TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.

Age-dependent regulation of ELP1 exon 20 splicing in Familial Dysautonomia by RNA Polymerase II kinetics and chromatin structure.

Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20.

Quantitative proteomics of dorsolateral prefrontal cortex reveals an early pattern of synaptic dysmaturation in children with idiopathic autism.

Autism spectrum disorder (ASD) is a developmental disorder with a rising prevalence and unknown etiology presenting with deficits in cognition and abnormal behavior. We hypothesized that the investigation of the synaptic component of prefrontal cortex may provide proteomic signatures that may identify the biological underpinnings of cognitive deficits in childhood ASD. Subcellular fractions of synaptosomes from prefrontal cortices of age-, brain area-, and postmortem-interval-matched samples from children and adults with idiopathic ASD vs. controls were subjected to HPLC-tandem mass spectrometry. Analysis of data revealed the enrichment of ASD risk genes that participate in slow maturation of the postsynaptic density (PSD) structure and function during early brain development. Proteomic analysis revealed down regulation of PSD-related proteins including AMPA and NMDA receptors, GRM3, DLG4, olfactomedins, Shank1-3, Homer1, CaMK2α, NRXN1, NLGN2, Drebrin1, ARHGAP32, and Dock9 in children with autism (FDR-adjusted P < 0.05). In contrast, PSD-related alterations were less severe or unchanged in adult individuals with ASD. Network analyses revealed glutamate receptor abnormalities. Overall, the proteomic data support the concept that idiopathic autism is a synaptopathy involving PSD-related ASD risk genes. Interruption in evolutionarily conserved slow maturation of the PSD complex in prefrontal cortex may lead to the development of ASD in a susceptible individual.

Physiological and pathological functions of TMEM106B in neurodegenerative diseases.

As an integral lysosomal transmembrane protein, transmembrane protein 106B (TMEM106B) regulates several aspects of lysosomal function and is associated with neurodegenerative diseases. The TMEM106B gene mutations lead to lysosomal dysfunction and accelerate the pathological progression of Neurodegenerative diseases. Yet, the precise mechanism of TMEM106B in Neurodegenerative diseases remains unclear. Recently, different research teams discovered that TMEM106B is an amyloid protein and the C-terminal domain of TMEM106B forms amyloid fibrils in various Neurodegenerative diseases and normally elderly individuals. In this review, we discussed the physiological functions of TMEM106B. We also included TMEM106B gene mutations that cause neurodegenerative diseases. Finally, we summarized the identification and cryo-electronic microscopic structure of TMEM106B fibrils, and discussed the promising therapeutic strategies aimed at TMEM106B fibrils and the future directions for TMEM106B research in neurodegenerative diseases.

The Role of SLIT3-ROBO4 Signaling in Endoplasmic Reticulum Stress-Induced Delayed Corneal Epithelial and Nerve Regeneration.

Purpose: In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration. Methods: Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells. Results: Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment. Conclusions: ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.

Hyperactivity of indirect pathway-projecting spiny projection neurons promotes compulsive behavior.

Compulsive behaviors are a hallmark symptom of obsessive compulsive disorder (OCD). Striatal hyperactivity has been linked to compulsive behavior generation in correlative studies in humans and causal studies in rodents. However, the contribution of the two distinct striatal output populations to the generation and treatment of compulsive behavior is unknown. These populations of direct and indirect pathway-projecting spiny projection neurons (SPNs) have classically been thought to promote or suppress actions, respectively, leading to a long-held hypothesis that increased output of direct relative to indirect pathway promotes compulsive behavior. Contrary to this hypothesis, here we find that indirect pathway hyperactivity is associated with compulsive grooming in the Sapap3-knockout mouse model of OCD-relevant behavior. Furthermore, we show that suppression of indirect pathway activity using optogenetics or treatment with the first-line OCD pharmacotherapy fluoxetine is associated with reduced grooming in Sapap3-knockouts. Together, these findings highlight the striatal indirect pathway as a potential treatment target for compulsive behavior.

BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation.

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.

Cartography of teneurin and latrophilin expression reveals spatiotemporal axis heterogeneity in the mouse hippocampus during development.

Synaptic adhesion molecules (SAMs) are evolutionarily conserved proteins that play an important role in the form and function of neuronal synapses. Teneurins (Tenms) and latrophilins (Lphns) are well-known cell adhesion molecules that form a transsynaptic complex. Recent studies suggest that Tenm3 and Lphn2 (gene symbol Adgrl2) are involved in hippocampal circuit assembly via their topographical expression. However, it is not known whether other teneurins and latrophilins display similar topographically restricted expression patterns during embryonic and postnatal development. Here, we reveal the cartography of all teneurin (Tenm1-4) and latrophilin (Lphn1-3 [Adgrl1-3]) paralog expression in the mouse hippocampus across prenatal and postnatal development as monitored by large-scale single-molecule RNA in situ hybridization mapping. Our results identify a striking heterogeneity in teneurin and latrophilin expression along the spatiotemporal axis of the hippocampus. Tenm2 and Tenm4 expression levels peak at the neonatal stage when compared to Tenm1 and Tenm3, while Tenm1 expression is restricted to the postnatal pyramidal cell layer. Tenm4 expression in the dentate gyrus (DG) exhibits an opposing topographical expression pattern in the embryonic and neonatal hippocampus. Our findings were validated by analyses of multiple RNA-seq datasets at bulk, single-cell, and spatial levels. Thus, our study presents a comprehensive spatiotemporal map of Tenm and Lphn expression in the hippocampus, showcasing their diverse expression patterns across developmental stages in distinct spatial axes.

Differential SNARE chaperoning by Munc13-1 and Munc18-1 dictates fusion pore fate at the release site.

The regulated release of chemical messengers is crucial for cell-to-cell communication; abnormalities in which impact coordinated human body function. During vesicular secretion, multiple SNARE complexes assemble at the release site, leading to fusion pore opening. How membrane fusion regulators act on heterogeneous SNARE populations to assemble fusion pores in a timely and synchronized manner, is unknown. Here, we demonstrate the role of SNARE chaperones Munc13-1 and Munc18-1 in rescuing individual nascent fusion pores from their diacylglycerol lipid-mediated inhibitory states. At the onset of membrane fusion, Munc13-1 clusters multiple SNARE complexes at the release site and synchronizes release events, while Munc18-1 stoichiometrically interacts with trans-SNARE complexes to enhance N- to C-terminal zippering. When both Munc proteins are present simultaneously, they differentially access dynamic trans-SNARE complexes to regulate pore properties. Overall, Munc proteins' direct action on fusion pore assembly indicates their role in controlling quantal size during vesicular secretion.

Investigating gene-environment interaction on attention in a double-hit model for Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a neurodevelopmental behavioral disorder characterized by social, communicative, and motor deficits. There is no single etiological cause for ASD, rather, there are various genetic and environmental factors that increase the risk for ASD. It is thought that some of these factors influence the same underlying neural mechanisms, and that an interplay of both genetic and environmental factors would better explain the pathogenesis of ASD. To better appreciate the influence of genetic-environment interaction on ASD-related behaviours, rats lacking a functional copy of the ASD-linked gene Cntnap2 were exposed to maternal immune activation (MIA) during pregnancy and assessed in adolescence and adulthood. We hypothesized that Cntnap2 deficiency interacts with poly I:C MIA to aggravate ASD-like symptoms in the offspring. In this double-hit model, we assessed attention, a core deficit in ASD due to prefrontal cortical dysfunction. We employed a well-established attentional paradigm known as the 5-choice serial reaction time task (5CSRTT). Cntnap2-/- rats exhibited greater perseverative responses which is indicative of repetitive behaviors. Additionally, rats exposed to poly I:C MIA exhibited premature responses, a marker of impulsivity. The rats exposed to both the genetic and environmental challenge displayed an increase in impulsive activity; however, this response was only elicited in the presence of an auditory distractor. This implies that exacerbated symptomatology in the double-hit model may situation-dependent and not generally expressed.

A new genetic diagnosis strategy for paroxysmal kinesigenic dyskinesia: Targeted high-throughput detection of PRRT2 gene c.649 locus.

BACKGROUND: Paroxysmal kinesigenic dyskinesia (PKD) is the most prevalent kind type of paroxysmal Dyskinesia, characterized by recurrent and transient episodes of involuntary movements. Most PKD cases were attributed to the proline-rich transmembrane protein 2 (PRRT2) gene, in which the c.649 region is a hotspot for known mutations. Even though some patients with PKD have been genetically diagnosed using whole-exome sequencing (WES) and Sanger sequencing, there are still cases of missed diagnoses due to the limitations of sequencing technology and analytic methods on throughput. METHODS: Patients meeting the diagnosis criteria of PKD with negative results of PRRT2-Sanger sequencing and WES were included in this study. Mutation screening and targeted high-throughput sequencing were performed to analyze and verify the sequencing results of the potential mutations. RESULTS: Six patients with PKD with high mutation ratios of c.649dupC were screened using our targeted high-throughput sequencing from 26 PKD patients with negative results of PRRT2-Sanger sequencing and WES (frequency = 23.1%), which compensated for the comparatively shallow sequencing depth and statistical flaws in this region. Compared with the local normal population and other patients with PKD, the mutation ratios of c.649dupC of these six patients with PKD were much higher and also had truncated protein structures and differentially altered mRNA expression. CONCLUSION: Based on the above studies, we emphasize the routine targeted high-throughput sequencing of the c.649 site in the PRRT2 gene in so-called genetic-testing-negative patients with PKD, and manually calculate the deletion and duplication mutations depth and ratios to lower the rate of clinical misdiagnosis.

A global gene regulatory program and its region-specific regulator partition neurons into commissural and ipsilateral projection types.

Understanding the genetic programs that drive neuronal diversification into classes and subclasses is key to understand nervous system development. All neurons can be classified into two types: commissural and ipsilateral, based on whether their axons cross the midline or not. However, the gene regulatory program underlying this binary division is poorly understood. We identified a pair of basic helix-loop-helix transcription factors, Nhlh1 and Nhlh2, as a global transcriptional mechanism that controls the laterality of all floor plate-crossing commissural axons in mice. Mechanistically, Nhlh1/2 play an essential role in the expression of Robo3, the key guidance molecule for commissural axon projections. This genetic program appears to be evolutionarily conserved in chick. We further discovered that Isl1, primarily expressed in ipsilateral neurons within neural tubes, negatively regulates the Robo3 induction by Nhlh1/2. Our findings elucidate a gene regulatory strategy where a conserved global mechanism intersects with neuron class-specific regulators to control the partitioning of neurons based on axon laterality.

Reelin Regulates Developmental Desynchronization Transition of Neocortical Network Activity.

During the first and second stages of postnatal development, neocortical neurons exhibit a wide range of spontaneous synchronous activity (SSA). Towards the end of the second postnatal week, the SSA is replaced by a more sparse and desynchronized firing pattern. The developmental desynchronization of neocortical spontaneous neuronal activity is thought to be intrinsically generated, since sensory deprivation from the periphery does not affect the time course of this transition. The extracellular protein reelin controls various aspects of neuronal development through multimodular signaling. However, so far it is unclear whether reelin contributes to the developmental desynchronization transition of neocortical neurons. The present study aims to investigate the role of reelin in postnatal cortical developmental desynchronization using a conditional reelin knockout (RelncKO) mouse model. Conditional reelin deficiency was induced during early postnatal development, and Ca2+ recordings were conducted from organotypic cultures (OTCs) of the somatosensory cortex. Our results show that both wild type (wt) and RelncKO exhibited an SSA pattern during the early postnatal week. However, at the end of the second postnatal week, wt OTCs underwent a transition to a desynchronized network activity pattern, while RelncKO activity remained synchronous. This changing activity pattern suggests that reelin is involved in regulating the developmental desynchronization of cortical neuronal network activity. Moreover, the developmental desynchronization impairment observed in RelncKO was rescued when RelncKO OTCs were co-cultured with wt OTCs. Finally, we show that the developmental transition to a desynchronized state at the end of the second postnatal week is not dependent on glutamatergic signaling. Instead, the transition is dependent on GABAAR and GABABR signaling. The results suggest that reelin controls developmental desynchronization through GABAAR and GABABR signaling.

Patient-derived castration-resistant prostate cancer model revealed CTBP2 upregulation mediated by OCT1 and androgen receptor.

BACKGROUND: Prostate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation. METHODS: In this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database. RESULTS: Genome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC. CONCLUSIONS: Our findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management.

Clinical, genetic, and cognitive correlates of seizure occurrences in Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid syndrome (PMS) is a genetic neurodevelopmental disorder caused by SHANK3 haploinsufficiency and is associated with an increased risk for seizures. Previous literature indicates that around one third of individuals with PMS also have epilepsy or seizures, with a wide range of types and ages of onset. Investigating the impact of seizures on intellectual and adaptive functioning for PMS is a primary concern for caregivers and is important to understanding the natural history of this syndrome. METHODS: We report on results from 98 individuals enrolled in a prospective, longitudinal study. We detailed seizure frequency, type, and age of onset, and we analyzed seizure occurrence with best estimate IQ, adaptive functioning, clinical features, and genotype. We conducted multiple linear regression analyses to assess the relationship between the presence of seizures and the Vineland Adaptive Behavior Scale, Second Edition (VABS-II) Adaptive Behavior Composite score and the best estimate full-scale IQ. We also performed Chi-square tests to explore associations between seizure prevalence and genetic groupings. Finally, we performed Chi-square tests and t-tests to explore the relationship between seizures and demographic features, features that manifest in infancy, and medical features. RESULTS: Seizures were present in 41% of the cohort, and age of onset was widely variable. The presence of seizures was associated with significantly lower adaptive and intellectual functioning. Genotype-phenotype analyses were discrepant, with no differences in seizure prevalence across genetic classes, but with more genes included in deletions of participants with 22q13 deletions and seizures compared to those with 22q13 deletions and no seizures. No clinical associations were found between the presence of seizures and sex, history of pre- or neonatal complications, early infancy, or medical features. In this cohort, generalized seizures were associated with developmental regression, which is a top concern for PMS caregivers. CONCLUSIONS: These results begin to eludicate correlates of seizures in individuals with PMS and highlight the importance of early seizure management. Importantly, presence of seizures was associated with adaptive and cognitive functioning. A larger cohort might be able to identify additional associations with medical features. Genetic findings suggest an increased capability to realize genotype-phenotype relationships when deletion size is taken into account.

Deciphering the dual nature of nesfatin-1: a tale of zinc ion's Janus-faced influence.

BACKGROUND: Nucleobindin-2 (Nucb2) and nesfatin-1 (N1) are widely distributed hormones that regulate numerous physiological processes, from energy homeostasis to carcinogenesis. However, the role of nesfatin-2 (N2), the second product of Nucb2 proteolytic processing, remains elusive. To elucidate the relationship between the structure and function of nesfatins, we investigated the properties of chicken and human homologs of N1, as well as a fragment of Nucb2 consisting of N1 and N2 conjoined in a head-to-tail manner (N1/2). RESULTS: Our findings indicate that Zn(II) sensing, in the case of N1, is conserved between chicken and human species. However, the data presented here reveal significant differences in the molecular features of the analyzed peptides, particularly in the presence of Zn(II). We demonstrated that Zn(II) has a Janus effect on the M30 region (a crucial anorexigenic core) of N1 and N1/2. In N1 homologs, Zn(II) binding results in the concealment of the M30 region driven by a disorder-to-order transition and adoption of the amyloid fold. In contrast, in N1/2 molecules, Zn(II) binding causes the exposure of the M30 region and its destabilization, resulting in strong exposure of the region recognized by prohormone convertases within the N1/2 molecule. CONCLUSIONS: In conclusion, we found that Zn(II) binding is conserved between chicken and human N1. However, despite the high homology of chicken and human N1, their interaction modes with Zn(II) appear to differ. Furthermore, Zn(II) binding might be essential for regulating the function of nesfatins by spatiotemporally hindering the N1 anorexigenic M30 core and concomitantly facilitating N1 release from Nucb2.

Neurexins control the strength and precise timing of glycinergic inhibition in the auditory brainstem.

Neurexins play diverse functions as presynaptic organizers in various glutamatergic and GABAergic synapses. However, it remains unknown whether and how neurexins are involved in shaping functional properties of the glycinergic synapses, which mediate prominent inhibition in the brainstem and spinal cord. To address these issues, we examined the role of neurexins in a model glycinergic synapse between the principal neuron in the medial nucleus of the trapezoid body (MNTB) and the principal neuron in the lateral superior olive (LSO) in the auditory brainstem. Combining RNAscope with stereotactic injection of AAV-Cre in the MNTB of neurexin1/2/3 conditional triple knockout mice, we showed that MNTB neurons highly express all isoforms of neurexins although their expression levels vary remarkably. Selective ablation of all neurexins in MNTB neurons not only reduced the amplitude but also altered the kinetics of the glycinergic synaptic transmission at LSO neurons. The synaptic dysfunctions primarily resulted from an impaired Ca2+ sensitivity of release and a loosened coupling between voltage-gated Ca2+ channels and synaptic vesicles. Together, our current findings demonstrate that neurexins are essential in controlling the strength and temporal precision of the glycinergic synapse, which therefore corroborates the role of neurexins as key presynaptic organizers in all major types of fast chemical synapses.

The tale of capturing Norrin.

Detailed binding experiments reveal new insights into the Norrin/Wnt signaling pathway that helps to control vascularization in the retina.

Exome sequencing implicates ancestry-related Mendelian variation at SYNE1 in childhood-onset essential hypertension.

Childhood-onset essential hypertension (COEH) is an uncommon form of hypertension that manifests in childhood or adolescence and, in the United States, disproportionately affects children of African ancestry. The etiology of COEH is unknown, but its childhood onset, low prevalence, high heritability, and skewed ancestral demography suggest the potential to identify rare genetic variation segregating in a Mendelian manner among affected individuals and thereby implicate genes important to disease pathogenesis. However, no COEH genes have been reported to date. Here, we identify recessive segregation of rare and putatively damaging missense variation in the spectrin domain of spectrin repeat containing nuclear envelope protein 1 (SYNE1), a cardiovascular candidate gene, in 3 of 16 families with early-onset COEH without an antecedent family history. By leveraging exome sequence data from an additional 48 COEH families, 1,700 in-house trios, and publicly available data sets, we demonstrate that compound heterozygous SYNE1 variation in these COEH individuals occurred more often than expected by chance and that this class of biallelic rare variation was significantly enriched among individuals of African genetic ancestry. Using in vitro shRNA knockdown of SYNE1, we show that reduced SYNE1 expression resulted in a substantial decrease in the elasticity of smooth muscle vascular cells that could be rescued by pharmacological inhibition of the downstream RhoA/Rho-associated protein kinase pathway. These results provide insights into the molecular genetics and underlying pathophysiology of COEH and suggest a role for precision therapeutics in the future.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.