T cells from patients with Parkinson's disease recognize α-synuclein peptides.

Genetic studies have shown the association of Parkinson's disease with alleles of the major histocompatibility complex. Here we show that a defined set of peptides that are derived from α-synuclein, a protein aggregated in Parkinson's disease, act as antigenic epitopes displayed by these alleles and drive helper and cytotoxic T cell responses in patients with Parkinson's disease. These responses may explain the association of Parkinson's disease with specific major histocompatibility complex alleles.

Erratum: T cells from patients with Parkinson's disease recognize α-synuclein peptides.

This corrects the article DOI: 10.1038/nature22815.

Genetic, Structural, and Functional Evidence Link TMEM175 to Synucleinopathies.

OBJECTIVE: The TMEM175/GAK/DGKQ locus is the 3rd strongest risk locus in genome-wide association studies of Parkinson disease (PD). We aimed to identify the specific disease-associated variants in this locus, and their potential implications. METHODS: Full sequencing of TMEM175/GAK/DGKQ followed by genotyping of specific associated variants was performed in PD (n = 1,575) and rapid eye movement sleep behavior disorder (RBD) patients (n = 533) and in controls (n = 1,583). Adjusted regression models and a meta-analysis were performed. Association between variants and glucocerebrosidase (GCase) activity was analyzed in 715 individuals with available data. Homology modeling, molecular dynamics simulations, and lysosomal localization experiments were performed on TMEM175 variants to determine their potential effects on structure and function. RESULTS: Two coding variants, TMEM175 p.M393T (odds ratio [OR] = 1.37, p = 0.0003) and p.Q65P (OR = 0.72, p = 0.005), were associated with PD, and p.M393T was also associated with RBD (OR = 1.59, p = 0.001). TMEM175 p.M393T was associated with reduced GCase activity. Homology modeling and normal mode analysis demonstrated that TMEM175 p.M393T creates a polar side-chain in the hydrophobic core of the transmembrane, which could destabilize the domain and thus impair either its assembly, maturation, or trafficking. Molecular dynamics simulations demonstrated that the p.Q65P variant may increase stability and ion conductance of the transmembrane protein, and lysosomal localization was not affected by these variants. INTERPRETATION: Coding variants in TMEM175 are likely to be responsible for the association in the TMEM175/GAK/DGKQ locus, which could be mediated by affecting GCase activity. ANN NEUROL 2020;87:139-153.

Elevated In Vitro Kinase Activity in Peripheral Blood Mononuclear Cells of Leucine-Rich Repeat Kinase 2 G2019S Carriers: A Novel Enzyme-Linked Immunosorbent Assay-Based Method.

BACKGROUND: Leucine-rich repeat kinase 2 kinase inhibitors are being vigorously pursued as potential therapeutic options; however, there is a critical need for sensitive and quantitative assays of leucine-rich repeat kinase 2 function and target engagement. OBJECTIVES: Our objective was to compare collection and storage protocols for peripheral blood mononuclear cells, and to determine the optimal conditions for downstream analyses of leucine-rich repeat kinase 2 in PD cohorts. METHODS: Here, we describe enzyme-linked immunosorbent assay-based assays capable of detecting multiple aspects of leucine-rich repeat kinase 2 function at endogenous levels in human tissues. RESULTS: In peripheral blood mononuclear cells from both healthy and affected carriers of the G2019S mutation in leucine-rich repeat kinase 2, we report, for the first time, significantly elevated in vitro kinase activity, while detecting a significant increase in pS935/leucine-rich repeat kinase 2 in idiopathic PD patients. CONCLUSIONS: Quantitative assays such as these described here could potentially uncover specific markers of leucine-rich repeat kinase 2 function that are predictive of disease progression, aid in patient stratification, and be a critical component of upcoming clinical trials. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

SMPD1 mutations, activity, and α-synuclein accumulation in Parkinson's disease.

BACKGROUND: SMPD1 (acid-sphingomyelinase) variants have been associated with Parkinson's disease in recent studies. The objective of this study was to further investigate the role of SMPD1 mutations in PD. METHODS: SMPD1 was sequenced in 3 cohorts (Israel Ashkenazi Jewish cohort, Montreal/Montpellier, and New York), including 1592 PD patients and 975 controls. Additional data were available for 10,709 Ashkenazi Jewish controls. Acid-sphingomyelinase activity was measured by a mass spectrometry-based assay in the New York cohort. α-Synuclein levels were measured in vitro following CRISPR/Cas9-mediated knockout and siRNA knockdown of SMPD1 in HeLa and BE(2)-M17 cells. Lysosomal localization of acid-sphingomyelinase with different mutations was studied, and in silico analysis of their effect on acid-sphingomyelinase structure was performed. RESULTS: SMPD1 mutations were associated with PD in the Ashkenazi Jewish cohort, as 1.4% of PD patients carried the p.L302P or p.fsP330 mutation, compared with 0.37% in 10,709 Ashkenazi Jewish controls (OR, 3.7; 95%CI, 1.6-8.2; P = 0.0025). In the Montreal/Montpellier cohort, the p.A487V variant was nominally associated with PD (1.5% versus 0.14%; P = 0.0065, not significant after correction for multiple comparisons). Among PD patients, reduced acid-sphingomyelinase activity was associated with a 3.5- to 5.8-year earlier onset of PD in the lowest quartile versus the highest quartile of acid-sphingomyelinase activity (P = 0.01-0.001). We further demonstrated that SMPD1 knockout and knockdown resulted in increased α-synuclein levels in HeLa and BE(2)-M17 dopaminergic cells and that the p.L302P and p.fsP330 mutations impair the traffic of acid-sphingomyelinase to the lysosome. CONCLUSIONS: Our results support an association between SMPD1 variants, acid-sphingomyelinase activity, and PD. Furthermore, they suggest that reduced acid-sphingomyelinase activity may lead to α-synuclein accumulation. © 2019 International Parkinson and Movement Disorder Society.

Association of Low Lysosomal Enzymes Activity With Brain Arterial Dilatation.

Background and Purpose- Absent or diminished α-galactosidase A (GLA) and acid α-glucosidase (GAA) enzyme activity are core features of Fabry and Pompe disease, respectively. Patients with Fabry or Pompe disease may have dilated intracranial arteries but whether lower GLA or GAA enzyme activity relates to brain arterial dilatation in other populations is unknown. Methods- Participants included Parkinson disease patients and nonblood-related controls, whose GLA and GAA enzymatic activities were measured in dried blood spots. Independent readers measured the axial arterial diameter of the ascending portion of the cavernous internal carotid arteries and the most proximal segment of the basilar artery in T2 black voids. Linear regression models were built to investigate the relationship between brain arterial diameters and lysosomal enzymatic activities. Results- The cohort included 107 participants (mean age, 66.5±10.3; 67% men). In an adjusted linear regression model, lower GLA activity was associated with larger brain arterial diameters (B=0.50±0.23, P=0.03). The strength of association was the greatest for the basilar artery diameter (B=0.80±0.33, P=0.02). Similarly, lower GAA activity was associated with an increased basilar arterial diameter (B=0.73±0.35, P=0.04). Conclusions- Lower GLA and GAA enzymatic activities were associated with larger brain arterial diameters, particularly the basilar artery diameter. Lower lysosomal enzymatic function in patients without Fabry or Pompe disease may play a role in brain arterial dilatation.

Tandem mass spectrometry assay of ß-glucocerebrosidase activity in dried blood spots eliminates false positives detected in fluorescence assay.

Deficiency of ß-Glucocerebrosidase (GBA) activity causes Gaucher Disease (GD). GD can be diagnosed by measuring GBA activity (Beutler and Kuhl, 1990). In this study, we assayed dried blood spots from a cohort (n=528) enriched for GBA mutation carriers (n=78) and GD patients (n=18) using both the tandem mass spectrometry (MS/MS) and fluorescence assays and their respective synthetic substrates. The MS/MS assay differentiated normal controls, which included GBA mutation carriers, from GD patients with no overlap. The fluorescence assay did not always differentiate normal controls including GBA mutation carriers from GD patients and false positives were observed. The MS/MS assay improved specificity compared to the fluorescence assay.

GBA-Associated Parkinson's Disease and Other Synucleinopathies.

PURPOSE OF REVIEW: GBA mutations are the most common known genetic cause of Parkinson's disease (PD). Its biological pathway may be important in idiopathic PD, since activity of the enzyme encoded by GBA, glucocerebrosidase, is reduced even among PD patients without GBA mutations. This article describes the structure and function of GBA, reviews recent literature on the clinical phenotype of GBA PD, and suggests future directions for research, counseling, and treatment. RECENT FINDINGS: Several longitudinal studies have shown that GBA PD has faster motor and cognitive progression than idiopathic PD and that this effect is dose dependent. New evidence suggests that GBA mutations may be important in multiple system atrophy. Further, new interventional studies focusing on GBA PD are described. These studies may increase the interest of PD patients and caregivers in genetic counseling. GBA mutation status may help clinicians estimate PD progression, though mechanisms underlying GBA and synucleinopathy require further understanding.

Drug Counselor Responses to Patients' Pain Reports: A Qualitative Investigation of Barriers and Facilitators to Treating Patients with Chronic Pain in Methadone Maintenance Treatment.

OBJECTIVE: To examine how drug counselors with no prior training in pain management respond to their patients' reports of chronic pain. DESIGN, SETTING, SUBJECTS, AND METHODS: We conducted individual interviews with 30 drug counselors in methadone maintenance treatment. Interviews were audiotaped, transcribed, and systematically coded using the constant comparative method. RESULTS: Participants identified counselor, patient, and logistical factors that serve as a barrier or facilitate their treatment of patients with chronic pain. Counselor barriers included lack of expertise in managing co-occurring chronic pain and opioid use disorder, complexity of patients' treatment needs, concerns about medication regimens, reliance on patient self-report, and absence of patient improvement. Counselor barriers facilitators included empathy, attending to small changes, and self-reflection. Counselors' perceptions of patient-related barriers included prior negative interactions with medical providers, diminished social roles, attenuated motivation, and negative attitudes toward opioid use disorder. Logistical barriers included lack of appropriate pain management referrals, limited counselor time, and attenuated treatment adherence; a logistical facilitator was consulting with medical providers. CONCLUSIONS: Perceived barriers to treating patients with chronic noncancer pain are common among drug counselors. Addressing these barriers in drug counselor training and in methadone maintenance treatment programs may benefit both methadone-maintained patients with chronic pain and their providers.

Physical activity, psychiatric distress, and interest in exercise group participation among individuals seeking methadone maintenance treatment with and without chronic pain.

BACKGROUND AND OBJECTIVES: Physical activity may improve chronic pain, anxiety, and depression, which are prevalent among patients in methadone maintenance treatment (MMT), but relatively little is known about the physical activity levels or interest in exercise of patients in MMT. METHODS: We used a brief self-report instrument to assess physical activity levels, chronic pain, psychiatric distress, and interest in exercise group participation among 303 adults seeking MMT. RESULTS: Most (73%) reported no moderate or vigorous intensity physical activity in the past week; 27% met recommended physical activity levels, and 24% reported interest in exercise group participation. Participants with (compared to those without) chronic pain had higher levels of psychiatric distress and were less likely to meet recommended levels of physical activity (p < .05), but did not differ significantly in their interest in participating in an exercise group. Participants who met recommended levels of physical activity in the past week were more likely to be men and had lower levels of depression than others (p < .05). CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: Low levels of physical activity and low interest in exercise group participation among patients entering MMT point to the need for and likely challenges of implementing exercise interventions in MMT.

SenseCam reminiscence and action recall in memory-unimpaired people.

Case studies of memory-impaired individuals consistently show that reminiscing with SenseCam images enhances event recall. This exploratory study examined whether a similar benefit would occur for the consolidation of memories in memory-unimpaired people. We tested delayed recall for atypical actions observed on a lengthy walk. Participants used SenseCam, a diary, or no external memory aid while walking, followed by reminiscence with SenseCam images, diary entries, or no aid, either alone (self-reminiscence) or with the experimenter (social reminiscence). One week later, when tested without SenseCam images or diary entries, prior social reminiscence produced greater recall than self-reminiscence, but there were no differences between memory aid conditions for action free recall or action order recall. When methodological variables were controlled, there was no recall advantage for SenseCam reminiscence with memory-unimpaired participants. The case studies and present study differ in multiple ways, making direct comparisons problematic. SenseCam is a valuable aid to the memory impaired, but its mnemonic value for non-clinical populations remains to be determined.

Psychiatric comorbidity and order of condition onset among patients seeking treatment for chronic pain and opioid use disorder.

BACKGROUND: The study objective was to compare psychiatric comorbidity among patients seeking treatment for chronic pain and opioid use disorder (OUD) by order of condition onset (i.e., "Pain First," "OUD First," "Same Time"). METHODS: Data from 170 patients entering two clinical trials of treatments for current comorbid chronic pain and OUD conducted between March 2009 and July 2013 were compared by order of condition onset. The Structured Clinical Interview for DSM-IV-TR Axis I Disorders and the Diagnostic Interview for DSM-IV Personality Disorders (Axis II) were performed by doctoral-level providers using a standardized training protocol. Age of onset group differences on specific diagnostic variables were examined using multinomial logistic regression. RESULTS: Fifty-two percent were in the "Pain First" group (n = 89), 35 % in the "OUD First" group (n = 59), and 13 % in the "Same Time" group (n = 22). Compared with the Pain First group, the Same Time group was less likely to report heroin (vs. prescription opioids) as the primary drug used (OR = 0.20, 95 % CI = 0.06-0.72) or meet criteria for an Axis II disorder (OR = 0.24, 95 % CI = 0.07-0.83). Compared with the Pain First group, the OUD First group was more likely to meet criteria for a current nonopioid substance use disorder (OR = 3.20, 95 % CI = 1.22-8.40). CONCLUSIONS: Our findings regarding differences in psychiatric comorbidity associated with order of condition onset indicate that varying pathways may exist for the emergence of chronic pain and OUD; further research should investigate potential treatment implications.

An Assessment of LRRK2 Serine 935 Phosphorylation in Human Peripheral Blood Mononuclear Cells in Idiopathic Parkinson's Disease and G2019S LRRK2 Cohorts.

The phosphorylated form of LRRK2, pS935 LRRK2, has been proposed as a target modulation biomarker for LRRK2 inhibitors. The primary aim of the study was to characterize and qualify this biomarker for therapeutic trials of LRRK2 inhibitors in Parkinson's disease (PD). To this end, analytically validated assays were used to monitor levels of pS935 LRRK2 and total LRRK2 in peripheral blood mononuclear cells (PBMCs) from the following donor groups: healthy controls, idiopathic PD, and G2019S carriers with and without PD. Neither analyte correlated with age, gender, or disease severity. While total LRRK2 levels were similar across the four groups, there was a significant reduction in pS935 LRRK2 levels in disease-manifesting G2019S carriers compared to idiopathic PD. In aggregate, these data indicate that phosphorylation of LRRK2 at S935 may reflect a state marker for G2019S LRRK2-driven PD, the underlying biology for which requires investigation in future studies. This study also provides critical foundational data to inform the integration of pS935 and total LRRK2 levels as biomarkers in therapeutic trials of LRRK2 kinase inhibitors.

An evaluation of the feasibility, acceptability, and preliminary efficacy of cognitive-behavioral therapy for opioid use disorder and chronic pain.

AIMS: The primary study aim was to evaluate the feasibility and acceptability of cognitive-behavioral therapy (CBT) for opioid use disorder and chronic pain. The secondary aim was to examine its preliminary efficacy. METHODS: In a 12-week pilot randomized clinical trial, 40 methadone-maintained patients were assigned to receive weekly manualized CBT (n = 21) or Methadone Drug Counseling (MDC) to approximate usual drug counseling (n = 19). RESULTS: Twenty of 21 patients assigned to CBT and 18 of 19 assigned to MDC completed the pilot study. Mean (SD) sessions attended were 8.4 (2.9) for CBT (out of 12 possible) and 3.8 (1.1) for MDC (out of 4 possible); mean (SD) patient satisfaction ratings (scored on 1-7 Likert-type scales) were 6.6 (0.5) for CBT and 6.0 (0.4) for MDC (p < .001). The proportion of patients abstinent during the baseline and each successive 4-week interval was higher for patients assigned to CBT than for those assigned to MDC [Wald χ2 (1) = 5.47, p = .02]; time effects (p = .69) and interaction effects between treatment condition and time (p = .10) were not significant. Rates of clinically significant change from baseline to end of treatment on pain interference (42.9% vs. 42.1%, [χ2 (1, N = 40) = 0.002, p = 0.96]) did not differ significantly for patients assigned to CBT or MDC. CONCLUSIONS: We found support for the feasibility, acceptability, and preliminary efficacy of cognitive-behavioral therapy relative to standard drug counseling in promoting abstinence from nonmedical opioid use among patients with opioid use disorder and chronic pain. Overall, patients exhibited improved pain outcomes, but these improvements did not differ significantly by treatment condition.

Common and rare GCH1 variants are associated with Parkinson's disease.

GCH1 encodes the enzyme guanosine triphospahte (GTP) cyclohydrolase 1, essential for dopamine synthesis in nigrostriatal cells, and rare mutations in GCH1 may lead to Dopa-responsive dystonia (DRD). While GCH1 is implicated in genomewide association studies in Parkinson's disease (PD), only a few studies examined the role of rare GCH1 variants in PD, with conflicting results. In the present study, GCH1 and its 5' and 3' untranslated regions were sequenced in 1113 patients with PD and 1111 controls. To examine the association of rare GCH1 variants with PD, burden analysis was performed. Three rare GCH1 variants, which were previously reported to be pathogenic in DRD, were found in five patients with PD and not in controls (sequence Kernel association test, p = 0.024). A common haplotype, tagged by rs841, was associated with a reduced risk for PD (OR = 0.71, 95% CI = 0.61-0.83, p = 1.24 × 10-4), and with increased GCH1 expression in brain regions relevant for PD (www.gtexportal.org). Our results support a role for rare, DRD-related variants, and common GCH1 variants in the pathogenesis of PD.

Increased yield of full GBA sequencing in Ashkenazi Jews with Parkinson's disease.

BACKGROUND: Variants in GBA are the most common genetic risk factor for Parkinson's disease (PD), and are especially prevalent in the Ashkenazi Jewish (AJ) population. However, most studies on GBA in AJ genotype only seven selected Gaucher-associated pathogenic variants rather than sequencing the whole gene, which may leave carriers of PD-associated GBA variants undiscovered. METHODS: GBA was fully sequenced using molecular inversion probes (MIPs) and Sanger sequencing in 735 AJ PD patients and 662 AJ controls, from Israel and New York. Additional AJ control data (n = 3044) from the Inflammatory Bowel Disease Exome Portal was used. RESULTS: Full GBA sequencing increased the number of variants discovered by 17.4%, compared to targeted genotyping. An additional 17 PD patients were identified with GBA-associated PD. The p.E326K variant was found in 1.6% of AJ PD patients, making it the second most common PD-associated GBA variant in AJ. GBA variants were found in 18% of PD patients and 7.5% of controls (OR = 2.7, 95%CI = 1.9-3.8, p < 0.0001). CONCLUSION: Without full sequencing of GBA, or at minimum including p.E326K in the genotyping panel, a significant proportion of variant carriers go undiscovered and may be incorrectly assigned as non-carriers in studies or clinical trials.

Sequencing of the GBA coactivator, Saposin C, in Parkinson disease.

Saposin C (SapC), encoded by PSAP, is required for the activity of glucocerebrosidase, encoded by GBA. Although GBA mutations have been studied thoroughly in Parkinson disease (PD), genetic studies on SapC are still lacking. PSAP was sequenced in 1123 PD patients and 1153 controls, and data from additional 1167 patients and 1685 controls were examined. A total of 6 patients had SapC mutations in the 2 combined cohorts, but no statistically significant association after correction for multiple comparisons was found. Larger studies are necessary to examine the role of very rare SapC variants in PD.

Parkinson's disease prevalence in Fabry disease: A survey study.

Recent research has suggested a possible link between Parkinson's disease (PD) and Fabry disease. To test this relationship, we administered a self-report and family history questionnaire to determine the prevalence of PD in Fabry disease patients and family members with likely pathogenic alpha-galactosidase A (GLA) mutations. A total of 90 Fabry patients (77 from the online survey and 13 from the Icahn School of Medicine at Mount Sinai (ISMMS)) were included in the analysis. Two of the Fabry disease patients who completed the online survey were diagnosed with PD (2/90, 2.2%). Among probands older than 60, 8.3% (2/24) were diagnosed with PD. Using Kaplan Meier survival analysis, the age-specific risk of PD by age 70 was 11.1%. Family history was available on 72 Fabry families from the online study and 9 Fabry families from ISMMS. Among these 81 families, 6 (7.4%) had one first degree relative who fit the criteria for a conservative diagnosis of PD. The results of this study suggest that there may be an increased risk of developing PD in individuals with GLA mutations, but these findings should be interpreted with caution given the limitations of the study design.

Frequency of GBA variants in autopsy-proven multiple system atrophy.

BACKGROUND: Multiple system atrophy (MSA) is marked by abnormal inclusions of alpha-synuclein in oligodendrogliocytes. Etiology remains unknown. Variants in the glucocerebrosidase gene have been associated with other synucleinopathies, dementia with Lewy bodies and Parkinson disease. It is unclear whether glucocerebrosidase variants are associated with MSA. OBJECTIVES: To analyze the frequency of glucocerebrosidase gene variants among autopsy-proven cases of MSA at a brain bank in New York City. METHODS: The glucocerebrosidase gene was fully sequenced in the 17 autopsy-proven MSA cases with extractable DNA at the Columbia University New York Brain Bank from 2002 to 2016. To test if the MSA cases in the brain bank are enriched for GBA variants, we compared the GBA variant frequency in MSA to all brain bank cases with pure Alzheimer's disease (AD) at Columbia University for whom GBA genotype was available (n=82). RESULTS: 4/17 (23.5%) MSA cases carried glucocerebrosidase gene variants, including an individual homozygous for N370S, and one each who were heterozygous carriers of N370S, T369M and R496H. Among the comparator cases with pure AD, 3 of the 82 autopsies (3.7%) carried GBA variants (P = 0.0127, Fisher exact test), including one case each of N370S homozygote, and R496H and T369M heterozygous variant. CONCLUSION: We found a higher frequency of glucocerebrosidase variants among pathologically diagnosed MSA cases in our brain bank compared to AD autopsies. This study demonstrates the need for further investigation into the role of glucocerebrosidase and lysosomal dysfunction in the etiology of MSA.

Elevated GM3 plasma concentration in idiopathic Parkinson's disease: A lipidomic analysis.

Parkinson's disease (PD) is a common neurodegenerative disease whose pathological hallmark is the accumulation of intracellular α-synuclein aggregates in Lewy bodies. Lipid metabolism dysregulation may play a significant role in PD pathogenesis; however, large plasma lipidomic studies in PD are lacking. In the current study, we analyzed the lipidomic profile of plasma obtained from 150 idiopathic PD patients and 100 controls, taken from the 'Spot' study at Columbia University Medical Center in New York. Our mass spectrometry based analytical panel consisted of 520 lipid species from 39 lipid subclasses including all major classes of glycerophospholipids, sphingolipids, glycerolipids and sterols. Each lipid species was analyzed using a logistic regression model. The plasma concentrations of two lipid subclasses, triglycerides and monosialodihexosylganglioside (GM3), were different between PD and control participants. GM3 ganglioside concentration had the most significant difference between PD and controls (1.531±0.037 pmol/µl versus 1.337±0.040 pmol/µl respectively; p-value = 5.96E-04; q-value = 0.048; when normalized to total lipid: p-value = 2.890E-05; q-value = 2.933E-03). Next, we used a collection of 20 GM3 and glucosylceramide (GlcCer) species concentrations normalized to total lipid to perform a ROC curve analysis, and found that these lipids compare favorably with biomarkers reported in previous studies (AUC = 0.742 for males, AUC = 0.644 for females). Our results suggest that higher plasma GM3 levels are associated with PD. GM3 lies in the same glycosphingolipid metabolic pathway as GlcCer, a substrate of the enzyme glucocerebrosidase, which has been associated with PD. These findings are consistent with previous reports implicating lower glucocerebrosidase activity with PD risk.