Brain-specific Isoform Research Articles

ZMYND11 Functions in Bimodal Regulation of Latent Genes and Brain-like Splicing to Safeguard Corticogenesis.

Despite the litany of pathogenic variants linked to neurodevelopmental disorders (NDD) including autism (ASD) and intellectual disability 1,2 , our understanding of the underlying mechanisms caused by risk genes remain unclear. Here, we leveraged a human pluripotent stem cell model to uncover the neurodevelopmental consequences of mutations in ZMYND11 , a newly implicated risk gene 3,4 . ZMYND11, known for its tumor suppressor function, encodes a histone-reader that recognizes sites of transcriptional elongation and acts as a co-repressor 5,6 . Our findings reveal that ZMYND11-deficient cortical neural stem cells showed upregulation of latent developmental pathways, impairing progenitor and neuron production. In addition to its role on histones, ZMYND11 controls a brain-specific isoform switch involving the splicing regulator RBFOX2. Extending our findings to other chromatin-related ASD risk factors revealed similar developmental pathway activation and splicing dysregulation, partially rescuable through ZMYND11's regulatory functions.

Effects of Tropomodulin 2 on Dendritic Spine Reorganization and Dynamics.

Dendritic spines are actin-rich protrusions that receive a signal from the axon at the synapse. Remodeling of cytoskeletal actin is tightly connected to dendritic spine morphology-mediated synaptic plasticity of the neuron. Remodeling of cytoskeletal actin is required for the formation, development, maturation, and reorganization of dendritic spines. Actin filaments are highly dynamic structures with slow-growing/pointed and fast-growing/barbed ends. Very few studies have been conducted on the role of pointed-end binding proteins in the regulation of dendritic spine morphology. In this study, we evaluated the role played by tropomodulin 2 (Tmod2)-a brain-specific isoform, on the dendritic spine re-organization. Tmod2 regulates actin nucleation and polymerization by binding to the pointed end via actin and tropomyosin (Tpm) binding sites. We studied the effects of Tmod2 overexpression in primary hippocampal neurons on spine morphology using confocal microscopy and image analysis. Tmod2 overexpression decreased the spine number and increased spine length. Destroying Tpm-binding ability increased the number of shaft synapses and thin spine motility. Eliminating the actin-binding abilities of Tmod2 increased the number of mushroom spines. Tpm-mediated pointed-end binding decreased F-actin depolymerization, which may positively affect spine stabilization; the nucleation ability of Tmod2 appeared to increase shaft synapses.

TPH2-703 G/T polymorphism is associated with stress, depression, and psychosocial symptoms in mentally healthy Mexicans

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step of serotonin synthesis. TPH2 is the brain-specific isoform of this enzyme, and genetic variations in the TPH2 gene have been shown to impact its transcription and enzymatic activity and are associated with mood disorders. In this study we focused on the rs4570625 (-703G/T) single nucleotide polymorphism of TPH2 gene. By using conventional polymerase chain reaction (PCR), we examined the effect of this polymorphism on stress, anxiety, and depression symptoms as well as quality of life, evaluated based on the Holmes–Rahe Inventory, the Beck Anxiety Inventory, the Beck Depression Inventory, and the World Health Organization Quality of Life – Short Version, respectively. We found that individuals with the homozygous recessive T/T genotype had lower stress and depression scores. In addition, the quality of life in the psychological health domain was better in males with the T/T genotype. These results suggest that T/T genotype could decrease the susceptibility to developing stress and depression in the Mexican population without a diagnosis for an emotional disorder.

Protein interaction studies in human induced neurons indicate convergent biology underlying autism spectrum disorders.

Autism spectrum disorders (ASDs) have been linked to genes with enriched expression in the brain, but it is unclear how these genes converge into cell-type-specific networks. We built a protein-protein interaction network for 13 ASD-associated genes in human excitatory neurons derived from induced pluripotent stem cells (iPSCs). The network contains newly reported interactions and is enriched for genetic and transcriptional perturbations observed in individuals with ASDs. We leveraged the network data to show that the ASD-linked brain-specific isoform of ANK2 is important for its interactions with synaptic proteins and to characterize a PTEN-AKAP8L interaction that influences neuronal growth. The IGF2BP1-3 complex emerged as a convergent point in the network that may regulate a transcriptional circuit of ASD-associated genes. Our findings showcase cell-type-specific interactomes as a framework to complement genetic and transcriptomic data and illustrate how both individual and convergent interactions can lead to biological insights into ASDs.

Dendritic spine formation and synapse maturation in transcription factor-induced human iPSC-derived neurons

Synaptic maturation is reportedly limited in human induced pluripotent stem cell (iPSC)-derived neurons. Notably, their ability to reach postnatal-like stages and form dendritic spines has been difficult to demonstrate unless using long-term cultured organoids. Recent transcription factor (TF)-based induction methods allow the accelerated generation of differentiated neurons, which offers an unprecedented opportunity to address further progression into late developmental stages. Herein, we report on a comprehensive time-course study of TF-induced iPSC neurons cultured invitro through an intrinsic maturation program following neurogenesis. Moreover, we determined the transcriptional and morphological sequences of key developmental events associated with spinogenesis, including the conversion of drebrin to its brain-specific isoform A and the N-methyl-D-aspartate (NMDA) receptor subunit switch. TF-induced iPSC neurons successfully acquired structural and functional synaptic maturity, which will critically expand their utility in modeling higher brain functions and disorders.

BIN1 K358R variant in 5xFAD mice ameliorates AB pathology and microgliosis

AbstractBackgroundGenome‐Wide Association Studies (GWAS) identified bridging integrator 1 (BIN1) as the most important susceptibility loci for late‐onset Alzheimer's disease (LOAD), second to apolipoprotein E (APOE). BIN1 is an important regulator of endocytosis and membrane dynamics. Neuronal BIN1 localizes to pre‐synaptic termini where it functions in excitatory synaptic transmission via regulation of neurotransmitter vesicle dynamics. In humans, the gene produces multiple isoforms with 7 brain specific isoforms. A missense variant in Bin1 (K358R) has been linked to increased risk of development of LOAD. Here, we introduced this variant in mice and evaluated its effects on plaque genesis and subsequent damage.MethodTo create the Bin1K358R allele, CRISPR/Cas9 endonuclease‐mediated genome editing of Bin1 was used to introduce a K426R mutation. This mutant allele models a SNP (rs138047593) found in human BIN1 that encodes a missense mutation associated with increased risk of sporadic Alzheimer's disease. The K358R variant in human BIN1 (K542R in the BIN1‐202 isoform) is located within the C‐terminal SH3 domain. Generated BIN1 K358R mice were crossed with 5xFAD mouse models, creating four groups (wild type (WT), BIN1K358R homozygous (HO), 5xFAD, and 5xFAD/BIN1 K358R HO), then aged to 4 months for analysis of AD‐related pathology. Coronal brain sections were immunolabeled to visualize dense‐core plaques (Thioflavin‐S), microglia (IBA1), reactive astrocytes (GFAP), axonal and neuritic damage (neurofilament light chain (NfL) and LAMP1, respectively). Confocal images of hippocampal and cortical regions were analyzed using Imaris software. In addition, Aβ and NfL levels were quantified in the brain tissue and plasma NfL measured using Meso Scale Discovery technology.ResultBIN1 K358R introduced in the 5xFAD background significantly reduces plaque densities and associated microgliosis in the hippocampal region, which matches insoluble Aβ42 levels. In contrast, GFAP levels are significantly increased in 5xFAD/BIN1 K358R mice compared to 5xFAD mice. Despite reduced plaque densities, dystrophic neurite area as measured by LAMP1 staining, as well as plasma NfL, are equivalent between 5xFAD/BIN1 K358R and 5xFAD mice, suggesting increased damage per plaque.ConclusionBIN1 K358R reduces Aβ plaque deposition, but exacerbates the damage caused by those plaques, as determined by the surrounding halo of dystrophic neurites, and plasma NfL.

Palmitoylation of the small GTPase Cdc42 by DHHC5 modulates spine formation and gene transcription

The small GTPase Cdc42 exists in the form of two alternatively spliced variants that are modified by hydrophobic chains: the ubiquitously expressed Cdc42-prenyl and a brain-specific isoform that can be palmitoylated, Cdc42-palm. Our previous work demonstrated that Cdc42-palm can be palmitoylated at two cysteine residues, Cys188 and Cys189, while Cys188 can also be prenylated. We showed that palmitoylation of Cys188 is essential for the plasma membrane localization of Cdc42-palm and is critically involved in Cdc42-mediated regulation of gene transcription and neuronal morphology. However, the abundance and regulation of this modification was not investigated. In the present study, we found that only a minor fraction of Cdc42 undergoes monopalmitoylation in neuroblastoma cells and in hippocampal neurons. In addition, we identified DHHC5 as one of the major palmitoyl acyltransferases that could physically interact with Cdc42-palm. We demonstrate that overexpression of dominant negative DHHC5 mutant decreased palmitoylation and plasma membrane localization of Cdc42-palm. In addition, knockdown of DHHC5 significantly reduced Cdc42-palm palmitoylation, leading to a decrease of Cdc42-mediated gene transcription and spine formation in hippocampal neurons. We also found that the expression of DHHC5 in the brain is developmentally regulated. Taken together, these findings suggest that DHHC5-mediated palmitoylation of Cdc42 represents an important mechanism for the regulation of Cdc42 functions in hippocampus.

P508. Assessing Isoform Diversity in the Context of Psychiatric Disorders

The genetic architecture of schizophrenia is dominated by common variants of small effects, similar to most complex human traits. The majority of the schizophrenia GWAS SNPs reside in non-coding regions where they could act through a variety of regulatory mechanisms, such as alternative splicing to generate schizophrenia-associated isoforms. The brain expresses the most diverse repertoire of isoforms, yet our understanding of brain-specific isoform expression and the mechanisms of their regulation by RNA binding proteins (RBPs) are incomplete.

Protein Profiling of RGS6, a Pleiotropic Gene Implicated in Numerous Neuropsychiatric Disorders, Reveals Multi-Isoformic Expression and a Novel Brain-Specific Isoform.

A metanalysis identified regulator of G-protein signaling 6 (RGS6) as one of 23 loci with pleiotropic effects on four or more human psychiatric disorders. This finding is significant as it confirms/extends the findings of numerous other studies implicating RGS6 in CNS function and pathology. RGS6 is a highly conserved member of the RGS protein family whose cellular roles are likely affected by mRNA splicing and alternative domain inclusion/exclusion. Indeed, we previously identified multiple RGS6 splice variants predicted to produce 36 distinct protein isoforms containing either long (RGS6L) or short (RGS6S) N-terminal domains, an incomplete or intact GGL domain, and nine alternative C termini. Unfortunately, sequence similarities between the isoforms have made it difficult to confirm their individual existence and/or to determine their unique functions. Here, we developed three RGS6-specific antibodies that recognize all RGS6 protein isoforms (RGS6-fl), the N-terminus of RGS6L isoforms (RGS6-L), and an 18-amino acid alternate C-terminal sequence (RGS6-18). Using these antibodies, we demonstrate that RGS6L(+GGL) isoforms, predominating in both mouse (both sexes) CNS and peripheral tissues, are most highly expressed in the CNS. We further identify three novel RGS6 protein bands that are larger (61, 65, and 69-kDa) than the ubiquitously expressed 53- to 57-kDa RGS6L(+GGL) proteins. Importantly, we show that the 69-kDa protein is a brain-specific dephospho form of the 65-kDa band, the first identified phosphorylated RGS6 isoform. Together, these data begin to define the functional significance behind the complexity of RGS6 gene processing and further clarifies RGS6’s physiological roles by resolving tissue-specific RGS6 protein expression.

ε-Sarcoglycan: Unraveling the Myoclonus-Dystonia Gene

Myoclonus-dystonia (MD) is a rare childhood-onset movement disorder, with an estimated prevalence of about 2 per 1,000,.000 in Europe, characterized by myoclonic jerks in combination with focal or segmental dystonia. Pathogenic variants in the gene encoding ε-sarcoglycan (SGCE), a maternally imprinted gene, are the most frequent genetic cause of MD. To date, the exact role of ε-sarcoglycan and the pathogenic mechanisms that lead to MD are still unknown. However, there are more than 40 reported isoforms of human ε-sarcoglycan, pointing to a complex biology of this protein. Additionally, some of these are brain-specific isoforms, which may suggest an important role within the central nervous system. In the present review, we aim to provide an overview of the current state of knowledge of ε-sarcoglycan. We will focus on the genetic landscape of SGCE and the presence and plausible role of ε-sarcoglycan in the brain. Finally, we discuss the importance of the brain-specific isoforms and hypothesize that SGCE may play essential roles in normal synaptic functioning and their alteration will be strongly related to MD.

Targeting ROCK2 isoform with its widely used inhibitors for faster post-stroke recovery

Recovery after ischemic stroke is slow and highly variable. Activated ROCK (Rho-associated coiled-coil kinase) pathway hampers recovery of impaired neurons. Though inhibiting ROCK pathway has shown therapeutic effects in vitro, the selectivity of most of the ROCK inhibitors is still not investigated. Present study aims to investigate the binding affinity in silico of nine widely used ROCK inhibitors with brainspecific ROCK2 isoform. Three-dimensional structures of ROCK2 and eight drugs were taken from Protein Data Bank and PubChem Chemical Compound Database, respectively, whereas, FSD-C10 structure was generated based on Xin et al., 2015. In docking, ROCK2 was set to be rigid and drugs were free to rotate. All simulations were carried out using AutoDock 4.2. This study demonstrated strong complexation between all ligands and ROCK2. All ROCK inhibitors, except FSD-C10, were able to bind to ROCK2 more strongly [Binding constant (Ka) between 2.6 – 36.7 × 105 M−1] than fasudil (Ka = 2.5 × 105 M−1). SLx-2119 (KD-025) had the highest binding constant (Ka = 36.7 × 105 M−1) thus succeeding as a better ROCK2 specific inhibitor. Selectivity of ROCK inhibitors (in silico) towards ROCK2 can be an indicative measure to estimate therapeutic benefits or adverse effects prior to in vitro study.

Deletion of the Brain-Specific α and δ Isoforms of Adapter Protein SH2B1 Protects Mice From Obesity.

Mice lacking SH2B1 and humans with variants of SH2B1 display severe obesity and insulin resistance. SH2B1 is an adapter protein that is recruited to the receptors of multiple hormones and neurotrophic factors. Of the four known alternatively spliced SH2B1 isoforms, SH2B1β and SH2B1γ exhibit ubiquitous expression, whereas SH2B1α and SH2B1δ are essentially restricted to the brain. To understand the roles for SH2B1α and SH2B1δ in energy balance and glucose metabolism, we generated mice lacking these brain-specific isoforms (αδ knockout [αδKO] mice). αδKO mice exhibit decreased food intake, protection from weight gain on standard and high-fat diets, and an adiposity-dependent improvement in glucose homeostasis. SH2B1 has been suggested to impact energy balance via the modulation of leptin action. However, αδKO mice exhibit leptin sensitivity that is similar to that of wild-type mice by multiple measures. Thus, decreasing the abundance of SH2B1α and/or SH2B1δ relative to the other SH2B1 isoforms likely shifts energy balance toward a lean phenotype via a primarily leptin-independent mechanism. Our findings suggest that the different alternatively spliced isoforms of SH2B1 perform different functions in vivo.

Expression of a novel brain specific isoform of C3G is regulated during development

Mice lacking C3G (RapGEF1), a ubiquitously expressed protein essential for neuronal differentiation, show multiple defects in brain development. Function of C3G in neurogenesis is poorly defined. Here, we identify brain specific expression of a novel C3G isoform in mice and humans. This isoform has an insert in the Crk-binding region, generating a polypeptide of 175 kDa, unlike the previously known 140 kDa form expressed in all other tissues. In the adult mouse brain, C3G expression is seen in neurons, but was not detectable in GFAP-positive cells. C3G levels were high in the CA3 region of hippocampus and in mitral cells of olfactory bulb. Neural progenitor cells positive for Doublecortin and Nestin, show expression of C3G. During development, C3G is expressed in precursor cells prior to their differentiation into mature neurons or astrocytes. The 175 kDa as well as 140 kDa forms are seen in embryonic mouse brain, while only the 175 kDa variant is seen in post-natal brain. Human cerebral organoids generated from induced pluripotent stem cells predominantly expressed the 140 kDa polypeptides, and the 175 kDa isoform appeared upon maturation. This study describes developmental regulation and neuronal expression of a brain specific isoform of C3G, a molecule essential for normal development of the mammalian brain.

Susceptibility of Mice Lacking Renin‐b to Chronic Angiotensin II Infusion

In addition to the classical endocrine renin angiotensin system (RAS), local activity of the RAS has been detected in many tissues including the brain. The brain renin angiotensin system is known for its importance in blood pressure regulation and water homeostasis. The brain specific isoform of renin, termed renin‐b, has been proposed as a negative regulator of the brain renin‐angiotensin system (RAS) since the selective ablation of ren‐b (Ren‐b KO) in mice results in brain RAS disinhibition leading to blood pressure (BP) elevation. New cohorts of Ren‐b KO mice, however, do not exhibit elevated BP and the retrospective analysis of five previous cohorts indicated that Ren‐b KO model exhibits a high degree of variability in BP, which may be attributed to the susceptibility to different stressors. Importantly, Ren‐b KO (including recent cohorts) exhibit suppressed plasma renin and angiotensin II (Ang II) implying that suppression of the endocrine RAS might compensate for the elevation of the brain RAS activity in those Ren‐b KO mice exhibiting normal BP. Therefore, we hypothesized that Ren‐b KO are sensitized to exogenous systemic infusion of Ang II. Wildtype (WT) or Ren‐b KO were infused with either vehicle or Ang II (400 ng/kg/min; sc.) via osmotic minipumps for 4 weeks. Ang II significantly increased BP in both WT and Ren‐b KO to the same degree (WT+veh: 103±4 vs WT+Ang: 120±4 mmHg; p = 0.01 (n=10–12) and KO+veh: 104±3 vs KO+Ang: 118±5 mmHg; p = 0.03 (n=10–14)). Although the BP between Ren‐b KO and WT mice was elevated equally, Ren‐b KO exhibited enhanced Ang II‐induced dipsogenic response (WT+Ang: 3.7±0.3 vs KO+Ang: 4.6±0.4 ml/day; p = 0. 05; n=10–12) and exhibited a trend toward increase in levels of urinary aldosterone (WT+Ang: 14.6±1.4 vs KO+Ang: 17.8±1.7 mmHg) and copeptin, a marker of arginine vasopressin (WT+Ang: 11.5±2.6 vs KO+Ang: 22.2±5.2 mmHg). BP, drinking, aldosterone, and copeptin data in Ang II‐treated Ren‐b KO exhibits a bi‐modal distribution indicating that some mice are, whereas others, are not sensitized to Ang II. Ang II‐treated Ren‐b KO exhibited elevated heart weight (WT+Ang: 4.8±0.2 vs KO±Ang: 5.2±0.1 mg/g of body weight; p = 0.03; n=10–12) concomitant with an increased cardiac collagen type I alpha‐2 mRNA expression (p = 0.03; n=7–9) and trend toward increase in tumor necrosis factor‐α. Ang II induced an exaggerated suppression of plasma renin in Ren‐b KO compared to WT (WT+Ang: 14.8±2.1 vs KO+Ang: 7.2±2.6 ng/ml; p = 0. 03; n=7–9). However, there was no difference in the renin mRNA and protein levels of renin in the renal cortex. We used in situ hybridization technique to localize renin expression in the renin cortex. We did not observe a shift from glomerular to tubular renin expression in Ren‐b KO. We conclude Ren‐b KO exhibits higher sensitivity to Ang II‐induced aldosterone secretion and drinking responses as well as elevated susceptibility to Ang II‐evoked cardiac remodeling. The mechanism causing suppressed plasma renin in Ren‐b KO remains unclear. Studies of a conditional Ren‐b KO model will be used to elucidate the physiological consequences of downregulating renin‐b in specific neuroanatomical regions.Support or Funding InformationP01 HL084207 to Sigmund and 5T32HL134643 to Nakagawa

OXR1A, a Coactivator of PRMT5 Regulating Histone Arginine Methylation

Oxidation resistance gene 1 (OXR1) protects cells against oxidative stress. We find that male mice with brain-specific isoform A knockout (Oxr1A-/-) develop fatty liver. RNA sequencing of male Oxr1A-/- liver indicates decreased growth hormone (GH) signaling, which is known to affect liver metabolism. Indeed, Gh expression is reduced in male mice Oxr1A-/- pituitary gland and in rat Oxr1A-/- pituitary adenoma cell-line GH3. Oxr1A-/- male mice show reduced fasting-blood GH levels. Pull-down and proximity ligation assays reveal that OXR1A is associated with arginine methyl transferase PRMT5. OXR1A-depleted GH3 cells show reduced symmetrical dimethylation of histone H3 arginine 2 (H3R2me2s), a product of PRMT5 catalyzed methylation, and chromatin immunoprecipitation (ChIP) of H3R2me2s shows reduced Gh promoter enrichment. Finally, we demonstrate with purified proteins that OXR1A stimulates PRMT5/MEP50-catalyzed H3R2me2s. Our data suggest that OXR1A is a coactivator of PRMT5, regulating histone arginine methylation and thereby GH production within the pituitary gland.

Genetic deletion of gpr27 alters acylcarnitine metabolism, insulin sensitivity, and glucose homeostasis in zebrafish.

G protein-coupled receptors (GPCRs) comprise the largest group of membrane receptors in eukaryotic genomes and collectively they regulate nearly all cellular processes. Despite the widely recognized importance of this class of proteins, many GPCRs remain understudied. G protein-coupled receptor 27 (Gpr27) is an orphan GPCR that displays high conservation during vertebrate evolution. Although, GPR27 is known to be expressed in tissues that regulate metabolism including the pancreas, skeletal muscle, and adipose tissue, its functions are poorly characterized. Therefore, to investigate the potential roles of Gpr27 in energy metabolism, we generated a whole body gpr27 knockout zebrafish line. Loss of gpr27 potentiated the elevation in glucose levels induced by pharmacological or nutritional perturbations. We next leveraged a mass spectrometry metabolite profiling platform to identify other potential metabolic functions of Gpr27. Notably, genetic deletion of gpr27 elevated medium-chain acylcarnitines, in particular C6-hexanoylcarnitine, C8-octanoylcarnitine, C9-nonanoylcarnitine, and C10-decanoylcarnitine, lipid species known to be associated with insulin resistance in humans. Concordantly, gpr27 deletion in zebrafish abrogated insulin-dependent Akt phosphorylation and glucose utilization. Finally, loss of gpr27 increased the expression of key enzymes in carnitine shuttle complex, in particular the homolog to the brain-specific isoform of CPT1C which functions as a hypothalamic energy senor. In summary, our findings shed light on the biochemical functions of Gpr27 by illuminating its role in lipid metabolism, insulin signaling, and glucose homeostasis.

Abstract 098: Susceptibility of Mice Lacking Renin-B to Chronic Angiotensin II Infusion

Prior experience to various hypertensive conditions enhances the response to subsequent hypertensive challenges and the activation of the brain renin angiotensin system (RAS) is required for the sensitization to these hypertensive challenges. We previously showed that the brain-specific renin alternative isoform (Ren-b) tonically inhibits the activation of the brain RAS and that ablation of Ren-b (Ren-b KO) results in brain RAS disinhibition leading to elevated blood pressure (BP). Retrospective analysis of five cohorts of mice indicated that Ren-b KO model exhibits a high degree of variability in BP, which may be attributed to the susceptibility to stress. Therefore, we hypothesized that Ren-b KO are sensitized to hypertensive challenge such as angiotensin (Ang) II. Wildtype (WT) or Ren-b KO were infused with either vehicle or Ang II (400 ng/kg/min; sc.) via osmotic minipumps for 4 weeks. Ang II significantly increased BP in both WT and Ren-b KO to the same degree (WT+veh: 103±4 vs WT+Ang: 120±4 mmHg; p = 0.01 (n=10-12) and KO+veh: 104±3 vs KO+Ang: 118±5 mmHg; p = 0.03 (n=10-14)). Although the BP between Ren-b KO and WT mice was elevated equally, Ang II induced an exaggerated suppression of plasma renin in Ren-b KO compared to WT (WT+Ang: 14.8±2.1 vs KO±Ang: 7.2±2.6 ng/ml; p = 0. 03; n=7-9). Ren-b KO exhibited enhanced Ang II-induced dipsogenic response (WT+Ang: 3.7±0.3 vs KO+Ang: 4.6±0.4 ml/day; p = 0. 05; n=10-12) and elevated heart weight (WT+Ang: 4.8±0.2 vs KO±Ang: 5.2±0.1 mg/g of body weight; p = 0.03; n=10-12) concomitant with an increased cardiac collagen type I alpha-2 mRNA expression (p = 0.03; n=7-9). Altogether, this data indicates that in the presence of Ang II, Ren-b KO exhibits higher susceptibility to cardiovascular disease. After successive breeding and backcrossing Ren-b KO developed a milder and more varied baseline BP elevation compared to our initial cohorts. This suggests that epigenetic silencing or other physiological and molecular mechanisms might compensate for some of the cardiovascular alterations driven by the genetic deletion of renin-b. Studies of a conditional Ren-b KO model will be used to elucidate the physiological and/or pathophysiological consequences of an acute downregulation of renin-b in adulthood.

SGIP1\u03b1 functions as a selective endocytic adaptor for the internalization of synaptotagmin 1 at synapses

Proper sorting of exocytosed synaptic vesicle (SV) proteins into individual SVs during endocytosis is of the utmost importance for the fidelity of subsequent neurotransmission. Recent studies suggest that each SV protein is sorted into individual SVs by its own dedicated adaptors as well as by association between SV proteins. The SH3-containing GRB2-like protein 3-interacting protein 1 (SGIP1), an ortholog of Fer/Cip4 homology domain-only (FCHo) proteins, contains a μ-homology domain (μHD) and binds AP-2 and Eps15, thus functioning as an endocytic regulator of clathrin-mediated endocytosis (CME). Its longest isoform SGIP1α is predominantly expressed in the brain but the functional significance of SGIP1 in SV recycling remains unknown. Here, we found that SGIP1α, a brain-specific long isoform of SGIP1 binds synaptotagmin1 (Syt1) via its μHD and promotes the internalization of Syt1 on the neuronal surface. The small hairpin RNA (shRNA)-mediated knockdown (KD) of SGIP1α caused selective impairment of Syt1 internalization at hippocampal synapses and it was fully rescued by coexpression of the shRNA-resistant form of SGIP1α in KD neurons. We further found that the μHD of SGIP1α is structurally similar to those of AP-2 and stonin2, and mutations at Trp771 and Lys781, which correspond to Syt1-recognition motifs of AP-2 and stonin2, to Ala bound less efficiently to Syt1 and failed to rescue the endocytic defect of Syt1 caused by KD. Our results indicate that SGIP1α is an endocytic adaptor dedicated to the retrieval of surface-stranded Syt1. Since endocytic sorting of Syt1 is also mediated by the overlapping activities of synaptic vesicle glycoprotein 2A/B (SV2A/B) and stonin2, our results suggest that complementary fail-safe mechanism by these proteins ensures high fidelity of Syt1 retrieval.

IQSEC2-related encephalopathy in males and females: a comparative study including 37 novel patients

PurposeVariants in IQSEC2, escaping X inactivation, cause X-linked intellectual disability with frequent epilepsy in males and females. We aimed to investigate sex-specific differences.MethodsWe collected the data of 37 unpublished patients (18 males and 19 females) with IQSEC2 pathogenic variants and 5 individuals with variants of unknown significance and reviewed published variants. We compared variant types and phenotypes in males and females and performed an analysis of IQSEC2 isoforms.ResultsIQSEC2 pathogenic variants mainly led to premature truncation and were scattered throughout the longest brain-specific isoform, encoding the synaptic IQSEC2/BRAG1 protein. Variants occurred de novo in females but were either de novo (2/3) or inherited (1/3) in males, with missense variants being predominantly inherited. Developmental delay and intellectual disability were overall more severe in males than in females. Likewise, seizures were more frequently observed and intractable, and started earlier in males than in females. No correlation was observed between the age at seizure onset and severity of intellectual disability or resistance to antiepileptic treatments.ConclusionThis study provides a comprehensive overview of IQSEC2-related encephalopathy in males and females, and suggests that an accurate dosage of IQSEC2 at the synapse is crucial during normal brain development.

Functional genomics analysis of Phelan-McDermid syndrome 22q13 region during human neurodevelopment.

Phelan-McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by varying degrees of intellectual disability, severely delayed language development and specific facial features, and is caused by a deletion within chromosome 22q13.3. SHANK3, which is located at the terminal end of this region, has been repeatedly implicated in other neurodevelopmental disorders and deletion of this gene specifically is thought to cause much of the neurologic symptoms characteristic of PMS. However, it is still unclear to what extent SHANK3 deletions contribute to the PMS phenotype, and what other genes nearby are causal to the neurologic disease. In an effort to better understand the functional landscape of the PMS region during normal neurodevelopment, we assessed RNA-sequencing (RNA-seq) expression data collected from post-mortem brain tissue from developmentally normal subjects over the course of prenatal to adolescent age and analyzed expression changes of 65 genes on 22q13. We found that the majority of genes within this region were expressed in the brain, with ATNX10, MLC1, MAPK8IP2, and SULT4A1 having the highest overall expression. Analysis of the temporal profiles of the highest expressed genes revealed a trend towards peak expression during the early post-natal period, followed by a drop in expression later in development. Spatial analysis revealed significant region specific differences in the expression of SHANK3, MAPK8IP2, and SULT4A1. Region specific expression over time revealed a consistently unique gene expression profile within the cerebellum, providing evidence for a distinct developmental program within this region. Exon-specific expression of SHANK3 showed higher expression within exons contributing to known brain specific functional isoforms. Overall, we provide an updated roadmap of the PMS region, implicating several genes and time periods as important during neurodevelopment, with the hope that this information can help us better understand the phenotypic heterogeneity of PMS.