Parkinson's Institute and Clinical Center

ABSTRACTBackgroundA common genetic mutation that causes Parkinson's disease (PD) is the G2019S LRRK2 mutation. A precision medicine approach that selectively blocks only excess kinase activity of the mutant allele could yield a safe and effective treatment for G2019S LRRK2 PD.ObjectiveTo determine the activity of a G2019S mutant selective leucine‐rich repeat kinase 2 (LRRK2) kinase inhibitor as compared to a nonselective inhibitor in blood of subjects with genetic and idiopathic PD on two LRRK2 biomarkers, pSer935 LRRK2 and pThr73 Rab10.MethodsBlood was collected from 13 subjects with or without a G2019S LRRK2 mutation with PD and one healthy control. Peripheral blood mononuclear cells were treated ex vivo with a novel G2019S LRRK2 inhibitor (EB‐42168) or the nonselective inhibitor MLi‐2. Quantitative western immunoblot analyses were performed.ResultsEB‐42168 was 100 times more selective for G2019S LRRK2 when compared to wild‐type (WT) LRRK2. Concentrations that inhibited phosphorylation of pSer935 LRRK2 by 90% in homozygous G2019S LRRK2 patients, inhibited pSer935 LRRK2 by 36% in heterozygous patients, and by only 5% in patients carrying only the WT allele. Similar selectivity was seen for pThr73 Rab10. MLi‐2 showed an equivalent level of inhibition across all genotypes.ConclusionsThese findings demonstrate that EB‐42168, a G2019S LRRK2 selective inhibitor, lowers mutant G2019S LRRK2 phosphorylated biomarkers while simultaneously sparing WT LRRK2. Selective targeting of G2019S LRRK2 with a small molecule lays the foundation for a precision medicine treatment of G2019S LRRK2 PD. © 2021 ESCAPE Bio, Inc. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

This study evaluated the efficacy and safety of repeated home use of an individualized, non-invasive neuromodulation therapy targeting the median and radial nerves for reducing hand tremor in essential tremor patients over three months.

Leucine-rich repeat kinase 2 is a potential therapeutic target for the treatment of Parkinson's disease, and clinical trials of leucine-rich repeat kinase 2 inhibitors are in development. The objective of this study was to evaluate phosphorylation of a new leucine-rich repeat kinase 2 substrate, Rab10, for potential use as a target engagement biomarker and/or patient enrichment biomarker for leucine-rich repeat kinase 2 inhibitor clinical trials. Peripheral blood mononuclear cells and neutrophils were isolated from Parkinson's disease patients and matched controls, and treated ex vivo with a leucine-rich repeat kinase 2 inhibitor. Immunoblotting was used to measure levels of leucine-rich repeat kinase 2 and Rab10 and their phosphorylation. Plasma inflammatory cytokines were measured by multiplex enzyme-linked immunosorbent assay. Mononuclear cells and neutrophils of both controls and Parkinson's disease patients responded the same to leucine-rich repeat kinase 2 inhibitor treatment. Leucine-rich repeat kinase 2 levels in mononuclear cells were the same in controls and Parkinson's disease patients, whereas leucine-rich repeat kinase 2 was significantly increased in Parkinson's disease neutrophils. Rab10 T73 phosphorylation levels were similar in controls and Parkinson's disease patients and did not correlate with leucine-rich repeat kinase 2 levels. Immune-cell levels of leucine-rich repeat kinase 2 and Rab10 T73 phosphorylation were associated with plasma inflammatory cytokine levels. Rab10 T73 phosphorylation appears to be a valid target engagement biomarker for potential use in leucine-rich repeat kinase 2 inhibitor clinical trials. However, a lack of association between leucine-rich repeat kinase 2 and Rab10 phosphorylation complicates the potential use of Rab10 phosphorylation as a patient enrichment biomarker. Although replication is required, increased leucine-rich repeat kinase 2 levels in neutrophils from Parkinson's disease patients may have the potential for patient stratification. leucine-rich repeat kinase 2 activity in peripheral immune cells may contribute to an inflammatory phenotype. © 2018 International Parkinson and Movement Disorder Society.

BackgroundFabry disease is an X-linked lysosomal storage disorder caused by GLA mutations, resulting in α-galactosidase (α-Gal) deficiency and accumulation of lysosomal substrates. Migalastat, an oral pharmacological chaperone being developed as an alternative to intravenous enzyme replacement therapy (ERT), stabilises specific mutant (amenable) forms of α-Gal to facilitate normal lysosomal trafficking.MethodsThe main objective of the 18-month, randomised, active-controlled ATTRACT study was to assess the effects of migalastat on renal function in patients with Fabry disease previously treated with ERT. Effects on heart, disease substrate, patient-reported outcomes (PROs) and safety were also assessed.ResultsFifty-seven adults (56% female) receiving ERT (88% had multiorgan disease) were randomised (1.5:1), based on a preliminary cell-based assay of responsiveness to migalastat, to receive 18 months open-label migalastat or remain on ERT. Four patients had non-amenable mutant forms of α-Gal based on the validated cell-based assay conducted after treatment initiation and were excluded from primary efficacy analyses only. Migalastat and ERT had similar effects on renal function. Left ventricular mass index decreased significantly with migalastat treatment (−6.6 g/m2 (−11.0 to −2.2)); there was no significant change with ERT. Predefined renal, cardiac or cerebrovascular events occurred in 29% and 44% of patients in the migalastat and ERT groups, respectively. Plasma globotriaosylsphingosine remained low and stable following the switch from ERT to migalastat. PROs were comparable between groups. Migalastat was generally safe and well tolerated.ConclusionsMigalastat offers promise as a first-in-class oral monotherapy alternative treatment to intravenous ERT for patients with Fabry disease and amenable mutations.Trial registration number:NCT00925301; Pre-results.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene were discovered in 2004 and have been found to be the most frequently mutated gene in Parkinson's disease. LRRK2 is a large multi-domain protein with a functional GTPase and kinase domain. The signal transduction pathways in which LRRK2 is dysfunctional in the disease state are only now being resolved, but we do know that LRRK2 is, itself, a substrate of multiple kinases and phosphatases and exists in variable phosphorylated states. Autophosphorylation of LRRK2 can impact GTPase and pathological outcomes. LRRK2 serines (910/935/955/973) are differentially phosphorylated in pathogenic PD mutations and after LRRK2 kinase inhibition. The phosphorylation status of LRRK2 can therefore provide key insight into the mechanisms of kinase dysfunction during disease. This chapter will describe the identification of LRRK2 phosphorylation sites and how phosphoregulation of LRRK2 reveals its own kinase activity and regulates its ubiquitination and localization in vitro, in cells, and in tissues.

The induced pluripotent stem (iPS) cell field holds promise for in vitro disease modeling. However, identifying innate cellular pathologies, particularly for age-related neurodegenerative diseases, has been challenging. Here, we exploited mutation correction of iPS cells and conserved proteotoxic mechanisms from yeast to humans to discover and reverse phenotypic responses to α-synuclein (αsyn), a key protein involved in Parkinson's disease (PD). We generated cortical neurons from iPS cells of patients harboring αsyn mutations, who are at high risk of developing PD dementia. Genetic modifiers from unbiased screens in a yeast model of αsyn toxicity led to identification of early pathogenic phenotypes in patient neurons. These included nitrosative stress, accumulation of endoplasmic reticulum (ER)-associated degradation substrates, and ER stress. A small molecule identified in a yeast screen (NAB2), and the ubiquitin ligase Nedd4 it affects, reversed pathologic phenotypes in these neurons.

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease(1). Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms(2) and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

OBJECTIVE: To determine if a Mediterranean-type diet (MeDi) modifies the time to clinical onset of HD ('phenoconversion') in premanifest carriers participating in Prospective Huntington At Risk Observational Study (PHAROS).BACKGROUND: We previously showed that in participants with CAG >37, higher caloric intake, but not body mass index (BMI), was associated with higher CAG (p=0.03) and increased 5-year probability of HD (p=0.013). Adherence to MeDi may delay onset of Alzheimer's and Parkinson's disease. Whether adherence to MeDi affects time to phenoconversion is unknown.DESIGN/METHODS: At baseline, participants were 26-57 years and had not undergone genetic testing. A semi-quantitative food frequency questionnaire (FFQ) was administered ∼30 months after baseline. We calculated daily gram intake for dairy, meat, fruit, vegetables, legumes, cereals, fish, monounsaturated and saturated fatty-acids, and alcohol, and constructed MeDi scores (0-9); higher scores indicate higher adherence. Demographics, medical history, BMI and Unified Huntington's Disease Rating Scale (UHDRS) were collected. We used Cox proportional hazards models to determine the association of MeDi and phenoconversion in participants with CAG >37.RESULTS: Age, caloric intake, gender, education, and UHDRS motor scores did not differ when compared among MeDi tertiles (0-3, 4-5, 6-9). The highest BMI was associated with lowest adherence to MeDi. Among 211 premanifest individuals who completed the FFQ, 31 phenoconverted during follow up. In a model adjusted for age, CAG, and caloric intake, MeDi was not associated with phenoconversion (p for trend=0.13 for tertile of MeDi, and p=0.22 for continuous MeDi). When individual diet components of MeDi were analyzed, high dairy consumption (hazard ratio 2.36; 1.0-5.57; p=0.051) and higher caloric intake (p=0.04) were associated with risk of phenoconversion in an adjusted model.CONCLUSIONS: MeDi was not associated with phenoconversion, however higher consumption of dairy products had a two-fold increased risk. A larger study of premanifest individuals is necessary to explore the relationship to dairy products.Supported by: National Institutes of Health (# 2 R01 HG002449-06), including support from the National Human Genome Research Institute and the National Institute of Neurological Disorders and Stroke.Disclosure: Dr. Marder has received personal compensation in an editorial capacity for Current Neuroscience. Dr. Marder has received research support from NIH, Michael J. Fox, Parkinson Disease Foundation, CHDI, and the Huntington's Disese Society of America. Dr. Gu has nothing to disclose. Dr. Eberly has received research support from Cephalon, Inc. and Lundbeck. Dr. Tanner has received personal compensation for activities with Adamas Pharmaceuticals as a consultant. Dr. Scarmeas has nothing to disclose. Dr. Oakes has received personal compensation in an editorial capacity for Lifetime Data Analysis. Dr. Shoulson has recieved personal compensation for activities with AstraZeneca, Keryx Pharmaceuticals, Jazz Pharmaceuticals, Neurogen Corp, Alexa Molecular Delivery Corp, Nouvel Pharma, Novartis, and Westat. Dr. Shoulson has received personal compensation in an editorial capacity for Archives of Neurology. Dr. Shouldson has received research support from NIH, NHGRI, NINDS, Cephalon, Inc., Pharmacia and Upjohn, Inc. (Pfizer), Parallel Group, and Prestwick Pharmaceuticals, Inc. Dr. Shoulson has recieved personal compensation for activities with AstraZeneca, Keryx Pharmaceuticals, Jazz Pharmaceuticals, Neurogen Corp, Alexa Molecular Delivery Corp, Nouvel Pharma, Novartis, and Westat. Dr. Shoulson has received personal compensation in an editorial capacity for Archives of Neurology. Dr. Shouldson has received research support from NIH, NHGRI, NINDS, Cephalon, Inc., Pharmacia and Upjohn, Inc. (Pfizer), Parallel Group, and Prestwick Pharmaceuticals, Inc.