PIP2 Is An Electrostatic Catalyst for Vesicle Fusion by Lowering the Hydration Energy: Arresting Vesicle Fusion by Masking PIP2.

Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.

Deletion of TRPC6, an Autism Risk Gene, Induces Hyperexcitability in Cortical Neurons Derived from Human Pluripotent Stem Cells.

Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder linked to numerous rare, inherited, and arising de novo genetic variants. ASD often co-occurs with attention-deficit hyperactivity disorder and epilepsy, which are associated with hyperexcitability of neurons. However, the physiological and molecular mechanisms underlying hyperexcitability in ASD remain poorly understood. Transient receptor potential canonical-6 (TRPC6) is a Ca2+-permeable cation channel that regulates store-operated calcium entry (SOCE) and is a candidate risk gene for ASD. Using human pluripotent stem cell (hPSC)-derived cortical neurons, single-cell calcium imaging, and electrophysiological recording, we show that TRPC6 knockout (KO) reduces SOCE signaling and leads to hyperexcitability of neurons by increasing action potential frequency and network burst frequency. Our data provide evidence that reduction of SOCE by TRPC6 KO results in neuronal hyperexcitability, which we hypothesize is an important contributor to the cellular pathophysiology underlying hyperactivity in some ASD.

Requirement of Cholesterol for Calcium-Dependent Vesicle Fusion by Strengthening Synaptotagmin-1-Induced Membrane Bending.

Cholesterol is essential for neuronal activity and function. Cholesterol depletion in the plasma membrane impairs synaptic transmission. However, the molecular mechanisms by which cholesterol deficiency leads to defects in vesicle fusion remain poorly understood. Here, it is shown that cholesterol is required for Ca2+ -dependent native vesicle fusion using the in vitro reconstitution of fusion and amperometry to monitor exocytosis in chromaffin cells. Purified native vesicles are crucial for the reconstitution of physiological Ca2+ -dependent fusion, because vesicle-mimicking liposomes fail to reproduce the cholesterol effect. Intriguingly, cholesterol has no effect on the membrane binding of synaptotagmin-1, a Ca2+ sensor for ultrafast fusion. Cholesterol strengthens local membrane deformation and bending induced by synaptotagmin-1, thereby lowering the energy barrier for Ca2+ -dependent fusion to occur. The data provide evidence that cholesterol depletion abolishes Ca2+ -dependent vesicle fusion by disrupting synaptotagmin-1-induced membrane bending, and suggests that cholesterol is an essential lipid regulator for Ca2+ -dependent fusion.

Electrostatic regulation of the cis- and trans-membrane interactions of synaptotagmin-1.

Synaptotagmin-1 is a vesicular protein and Ca2+ sensor for Ca2+-dependent exocytosis. Ca2+ induces synaptotagmin-1 binding to its own vesicle membrane, called the cis-interaction, thus preventing the trans-interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of the cis- and trans-membrane interaction of synaptotagmin-1 was poorly understood in different Ca2+-buffering conditions. Here we provide an assay to monitor the cis- and trans-membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse the cis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10-100 µM. High PIP2 concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP2 concentrations control the Ca2+-cooperativity of synaptotagmin-1. Our data provide evidence that Ca2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1.

Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers.

Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility. In this study, using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. As a neuronal EV marker, neural cell adhesion molecule (NCAM) is highly enriched in EVs released from induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis. Our results suggest that plasma NCAM-positive neuronal EVs can be used to discover biomarkers for neurological disorders.

Cerebrospinal α-Synuclein Oligomers Reflect Disease Motor Severity in DeNoPa Longitudinal Cohort.

BACKGROUND: Tangible efforts have been made to identify biomarkers for Parkinson's disease (PD) diagnosis and progression, with α-synuclein (α-syn) related biomarkers being at the forefront. OBJECTIVES: The objectives of this study were to explore whether cerebrospinal fluid (CSF) levels of total, oligomeric, phosphorylated Ser 129 α-synuclein, along with total tau, phosphorylated tau 181, and ß-amyloid 1-42 are (1) informative as diagnostic markers for PD, (2) changed over disease progression, and/or (3) correlated with motor and cognitive indices of disease progression in the longitudinal De Novo Parkinson cohort. METHODS: A total of 94 de novo PD patients and 52 controls at baseline and 24- and 48-month follow-up were included, all of whom had longitudinal lumbar punctures and clinical assessments for both cognitive and motor functions. Using our in-house enzymelinked immunosorbent assays and commercially available assays, different forms of α-synuclein, tau, and ß-amyloid 1-42 were quantified in CSF samples from the De Novo Parkinson cohort. RESULTS: Baseline CSF total α-synuclein was significantly lower in early de novo PD compared with healthy controls, whereas the ratio of oligomeric/total and phosphorylated/total were significantly higher in the PD group. CSF oligomeric-α-synuclein longitudinally increased over the 4-year follow-up in the PD group and correlated with PD motor progression. Patients at advanced stages of PD presented with elevated CSF oligomeric-α-synuclein levels compared with healthy controls. CONCLUSIONS: Longitudinal transitions of CSF biomarkers over disease progression might not occur linearly and are susceptible to disease state. CSF oligomeric-α-synuclein levels appear to increase with diseases severity and reflect PD motor rather than cognitive trajectories. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

First study in Qatar to reveal high Legionella counts in cooling towers.

Legionella spp. is transmitted from water to humans by aerosol-generating devices, including cooling towers (CTs). There have not been published reports about Legionella in these systems in Qatar. Ten CTs in Qatar University were sampled on a monthly basis. Bacteria were recovered from 90 water samples by filtration and concentration. Legionella DNA copy number (CN) was assessed by quantitative RT-PCR. Legionella DNA was detected in 100% of the samples. The bacterial counts ranged from 0.006 to 199.56 CFU/mL, and critical counts were found in 51 (56.7 %) samples. Moreover, 7 (7.8%) samples showed a count of more than 100 CFU/mL. The highest counts were found in the months of May and June. These results suggest that this organism is found in high number in tested CTs, presenting a potential health risk to the local population.