RESUMEN
Blood-CNS barrier disruption is a hallmark of numerous neurological disorders, yet whether barrier breakdown is sufficient to trigger neurodegenerative disease remains unresolved. Therapeutic strategies to mitigate barrier hyperpermeability are also limited. Dominant missense mutations of the cation channel transient receptor potential vanilloid 4 (TRPV4) cause forms of hereditary motor neuron disease. To gain insights into the cellular basis of these disorders, we generated knock-in mouse models of TRPV4 channelopathy by introducing two disease-causing mutations (R269C and R232C) into the endogenous mouse Trpv4 gene. TRPV4 mutant mice exhibited weakness, early lethality, and regional motor neuron loss. Genetic deletion of the mutant Trpv4 allele from endothelial cells (but not neurons, glia, or muscle) rescued these phenotypes. Symptomatic mutant mice exhibited focal disruptions of blood-spinal cord barrier (BSCB) integrity, associated with a gain of function of mutant TRPV4 channel activity in neural vascular endothelial cells (NVECs) and alterations of NVEC tight junction structure. Systemic administration of a TRPV4-specific antagonist abrogated channel-mediated BSCB impairments and provided a marked phenotypic rescue of symptomatic mutant mice. Together, our findings show that mutant TRPV4 channels can drive motor neuron degeneration in a non-cell autonomous manner by precipitating focal breakdown of the BSCB. Further, these data highlight the reversibility of TRPV4-mediated BSCB impairments and identify a potential therapeutic strategy for patients with TRPV4 mutations.
Asunto(s)
Barrera Hematoencefálica , Células Endoteliales , Mutación con Ganancia de Función , Neuronas Motoras , Canales Catiónicos TRPV , Animales , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Neuronas Motoras/patología , Neuronas Motoras/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Ratones , Degeneración Nerviosa/patología , Degeneración Nerviosa/genética , Fenotipo , Médula Espinal/patología , Médula Espinal/metabolismoAsunto(s)
Iluminación , Mentores , Humanos , Estudiantes , Encuestas y Cuestionarios , Selección de ProfesiónRESUMEN
Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. Here, we show that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.
Asunto(s)
Canales Catiónicos TRPV , Ubiquitinación , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Drosophila/genética , Drosophila/metabolismo , Humanos , Ratones , Mutación , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Ubiquitina/metabolismoRESUMEN
Transient receptor potential vanilloid 4 (TRPV4), a member of the TRP superfamily, is a broadly expressed, cell surface-localized cation channel that is activated by a variety of environmental stimuli. Importantly, TRPV4 has been increasingly implicated in the regulation of cellular morphology. Here we propose that TRPV4 and the cytoskeletal remodeling small GTPase RhoA together constitute an environmentally sensitive signaling complex that contributes to pathological cell cytoskeletal alterations during neurological injury and disease. Supporting this hypothesis is our recent work demonstrating direct physical and bidirectional functional interactions of TRPV4 with RhoA, which can lead to activation of RhoA and reorganization of the actin cytoskeleton. Furthermore, a confluence of evidence implicates TRPV4 and/or RhoA in pathological responses triggered by a range of acute neurological insults ranging from stroke to traumatic injury. While initiated by a variety of insults, TRPV4-RhoA signaling may represent a common pathway that disrupts axonal regeneration and blood-brain barrier integrity. These insights also suggest that TRPV4 inhibition may represent a safe, feasible, and precise therapeutic strategy for limiting pathological TRPV4-RhoA activation in a range of neurological diseases.
Asunto(s)
Citoesqueleto , Canales Catiónicos TRPV , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Transducción de Señal , Canales Catiónicos TRPV/metabolismoRESUMEN
Autism spectrum disorders (ASD) are associated with defects in neuronal connectivity and are highly heritable. Genetic findings suggest that there is an overrepresentation of chromatin regulatory genes among the genes associated with ASD. ASH1 like histone lysine methyltransferase (ASH1L) was identified as a major risk factor for ASD. ASH1L methylates Histone H3 on Lysine 36, which is proposed to result primarily in transcriptional activation. However, how mutations in ASH1L lead to deficits in neuronal connectivity associated with ASD pathogenesis is not known. We report that ASH1L regulates neuronal morphogenesis by counteracting the catalytic activity of Polycomb Repressive complex 2 group (PRC2) in stem cell-derived human neurons. Depletion of ASH1L decreases neurite outgrowth and decreases expression of the gene encoding the neurotrophin receptor TrkB whose signaling pathway is linked to neuronal morphogenesis. The neuronal morphogenesis defect is overcome by inhibition of PRC2 activity, indicating that a balance between the Trithorax group protein ASH1L and PRC2 activity determines neuronal morphology. Thus, our work suggests that ASH1L may epigenetically regulate neuronal morphogenesis by modulating pathways like the BDNF-TrkB signaling pathway. Defects in neuronal morphogenesis could potentially impair the establishment of neuronal connections which could contribute to the neurodevelopmental pathogenesis associated with ASD in patients with ASH1L mutations.
Asunto(s)
Proteínas de Unión al ADN , N-Metiltransferasa de Histona-Lisina , Proteínas de Unión al ADN/genética , Epigénesis Genética/genética , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Neuronas/metabolismoRESUMEN
Stem cells are capable of unlimited proliferation but can be induced to form brain cells. Factors that specifically regulate human development are poorly understood. We found that human stem cells expressed high levels of the envelope protein of an endogenized human-specific retrovirus (HERV-K, HML-2) from loci in chromosomes 12 and 19. The envelope protein was expressed on the cell membrane of the stem cells and was critical in maintaining the stemness via interactions with CD98HC, leading to triggering of human-specific signaling pathways involving mammalian target of rapamycin (mTOR) and lysophosphatidylcholine acyltransferase (LPCAT1)-mediated epigenetic changes. Down-regulation or epigenetic silencing of HML-2 env resulted in dissociation of the stem cell colonies and enhanced differentiation along neuronal pathways. Thus HML-2 regulation is critical for human embryonic and neurodevelopment, while it's dysregulation may play a role in tumorigenesis and neurodegeneration.
Asunto(s)
Diferenciación Celular , Retrovirus Endógenos/fisiología , Neuronas/metabolismo , Transducción de Señal , Células Madre/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Biomarcadores , Diferenciación Celular/genética , Autorrenovación de las Células/genética , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Unión Proteica , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
Vibrio vulnificus, a gram-negative bacterium, is the leading cause of seafood-borne illnesses and mortality in the United States. Previous studies have identified metabolites 2-C-methylerythritol 4-phosphate (MEP) as being essential for V. vulnificus growth and function. It was shown that 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) is a critical enzyme in the viability of V. vulnificus, and many other bacteria, as it catalyzes the rearrangement of 1-deoxy-D-xylulose-5-phosphate (Dxp) to 2-C-methylerythritol 4-phosphate (MEP) within the MEP pathway, found in plants and bacteria. The MEP pathway produces the isoprenoids, isopentenyl diphosphate and dimethylallyl pyrophosphate. In this study, we produced and structurally characterized V. vulnificus Dxr. The enzyme forms a dimeric assembly and contains a metal ion in the active site. Protein produced in Escherichia coli co-purifies with Mg2+ ions, however the Mg2+ cations may be substituted with Mn2+, as both of these metals may be utilized by Dxrs. These findings will provide a basis for the design of Dxr inhibitors that may find application as antimicrobial compounds.