Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity.

In Parkinson's disease and other synucleinopathies, the elevation of α-synuclein phosphorylated at Serine129 (pS129) is a widely cited marker of pathology. However, the physiological role for pS129 has remained undefined. Here we use multiple approaches to show for the first time that pS129 functions as a physiological regulator of neuronal activity. Neuronal activity triggers a sustained increase of pS129 in cultured neurons (200% within 4 h). In accord, brain pS129 is elevated in environmentally enriched mice exhibiting enhanced long-term potentiation. Activity-dependent α-synuclein phosphorylation is S129-specific, reversible, confers no cytotoxicity, and accumulates at synapsin-containing presynaptic boutons. Mechanistically, our findings are consistent with a model in which neuronal stimulation enhances Plk2 kinase activity via a calcium/calcineurin pathway to counteract PP2A phosphatase activity for efficient phosphorylation of membrane-bound α-synuclein. Patch clamping of rat SNCA-/- neurons expressing exogenous wild-type or phospho-incompetent (S129A) α-synuclein suggests that pS129 fine-tunes the balance between excitatory and inhibitory neuronal currents. Consistently, our novel S129A knock-in (S129AKI) mice exhibit impaired hippocampal plasticity. The discovery of a key physiological function for pS129 has implications for understanding the role of α-synuclein in neurotransmission and adds nuance to the interpretation of pS129 as a synucleinopathy biomarker.

Impact of leukoaraiosis in patients with acute ischemic stroke treated with thrombectomy: a post hoc analysis of the DIRECT-MT trial.

BACKGROUND: The influence of leukoaraiosis in patients with acute ischemic stroke (AIS) given intra-arterial treatment (IAT) with or without preceding intravenous thrombolysis (IVT) remains unknown. OBJECTIVE: To assess the clinical and radiological outcomes of IAT in patients with or without leukoaraiosis. METHODS: Patients of the direct mechanical thrombectomy trial (DIRECT-MT) whose leukoaraiosis grade could be assessed were included. DIRECT-MT was a randomized clinical trial performed in China to assess the effect of direct IAT compared with intravenous thrombolysis plus IAT. We employed the Age-Related White Matter Changes Scale for grading leukoaraiosis (ARWMC, 0 indicates no leukoaraiosis, 1-2 indicates mild-to-moderate leukoaraiosis, and 3 indicates severe leukoaraiosis) based on brain CT. The primary outcome was the score on the modified Rankin Scale (mRS) assessed at 90 days. RESULTS: There were 656 patients in the trial, 649 patients who were included, with 432 patients without leukoaraiosis, and 217 (33.4%) patients with leukoaraiosis divided into mild-to-moderate (n=139) and severe groups (n=78). Leukoaraiosis was a predictor of a worse mRS score (adjusted OR (aOR)=0.7 (95% CI 0.5 to 0.8)) and higher mortality (aOR=1.4 (1.1 to 1.9)), but it was not associated with symptomatic intracranial hemorrhage (sICH) (aOR=0.9 (0.5 to 1.5)). IVT preceding IAT did not increase sICH risk for patients with no (aOR=1.4 (0.6 to 3.4)), mild-to-moderate (aOR=1.5 (0.3 to 7.8)), or severe (aOR=1.5 (0.1 to 21.3)) leukoaraiosis. CONCLUSION: Patients with leukoaraiosis with AIS due to large vessel occlusion are at increased risk of a poor functional outcome after IAT but demonstrate similar sICH rates, and IVT preceding IAT does not increase the risk of sICH in Chinese patients with leukoaraiosis.

Predictors of futile recanalization in basilar artery occlusion patients undergoing endovascular treatment: a post hoc analysis of the ATTENTION trial.

Background: Few studies have focused on factors associated with futile recanalization in patients with an acute basilar artery occlusion (BAO) that was treated with modern endovascular therapy (EVT). The aim of this study was to explore the factors associated with futile recanalization in patients with an acute BAO presented within 12 h. Methods: This is a post-hoc analysis of the ATTENTION trial (The Trial of Endovascular Treatment of Acute Basilar-Artery Occlusion, ClinicalTrials.gov, number NCT04751708). Demographics, clinical characteristics, acute stroke workflow interval times, and imaging characteristics were compared between the futile recanalization and favorable recanalization groups. The favorable outcome was defined as a modified Rankin scale (mRS) score of 0-3 at 90 days, successful reperfusion was defined as thrombolysis in cerebral infarction (TICI) 2b and 3 on the final angiogram, and futile recanalization was defined as failure to achieve a favorable outcome despite successful reperfusion. A multivariate analysis was performed to identify the predictors of futile recanalization. Results: In total, 185 patients were included in the final analysis: 89 (48.1%) patients had futile recanalization and 96 (51.9%) patients had favorable recanalization. In the multivariable logistic regression analysis, older age (OR 1.04, 95% CI 1.01 to 1.08, p = 0.01) and diabetes mellitus (OR 3.35, 95% CI 1.40 to 8.01, p = 0.007) were independent predictors of futile recanalization. Conclusion: Futile recanalization occurred in nearly half of patients with acute BAO following endovascular treatment. Old age and diabetes mellitus were identified as independent predictors of futile recanalization after endovascular therapy for acute BAO.

Application of Balloon AngioplaSty with the dIstal protection of Stent Retriever (BASIS) technique for acute intracranial artery atherosclerosis-related occlusion.

Background: Endovascular therapy (EVT) is complex in the context of intracranial atherosclerosis (ICAS)-related large vessel occlusion (LVO) and the re-occlusion rates are high due to residual stenosis, the procedure time is long and the optimal EVT technique is unclear. The Balloon AngioplaSty with the dIstal protection of Stent Retriever (BASIS) technique is a novel thrombectomy technique that allows emergent balloon angioplasty to be performed via the wire of the retrieval stent. Our study presents our initial experience with the BASIS technique in ICAS-related LVO and assesses its feasibility. Method: In patients with ICAS-related LVO treated with BASIS, clinical and angiographic data were retrospectively analyzed. Angiographic data included first-pass reperfusion (PFR), the rate of residual stenosis, distal emboli, and re-occlusion post-procedure. The Extended Thrombolysis in Cerebral Infarction (eTICI) scale was used to assess reperfusion extent, and an eTICI score ≥2b was defined as successful perfusion. Clinical outcome was evaluated at 3 months (modified Rankin score [mRS]), and an mRS ≤ 2 was defined as a good clinical outcome. Results: A total of seven patients with ICAS-related LVO were included, and the median age of the patients was 76 years. All patients achieved eTICI 3 reperfusion and FPR. The residual stenosis rate ranged from 5 to 10%. None of the patients had re-occlusion post-procedure. The median puncture-to-reperfusion time was 51 min. None of the patients had a symptomatic cerebral hemorrhage, re-occlusion, distal embolism, and dissection. Good clinical outcomes were observed in four patients (4/7, 57.1%), and 1 patient (1/7, 14.3%) died. Conclusion: The BASIS technique is feasible and safe for treating acute ICAS-related LVO.

Integrin α5 Is Regulated by miR-218-5p in Endothelial Progenitor Cells.

BACKGROUND: Endothelial cell injury is a common nidus of renal injury in patients and consistent with the high prevalence of AKI reported during the coronavirus disease 2019 pandemic. This cell type expresses integrin α5 (ITGA5), which is essential to the Tie2 signaling pathway. The microRNA miR-218-5p is upregulated in endothelial progenitor cells (EPCs) after hypoxia, but microRNA regulation of Tie2 in the EPC lineage is unclear. METHODS: We isolated human kidney-derived EPCs (hkEPCs) and surveyed microRNA target transcripts. A preclinical model of ischemic kidney injury was used to evaluate the effect of hkEPCs on capillary repair. We used a genetic knockout model to evaluate the effect of deleting endogenous expression of miR-218 specifically in angioblasts. RESULTS: After ischemic in vitro preconditioning, miR-218-5p was elevated in hkEPCs. We found miR-218-5p bound to ITGA5 mRNA transcript and decreased ITGA5 protein expression. Phosphorylation of 42/44 MAPK decreased by 73.6% in hkEPCs treated with miR-218-5p. Cells supplemented with miR-218-5p downregulated ITGA5 synthesis and decreased 42/44 MAPK phosphorylation. In a CD309-Cre/miR-218-2-LoxP mammalian model (a conditional knockout mouse model designed to delete pre-miR-218-2 exclusively in CD309+ cells), homozygotes at e18.5 contained avascular glomeruli, whereas heterozygote adults showed susceptibility to kidney injury. Isolated EPCs from the mouse kidney contained high amounts of ITGA5 and showed decreased migratory capacity in three-dimensional cell culture. CONCLUSIONS: These results demonstrate the critical regulatory role of miR-218-5p in kidney EPC migration, a finding that may inform efforts to treat microvascular kidney injury via therapeutic cell delivery.

Association of baseline core volume and early midline shift in acute stroke patients with a large ischaemic core.

Background: Midline shift (MLS) is troublesome problem that may occur in patients with a large infarct core (LIC) and may be related to the baseline infarct core volume. The purpose of this study was to explore the relationship between baseline infarct core volume and early MLS presence. Materials and methods: Patients with acute intracranial large artery occlusion and a pretreatment relative cerebral blood flow (rCBF) <30% volume ≥50 ml on CT perfusion (CTP) were included, clinical outcomes following endovascular treatment (EVT) were retrospectively analyzed. The primary endpoint was MLS within 48 h (early MLS presence). The association between baseline ICV and early MLS presence was evaluated with multivariable regression. Results: Ultimately, 95 patients were included, and 29.5% (28/95) of the patients had early MLS. The number of patients with a baseline rCBF < 15% volume (median [interquartile range], 46 [32-60] vs. 29 [19-40]; P < 0.001) was significantly larger in the early severe MLS presence group. A baseline rCBF < 15% volume showed significantly better predictive accuracy for early MLS presence than an rCBF < 30% volume (area under the curve, 0.74 vs. 0.64, P = 0.0023). In addition, an rCBF < 15% volume ≥40 ml (odds ratio, 4.34 [95% CI, 1.571-11.996]) was associated with early MLS presence after adjustment for sex, age, baseline National Institutes of Health Stroke Scale score, onset-to-recanalization time. Conclusion: In patients with an acute LIC following EVT, a pretreatment infarct core volume > 40 ml based on an rCBF < 15% showed good predictive value for early MLS occurrence.

ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain.

OCT4 and SOX2 confer pluripotency by recruiting coactivators to activate stem cell­specific transcription. However, the composition of coactivator complexes and their roles in maintaining stem cell fidelity remain unclear. Here, we report the ATP-binding cassette subfamily F member 1 (ABCF1) as a coactivator for OCT4/SOX2 critical for stem cell self-renewal. The intrinsically disordered low-complexity domain (LCD) of ABCF1 contributes to phase separation in vitro and transcriptional activation of pluripotency genes by mediating multivalent interactions with SOX2 and co-dependent coactivators XPC and DKC1. These LCD-driven transcription factor­coactivator interactions critical for pluripotency gene expression are disrupted by DNA damage, likely due to LCD-dependent binding of ABCF1 to damage-generated intracellular DNA fragments instead of SOX2. This study identifies a transcriptional coactivator that uses its LCD to form selective multivalent interactions to regulate stem cell self-renewal and exit from pluripotency when genome integrity is compromised.

Initial Clinical Experience of Repeat Thrombectomy with a Retrieval Stent (RTRS) with Continuous Proximal Flow Arrest by Balloon Guide Catheter for Acute Intracranial Carotid Occlusion.

BACKGROUND: Balloon guide catheters (BGCs) have good performance in terms of radiological outcomes in acute ischemic thrombectomy. It is not uncommon for BGCs to be blocked by thrombi, especially in cases with acute intracranial internal carotid artery (ICA) occlusion. Our initial experience using repeat thrombectomy with a retrieval stent (RTRS) with continuous proximal flow arrest by BGC for acute intracranial ICA occlusion is presented. METHODS: In patients with acute intracranial ICA occlusion treated with RTRS, clinical data, including the National Institutes of Health Stroke Scale (NIHSS) score at admission and modified Rankin Scale (mRS) score at 90 days, and procedural data, including the Extended treatment in Cerebral Infarction (eTICI) score, procedural time, and complications, were analyzed. RESULTS: Thirty-two consecutive patients (12 men (37.5%); mean age: 73 years) were treated with RTRS using a BGC. The median NIHSS score was 19. The median puncture-to-reperfusion time was 46 minutes (range: 22-142 minutes). All patients were successfully revascularized; eTICI 2c or better recanalization was achieved in 30 (93.8%) patients. No procedure-related complications or symptomatic intracranial hemorrhage occurred. Two cases (6.3%) had distal emboli, but none had emboli to the anterior cerebral artery. Fourteen patients (43.8%) achieved a good outcome with an mRS score of 0-2 at 90 days, and 8 patients (25.0%) died. CONCLUSIONS: In patients with intracranial ICA occlusion, RTRS with proximal flow arrest by BGC is effective and safe, achieving good clinical and angiographic outcomes. This method may reduce the incidence of distal emboli in thrombectomy with stent retrievers.

Kymolyzer, a Semi-Autonomous Kymography Tool to Analyze Intracellular Motility.

The movement of intracellular cargo, such as transcripts, proteins, and organelles, is fundamental to cellular function. Neurons, due to their long axons and dendrites, are particularly dependent on proper intracellular trafficking and vulnerable to defects in the movement of intracellular cargo that are noted in neurodegenerative and neurodevelopmental disorders. Accurate quantification of intracellular transport is therefore needed for studying the mechanisms of cargo trafficking, the influence of mutations, and the effects of potentially therapeutic pharmaceuticals. In this article, we introduce an algorithm called "Kymolyzer." The algorithm can quantify intracellular trafficking along a defined path, such as that formed by the aligned microtubules of axons and dendrites. Kymolyzer works as a semi-autonomous kymography software application. It constructs and analyzes kymographs to measure the movement and distribution of fluorescently tagged objects along a user-defined path. The algorithm can be used under a wide variety of experimental conditions and can extract a diverse array of motility parameters describing intracellular movement, including time spent in motion, percentage of objects in motion, percentage of objects that are stationary, and velocities of motile objects. This article serves as a user manual describing the design of Kymolyzer, providing a stepwise protocol for its use and illustrating its functions with common examples. © 2020 Wiley Periodicals LLC Basic Protocol: Kymolyzer, a semi-autonomous kymography tool to analyze intracellular motility.

Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis.

BACKGROUND: Genetic mutations in α-actinin-4 (ACTN4)-an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes-have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known. METHODS: Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo. We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF-ß levels increase phosphorylation of ACTN4. RESULTS: Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro. Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF-ß stimulates phosphorylation of ACTN4 at S159 in podocytes. CONCLUSIONS: These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation.