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Background This study aimed to investigate the predictive value of long non‐coding RNA intersectin 1‐2 (lnc‐ITSN1‐2) for severe acute pancreatitis (SAP) risk, and its correlation with disease severity and in‐hospital mortality in SAP patients. Methods Plasma samples from 60 SAP, 60 moderate‐severe acute pancreatitis (MSAP) and 60 mild acute pancreatitis (MAP) patients were collected within 24 hours, and plasma samples from 60 age and gender‐matched healthy controls (HCs) were collected when enrollment. Lnc‐ITSN1‐2 was detected by reverse transcription‐quantitative polymerase chain reaction. In AP patients, disease severity was evaluated and in‐hospital deaths were recorded. Results Lnc‐ITSN1‐2 was increased in SAP patients compared with MSAP, MAP patients, and HCs, and it is well‐discriminated SAP patients from MSAP patients (area under curve (AUC): 0.699, 95% confidence interval (CI): 0.605‐0.792), MAP patients (AUC: 0.862, 95% CI: 0.798‐0.926), and HCs (AUC: 0.958, 95% CI: 0.925‐0.990). For disease severity, lnc‐ITSN1‐2 was positively correlated with Ranson's score, acute pathologic and chronic health evaluation (APACHE) II score, sequential organ failure assessment (SOFA) score, and C‐reactive protein (CRP) in SAP patients, MSAP patients, and MAP patients; meanwhile, the correlation coefficients were highest in SAP patients. Furthermore, lnc‐ITSN1‐2 displayed a good predictive value for increased in‐hospital mortality in SAP (AUC: 0.803, 95% CI: 0.673‐0.933) and MSAP (AUC: 0.854, 95% CI: 0.752‐0.956) patients, which was similar with several common prognostic factors (including Ranson's score, APACHE II score, SOFA score, and CRP). Conclusion Lnc‐ITSN1‐2 might be a potential biomarker for discrimination of SAP to improve the prognosis of SAP patients.

Background This study aimed to evaluate the predictive value of long non‐coding RNA intersectin 1‐2 (lnc‐ITSN1‐2) for acute ischemic stroke (AIS) risk, and investigate its correlation with disease severity, inflammation, and recurrence‐free survival (RFS) in AIS patients. Methods Three hundred and twenty AIS patients were recruited, and plasma samples were collected within 24 hours after admission. lnc‐ITSN1‐2 expression form plasma was detected by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). The National Institute of Health Stroke Scale (NIHSS) score was assessed, and RFS was calculated. Meanwhile, 320 controls were enrolled and plasma samples were collected on the enrollment, and lnc‐ITSN1‐2 expression was detected by RT‐qPCR. Results lnc‐ITSN1‐2 expression was increased in AIS patients compared to controls (P < .001), and receiver operating characteristic curve revealed its predictive value for AIS risk (area under the curve: 0.804, 95% confidence interval, 0.763‐0.845). In AIS patients, lnc‐ITSN1‐2 expression was positively correlated with NIHSS score (r = 0.464, P < .001). For inflammation, lnc‐ITSN1‐2 expression was positively correlated with CRP (r = 0.398, P < .001), TNF‐α (r = 0.502, P < .001), IL‐1β (r = 0.313, P < .001), IL‐6 (r = 0.207, P < .001), IL‐8 (r = 0.400, P < .001), IL‐17 (r = 0.272, P < .001), and IL‐22 (r = 0.222, P < .001). In terms of predicted target microRNAs, lnc‐ITSN1‐2 expression was negatively correlated with microRNA (miR)‐107 (r = −0.467, P < .001), miR‐125a (r = −0.494, P < .001), and miR‐146a (r = −0.126, P = .025). For prognosis, high lnc‐ITSN1‐2 expression was correlated with worse RFS in AIS patients. Conclusion lnc‐ITSN1‐2 exerts a good predictive value for AIS risk; meanwhile, its increased expression is correlated with enhanced disease severity, elevated inflammation, and worse RFS in AIS patients.

Dozens of proteins capture, polymerize and reshape the clathrin lattice during clathrin-mediated endocytosis (CME). How or if this ensemble of proteins is organized in relation to the clathrin coat is unknown. Here, we map key molecules involved in CME at the nanoscale using correlative super-resolution light and transmission electron microscopy. We localize 19 different endocytic proteins (amphiphysin1, AP2, β2-arrestin, CALM, clathrin, DAB2, dynamin2, EPS15, epsin1, epsin2, FCHO2, HIP1R, intersectin, NECAP, SNX9, stonin2, syndapin2, transferrin receptor, VAMP2) on thousands of individual clathrin structures, generating a comprehensive molecular architecture of endocytosis with nanoscale precision. We discover that endocytic proteins distribute into distinct spatial zones in relation to the edge of the clathrin lattice. The presence or concentrations of proteins within these zones vary at distinct stages of organelle development. We propose that endocytosis is driven by the recruitment, reorganization and loss of proteins within these partitioned nanoscale zones. Using large-scale, super-resolution microscopy, Sochacki et al. define the spatial organization of 19 proteins within clathrin-coated pits during distinct stages of clathrin-mediated endocytosis.

Alzheimer’s disease (AD) is the most common form of dementia and is characterized by the accumulation of amyloid-beta (Aβ) plaques and neurofibrillary Tau tangles in the brain. We previously identified a set of candidate AD microRNAs (miRNAs) in human cerebrospinal fluid (CSF) and used a target prediction pipeline to identify mRNAs and pathways that could potentially be regulated by the miRNAs. Of these pathways, clathrin mediated endocytosis (CME) was selected for further investigation. CME is altered in multiple brain cell types in AD and is implicated in early cellular phenotypes such as enlarged early endosomes and pathogenic processing of Aβ. However, a comprehensive evaluation of major CME hub proteins in humans with AD across multiple brain regions is lacking. Thus, we used immunoblots to evaluate human post-mortem AD and control (CTL) frontal cortex (FC; AD n  = 22, CTL n  = 23) and hippocampus (HP; AD n  = 34, CTL n  = 22) for changes in Intersectin 1 (ITSN1), Phosphatidylinositol Binding Clathrin Assembly Protein gene (PICALM), Clathrin Light Chain (CLT), FCH and Mu Domain Containing Endocytic Adaptor 1 (FCHO1), Adaptor Related Protein Complex 2 (AP2) Subunit Alpha 1 (AP2A1), and Dynamin 2 (DNM2). Of these, we found that in AD, ITSN1-long (ITSN1-L) was decreased in the FC of males and HP of females, while ITSN1-short was increased in the HP of both males and females. We further evaluated ITSN1-L levels in cortex (CTX) and HP of the 5xFAD mouse model of Aβ pathology at different timepoints during aging and disease progression by immunoblot ( n  = 5–8 per group). At 3 months, female 5xFAD exhibited an increase of ITSN1-L in CTX but a decrease at 6 and 9 months. Additionally, immunofluorescent staining of 5xFAD primary HP neurons showed an increase of ITSN1-L in matured 5xFAD neurons at 21 and 28 days in vitro . Together, our studies show that in AD, isoforms of ITSN1 change in a brain region-and sex-dependent manner. Further, changes in ITSN1-L are transient with levels increasing during early Aβ accumulation and decreasing during later progression. These findings suggest that ITSN1 expression, and consequently CME activity, may change depending on the stage of disease progression.

Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery.

Membrane vesicles are considered virulence cargoes as they carry capsular and melanin components whose secretory transport is critical for the virulence of the human fungal pathogen species. However, other components of the vesicles and their function in the growth and virulence of the fungus remain unclear. We have previously found that the cryptococcal intersectin protein Cin1 governs a unique Cin1-Wsp1-Cdc42 endocytic pathway required for intracellular transport and virulence. Using RNA sequencing, we compared the profiles of extracellular RNA (exRNA), including microRNA (miRNA), small interference RNA (siRNA), long noncoding RNA (lncRNA), and messenger RNA (mRNA) between the wild-type (WT), and derived Δ mutant strains of . Seven hundred twelve miRNAs and 88 siRNAs were identified from WT, whereas 799 miRNAs and 66 siRNAs were found in Δ . Also, 572 lncRNAs and 7,721 mRNAs were identified from WT and 584 lncRNAs and 7,703 mRNAs from Δ . Differential expression analysis revealed that the disruption of results in many important cellular changes, including those in exRNA expression, transport, and function. First, for miRNA target genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that cellular processes, components, and macromolecular functions are the most affected pathways. A higher number of genes were involved in the intracellular transport of endocytosis. Second, the results of GO term and KEGG analysis of differentially expressed lncRNA target genes and mRNA genes were consistent with those of miRNA targets. In particular, protein export is the topmost affected pathway among lncRNA target genes and one of the affected pathways among mRNA genes. The result of quantitative real-time reverse transcription PCR (qRT-PCR) from 12 mRNAs tested is largely agreeable with that of RNA-Seq. Taken together, our studies provide a comprehensive reference that secretes abundant RNAs and that Cin1 plays a critical role in regulating their secretion. Given the growing clinical importance of exRNAs, our studies illuminate the significance of exploring this cutting-edge technology in studies of cryptococcal pathogenesis for the discovery of novel therapeutic strategies.

Endophilins-A are conserved endocytic adaptors with membrane curvature-sensing and -inducing properties. We show here that, independently of their role in endocytosis, endophilin-A1 and endophilin-A2 regulate exocytosis of neurosecretory vesicles. The number and distribution of neurosecretory vesicles were not changed in chromaffin cells lacking endophilin-A, yet fast capacitance and amperometry measurements revealed reduced exocytosis, smaller vesicle pools and altered fusion kinetics. The levels and distributions of the main exocytic and endocytic factors were unchanged, and slow compensatory endocytosis was not robustly affected. Endophilin-A’s role in exocytosis is mediated through its SH3-domain, specifically via a direct interaction with intersectin-1, a coordinator of exocytic and endocytic traffic. Endophilin-A not able to bind intersectin-1, and intersectin-1 not able to bind endophilin-A, resulted in similar exocytic defects in chromaffin cells. Altogether, we report that two endocytic proteins, endophilin-A and intersectin-1, are enriched on neurosecretory vesicles and regulate exocytosis by coordinating neurosecretory vesicle priming and fusion. Endophilins-A are conserved membrane-associated proteins required for endocytosis. Here, the authors report that endophilins-A also promote exocytosis of neurosecretory vesicles by coordinating priming and fusion through intersectin-1, independently of their roles in different types of endocytosis.

Prenatal valproic acid (VPA) exposure increases the risk of neurodevelopmental disorders, though its synaptic mechanisms remain unclear. Using multi-omics analyses, we identified Dlgap2 as a consistently dysregulated protein in VPA models. Mice with Dlgap2 knockdown exhibited synaptic deficits and autism-like behaviors, including social and cognitive impairments. Proteomics of postsynaptic density following Dlgap2 knockdown revealed disruption of synaptic organization and a specific reduction in Intersectin-1 (Itsn1), which interacts with Dlgap2 and undergoes ubiquitin-mediated degradation upon Dlgap2 deficiency. Our study defines a Dlgap2-Itsn1 regulatory axis that underlies VPA-induced synaptic dysfunction.

The development and maintenance of drug addiction triggered by psychoactive substances, such as methamphetamine (Meth), is strongly influenced by synaptic and morphological adaptations in the brain. The hippocampus has a key role in the formation of maladaptive drug-context associations and relapse. However, the mechanisms regulating this complex process are not completely understood. Recent studies highlight that the actin cytoskeleton and its regulatory proteins play a fundamental role in morphological and behavioral plasticity associated with drug use. Here, we show that a binge pattern of Meth administration is sufficient to increase hippocampal neurite outgrowth and dendritic spine density leading to augmented basal synaptic transmission and impaired long-term potentiation (LTP) response. To study the molecular pathways affected by Meth, we used embryonic primary neuron cultures from the hippocampus and performed RNA Sequencing from isolated soma or neurites compartments, FRET microscopy, biochemical analyses, and morphometric measurements of dendrites to demonstrate that intersectin (Itsn)1/cdc42 signaling is an important mediator of Meth-induced morphological adaptations, specifically in the synaptic compartment. Furthermore, AAV-mediated depletion of neuronal cdc42 in the hippocampus prevented Meth-induced changes in cytoarchitecture and their consequent impact on neuronal functionality. Our results highlight relevant compartment-associated changes in the activity of Itsn1/cdc42 pathway that critically regulate Meth-induced neuronal remodeling and plasticity.

Environmental enrichment (EE) through combination of social and non-biological stimuli enhances activity-dependent synaptic plasticity and improves behavioural performance. Our earlier studies have suggested that EE resilience the stress induced depression/ anxiety-like behaviour in Indian field mice Mus booduga . This study was designed to test whether EE reverses the social isolation (SI) induced effect and improve memory. Field-caught mice M . booduga were subjected to behaviour test (Direct wild, DW), remaining animals were housed under SI for ten days and then housed for short-term at standard condition (STSC)/ long-term at standard condition (LTSC) or as group in EE cage. Subsequently, we have examined reference, working memory and expression of genes associated with synaptic plasticity. Our analysis have shown that EE reversed SI induced impairment in reference, working memory and other accompanied changes i.e. increased level of Intersectin 1 (ITSN1), Huntingtin (Htt), Synaptotagmin -IV (SYT4), variants of brain-derived neurotrophic factor (Bdnf - III), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (GluR1) expression, and decreased variants of Bdnf (IV), BDNF, Reelin, Apolipoprotein E receptor 2 (ApoER2), very low-density lipoprotein receptor (VLDLR), Src family tyrosine kinase (SFKs), Disabled protein (Dab)-1, Protein kinase B (PKB/Akt), GluR2, Mitogen-activated protein kinase (MAPK) and Extracellular signal-regulated kinase (ERK1/2) expression. In addition, SI induced reduction in BDNF expressing neurons in dentate gyrus of hippocampus reversed by EE. Further, we found that SI decreases small neuro-active molecules such as Benzenedicarboxylic acid, and increases 2-Pregnene in the hippocampus and feces reversed by EE. Overall, this study demonstrated that EE is effectively reversed the SI induced memory impairment by potentially regulating the molecules associated with the ITSN1-Reelin–AMPA receptor pathway to increase synaptic plasticity.