Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM).

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins. In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart. To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired

Functional characterization of the schizophrenia associated gene AS3MT identifies a role in neuronal development

This is the final version. Available from Wiley via the DOI in this record. DATA AVAILABILITY STATEMENT: All raw RNAseq files are available at GSE182157 and generated cell lines available on request.Genome-wide association studies (GWAS) have identified multiple genomic regions associated with schizophrenia, although many variants reside in noncoding regions characterized by high linkage disequilibrium (LD) making the elucidation of molecular mechanisms challenging. A genomic region on chromosome 10q24 has been consistently associated with schizophrenia with risk attributed to the AS3MT gene. Although AS3MT is hypothesized to play a role in neuronal development and differentiation, work to fully understand the function of this gene has been limited. In this study we explored the function of AS3MT using a neuronal cell line (SH-SY5Y). We confirm previous findings of isoform specific expression of AS3MT during SH-SY5Y differentiation toward neuronal fates. Using CRISPR-Cas9 gene editing we generated AS3MT knockout SH-SY5Y cell lines and used RNA-seq to identify significant changes in gene expression in pathways associated with neuronal development, inflammation, extracellular matrix formation, and RNA processing, including dysregulation of other genes strongly implicated in schizophrenia. We did not observe any morphological changes in cell size and neurite length following neuronal differentiation and MAP2 immunocytochemistry. These results provide novel insights into the potential role of AS3MT in brain development and identify pathways through which genetic variation in this region may confer risk for schizophrenia.Academy of Medical SciencesBiotechnology and Biological Sciences Research CouncilMedical Research CouncilMedical Research CouncilWellcome TrustWellcome TrustUniversity of Exete

Regulation of astrocyte metabolism by mitochondrial translocator protein 18 kDa

This is the final version. Available on open access from Wiley via the DOI in this recordData availability statement: The datasets used and analyzed during this study are available from the corresponding author upon receipt of reasonable request.The mitochondrial translocator protein 18 kDa (TSPO) has been linked to functions from steroidogenesis to regulation of cellular metabolism and is an attractive therapeutic target for chronic CNS inflammation. Studies in Leydig cells and microglia indicate that TSPO function may vary between cells depending on their specialized roles. Astrocytes are critical for providing trophic and metabolic support in the brain. Recent work has highlighted that TSPO expression increases in astrocytes under inflamed conditions and may drive astrocyte reactivity. Relatively little is known about the role TSPO plays in regulating astrocyte metabolism and whether this protein is involved in immunometabolic processes in these cells. Using TSPO-deficient (TSPO-/- ) mouse primary astrocytes in vitro (MPAs) and a human astrocytoma cell line (U373 cells), we performed extracellular metabolic flux analyses. We found that TSPO deficiency reduced basal cellular respiration and attenuated the bioenergetic response to glucopenia. Fatty acid oxidation was increased, and lactate production was reduced in TSPO-/- MPAs and U373 cells. Co-immunoprecipitation studies revealed that TSPO forms a complex with carnitine palmitoyltransferase 1a in U373 and MPAs, presenting a mechanism wherein TSPO may regulate FAO in these cells. Compared to TSPO+/+ cells, in TSPO-/- MPAs we observed attenuated tumor necrosis factor release following 3 h lipopolysaccharide (LPS) stimulation, which was enhanced at 24 h post-LPS stimulation. Together these data suggest that while TSPO acts as a regulator of metabolic flexibility, TSPO deficiency does not appear to modulate the metabolic response of MPAs to inflammation, at least in response to the model used in this study.British Society for NeuroendocrinologyResearch EnglandUniversity of Exete

<i>Cntn4</i>, a risk gene for neuropsychiatric disorders, modulates hippocampal synaptic plasticity and behavior

Neurodevelopmental and neuropsychiatric disorders, such as autism spectrum disorders (ASD), anorexia nervosa (AN), Alzheimer’s disease (AD), and schizophrenia (SZ), are heterogeneous brain disorders with unknown etiology. Genome wide studies have revealed a wide variety of risk genes for these disorders, indicating a biological link between genetic signaling pathways and brain pathology. A unique risk gene is Contactin 4 (Cntn4), an Ig cell adhesion molecule (IgCAM) gene, which has been associated with several neuropsychiatric disorders including ASD, AN, AD, and SZ. Here, we investigated the Cntn4 gene knockout (KO) mouse model to determine whether memory dysfunction and altered brain plasticity, common neuropsychiatric symptoms, are affected by Cntn4 genetic disruption. For that purpose, we tested if Cntn4 genetic disruption affects CA1 synaptic transmission and the ability to induce LTP in hippocampal slices. Stimulation in CA1 striatum radiatum significantly decreased synaptic potentiation in slices of Cntn4 KO mice. Neuroanatomical analyses showed abnormal dendritic arborization and spines of hippocampal CA1 neurons. Short- and long-term recognition memory, spatial memory, and fear conditioning responses were also assessed. These behavioral studies showed increased contextual fear conditioning in heterozygous and homozygous KO mice, quantified by a gene-dose dependent increase in freezing response. In comparison to wild-type mice, Cntn4-deficient animals froze significantly longer and groomed more, indicative of increased stress responsiveness under these test conditions. Our electrophysiological, neuro-anatomical, and behavioral results in Cntn4 KO mice suggest that Cntn4 has important functions related to fear memory possibly in association with the neuronal morphological and synaptic plasticity changes in hippocampus CA1 neurons

Identification and single-base gene-editing functional validation of a cis-EPO variant as a genetic predictor for EPO-increasing therapies

Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are currently under clinical development for treating anemia in chronic kidney disease (CKD), but it is important to monitor their cardiovascular safety. Genetic variants can be used as predictors to help inform the potential risk of adverse effects associated with drug treatments. We therefore aimed to use human genetics to help assess the risk of adverse cardiovascular events associated with therapeutically altered EPO levels to help inform clinical trials studying the safety of HIF-PHIs. By performing a genome-wide association meta-analysis of EPO (n = 6,127), we identified a cis-EPO variant (rs1617640) lying in the EPO promoter region. We validated this variant as most likely causal in controlling EPO levels by using genetic and functional approaches, including single-base gene editing. Using this variant as a partial predictor for therapeutic modulation of EPO and large genome-wide association data in Mendelian randomization tests, we found no evidence (at p < 0.05) that genetically predicted long-term rises in endogenous EPO, equivalent to a 2.2-unit increase, increased risk of coronary artery disease (CAD, OR [95% CI] = 1.01 [0.93, 1.07]), myocardial infarction (MI, OR [95% CI] = 0.99 [0.87, 1.15]), or stroke (OR [95% CI] = 0.97 [0.87, 1.07]). We could exclude increased odds of 1.15 for cardiovascular disease for a 2.2-unit EPO increase. A combination of genetic and functional studies provides a powerful approach to investigate the potential therapeutic profile of EPO-increasing therapies for treating anemia in CKD

Genomics in neurodevelopmental disorders: an avenue to personalized medicine

Despite the remarkable number of scientific breakthroughs of the last 100 years, the treatment of neurodevelopmental disorders (e.g., autism spectrum disorder, intellectual disability) remains a great challenge. Recent advancements in genomics, such as whole-exome or whole-genome sequencing, have enabled scientists to identify numerous mutations underlying neurodevelopmental disorders. Given the few hundred risk genes that have been discovered, the etiological variability and the heterogeneous clinical presentation, the need for genotype — along with phenotype- based diagnosis of individual patients has become a requisite. In this review we look at recent advancements in genomic analysis and their translation into clinical practice

A current view on contactin-4, -5, and -6: Implications in neurodevelopmental disorders.

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.Contactins (Cntns) are a six-member subgroup of the immunoglobulin cell adhesion molecule superfamily (IgCAMs) with pronounced brain expression and function. Recent genetic studies of neuropsychiatric disorders have pinpointed contactin-4 (CNTN4), contactin-5 (CNTN5) and contactin-6 (CNTN6) as candidate genes in neurodevelopmental disorders, particularly in autism spectrum disorders (ASDs), but also in intellectual disability, schizophrenia (SCZ), attention-deficit hyperactivity disorder (ADHD), bipolar disorder (BD), alcohol use disorder (AUD) and anorexia nervosa (AN). This suggests that they have important functions during neurodevelopment. This suggestion is supported by data showing that neurite outgrowth, cell survival and neural circuit formation can be affected by disruption of these genes. Here, we review the current genetic data about their involvement in neuropsychiatric disorders and explore studies on how null mutations affect mouse behavior. Finally, we highlight to role of protein-protein interactions in the potential mechanism of action of Cntn4, -5 and -6 and emphasize that complexes with other membrane proteins may play a role in neuronal developmental functions.Authors of this review were supported by EU-AIMS (European Autism Interventions), which receives support from the Innovative Medicines Initiative Joint Undertaking under Grant agreement no.115300, there sources of which are composed of financial contributions from the European Union's Seventh Framework Programed Grant (P7/2007–2013), from the European Federation of Pharmaceutical Industries and Associations Companies' in-kind contributions, and from Autism Speaks. (K.T.E.K. and J.P.H.B.), by a Fellowship from JSPS (A.O.A. and A.Z)

Study of the functions and activities of neuronal K-Cl co-transporter KCC2 using western blotting

This is the final version. Available from MyJove Corporation via the DOI in this recordPotassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.Royal Society (Charity)National Institutes of Healt