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.

[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.

[The use of Akatinol Memantine in the treatment of gait disturbances in Parkinson's disease]. / Vliyanie Akatinola Memantina na narusheniya khod'by na razvernutykh stadiyakh bolezni Parkinsona.

OBJECTIVE: To assess the efficacy of memantine hydrochloride in the treatment of GD in PD patients. MATERIALS AND METHODS: Patients of the main group (n=30) received memantine hydrochloride (akatinol memantine) in a dose of 20 mg/day for 3 months in addition to antiparkinsonian therapy. Patients of the comparison group (n=25) received only antiparkinsonian drugs. Cognitive rating scales and computerized gait assessment protocol were performed in both groups twice in 3 months interval in order to examine cognitive deficit and gait parameters. RESULTS: The increase in MMSE scores, improvement in gait cycle phases ratio and increase of cadence according to computerized gait analysis were observed in the main group compared to the comparison group. CONCLUSION: The improvement in gait achieved during the study confirms that the treatment of cortical gait disturbances in patients with PD using memantine hydrochloride is a promising area of therapy.

Plasma cytokine profile in synucleinophaties with dementia.

Immune response may play a pivotal role in the pathogenesis of the common synucleinopathy as Parkinson's disease (PD) and could be mediated with the accumulation of neurotoxic alpha-synuclein. There is limited evidence for immune response in another synucleinopathy as dementia with Lewy bodies (DLB). Recent data suggest that immune response may contribute to cognitive impairment. We aimed to estimate plasma cytokine profile in patients with synucleinopathies with dementia (PD dementia (PDD), DLB). Plasma cytokine levels (interferon-gamma (IFN-gamma), interleukin (IL)-4 (IL-4), IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1)). were estimated in 16 patients with DLB, 19 patients with PDD, 28 patients with PD without dementia (PD) and 19 individuals without neurological disorders (controls) using Luminex array system. Cognitive status was assessed with the Mini-Mental State Examination (MMSE). TNF-alpha and IL-6 plasma levels were elevated in patients with synucleinopathies with dementia (DLB, PDD) compared to controls and IL-10 plasma level was increased in PDD compared to controls (p < 0.05). IFN-gamma levels were decreased in PD and PDD patients compared to controls (p < 0.001, p = 0.026, respectively) and in PD patients than in DLB patients (p = 0.032). Patients with PD, PDD, and DLB were characterized by increased plasma levels of MCP-1 compared to controls (p < 0.001). At the same time, no differences in TNF-alpha, IL-10, IL-6 plasma levels in PD patients compared to controls were found. Our study demonstrated more pronounced immune response in synucleinopathies associated with dementia compared to PD without demetia.

Plasma Cytokines Profile in Patients with Parkinson's Disease Associated with Mutations in GBA Gene.

Plasma cytokine concentration in patients with Parkinson's disease and mutation in GBA gene, in patients with sporadic Parkinson's disease, and in healthy volunteers were measured by ELISA and multiplex analysis. In patients with Parkinson's disease and mutation in GBA gene, elevated plasma concentrations of IL-1ß and TNFα were revealed by ELISA in comparison with both controls and patients with sporadic form of Parkinson's disease. Multiplex analysis revealed enhanced secretion of IL-1ß, IL-2, IFNγ and reduced plasma levels of monocyte chemoattractant protein-1 (MCP-1) in patients with Parkinson's disease and mutation in GBA gene (in comparison with other groups) and increased plasma levels of IL-13 (only in comparison with the healthy volunteers). Our results support the hypothesis that the concentrations of inflammatory mediators are increased in patients with Parkinson's disease and mutation in GBA gene.

[The genetic predictors of cognitive impairment in Parkinson's disease]. / Geneticheskie prediktory kognitivnogo defitsita pri bolezni Parkinsona.

Parkinson's disease (PD) is a common neurodegenerative disorder that can be both sporadic and familial. A number of studies are devoted to the study of non-motor symptoms in PD today. Cognitive deficits, and especially dementia, are one of the most severe and disabling non-motor symptoms of PD. More than a quarter of patients in the early stages of PD have a moderate cognitive impairment, more than half of patients with PD develop dementia within 10 years from the date of diagnosis. Using genome-wide association studies (GWAS), a number of genes associated with cognitive impairment have been identified based on a comparison of genetic and clinical phenotypes. These genes can be divided into three groups: genes that lead to the development of PD and are inherited according to the laws of Mendel (SNCA), genes that are risk factors for PD development (GBA, MAPT) and genes associated with the development of cognitive impairment, but not with PD (COMT, APOE, BDNF). This review examines the effect of genetic variants in the above-mentioned genes on cognitive functions in patients with PD. The elucidation of the genetic basis of cognitive deficits in PD could help in choice of treatment tactics and in development of new therapeutic strategies.

[Modern views on gait impairment in Parkinson`s disease and its correction]. / Sovremennye predstavleniia o narusheniiakh khod'by pri bolezni Parkinsona i metodakh ikh korrektsii.

The article reviews the causes of gait impairment in patients with Parkinson's disease (PD). The emphasis is made on modern ideas, according to which gait impairment in PD is caused by a multisystem lesion and non-dopaminergic dependent mechanisms play the leading role. It is highlighted that gait impairment in PD is associated with the disruption of frontal/subcortical neural pathways which requires a special approach to pharmacological and non-pharmacological therapy. Based on pathogenetic mechanisms, much attention is paid to anti-dementia medications. Attention is drawn to the fact that the use of memantine hydrochloride (akatinol memantine) is a promising direction for gait impairment correction in the advanced and late stages of PD due to the improvement of glutamatergic transfer from the striatum to the specific areas of the cerebral cortex involved in gait control. The results of the latest clinical trials are analyzed.

Interleukin-1ß, interleukin-1 receptor antagonist, interleukin-6, interleukin-10, and tumor necrosis factor-α levels in CSF and serum in relation to the clinical diversity of Parkinson's disease.

Several parameters representing the clinical diversity of Parkinson's disease (PD), including severity, phenotypes, cognitive decline, anxiety and depression were analyzed to examine the link with interleukin-1ß (IL-1ß), the interleukin-1 receptor antagonist (IL-1RA), IL-6, IL-10, and tumor necrosis factor-α (TNFα) and also to determine the relationship between levels of these factors in serum and cerebrospinal fluid (CSF). Significantly elevated serum IL-1ß and IL-6 and reduced IL-1RA levels were found in the PD group. In CSF and serum, inflammatory factors behaved differently, with increased CSF TNFα indicating rapid PD progression, and increased IL-1ß in serum. A low level of IL-6 was associated with a longer duration of PD. Anxiety, depression, non-tremor phenotype and late-onset PD correlated with a high serum level of IL-10. The serum TNFα level was lower in PD patients with mild cognitive impairment compared to controls. Serum IL-1ß, IL-6 and IL-10 levels correlated with CSF markers.

[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.

Oligomeric α-synuclein and glucocerebrosidase activity levels in GBA-associated Parkinson's disease.

Alpha-synuclein oligomerization plays a key role in the development of Parkinson's disease (PD). Being the most common genetic contributor to PD, glucocerebrosidase 1 (GBA) mutations have been associated with decreased GBA enzymatic activity in PD patients with mutations in the GBA gene (GBA-PD). However, it is unknown whether the activities of other lysosomal hydrolases are being altered in GBA-PD patients and are accompanied by an increase in alpha-synuclein oligomerization. The aim of our study was to estimate GBA enzymatic activity as well as the activities of five other lysosomal hydrolases (galactocerebrosidase, alpha-glucosidase, alpha-galactosidase, sphingomyelinase, alpha-iduronidase) in dried blood spots with assessing plasma oligomeric alpha-synuclein levels in sporadic PD (sPD) patients, in GBA-PD patients and in controls. GBA enzymatic activity and plasma oligomeric alpha-synuclein levels were assessed in sPD patients (N=84), in GBA-PD patients (N=21) and controls (N=62) by LC-MS/MS and ELISA methods accordingly. GBA-PD patients showed lower GBA enzymatic activity compared to controls (p=0.001) and to sPD (p=0.0001). We also found the reduction of GLA enzymatic activity (but not of other lysosomal hydrolases) in GBA-PD (p=0.001). At the same time plasma oligomeric alpha-synuclein levels were increased in GBA-PD group compared to sPD and controls (p=0.002 and p<0.0001, respectively). Our results suggest that the decrease in enzymatic activity of lysosomal hydrolases in GBA mutation carriers may contribute to PD pathogenesis by increasing the level of neurotoxic oligomeric alpha-synuclein species.

[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.

[The clinical heterogenity of Parkinson's disease].

OBJECTIVE: To identify clinical signs that determine a phenotypic heterogeneity of idiopathic Parkinson's disease (PD) using cluster analysis. MATERIAL AND METHODS: Cluster analysis was applied to main clinical signs characteristic of disease course and to the results of examination of 72 PD patients based on 5 scales. RESULTS: Clinical signs that determine a phenotypic heterogeneity of PD were identified. The most significant variables were a form of disease, predominance of akinetic/rigid syndrome or tremor syndrome as well as the age at disease onset, severity of motor deficit and autonomic symptoms. CONCLUSION: The definition "a form of PD" has a wider meaning compared to established one. To determine a form of disease, we recommend to consider both the predominance of a definite motor symptom and a set of additional clinical parameters.

[Symptomatic epilepsy in inflammatory demyelinating diseases].

Based on the own observations, we have summarized the features of symptomatic epilepsy in multiple sclerosis and conducted a clinical analysis of the most diagnostically difficult cases of inflammatory and demyelinating diseases in which epilepsy was the major clinical syndrome. The cases of post-infectious acute disseminated encephalomyelitis, idiopathic cerebral angiitis, Rasmussen's encephalitis are reviewed. Peculiarities of MRI structural brain lesions in these patients are discussed. The use of additional laboratory studies, including a study of cerebrospinal fluid, is considered.