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AnkyrinG (ankG) is highly enriched in neurons at axon initial segments (AISs) where it clusters Na(+) and K(+) channels and maintains neuronal polarity. How ankG becomes concentrated at the AIS is unknown. Here, we show that as neurons break symmetry, they assemble a distal axonal submembranous cytoskeleton, comprised of ankyrinB (ankB), αII-spectrin, and ßII-spectrin, that defines a boundary limiting ankG to the proximal axon. Experimentally moving this boundary altered the length of ankG staining in the proximal axon, whereas disruption of the boundary through silencing of ankB, αII-spectrin, or ßII-spectrin expression blocked AIS assembly and permitted ankG to redistribute throughout the distal axon. In support of an essential role for the distal cytoskeleton in ankG clustering, we also found that αII and ßII-spectrin-deficient mice had disrupted AIS. Thus, the distal axonal cytoskeleton functions as an intra-axonal boundary restricting ankG to the AIS.
Assuntos
Axônios/metabolismo , Citoesqueleto/metabolismo , Neurônios/metabolismo , Animais , Anquirinas/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Neurônios/citologia , Espectrina/metabolismoRESUMO
PURPOSE: Nonerythrocytic αII-spectrin (SPTAN1) variants have been previously associated with intellectual disability and epilepsy. We conducted this study to delineate the phenotypic spectrum of SPTAN1 variants. METHODS: We carried out SPTAN1 gene enrichment analysis in the rare disease component of the 100,000 Genomes Project and screened 100,000 Genomes Project, DECIPHER database, and GeneMatcher to identify individuals with SPTAN1 variants. Functional studies were performed on fibroblasts from 2 patients. RESULTS: Statistically significant enrichment of rare (minor allele frequency < 1 × 10-5) probably damaging SPTAN1 variants was identified in families with hereditary ataxia (HA) or hereditary spastic paraplegia (HSP) (12/1142 cases vs 52/23,847 controls, p = 2.8 × 10-5). We identified 31 individuals carrying SPTAN1 heterozygous variants or deletions. A total of 10 patients presented with pure or complex HSP/HA. The remaining 21 patients had developmental delay and seizures. Irregular αII-spectrin aggregation was noted in fibroblasts derived from 2 patients with p.(Arg19Trp) and p.(Glu2207del) variants. CONCLUSION: We found that SPTAN1 is a genetic cause of neurodevelopmental disorder, which we classified into 3 distinct subgroups. The first comprises developmental epileptic encephalopathy. The second group exhibits milder phenotypes of developmental delay with or without seizures. The final group accounts for patients with pure or complex HSP/HA.
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Epilepsia , Paraplegia Espástica Hereditária , Humanos , Espectrina/genética , Mutação , Epilepsia/genética , Fenótipo , Ataxia , Paraplegia Espástica Hereditária/genética , Convulsões , Paraplegia , LinhagemRESUMO
Dendritic spines are postsynaptic structures in neurons often having a mushroom-like shape. Physiological significance and cytoskeletal mechanisms that maintain this shape are poorly understood. The spectrin-based membrane skeleton maintains the biconcave shape of erythrocytes, but whether spectrins also determine the shape of nonerythroid cells is less clear. We show that ßIII spectrin in hippocampal and cortical neurons from rodent embryos of both sexes is distributed throughout the somatodendritic compartment but is particularly enriched in the neck and base of dendritic spines and largely absent from spine heads. Electron microscopy revealed that ßIII spectrin forms a detergent-resistant cytoskeletal network at these sites. Knockdown of ßIII spectrin results in a significant decrease in the density of dendritic spines. Surprisingly, the density of presynaptic terminals is not affected by ßIII spectrin knockdown. However, instead of making normal spiny synapses, the presynaptic structures in ßIII spectrin-depleted neurons make shaft synapses that exhibit increased amplitudes of miniature EPSCs indicative of excessive postsynaptic excitation. Thus, ßIII spectrin is necessary for formation of the constricted shape of the spine neck, which in turn controls communication between the synapse and the parent dendrite to prevent excessive excitation. Notably, mutations of SPTNB2 encoding ßIII spectrin are associated with neurodegenerative syndromes, spinocerebellar ataxia Type 5, and spectrin-associated autosomal recessive cerebellar ataxia Type 1, but molecular mechanisms linking ßIII spectrin functions to neuronal pathologies remain unresolved. Our data suggest that spinocerebellar ataxia Type 5 and spectrin-associated autosomal recessive cerebellar ataxia Type 1 pathology likely arises from poorly controlled synaptic activity that leads to excitotoxicity and neurodegeneration.SIGNIFICANCE STATEMENT Dendritic spines are small protrusions from neuronal dendrites that make synapses with axons of other neurons in the brain. Dendritic spines usually have a mushroom-like shape, which is essential for brain functions, because aberrant spine morphology is associated with many neuropsychiatric disorders. The bulbous head of a mushroom-shaped spine makes the synapse, whereas the narrow neck transmits the incoming signals to the dendrite and supposedly controls the signal propagation. We show that a cytoskeletal protein ßIII spectrin plays a key role for the formation of narrow spine necks. In the absence of ßIII spectrin, dendritic spines collapse onto dendrites. As a result, synaptic strength exceeds acceptable levels and damages neurons, explaining pathology of human syndromes caused by ßIII spectrin mutations.
Assuntos
Espinhas Dendríticas/fisiologia , Espinhas Dendríticas/ultraestrutura , Neurogênese/fisiologia , Neurônios/fisiologia , Espectrina/metabolismo , Transmissão Sináptica/fisiologia , Animais , Encéfalo/fisiologia , Encéfalo/ultraestrutura , Células Cultivadas , Masculino , Neurônios/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Calpain, a family of calcium-dependent neutral proteases, plays important roles in neurophysiology and pathology through the proteolytic modification of cytoskeletal proteins, receptors and kinases. Alpha 2 spectrin (αII spectrin) is a major substrate for this protease family, and the presence of the αII spectrin breakdown product (αΙΙ spectrin BDP) in a cell is evidence of calpain activity triggered by enhanced intracytoplasmic Ca(2+) concentrations. Astrocytes, the most dynamic CNS cells, respond to micro-environmental changes or noxious stimuli by elevating intracytoplasmic Ca(2+) concentration to become activated. As one measure of whether calpains are involved with reactive glial transformation, we examined paraffin sections of the human cerebral cortex and white matter by immunohistochemistry with an antibody specific for the calpain-mediated αΙΙ spectrin BDP. We also performed conventional double immunohistochemistry as well as immunofluorescent studies utilizing antibodies against αΙΙ spectrin BDP as well as glial fibrillary acidic protein (GFAP). We found strong immunopositivity in selected protoplasmic and fibrous astrocytes, and in transitional forms that raise the possibility of some of fibrous astrocytes emerging from protoplasmic astrocytes. Immunoreactive astrocytes were numerous in brain sections from cases with severe cardiac and/or respiratory diseases in the current study as opposed to our previous study of cases without significant clinical conditions that failed to reveal such remarkable immunohistochemical alterations. Our study suggests that astrocytes become αΙΙ spectrin BDP immunopositive in various stages of activation, and that spectrin cleavage product persists even in fully reactive astrocytes. Immunohistochemistry for αΙΙ spectrin BDP thus marks reactive astrocytes, and highlights the likelihood that calpains and their proteolytic processing of spectrin participate in the morphologic and physiologic transition from resting protoplasmic astrocytes to reactive fibrous astrocytes.
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Astrócitos/metabolismo , Encéfalo/metabolismo , Calpaína/metabolismo , Citoplasma/metabolismo , Espectrina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-IdadeRESUMO
We recently identified alpha II spectrin as a Tissue Degradation Indicator (TDI) and demonstrated that intrinsic spectrin-breakdown levels reliably reveal tissue degradation status in biospecimens. With the present study, we introduce an in vitro biological assay to mimic the endogenous spectrin-breakdown process and serve as degradation monitor (DM). By initiating the DM at the time of specimen collection and by attaching the DM to respective specimens, specimen degradation can be assessed by DM readout without specimen consumption. Using a protease inhibitory assay and protease-targeted immunoassays, we identified calpain as the protease responsible for degradation-induced spectrin breakdown. To recapitulate spectrin degradation in vitro, we developed several enzymatic assays in test tubes by incubating recombinant spectrins and synthetic Fluorescence Resonance Energy Transfer (FRET)-based spectrin peptides with purified human and porcine calpains. The in vitro assays reliably performed in different environments for a limited time due to loss of calpain activity. To maintain longer calpain activity, we introduced cultured cells as calpain providers into the in vitro assays. Under a variety of degradative conditions, including 4°C, 13°C, 23°C, 29°C, 37°C, freezing, and freeze-thaw steps, we compared the use of this prototype DM to the intrinsic spectrin cleavage assay (ISCA) in specimen degradation assessment using animal models. A strong correlation (r=0.9895) was detected between the DM-revealed degradation and the ISCA-revealed degradation. Notably, the DM-based degradation assessment takes only 10min and does not jeopardize the tissue itself, whereas the ISCA-based degradation assessment needs to sacrifice tissues and takes several hours to accomplish. Our data suggests the application of an in vitro degradation monitor for fast, real time, and non-invasive assessment of specimen degradation. This observation could lead to a transformative product dedicated to biospecimen quality control. This study also addresses critical, yet unmet needs for developing a universal standard for specimen degradation measurement.
Assuntos
Calpaína/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Análise em Microsséries/métodos , Manejo de Espécimes , Espectrina/metabolismo , Animais , Células Cultivadas , Exoma , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de DNA , SuínosRESUMO
During peripheral nerve development, Schwann cells ensheathe axons and form myelin to enable rapid and efficient action potential propagation. Although myelination requires profound changes in Schwann cell shape, how neuron-glia interactions converge on the Schwann cell cytoskeleton to induce these changes is unknown. Here, we demonstrate that the submembranous cytoskeletal proteins αII and ßII spectrin are polarized in Schwann cells and colocalize with signaling molecules known to modulate myelination in vitro. Silencing expression of these spectrins inhibited myelination in vitro, and remyelination in vivo. Furthermore, myelination was disrupted in motor nerves of zebrafish lacking αII spectrin. Finally, we demonstrate that loss of spectrin significantly reduces both F-actin in the Schwann cell cytoskeleton and the Nectin-like protein, Necl4, at the contact site between Schwann cells and axons. Therefore, we propose αII and ßII spectrin in Schwann cells integrate the neuron-glia interactions mediated by membrane proteins into the actin-dependent cytoskeletal rearrangements necessary for myelination.
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Bainha de Mielina/fisiologia , Nervos Periféricos/fisiologia , Células de Schwann/fisiologia , Espectrina/fisiologia , Actinas/antagonistas & inibidores , Actinas/fisiologia , Animais , Sequência de Bases , Polaridade Celular , Citoesqueleto/fisiologia , Técnicas de Silenciamento de Genes , Mutação , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Células de Schwann/citologia , Nervo Isquiático/citologia , Nervo Isquiático/lesões , Nervo Isquiático/fisiologia , Espectrina/antagonistas & inibidores , Espectrina/deficiência , Espectrina/genética , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Introduction: Deletion or mutation of members of the spectrin gene family contributes to many neurologic and neuropsychiatric disorders. While each spectrinopathy may generate distinct neuropathology, the study of ßΙ spectrin's role (Sptb) in the brain has been hampered by the hematologic consequences of its loss. Methods: Jaundiced mice (ja/ja) that lack ßΙ spectrin suffer a rapidly fatal hemolytic anemia. We have used exchange transfusion of newborn ja/ja mice to blunt their hemolytic pathology, enabling an examination of ßΙ spectrin deficiency in the mature mouse brain by ultrastructural and biochemical analysis. Results: ßΙ spectrin is widely utilized throughout the brain as the ßΙΣ2 isoform; it appears by postnatal day 8, and concentrates in the CA1,3 region of the hippocampus, dentate gyrus, cerebellar granule layer, cortical layer 2, medial habenula, and ventral thalamus. It is present in a subset of dendrites and absent in white matter. Without ßΙ spectrin there is a 20% reduction in postsynaptic density size in the granule layer of the cerebellum, a selective loss of ankyrinR in cerebellar granule neurons, and a reduction in the level of the postsynaptic adhesion molecule NCAM. While we find no substitution of another spectrin for ßΙ at dendrites or synapses, there is curiously enhanced ßΙV spectrin expression in the ja/ja brain. Discussion: ßΙΣ2 spectrin appears to be essential for refining postsynaptic structures through interactions with ankyrinR and NCAM. We speculate that it may play additional roles yet to be discovered.
RESUMO
Variable degrees of molecular degradation occur in human surgical specimens before clinical examination and severely affect analytical results. We therefore initiated an investigation to identify protein markers for tissue degradation assessment. We exposed 4 cell lines and 64 surgical/autopsy specimens to defined periods of time at room temperature before procurement (experimental cold ischemic time (CIT)-dependent tissue degradation model). Using two-dimensional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, we performed comparative proteomic analyses on cells at different CIT exposures and identified proteins with CIT-dependent changes. The results were validated by testing clinical specimens with western blot analysis. We identified 26 proteins that underwent dynamic changes (characterized by continuous quantitative changes, isoelectric changes, and/or proteolytic cleavages) in our degradation model. These changes are strongly associated with the length of CIT. We demonstrate these proteins to represent universal tissue degradation indicators (TDIs) in clinical specimens. We also devised and implemented a unique degradation measure by calculating the quantitative ratio between TDIs' intact forms and their respective degradation-modified products. For the first time, we have identified protein TDIs for quantitative measurement of specimen degradation. Implementing these indicators may yield a potentially transformative platform dedicated to quality control in clinical specimen analyses.
Assuntos
Isquemia Fria/métodos , Modelos Biológicos , Proteínas/metabolismo , Manejo de Espécimes/métodos , Sobrevivência de Tecidos/fisiologia , Biomarcadores/metabolismo , Western Blotting , Linhagem Celular , Eletroforese em Gel Bidimensional , Fluorescência , Humanos , Espectrometria de Massas , Proteômica , Análise de Sequência de ProteínaRESUMO
Spectrin α2 (αII-spectrin) is a scaffolding protein encoded by the Spna2 gene and constitutively expressed in most tissues. Exon trapping of Spna2 in C57BL/6 mice allowed targeted disruption of αII-spectrin. Heterozygous animals displayed no phenotype by 2 years of age. Homozygous deletion of Spna2 was embryonic lethal at embryonic day 12.5 to 16.5 with retarded intrauterine growth, and craniofacial, neural tube and cardiac anomalies. The loss of αII-spectrin did not alter the levels of αI- or ßI-spectrin, or the transcriptional levels of any ß-spectrin or any ankyrin, but secondarily reduced by about 80% the steady state protein levels of ßII- and ßIII-spectrin. Residual ßII- and ßIII-spectrin and ankyrins B and G were concentrated at the apical membrane of bronchial and renal epithelial cells, without impacting cell morphology. Neuroepithelial cells in the developing brain were more concentrated and more proliferative in the ventricular zone than normal; axon formation was also impaired. Embryonic fibroblasts cultured on fibronectin from E14.5 (Spna2(-/-)) animals displayed impaired growth and spreading, a spiky morphology, and sparse lamellipodia without cortical actin. These data indicate that the spectrin-ankyrin scaffold is crucial in vertebrates for cell spreading, tissue patterning and organ development, particularly in the developing brain and heart, but is not required for cell viability.
Assuntos
Proteínas de Transporte/metabolismo , Cardiopatias Congênitas/patologia , Proteínas dos Microfilamentos/metabolismo , Defeitos do Tubo Neural/patologia , Espectrina/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Anquirinas/genética , Anquirinas/metabolismo , Axônios/metabolismo , Axônios/fisiologia , Padronização Corporal , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Polaridade Celular , Proliferação de Células , Anormalidades Craniofaciais/embriologia , Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/patologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Fenótipo , Estabilidade Proteica , Pseudópodes/metabolismo , Espectrina/genéticaRESUMO
The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain betaIII spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in betaIII spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact betaIII spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most Spnb3(-/-) animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluRdelta, IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of alphaII spectrin. It appears that betaIII spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y.
Assuntos
Ataxia/etiologia , Convulsões/etiologia , Espectrina/deficiência , Animais , Ataxia/genética , Ataxia/fisiopatologia , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Encéfalo/ultraestrutura , Primers do DNA/genética , Modelos Animais de Doenças , Transportador 4 de Aminoácido Excitatório/metabolismo , Feminino , Marcação de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Fenótipo , Convulsões/genética , Convulsões/fisiopatologia , Espectrina/genética , Espectrina/fisiologia , Ataxias Espinocerebelares/etiologia , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/fisiopatologia , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
Reports have proposed a putative role for ßV spectrin in outer hair cells (OHCs) of the cochlea. In an ongoing investigation of the role of the cytoskeleton in electromotility, we tested mice with a targeted exon deletion of ßV spectrin (Spnb5), and unexpectedly find that Spnb5(-/-) animals' auditory thresholds are unaffected. Similarly, these mice have normal OHC electromechanical activity (otoacoustic emissions) and non-linear capacitance. In contrast, magnitudes of auditory brainstem response (ABR) wave 1-amplitudes are significantly reduced. Evidence of a synaptopathy was absent with normal hair cell CtBP2 counts. In Spnb5(-/-) mice, the number of afferent and efferent nerve fibers is decreased. Consistent with this data, Spnb5 mRNA is present in Type I and II spiral ganglion neurons, but undetectable in OHCs. Together, these data establish that ßV spectrin is important for hearing, affecting neuronal structure and function. Significantly, these data support that ßV spectrin as is not functionally important to OHCs as has been previously suggested.
Assuntos
Células Ciliadas Auditivas Externas , Espectrina , Animais , Cóclea/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico , Células Ciliadas Auditivas Externas/fisiologia , Camundongos , Camundongos Knockout , Emissões Otoacústicas Espontâneas , Espectrina/genética , Espectrina/metabolismoRESUMO
Experimental and hereditary defects in the ubiquitous scaffolding proteins of the spectrin gene family cause an array of neuropathologies. Most recognized are ataxias caused by missense, deletions, or truncations in the SPTBN2 gene that encodes beta III spectrin. Such mutations disrupt the organization of post-synaptic receptors, their active transport through the secretory pathway, and the organization and dynamics of the actin-based neuronal skeleton. Similar mutations in SPTAN1 that encodes alpha II spectrin cause severe and usually lethal neurodevelopmental defects including one form of early infantile epileptic encephalopathy type 5 (West syndrome). Defects in these and other spectrins are implicated in degenerative and psychiatric conditions. In recent published work, we describe in mice a novel variant of alpha II spectrin that results in a progressive ataxia with widespread neurodegenerative change. The action of this variant is distinct, in that rather than disrupting a constitutive ligand-binding function of spectrin, the mutation alters its response to calcium and calmodulin-regulated signaling pathways including its response to calpain activation. As such, it represents a novel spectrinopathy that targets a key regulatory pathway where calcium and tyrosine kinase signals converge. Here we briefly discuss the various roles of spectrin in neuronal processes and calcium activated regulatory inputs that control its participation in neuronal growth, organization, and remodeling. We hypothesize that damage to the neuronal spectrin scaffold may be a common final pathway in many neurodegenerative disorders. Targeting the pathways that regulate spectrin function may thus offer novel avenues for therapeutic intervention.
RESUMO
The neuronal membrane-associated periodic spectrin skeleton (MPS) contributes to neuronal development, remodeling, and organization. Post-translational modifications impinge on spectrin, the major component of the MPS, but their role remains poorly understood. One modification targeting spectrin is cleavage by calpains, a family of calcium-activated proteases. Spectrin cleavage is regulated by activated calpain, but also by the calcium-dependent binding of calmodulin (CaM) to spectrin. The physiologic significance of this balance between calpain activation and substrate-level regulation of spectrin cleavage is unknown. We report a strain of C57BL/6J mice harboring a single αII spectrin point mutation (Sptan1 c.3293G > A:p.R1098Q) with reduced CaM affinity and intrinsically enhanced sensitivity to calpain proteolysis. Homozygotes are embryonic lethal. Newborn heterozygotes of either gender appear normal, but soon develop a progressive ataxia characterized biochemically by accelerated calpain-mediated spectrin cleavage and morphologically by disruption of axonal and dendritic integrity and global neurodegeneration. Molecular modeling predicts unconstrained exposure of the mutant spectrin's calpain-cleavage site. These results reveal the critical importance of substrate-level regulation of spectrin cleavage for the maintenance of neuronal integrity. Given that excessive activation of calpain proteases is a common feature of neurodegenerative disease and traumatic encephalopathy, we propose that damage to the spectrin MPS may contribute to the neuropathology of many disorders.
Assuntos
Ataxia Cerebelar/genética , Espectrina/genética , Animais , Calpaína/metabolismo , Cerebelo/metabolismo , Cerebelo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação Puntual , Ligação Proteica , Proteólise , Espectrina/química , Espectrina/metabolismoRESUMO
SPTBN1 encodes ßII-spectrin, the ubiquitously expressed ß-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal ßII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect ßII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of ßII-spectrin in the central nervous system.
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Genes Dominantes , Predisposição Genética para Doença , Variação Genética , Transtornos do Neurodesenvolvimento/genética , Espectrina/genética , Animais , Estudos de Associação Genética/métodos , Heterozigoto , Humanos , Camundongos , Transtornos do Neurodesenvolvimento/diagnóstico , Fenótipo , Espectrina/metabolismoRESUMO
Clustered ion channels at nodes of Ranvier are critical for fast action potential propagation in myelinated axons. Axon-glia interactions converge on ankyrin and spectrin cytoskeletal proteins to cluster nodal Na+ channels during development. However, how nodal ion channel clusters are maintained is poorly understood. Here, we generated mice lacking nodal spectrins in peripheral sensory neurons to uncouple their nodal functions from their axon initial segment functions. We demonstrate a hierarchy of nodal spectrins, where ß4 spectrin is the primary spectrin and ß1 spectrin can substitute; each is sufficient for proper node organization. Remarkably, mice lacking nodal ß spectrins have normal nodal Na+ channel clustering during development, but progressively lose Na+ channels with increasing age. Loss of nodal spectrins is accompanied by an axon injury response and axon deformation. Thus, nodal spectrins are required to maintain nodal Na+ channel clusters and the structural integrity of axons.
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Nós Neurofibrosos/metabolismo , Canais de Sódio/metabolismo , Espectrina/metabolismo , Animais , Encéfalo/metabolismo , Citoesqueleto/metabolismo , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Espectrina/genéticaRESUMO
Previously, we showed that a hierarchy of spectrin cytoskeletal proteins maintains nodal Na+ channels (Liu et al., 2020). Here, using mice lacking ß1, ß4, or ß1/ß4 spectrins, we show this hierarchy does not function at axon initial segments (AIS). Although ß1 spectrin, together with AnkyrinR (AnkR), compensates for loss of nodal ß4 spectrin, it cannot compensate at AIS. We show AnkR lacks the domain necessary for AIS localization. Whereas loss of ß4 spectrin causes motor impairment and disrupts AIS, loss of ß1 spectrin has no discernable effect on central nervous system structure or function. However, mice lacking both neuronal ß1 and ß4 spectrin show exacerbated nervous system dysfunction compared to mice lacking ß1 or ß4 spectrin alone, including profound disruption of AIS Na+ channel clustering, progressive loss of nodal Na+ channels, and seizures. These results further define the important role of AIS and nodal spectrins for nervous system function.
Assuntos
Segmento Inicial do Axônio/metabolismo , Proteínas de Transporte/metabolismo , Hipocampo/metabolismo , Proteínas dos Microfilamentos/metabolismo , Espectrina/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Anquirinas/metabolismo , Comportamento Animal , Proteínas de Transporte/genética , Células Cultivadas , Feminino , Hipocampo/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Atividade Motora , Domínios Proteicos , Teste de Desempenho do Rota-Rod , Convulsões/genética , Convulsões/metabolismo , Convulsões/fisiopatologia , Espectrina/deficiência , Espectrina/genéticaRESUMO
Paranodal junctions of myelinated nerve fibers are important for saltatory conduction and function as paracellular and membrane protein diffusion barriers flanking nodes of Ranvier. The formation of these specialized axoglial contacts depends on the presence of three cell adhesion molecules: neurofascin 155 on the glial membrane and a complex of Caspr and contactin on the axon. We isolated axonal and glial membranes highly enriched in these paranodal proteins and then used mass spectrometry to identify additional proteins associated with the paranodal axoglial junction. This strategy led to the identification of three novel components of the paranodal cytoskeleton: ankyrinB, alphaII spectrin, and betaII spectrin. Biochemical and immunohistochemical analyses revealed that these proteins associate with protein 4.1B in a macromolecular complex that is concentrated at central and peripheral paranodal junctions in the adult and during early myelination. Furthermore, we show that the paranodal localization of ankyrinB is disrupted in Caspr-null mice with aberrant paranodal junctions, demonstrating that paranodal neuron-glia interactions regulate the organization of the underlying cytoskeleton. In contrast, genetic disruption of the juxtaparanodal protein Caspr2 or the nodal cytoskeletal protein betaIV spectrin did not alter the paranodal cytoskeleton. Our results demonstrate that the paranodal junction contains specialized cytoskeletal components that may be important to stabilize axon-glia interactions and contribute to the membrane protein diffusion barrier found at paranodes.
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Anquirinas/metabolismo , Axônios/metabolismo , Junções Comunicantes/metabolismo , Neuroglia/metabolismo , Nós Neurofibrosos/metabolismo , Espectrina/metabolismo , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Camundongos , RatosRESUMO
The kidney displays specialized regions devoted to filtration, selective reabsorption, and electrolyte and metabolite trafficking. The polarized membrane pumps, channels, and transporters responsible for these functions have been exhaustively studied. Less examined are the contributions of spectrin and its adapter ankyrin to this exquisite functional topography, despite their established contributions in other tissues to cellular organization. We have examined in the rodent kidney the expression and distribution of all spectrins and ankyrins by qPCR, Western blotting, immunofluorescent and immuno electron microscopy. Four of the seven spectrins (αΙΙ, ßΙ, ßΙΙ, and ßΙΙΙ) are expressed in the kidney, as are two of the three ankyrins (G and B). The levels and distribution of these proteins vary widely over the nephron. αΙΙ/ßΙΙ is the most abundant spectrin, found in glomerular endothelial cells; on the basolateral membrane and cytoplasmic vesicles in proximal tubule cells and in the thick ascending loop of Henle; and less so in the distal nephron. ßΙΙΙ spectrin largely replaces ßΙΙ spectrin in podocytes, Bowman's capsule, and throughout the distal tubule and collecting ducts. ßΙ spectrin is only marginally expressed; its low abundance hinders a reliable determination of its distribution. Ankyrin G is the most abundant ankyrin, found in capillary endothelial cells and all tubular segments. Ankyrin B populates Bowman's capsule, podocytes, the ascending thick loop of Henle, and the distal convoluted tubule. Comparison to the distribution of renal protein 4.1 isoforms and various membrane proteins indicates a complex relationship between the spectrin scaffold, its adapters, and various membrane proteins. While some proteins (e.g. ankyrin B, ßΙΙΙ spectrin, and aquaporin 2) tend to share a similar distribution, there is no simple mapping of different spectrins or ankyrins to most membrane proteins. The implications of this data are discussed.
Assuntos
Anquirinas/análise , Rim/química , Espectrina/análise , Animais , Anquirinas/genética , Western Blotting , Citoesqueleto/ultraestrutura , Éxons/genética , Rim/fisiologia , Rim/ultraestrutura , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Reação em Cadeia da Polimerase , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Organismos Livres de Patógenos Específicos , Espectrina/genéticaRESUMO
Axon initial segments (AIS) and nodes of Ranvier are highly specialized axonal membrane domains enriched in Na+ channels. These Na+ channel clusters play essential roles in action potential initiation and propagation. AIS and nodal Na+ channel complexes are linked to the actin cytoskeleton through ßIV spectrin. However, neuronal ßIV spectrin exists as two main splice variants: a longer ßIVΣ1 variant with canonical N-terminal actin and αII spectrin-binding domains, and a shorter ßIVΣ6 variant lacking these domains. Here, we show that the predominant neuronal ßIV spectrin splice variant detected in the developing brain switches from ßIVΣ1 to ßIVΣ6, and that this switch is correlated with expression changes in ankyrinG (ankG) splice variants. We show that ßIVΣ1 is the predominant splice variant at nascent and developing AIS and nodes of Ranvier, but with increasing age and in adults ßIVΣ6 becomes the main splice variant. Remarkably, super-resolution microscopy revealed that the spacing of spectrin tetramers between actin rings remains unchanged, but that shorter spectrin tetramers may also be present. Thus, during development ßIV spectrin may undergo a switch in the splice variants found at AIS and nodes of Ranvier.