-2), encoded by the SCARB2 gene, is the specific lysosomal receptor for glucocerebrosidase enzyme. Association between rs6812193 and rs68250047 of SCARB2 with PD has been shown in genetic studies, including large genome-wide association studies. The aim of the current study was to determine whether rs6812193 and rs8475 are associated with PD in Russia. rs6812193 and rs8475 were genotyped in a total of 604 PD patients (65 PD patients with positive (fPD) and 539 PD patients with negative family history (sPD)) and 413 controls and also in 17 patients with PD associated with GBA mutations (PD-GBA) and 18 asymptomatic GBA mutation carriers (GBA-Carriers). SCARB2 expression was measured by real-time PCR in CD45+ blood cells in part of individuals in the studied groups. No linkage disequilibrium was shown between rs6812193 and rs8475 in Russian population. Increased PD risk for TT variant of rs8475 (OR = 2.02; p < 0.001) was found in sPD patients but not in fPD. rs6812193 and rs8475 were not associated with age at onset (AAO) of PD. SCARB2 expression level was decreased in GBA-PD patients and GBA-Carriers compared to PD patients (padjusted = 0.02, padjusted = 0.003, respectively) and GBA-Carriers compared to controls (padjusted = 0.013) with no significant difference in PD patients and controls. SCARB2 expression was not modified with rs6812193 and rs8475. In conclusion, rs8475 was associated with PD status. rs6812193 and rs8475 are not genetic modifier of AAO of PD and do not influence on SCARB2 mRNA level in CD45+ blood cells in studied groups. SCARB2 expression could be modified with GBA mutations and is independent of PD status.

Dur3 and nrt2 genes in the bloom-forming dinoflagellate Prorocentrum minimum: Transcriptional responses to available nitrogen sources.

The increasing inflow of nitrogen (N) substrates into marine nearshore ecosystems induces proliferation of harmful algal blooms (HABs) of dinoflagellates, such as potentially toxic invasive species Prorocentrum minimum. In this study, we estimated the influence of NO3-, NH4+ and urea on transcription levels and urea transporter dur3 and nitrate transporter nrt2 genes expression in these dinoflagellates. We identified dur3 and nrt2 genes sequences in unannotated transcriptomes of P. minimum and other dinoflagellates presented in MMETSP database. Phylogenetic analysis showed that these genes of dinoflagellates clustered to the distinct clade demonstrating evolutionary relationship with the other known dur3 and nrt2 genes of microalgae. The evaluation of expression levels of dur3 and nrt2 genes by RT-qPCR revealed their sensitivity to input of the studied N sources. Dur3 expression levels were downregulated after the supplementation of additional N sources and were 1.7-2.6-fold lower than in the nitrate-grown culture. Nrt2 expression levels decreased 1.9-fold in the presence of NH4+. We estimated total RNA and DNA synthesis rates by the analysis of incorporation of 3H-thymidine and 3H-uridine in batch and continuous cultures. Addition of N compounds did not affect the DNA synthesis rates. Transcription levels increased up to 12.5-fold after the N supplementation in urea-limited treatments. Investigation of various nitrogen sources as biomarkers of dinoflagellate proliferation due to their differentiated impact on expression of dur3 and nrt2 genes and transcription rates in P. minimum cells allowed concluding about high potential of the studied parameters for future modeling of HABs under global N pollution.

[Prion Properties of Alpha-Synuclein].

The prion properties of alpha-synuclein, a key aggregating protein involved in the pathogenesis of so-called synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies, multiple system atrophy, and its various conformers are discussed. It is shown that alpha-synuclein may be transferred between cells by prion-like propagation. Similarly to other prions, alpha-synuclein aggregation develops from the initial lag-phase (nucleation) to the subsequent growth phase (elongation), and to the stationary phase where the aggregates and monomers exist in equilibrium. Similarly to prions, alpha-synuclein undergoes conformational changes from an alpha-helix to its beta-folded structure. However, there is currently no evidence that alpha-synuclein-dependent PD can be transmitted from person-to-person. This review describes the prion properties of alpha-synuclein, possible ways of its intercellular propagation, and novel approaches to PD diagnostics.

[The clinical features of Parkinson's disease in patients with mutations and polymorphic variants of GBA gene]. / Klinicheskie osobennosti bolezni Parkinsona u patsientov s mutatsiiami i polimorfnymi variantami gena GBA.

BACKGROUND: Mutations in the glucocerebrosidase gene (GBA) increase the risk of Parkinson's disease (PD) by 6-10 times in all populations and are associated with the early-onset of PD, development of cognitive impairment and presence of psychotic disorders. At the same time, polymorphic variants associated with the twofold increase in the risk of PD were also described in the GBA gene. AIM: To estimate the clinical features of PD in patients with mutations and polymorphic variants of the GBA gene. MATERIAL AND METHODS: Evaluation of motor, cognitive, emotional, psychotic and autonomic dysfunctions in patients with mutations (N370S, L444P) and polymorphic variants (E326K, T369M) in the GBA gene was performed using clinical scales. RESULTS: Patients with mutations (mGBA-PD), and with polymorphic variants (pGBA-PD) in the GBA gene were compared with the group of patients with sporadic PD (sPD). Compared to sPD, affective disorders (depression and anxiety) were more expressed in the mGBA-PD group (p=0.001) and the general GBA-PD group (p=0.001) assessed with Sheehan anxiety rating scale, in the pGBA-PD group (p=0.012) and the general GBA-PD group (p=0.05) assessed with the NPI, in the mGBA-PD (p=0.003), pGBA-PD (p=0.022), and general GBA-PD groups (p=0.001) assessed with the Hospital Anxiety and Depression scale (HADS 'A'), and in the pGBA-PD group (p=0.005) assessed with the HADS 'D'. Non-motor symptoms assessed with the PD-NMS were more expressed in the pGBA-PD patients (p=0.007) and in the total group with GBA-PD (p=0,014) compared to sPD. Cognitive impairment measured with MMSE was more marked in mGBA-PD patients (p=0.022). Differences in motor and non-motor clinical symptoms between pGBA-PD and mGBA-PD groups were not found. CONCLUSION: Thus, clinical features of non-motor symptoms were described both in carriers of GBA mutations and polymorphisms. Identification of the specific clinical phenotype of PD in carriers of GBA polymorphic variants is important due to their relatively high prevalence in PD patients.

[Mutation del 1,02kb in the CLN3 gene and extrapyramidal syndrome]. / Mutatsiya del 1,02 kb v gene CLN3 i sindrom ekstrapiramidnykh rasstroistv.

Mutations in the GBA and SMPD1 genes, which lead to the development of lysosomal storage diseases, are high risk factors for Parkinson's disease and dementia with Lewy bodies. We screened the mutations in the GALC and CLN3 genes in patients with Parkinson's disease and control subjects. A heterozygous CLN3 mutation (del 1.02 kb) carrier with clinical features of the unusual extrapyramidal syndrome was identified. A role of CLN3 mutations in the development of neurodegenerative disorders is discussed.

[SNCA rs356219 AND rs356165 VARIANTS ARE ASSOCIATED WITH PARKINSON'S DISEASE AND INCREASED ALPHA-SYNUCLEIN GENE EXPRESSION IN THE CD45(+)-BLOOD CELLS].

Impaired metabolism of alpha-synuclein (SNCA) and its aggregation are now implicated in the pathogenesis of Parkinson's disease (PD). Previous studies have found association between PD and gene locus, containing the SNCA gene. Meta-analysis have shown high significant association of single nucleotide polymorphisms (SNPs) rs356165 (A/G) and rs356219 (A/G) in the SNCA gene with PD. We genotyped these SNPs in 260 PD patients and 262 controls from north-western region of Russia. Alleles "G" of rs356165 and rs356219 were associated with increased risk of PD development. Linkage disequilibrium was shown between associated marker alleles. We studied the relationship between rs356165 and rs356219 and levels of mRNA SNCA and alpha-synuclein in CD45+ peripheral blood cells in drug-naive PD patients (n = 43) and controls (n = 39). Alleles "G" of rs356165 and rs356219 were associated with increased levels of SNCA expression (p = 0.046) and high alpha-synuclein levels (p = 0.039) in controls. Our data suggest that rs356165 and rs356219 variants might influence on PD development by upregulating SNCA expression.

[Increase in GP IIb-IIIa and P2Y12 Receptors in Activated Platelets as the Possible Indicator of de novo Protein Synthesis].

Although platelets lack nuclei, they are capable of de novo protein synthesis. We speculate that key platelet receptors are involved in the regulation of this process, and the changes in their number indicate the de novo protein synthesis in platelets. The object of our study was native platelets obtained from healthy donors. Using flow cytometry and Western blot, we determined the number of GP IIb-IIIa receptors (fibrinogen receptor) and P2Y12 receptors (ADP receptor) on the surface of platelets upon their activation with ADP and collagen. To verify the approaches and techniques used, we studied IL-1ß protein, which was previously shown to be synthesized de novo in activated platelets. GP IIb-IIIa receptor numbers correlate with the number of P2Y12 receptors on the cell surface (R = 0.45, p = 0.03). It was demonstrated that the platelet receptor numbers are higher on the surface of the cells with high functional activity. According to the data obtained by Western blot, upon the cell activation with ADP, the number of GP IIb-IIIa and P2Y12 receptors increases, which may serve as evidence of these proteins being synthesized in the activated platelets. It was observed that the level of P2Y12 and IL-1ß was lower in the samples where GP IIb-IIIa receptor was blocked by the selective inhibitor, i.e., the Fab fragment of the antibodies that specifically recognizes the GP IIb-IIIa complex. This suggests the important role of GP IIb-IIIa receptor in the regulation of protein synthesis.

Increased plasma oligomeric alpha-synuclein in patients with lysosomal storage diseases.

A link between lysosomal storage diseases (LSDs) and neurodegenerative disorders associated with accumulation of presynaptic protein alpha-synuclein has been shown. Particularly, Gaucher disease (GD) patients with a deficiency of the lysosomal enzyme glucocerebrosidase (GBA) and carriers of GBA mutations are at increased risk of Parkinson's disease (PD). It remains unclear whether this link is due to increased alpha-synuclein oligomerization. Here we show that level of oligomeric alpha-synuclein form, associated with PD development, is increased in plasma of GD patients (n=41, median=22.9pg/mL, range1.57-444.58pg/mL; controls (n=40, median=6.02pg/mL, range 1.05-103.14pg/mL, p<0.0001). This difference is absent in GD patients receiving enzyme replacement therapy (ERT) for more than 5 years. Moreover, the levels of alpha-synuclein oligomers in plasma are also higher in patients with other LSDs (Niemann-Pick type C, Krabbe disease, Wolman disease) compared to the median value in controls. Therefore, we suggest that mutations in the GBA gene and at least in several other LSDs genes may be associated with an increase in oligomeric alpha-synuclein in plasma. ERT applied for recovering of GBA functions in GD treatment might decrease formation of plasma oligomeric alpha-synuclein.

Reduced content of α-synuclein in peripheral blood leukocytes of patients with LRRK2-associated Parkinson's disease.

Measurement of α-synuclein level in the peripheral blood was proposed as a diagnostic test for Parkinson's disease. However, the results of these studies remain contradictory, probably because the examined samples included patients with different etiology of Parkinson's disease. To verify this assumption we studied the levels of α-synuclein in peripheral blood leukocytes of patients with Parkinson's disease associated with mutations in the gene of leucine-rich kinase 2 (LRRK2). The mean α-synuclein level was significantly lower in patients with LRRK2-associated Parkinson's disease (N=8) than in patients with sporadic form of the disease (N=33; p<0.02) and in controls (N=18; p<0.05). On the other hand, we found no differences in the level of α-synuclein level between patients with sporadic form of the disease and controls. We hypothesize that the level of α-synuclein in the peripheral blood largely depends on the etiology of the disease and cannot be used as a universal diagnostic test for Parkinson's disease.

Screening for LRRK2 mutations in patients with Parkinson's disease in Russia: identification of a novel LRRK2 variant.

BACKGROUND AND PURPOSE: Mutations in LRRK2, encoding leucine-rich repeat kinase 2 (or Dardarin), cause autosomal dominant Parkinson's disease (AdPD) and are also found in sporadic PD (sPD). To investigate the frequency of LRRK2 mutations in a sample of Russian PD patients. METHODS: We sequenced the complete coding region of LRRK2 in 65 patients with AdPD and in 30 patients with sPD. Furthermore, in 20 patients with AdPD and in 159 patients with sPD we screened several common LRRK2 mutations (G2019S, R1441C/G/H, I2012T and I2020T). RESULTS: Five AdPD patients had the LRRK2 G2019S mutation (5.9%, 5/85). In addition, we discovered a novel LRRK2 variant V1613A in a family with a tremor dominant form of AdPD; this variant was not present in controls. We identified two patients with LRRK2 mutations in sPD: one with the G2019S mutation (0.5; 1/189) and another with the previously described R1441C mutation (0,5; 1/189). CONCLUSIONS: LRRK2 mutations are common amongst patients with PD in Russia. The results also show that the G2019S mutation is the most frequent. We identified one novel mutation in a functional region of LRRK2.