Samuel Evetts

ER Stress and Autophagic Perturbations Lead to Elevated Extracellular α-Synuclein in GBA-N370S Parkinson's iPSC-Derived Dopamine Neurons

Summary Heterozygous mutations in the glucocerebrosidase gene (GBA) represent the strongest common genetic risk factor for Parkinsons disease (PD), the second most common neurodegenerative disorder. However, the molecular mechanisms underlying this association are still poorly understood. Here, we have analyzed ten independent induced pluripotent stem cell (iPSC) lines from three controls and three unrelated PD patients heterozygous for the GBA-N370S mutation, and identified relevant disease mechanisms. After differentiation into dopaminergic neurons, we observed misprocessing of mutant glucocerebrosidase protein in the ER, associated with activation of ER stress and abnormal cellular lipid profiles. Furthermore, we observed autophagic perturbations and an enlargement of the lysosomal compartment specifically in dopamine neurons. Finally, we found increased extracellular α-synuclein in patient-derived neuronal culture medium, which was not associated with exosomes. Overall, ER stress, autophagic/lysosomal perturbations, and elevated extracellular α-synuclein likely represent critical early cellular phenotypes of PD, which might offer multiple therapeutic targets.

Identification of distinct circulating exosomes in Parkinson's disease

Whether circulating microvesicles convey bioactive signals in neurodegenerative diseases remains currently unknown. In this study, we investigated the biochemical composition and biological function of exosomes isolated from sera of patients with Parkinsons disease (PD).

Alpha‐synuclein RT‐QuIC in the CSF of patients with alpha‐synucleinopathies

We have developed a novel real‐time quaking‐induced conversion RT‐QuIC‐based assay to detect alpha‐synuclein aggregation in brain and cerebrospinal fluid from dementia with Lewy bodies and Parkinsons disease patients. This assay can detect alpha‐synuclein aggregation in Dementia with Lewy bodies and Parkinsons disease cerebrospinal fluid with sensitivities of 92% and 95%, respectively, and with an overall specificity of 100% when compared to Alzheimer and control cerebrospinal fluid. Patients with neuropathologically confirmed tauopathies (progressive supranuclear palsy; corticobasal degeneration) gave negative results. These results suggest that RT‐QuiC analysis of cerebrospinal fluid is potentially useful for the early clinical assessment of patients with alpha‐synucleinopathies.

Prodromal Parkinsonism and Neurodegenerative Risk Stratification in REM Sleep Behavior Disorder

Objectives Rapid eye movement (REM) sleep behavior disorder (RBD) is the most specific marker of prodromal alpha‐synucleinopathies. We sought to delineate the baseline clinical characteristics of RBD and evaluate risk stratification models. Methods Clinical assessments were performed in 171 RBD, 296 control, and 119 untreated Parkinsons (PD) participants. Putative risk measures were assessed as predictors of prodromal neurodegeneration, and Movement Disorders Society (MDS) criteria for prodromal PD were applied. Participants were screened for common leucine‐rich repeat kinase 2 (LRRK2)/glucocerebrosidase gene (GBA) gene mutations. Results Compared to controls, participants with RBD had higher rates of solvent exposure, head injury, smoking, obesity, and antidepressant use. GBA mutations were more common in RBD, but no LRRK2 mutations were found. RBD participants performed significantly worse than controls on Unified Parkinsons Disease Rating Scale (UPDRS)‐III, timed “get‐up‐and‐go”, Flamingo test, Sniffin Sticks, and cognitive tests and had worse measures of constipation, quality of life (QOL), and orthostatic hypotension. For all these measures except UPDRS‐III, RBD and PD participants were equally impaired. Depression, anxiety, and apathy were worse in RBD compared to PD participants. Stratification of people with RBD according to antidepressant use, obesity, and age altered the odds ratio (OR) of hyposmia compared to controls from 3.4 to 45.5. 74% (95% confidence interval [CI] 66%, 80%) of RBD participants met the MDS criteria for probable prodromal Parkinsons compared to 0.3% (95% CI 0.009%, 2%) of controls. Conclusions RBD are impaired across a range of clinical measures consistent with prodromal PD and suggestive of a more severe nonmotor subtype. Clinical risk stratification has the potential to select higher risk patients for neuroprotective interventions.

Delineating Nonmotor Symptoms in Early Parkinson's Disease and First-Degree Relatives

Nonmotor symptoms (NMS) are an important prodromal feature of Parkinsons disease (PD). However, their frequency, treatment rates, and impact on health‐related quality of life (HRQoL) in the early motor phase is unclear. Rates of NMS in enriched at‐risk populations, such as first‐degree PD relatives, have not been delineated. We assessed NMS in an early cohort of PD, first‐degree PD relatives and control subjects to address these questions. In total, 769 population‐ascertained PD subjects within 3.5 years of diagnosis, 98 first‐degree PD relatives, and 287 control subjects were assessed at baseline across the following NMS domains: (1) neuropsychiatric; (2) gastrointestinal; (3) sleep; (4) sensory; (5) autonomic; and (6) sexual. NMS were much more common in PD, compared to control subjects. More than half of the PD cases had hyposmia, pain, fatigue, sleep disturbance, or urinary dysfunction. NMS were more frequent in those with the postural instability gait difficulty phenotype, compared to the tremor dominant (mean total number of NMS 7.8 vs. 6.2; P < 0.001). PD cases had worse HRQoL scores than controls (odds ratio: 4.1; P < 0.001), with depression, anxiety, and pain being stronger drivers than motor scores. NMS were rarely treated in routine clinical practice. First‐degree PD relatives did not significantly differ in NMS, compared to controls, in this baseline study. NMS are common in early PD and more common in those with postural instability gait difficulty phenotype or on treatment. Despite their major impact on quality of life, NMS are usually under‐recognized and untreated.

Cerebrospinal fluid macrophage biomarkers in amyotrophic lateral sclerosis.

The neurodegenerative disease, amyotrophic lateral sclerosis (ALS), is a heterogeneous clinical syndrome involving multiple molecular pathways. The development of biomarkers for use in therapeutic trials is a priority. We sought to use a high‐throughput proteomic method to identify novel biomarkers in individual cerebrospinal fluid (CSF) samples.

An impedimetric assay of α-synuclein autoantibodies in early stage Parkinson's disease

α-Synuclein (α-Syn), a protein synthesized by neurons, as the major protein component of Lewy body inclusions, undoubtedly has a prominent role in the pathogenesis of Parkinsons Disease (PD). In an attempt to enable pre-symptomatic and definitive diagnosis, numerous attempts have been made to align assayed total α-Syn levels in serum (where there is a native presence) to PD disease status. Results have been conflicting. The status of circulating and potentially neuroprotective α-Syn autoantibodies in PD subjects is also unclear. In previous work we demonstrated that electrochemically assayed autoantibody levels were higher in PD patients compared to controls and, significantly, noted that this differentiation was most marked early in disease. Herein we report a robust (coefficient of variation 3.0%) single step and label free analysis of 90 subjects, including 60 PD patients, with a mean disease duration of 1.4 years and 29 control subjects. In this cross sectional cohort we observe a statistically significant (p 0.05; Kruskal–Wallis test).

Mitochondrial dysfunction and increased glycolysis in prodromal and early Parkinson's blood cells: Mitochondria and Glycolysis In PD Blood Cells

Background: Although primarily a neurodegenerative process, there is increasing awareness of peripheral disease mechanisms in Parkinsons disease. To investigate disease processes in accessible patient cells, we studied peripheral blood mononuclear cells in recently diagnosed PD patients and rapid eye movement‐sleep behavior disorder patients who have a greatly increased risk of developing PD. We hypothesized that peripheral blood mononuclear cells may recapitulate cellular pathology found in the PD brain and investigated these cells for mitochondrial dysfunction and oxidative stress.

Potential Metabolomic Linkage in Blood between Parkinson’s Disease and Traumatic Brain Injury

The etiologic basis for sporadic forms of neurodegenerative diseases has been elusive but likely represents the product of genetic predisposition and various environmental factors. Specific gene-environment interactions have become more salient owing, in part, to the elucidation of epigenetic mechanisms and their impact on health and disease. The linkage between traumatic brain injury (TBI) and Parkinson’s disease (PD) is one such association that currently lacks a mechanistic basis. Herein, we present preliminary blood-based metabolomic evidence in support of potential association between TBI and PD. Using untargeted and targeted high-performance liquid chromatography-mass spectrometry we identified metabolomic biomarker profiles in a cohort of symptomatic mild TBI (mTBI) subjects (n = 75) 3–12 months following injury (subacute) and TBI controls (n = 20), and a PD cohort with known PD (n = 20) or PD dementia (PDD) (n = 20) and PD controls (n = 20). Surprisingly, blood glutamic acid levels in both the subacute mTBI (increased) and PD/PDD (decreased) groups were notably altered from control levels. The observed changes in blood glutamic acid levels in mTBI and PD/PDD are discussed in relation to other metabolite profiling studies. Should our preliminary results be replicated in comparable metabolomic investigations of TBI and PD cohorts, they may contribute to an “excitotoxic” linkage between TBI and PD/PDD.

SERUM BIOMARKERS FOR PARKINSON'S DISEASE

Objectives There is a critical need to develop robust biomarkers for Parkinsons diasease (PD) research. We aimed to determine whether a serum biomarker could be used: to predict cognitive decline in PD or a diagnosis of PD. Five of the most promising serum biomarkers were identified based on recent and current evidence: Vitamin D, Apolipoprotein A1 (ApoA1), Uric Acid, C-reactive protein and Epidermal Growth Factor. Method A peripheral blood sample was taken at baseline, spun in a centrifuge and then stored at −80°C. PD patients (n=641) diagnosed within 3.5 years, a control population (n=146) and patients with REM sleep behaviour disorder (n=74) were included at baseline. 3 year follow up data was available from 229 PD patients. Serum analysis was conducted either using Abbott systems or ELISA techniques. Age, gender, disease duration and seasonality (for vitamin D) were adjusted for using statistical models. Results: There was no strong evidence that the potential biomarkers predicted either baseline or change in cognition. Uric acid was lower in the PD group compared to controls (p=0.008), though this effect was not replicated in the RBD group. EGF (p<0.001) and ApoA1 (p=0.04) were both lower in RBD cases compared to controls, and Vitamin D levels were higher (p=0.009). These results should be viewed with caution however as they were not replicated in the PD group so may relate to type I errors and require further replication.Conclusion: None of the biomarkers predicted cognitive decline in the PD group. This may be because there is not yet enough follow up data to detect a signal, but they do not predict baseline cognition. EGF, Vitamin D and ApoA1 show some differences in the RBD group compared to controls, while Uric Acid was lower in the PD group. Individually, the candidate biomarkers did not predict cognitive decline or patient group. Additional data will be available by the time of the conference as data collection is.