Activation of endogenous PRKN by structural derepression is linked to increased turnover of the E3 ubiquitin ligase

Loss-of-function mutations in the PINK1 and PRKN genes are the most common cause of early-onset Parkinson disease (PD). The encoded enzymatic pair selectively identifies, labels, and targets damaged mitochondria for degradation via the macroautophagy/autophagy-lysosome system (mitophagy). This pathway is cytoprotective and efforts to activate mitophagy are pursued as therapeutic avenues to combat PD and other neurodegenerative disorders. When mitochondria are damaged, the ubiquitin kinase PINK1 accumulates and recruits PRKN from the cytosol to activate the E3 ubiquitin ligase from its auto-inhibited conformation. We have previously designed several mutations that effectively derepress the structure of PRKN and activate its enzymatic functions in vitro. However, it remained unclear how these PRKN-activating mutations would perform endogenously in cultured neurons or in vivo in the brain. Here, we gene-edited neural progenitor cells and induced pluripotent stem cells to express PRKN-activating mutations in dopaminergic cultures. All tested PRKN-activating mutations indeed enhanced the enzymatic activity of PRKN in the absence of exogenous stress, but their hyperactivity was linked to their own PINK1-dependent degradation. Strikingly, in vivo in a mouse model expressing an equivalent activating mutation, we find the same relationship between PRKN enzymatic activity and protein stability. We conclude that PRKN degradation is the consequence of its structural derepression and enzymatic activation, thus resulting only in a temporary gain of activity. Our findings imply that pharmacological activation of endogenous PRKN will lead to increased turnover and suggest that additional considerations might be necessary to achieve sustained E3 ubiquitin ligase activity for disease treatment. Abbreviations: BSA: bovine serum album, CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECL: electrochemiluminescence; EGF: epidermal growth factor; ELISA: enzyme-linked immunosorbent assay; FGF: fibroblast growth factor; iPSC: induced pluripotent stem cell; KI: knock-in; KO: knockout; MAP2: microtubule associated protein 2; MFN2: mitofusin 2; MSD: Meso Scale Discovery; mt-Keima: mitochondrial targeted Keima; NPC: neural progenitor cell; PD: Parkinson disease; PDH: pyruvate dehydrogenase; p-S65-PRKN: Serine 65 phosphorylated PRKN; p-S65-Ub: Serine 65 phosphorylated ubiquitin; REP: repressor element of PRKN; TH: tyrosine hydroxylase; TX: Triton X-100, Ub: ubiquitin; UBL: ubiquitin-like; WT: wild-type.</p

Soluble intercellular adhesion molecule-1 in patients with recent cerebral ischemic events

P46 Objective: To determine whether serum levels of soluble intercellular adhesion molecule-1 (sICAM) differ between individuals with recent acute ischemic stroke (AIS) or transient ischemic attack (TIA) and control subjects. Methods: sICAM levels were measured in patients with recent (≤ 7 days) AIS or TIA and in patients with asymptomatic carotid stenosis and control subjects. Demographic information was collected and all subjects underwent complete neurological evaluations, routine blood tests and baseline cervical ultrasonography. Subjects with either a qualifying AIS or TIA also underwent cranial CT. sICAM levels were compared between different patient groups and correlated with carotid stenosis in those with AIS or TIA using analysis of variance (ANOVA) and logistic regression. Results: sICAM levels were measured in 288 patients (48.6% males). Of these, 91 had either a recent AIS or TIA. There were 75 control subjects. The remainder (n=122) had an asymptomatic carotid stenosis. Mean follow-up for all patients was 25.1 months. Mean age and distribution of cardiovascular risk factors were similar across all groups of patients. Mean sICAM levels were higher in those with recent AIS or TIA than in control subjects but without quite reaching statistical significance (p=0.10). For those with either AIS or TIA, mean sICAM levels did not differ on repeat measurement nor did initial levels correlate with the degree of carotid stenosis homolateral to the side of the ischemic event. During follow-up, 30 patients overall, (10.4%), experienced a recurrent ischemic event (either TIA, AIS, myocardial infarction and/or vascular death). Patients with either AIS or TIA, experienced a higher recurrence rate (24.6%). Mean sICAM levels were higher in those patients with a recurrent ischemic event (375.9 ng/mL) than in those without (332.1 ng/mL). Conclusion: There was a trend for higher sICAM levels in patients with recent AIS or TIA compared with control subjects. In addition, higher initial sICAM levels were associated with an increased risk of recurrent ischemic events for all patients. </jats:p

Prevalence of Undiagnosed Risk Factors in Patients with First-Ever Ischemic Stroke Treated at MUHC: A Retrospective Analysis

Background: Ischemic stroke is a leading cause of morbidity and mortality worldwide. Despite established prevention strategies, many patients present with previously undiagnosed vascular risk factors (URFs) at the time of their first-ever ischemic stroke, suggesting missed opportunities for early detection. In Canada, particularly in Quebec, access to primary care is inconsistent, and a substantial proportion of the population lacks attachment to a family doctor (FD). Objective: This study aimed to determine the prevalence of URFs among patients with first-ever ischemic stroke and to evaluate the relationship between URFs, geographic region, and access to primary care in Quebec, Canada. We hypothesized that patients without an FD would have a higher prevalence of URFs. Methods: We conducted a retrospective chart review of 610 patients admitted with first-ever ischemic stroke to the McGill University Health Center (MUHC) between 2014 and 2017. Data collected included demographics; known and undiagnosed stroke risk factors such as hypertension (HTN), diabetes mellitus (DM), hyperlipidemia (HLD), and atrial fibrillation (AF); FD status; and geographic location based on postal code. Results: Among the 610 patients, 136 (22.3%) had at least one URF. The most common URF was HLD (14.3%), followed by HTN (6.2%), AF (1.6%), and DM (0.1%). Of 609 patients with available data, 146 (23.97%) lacked an FD. Patients without an FD were significantly more likely to have undiagnosed HTN (7.6% vs. 2.1%, p = 0.008). No significant differences were observed for the other URFs. Geographic variation was noted in both URF prevalence and FD access, but regional differences were not statistically significant. Conclusions: Our findings support the hypothesis that a lack of an FD is associated with a higher prevalence of undiagnosed HTN in ischemic stroke patients. Targeted screening and improved access to primary care, particularly in underserved regions, may help to reduce the burden of preventable stroke by facilitating the earlier identification and management of modifiable risk factors

Disruption of GRIN2B Impairs Differentiation in Human Neurons

Summary: Heterozygous loss-of-function mutations in GRIN2B, a subunit of the NMDA receptor, cause intellectual disability and language impairment. We developed clonal models of GRIN2B deletion and loss-of-function mutations in a region coding for the glutamate binding domain in human cells and generated neurons from a patient harboring a missense mutation in the same domain. Transcriptome analysis revealed extensive increases in genes associated with cell proliferation and decreases in genes associated with neuron differentiation, a result supported by extensive protein analyses. Using electrophysiology and calcium imaging, we demonstrate that NMDA receptors are present on neural progenitor cells and that human mutations in GRIN2B can impair calcium influx and membrane depolarization even in a presumed undifferentiated cell state, highlighting an important role for non-synaptic NMDA receptors. It may be this function, in part, which underlies the neurological disease observed in patients with GRIN2B mutations. : Mutations in GRIN2B cause intellectual disability and language impairments. In this study, Bell and colleagues engineer mutations in GRIN2B and repair a patient missense mutation to show an important role for GRIN2B and non-synaptic NMDA receptors in differentiating neural stem cells. Keywords: GRIN2B, NMDAR2B, NMDA, glutamate, iPSCs, CRISPR, CRISPR-Cas9, neurodevelopment, NPCs, neural stem cel

Stimulation of L-type calcium channels increases tyrosine hydroxylase and dopamine in ventral midbrain cells induced from somatic cells

Abstract Making high-quality dopamine (DA)-producing cells for basic biological or small molecule screening studies is critical for the development of novel therapeutics for disorders of the ventral midbrain. Currently, many ventral midbrain assays have low signal-to-noise ratio due to low levels of cellular DA and the rate-limiting enzyme of DA synthesis, tyrosine hydroxylase (TH), hampering discovery efforts. Using intensively characterized ventral midbrain cells derived from human skin, which demonstrate calcium pacemaking activity and classical electrophysiological properties, we show that an L-type calcium agonist can significantly increase TH protein levels and DA content and release. Live calcium imaging suggests that it is the immediate influx of calcium occurring simultaneously in all cells that drives this effect. Genome-wide expression profiling suggests that L-type calcium channel stimulation has a significant effect on specific genes related to DA synthesis and affects expression of L-type calcium receptor subunits from the CACNA1 and CACNA2D families. Together, our findings provide an advance in the ability to increase DA and TH levels to improve the accuracy of disease modeling and small molecule screening for disorders of the ventral midbrain, including Parkinson’s disease. Significance statement A single molecule provides a major boost to both tyrosine hydroxylase and dopamine in stem cell-derived human ventral midbrain cells. </jats:sec