RESUMO
BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is predominantly associated with motor cortex, corticospinal tract (CST), brainstem, and spinal cord degeneration, and cerebellar involvement is much less well characterized. However, some of the cardinal clinical features of ALS, such as dysarthria, dysphagia, gait impairment, falls, and impaired dexterity, are believed to be exacerbated by coexisting cerebellar pathology. Cerebellar pathology may also contribute to cognitive, behavioral, and pseudobulbar manifestations. Our objective was to systematically assess both intracerebellar pathology and cerebrocerebellar connectivity alterations in a genetically stratified cohort of ALS. METHODS: A prospective, multimodal neuroimaging study was conducted to evaluate the longitudinal evolution of intracerebellar pathology and cerebrocerebellar connectivity, using structural and functional measures. RESULTS: A total of 113 healthy controls and 212 genetically stratified individuals with ALS were included: (1) C9orf72 hexanucleotide carriers ("C9POS"), (2) sporadic patients who tested negative for ALS-associated genetic variants, and (3) intermediate-length CAG trinucleotide carriers in ATXN2 ("ATXN2"). Flocculonodular lobule (padj = 0.014, 95% CI -5.06e-5 to -3.98e-6) and crura (padj = 0.031, 95% CI -1.63e-3 to -5.55e-5) volume reductions were detected at baseline in sporadic patients. Cerebellofrontal and cerebelloparietal structural connectivity impairment was observed in both C9POS and sporadic patients at baseline, and both projections deteriorated further over time in sporadic patients (padj = 0.003, t(249) = 3.04 and padj = 0.05, t(249) = 1.93). Functional cerebelloparietal uncoupling was evident in sporadic patients at baseline (padj = 0.004, 95% CI -0.19 to -0.03). ATXN2 patients exhibited decreased cerebello-occipital functional connectivity at baseline (padj = 0.004, 95% CI -0.63 to -0.06), progressive cerebellotemporal functional disconnection (padj = 0.025, t(199) = -2.26), and progressive flocculonodular lobule degeneration (padj = 0.017, t(249) = -2.24). C9POS patients showed progressive ventral dentate atrophy (padj = 0.007, t(249) = -2.75). The CSTs (padj < 0.001, 95% CI 4.89e-5 to 1.14e-4) and transcallosal interhemispheric fibers (padj < 0.001, 95% CI 5.21e-5 to 1.31e-4) were affected at baseline in C9POS and exhibited rapid degeneration over the 4 time points. The rate of decline in CST and corpus callosum integrity was faster than the rate of cerebrocerebellar disconnection (padj = 0.001, t(190) = 6.93). DISCUSSION: ALS is associated with accruing intracerebellar disease burden as well as progressive corticocerebellar uncoupling. Contrary to previous suggestions, we have not detected evidence of compensatory structural or functional changes in response to supratentorial degeneration. The contribution of cerebellar disease burden to dysarthria, dysphagia, gait impairment, pseudobulbar affect, and cognitive deficits should be carefully considered in clinical assessments, monitoring, and multidisciplinary interventions.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Cerebelo , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Masculino , Feminino , Pessoa de Meia-Idade , Cerebelo/diagnóstico por imagem , Cerebelo/patologia , Idoso , Proteína C9orf72/genética , Estudos Prospectivos , Ataxina-2/genética , Imageamento por Ressonância Magnética , Progressão da Doença , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Adulto , Estudos LongitudinaisRESUMO
In Metazoa, the diversity of transcripts produced by the RNA Polymerase II is generated essentially through post-transcriptional processing of the nascent transcripts. The regulation of exon inclusion by alternative splicing is one of the main sources of this diversity, which leads to the expansion of the proteome. The portfolio of alternative transcripts remains largely underestimated. Improvement of the sequencing technologies has enhanced the characterization of RNA isoforms and led to the perpetual incrementation of gene expression diversity. Here, we describe a high throughput approach to assess in-depth the splicing regulation of target gene(s) using the third-generation sequencing (TGS) technologies.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Splicing de RNA , Processamento Alternativo , Isoformas de RNA/genética , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
The cerebellum, a primary brain structure involved in the control of sensorimotor tasks, also contributes to higher cognitive functions including reward, emotion and social interaction. Although the regulation of these behaviors has been largely ascribed to the monoaminergic system in limbic regions, the contribution of cerebellar dopamine signaling in the modulation of these functions remains largely unknown. By combining cell-type-specific transcriptomics, histological analyses, three-dimensional imaging and patch-clamp recordings, we demonstrate that cerebellar dopamine D2 receptors (D2Rs) in mice are preferentially expressed in Purkinje cells (PCs) and regulate synaptic efficacy onto PCs. Moreover, we found that changes in D2R levels in PCs of male mice during adulthood alter sociability and preference for social novelty without affecting motor functions. Altogether, these findings demonstrate novel roles for D2R in PC function and causally link cerebellar D2R levels of expression to social behaviors.
Assuntos
Células de Purkinje , Receptores de Dopamina D2 , Animais , Cerebelo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células de Purkinje/fisiologia , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Comportamento SocialRESUMO
Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse and human brain development. Proteomic analysis reveals at least a hundred isoforms of GPHN incorporated at inhibitory Glycine and gamma-aminobutyric acid A receptors containing synapses. They exhibit different localization and postsynaptic clustering properties, and altering the expression level of one isoform is sufficient to affect the number, size, and density of inhibitory synapses in cerebellar Purkinje cells. Furthermore, we discovered that splicing defects reported in neuropsychiatric disorders are carried by multiple alternative GPHN transcripts, demonstrating the need for a thorough analysis of the GPHN transcriptome in patients. Overall, we show that alternative splicing of GPHN is an important genetic variation to consider in neurological diseases and a determinant of the diversity of postsynaptic inhibitory synapses.
Assuntos
Proteínas de Transporte , Proteômica , Proteínas de Transporte/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de GABA/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/genética , Sinapses/metabolismoRESUMO
Amyotrophic lateral sclerosis is a relentlessly progressive multi-system condition. The clinical picture is dominated by upper and lower motor neuron degeneration, but extra-motor pathology is increasingly recognized, including cerebellar pathology. Post-mortem and neuroimaging studies primarily focus on the characterization of supratentorial disease, despite emerging evidence of cerebellar degeneration in amyotrophic lateral sclerosis. Cardinal clinical features of amyotrophic lateral sclerosis, such as dysarthria, dysphagia, cognitive and behavioral deficits, saccade abnormalities, gait impairment, respiratory weakness and pseudobulbar affect are likely to be exacerbated by co-existing cerebellar pathology. This review summarizes in vivo and post mortem evidence for cerebellar degeneration in amyotrophic lateral sclerosis. Structural imaging studies consistently capture cerebellar grey matter volume reductions, diffusivity studies readily detect both intra-cerebellar and cerebellar peduncle white matter alterations and functional imaging studies commonly report increased functional connectivity with supratentorial regions. Increased functional connectivity is commonly interpreted as evidence of neuroplasticity representing compensatory processes despite the lack of post-mortem validation. There is a scarcity of post-mortem studies focusing on cerebellar alterations, but these detect pTDP-43 in cerebellar nuclei. Cerebellar pathology is an overlooked facet of neurodegeneration in amyotrophic lateral sclerosis despite its contribution to a multitude of clinical symptoms, widespread connectivity to spinal and supratentorial regions and putative role in compensating for the degeneration of primary motor regions.
RESUMO
G-protein-coupled receptors can be constitutively activated following physical interaction with intracellular proteins. The first example described was the constitutive activation of Group I metabotropic glutamate receptors (mGluR: mGluR1,5) following their interaction with Homer1a, an activity-inducible early-termination variant of the scaffolding protein Homer that lacks dimerization capacity (Ango et al., 2001). Homer1a disrupts the links, maintained by the long form of Homer (cross-linking Homers), between mGluR1,5 and the Shank-GKAP-PSD-95-ionotropic glutamate receptor network. Two characteristics of the constitutive activation of the Group I mGluR-Homer1a complex are particularly interesting: (1) it affects a large number of synapses in which Homer1a is upregulated following enhanced, long-lasting neuronal activity; and (2) it mainly depends on Homer1a protein turnover. The constitutively active Group I mGluR-Homer1a complex is involved in the two main forms of non-Hebbian neuronal plasticity: "metaplasticity" and "homeostatic synaptic scaling," which are implicated in a large series of physiological and pathologic processes. Those include non-Hebbian plasticity observed in visual system, synapses modulated by addictive drugs (rewarded synapses), chronically overactivated synaptic networks, normal sleep, and sleep deprivation.
Assuntos
Encéfalo/fisiologia , Homeostase/fisiologia , Proteínas de Arcabouço Homer/metabolismo , Plasticidade Neuronal/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Animais , HumanosRESUMO
One of the most intriguing features of inhibitory synapses is the precision by which they innervate their target, not only at the cellular level but also at the subcellular level (i.e. axo-dendritic, axo-somatic, or axo-axonic innervation). In particular, in the cerebellum, cortex, and spinal cord, distinct and highly specialized GABAergic interneurons, such as basket cells, chandelier cells, and GABApre interneurons, form precise axo-axonic synapses, allowing them to directly regulate neuronal output and circuit function. In this article, we summarize our latest knowledge of the cellular and molecular mechanisms that regulate the establishment and maintenance of axo-axonic synapses in these regions of the CNS. We also detail the key roles of the L1CAM family of cell adhesion molecules in such GABAergic subcellular target recognition.
Assuntos
Axônios , Interneurônios , Córtex Cerebral , SinapsesRESUMO
GABAergic interneurons migrate long distances through stereotyped migration programs toward specific laminar positions. During their migration, GABAergic interneurons are morphologically alike but then differentiate into a rich array of interneuron subtypes critical for brain function. How interneuron subtypes acquire their final phenotypic traits remains largely unknown. Here, we show that cerebellar molecular layer GABAergic interneurons, derived from the same progenitor pool, use separate migration paths to reach their laminar position and differentiate into distinct basket cell (BC) and stellate cell (SC) GABAergic interneuron subtypes. Using two-photon live imaging, we find that SC final laminar position requires an extra step of tangential migration supported by a subpopulation of glutamatergic granule cells (GCs). Conditional depletion of GCs affects SC differentiation but does not affect BCs. Our results reveal how timely feedforward control of inhibitory interneuron migration path regulates their terminal differentiation and, thus, establishment of the local inhibitory circuit assembly.
Assuntos
Diferenciação Celular , Grânulos Citoplasmáticos/metabolismo , Interneurônios/citologia , Animais , Axônios/metabolismo , Movimento Celular , Neurônios GABAérgicos/citologia , Interneurônios/metabolismo , Camundongos TransgênicosRESUMO
Cannabis abuse during adolescence confers an increased risk for developing later in life cognitive deficits reminiscent of those observed in schizophrenia, suggesting common pathological mechanisms that remain poorly characterized. In line with previous findings that revealed a role of 5-HT6 receptor-operated mTOR activation in cognitive deficits of rodent developmental models of schizophrenia, we show that chronic administration of ∆9-tetrahydrocannabinol (THC) to mice during adolescence induces a long-lasting activation of mTOR in prefrontal cortex (PFC), alterations of excitatory/inhibitory balance, intrinsic properties of layer V pyramidal neurons, and long-term depression, as well as cognitive deficits in adulthood. All are prevented by administrating a 5-HT6 receptor antagonist or rapamycin, during adolescence. In contrast, they are still present 2 weeks after the same treatments delivered at the adult stage. Collectively, these findings suggest a role of 5-HT6 receptor-operated mTOR signaling in abnormalities of cortical network wiring elicited by THC at a critical period of PFC maturation and highlight the potential of 5-HT6 receptor antagonists as early therapy to prevent cognitive symptom onset in adolescent cannabis abusers.
Assuntos
Abuso de Maconha , Animais , Dronabinol , Camundongos , Córtex Pré-Frontal , Receptores de SerotoninaRESUMO
Dopamine receptor D1 modulates glutamatergic transmission in cortico-basal ganglia circuits and represents a major target of L-DOPA therapy in Parkinson's disease. Here we show that D1 and metabotropic glutamate type 5 (mGlu5) receptors can form previously unknown heteromeric entities with distinctive functional properties. Interacting with Gq proteins, cell-surface D1-mGlu5 heteromers exacerbated PLC signaling and intracellular calcium release in response to either glutamate or dopamine. In rodent models of Parkinson's disease, D1-mGlu5 nanocomplexes were strongly upregulated in the dopamine-denervated striatum, resulting in a synergistic activation of PLC signaling by D1 and mGlu5 receptor agonists. In turn, D1-mGlu5-dependent PLC signaling was causally linked with excessive activation of extracellular signal-regulated kinases in striatal neurons, leading to dyskinesia in animals treated with L-DOPA or D1 receptor agonists. The discovery of D1-mGlu5 functional heteromers mediating maladaptive molecular and motor responses in the dopamine-denervated striatum may prompt the development of new therapeutic principles for Parkinson's disease.
Assuntos
Corpo Estriado/metabolismo , Sistema de Sinalização das MAP Quinases , Neurônios/metabolismo , Doença de Parkinson Secundária/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Corpo Estriado/patologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Complexos Multiproteicos/agonistas , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neurônios/patologia , Doença de Parkinson Secundária/tratamento farmacológico , Doença de Parkinson Secundária/genética , Doença de Parkinson Secundária/patologia , Ratos , Receptor de Glutamato Metabotrópico 5/genética , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/genéticaRESUMO
How does the inner ear communicate with the cerebellar cortex to maintain balance and posture?
Assuntos
Córtex Cerebelar , Fibras NervosasRESUMO
By virtue of their extensive axonal arborization and perisomatic synaptic targeting, cortical inhibitory parvalbumin (PV) cells strongly regulate principal cell output and plasticity and modulate experience-dependent refinement of cortical circuits during development. An interesting aspect of PV cell connectivity is its prolonged maturation time course, which is completed only by end of adolescence. The p75 neurotrophin receptor (p75NTR) regulates numerous cellular functions; however, its role on cortical circuit development and plasticity remains elusive, mainly because localizing p75NTR expression with cellular and temporal resolution has been challenging. By using RNAscope and a modified version of the proximity ligation assay, we found that p75NTR expression in PV cells decreases between the second and fourth postnatal week, at a time when PV cell synapse numbers increase dramatically. Conditional knockout of p75NTR in single PV neurons in vitro and in PV cell networks in vivo causes precocious formation of PV cell perisomatic innervation and perineural nets around PV cell somata, therefore suggesting that p75NTR expression modulates the timing of maturation of PV cell connectivity in the adolescent cortex. Remarkably, we found that PV cells still express p75NTR in adult mouse cortex of both sexes and that its activation is sufficient to destabilize PV cell connectivity and to restore cortical plasticity following monocular deprivation in vivo Together, our results show that p75NTR activation dynamically regulates PV cell connectivity, and represent a novel tool to foster brain plasticity in adults.SIGNIFICANCE STATEMENT In the cortex, inhibitory, GABA-releasing neurons control the output and plasticity of excitatory neurons. Within this diverse group, parvalbumin-expressing (PV) cells form the larger inhibitory system. PV cell connectivity develops slowly, reaching maturity only at the end of adolescence; however, the mechanisms controlling the timing of its maturation are not well understood. We discovered that the expression of the neurotrophin receptor p75NTR in PV cells inhibits the maturation of their connectivity in a cell-autonomous fashion, both in vitro and in vivo, and that p75NTR activation in adult PV cells promotes their remodeling and restores cortical plasticity. These results reveal a new p75NTR function in the regulation of the time course of PV cell maturation and in limiting cortical plasticity.
Assuntos
Envelhecimento/fisiologia , Interneurônios/fisiologia , Plasticidade Neuronal/fisiologia , Receptores de Fator de Crescimento Neural/fisiologia , Maturidade Sexual/fisiologia , Córtex Visual/crescimento & desenvolvimento , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Conectoma , Potenciais Evocados Visuais , Feminino , Neurônios GABAérgicos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Interneurônios/química , Interneurônios/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Parvalbuminas/análise , Precursores de Proteínas/farmacologia , Distribuição Aleatória , Receptores de Fator de Crescimento Neural/biossíntese , Receptores de Fator de Crescimento Neural/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sinapses/fisiologia , Visão Monocular/fisiologia , Córtex Visual/citologia , Córtex Visual/metabolismoRESUMO
Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS-expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS-immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα-immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFPVglut2 , EYFPVgat , and GFPGad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes.
Assuntos
Hipotálamo/crescimento & desenvolvimento , Hipotálamo/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Hipotálamo/citologia , Imuno-Histoquímica , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
In cerebellar cortex, mGlu4 receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu4 receptors control synaptic transmission through an atypical activation of Gαq proteins. First, the Gαq subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu4 receptor. The i2 loop and the C terminal domain were used as baits, two domains that are known to play a pivotal role in coupling selectivity and efficacy. Second, in situ proximity ligation assays show that native mGlu4 receptors and Gαq subunits are in close physical proximity in cerebellar cortical slices. Finally, electrophysiological experiments demonstrate that the molecular mechanisms underlying mGlu4 receptor-mediated inhibition of transmitter release at cerebellar Parallel Fiber (PF) - Molecular Layer Interneuron (MLI) synapses involves the Gαq-PLC signaling pathway. Taken together, our results provide compelling evidence that, in the rodent cerebellar cortex, mGlu4 receptors act by coupling to the Gαq protein and PLC effector system to reduce glutamate synaptic transmission.
Assuntos
Córtex Cerebelar/citologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Benzopiranos/farmacologia , Citoplasma/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Rede Nervosa/efeitos dos fármacos , Propionatos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/genética , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genéticaRESUMO
The olfacto-genital syndrome (Kallmann syndrome) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. This is a genetically heterogeneous developmental disease with various modes of transmission, including oligogenic inheritance. Previous reports have involved defective cell signaling by semaphorin-3A in the disease pathogenesis. Here, we report that the embryonic phenotype of Plxna1-/- mutant mice lacking plexin-A1 (a major receptor of class 3 semaphorins), though not fully penetrant, resembles that of Kallmann syndrome fetuses. Pathohistological analysis indeed showed a strongly abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the hypothalamic brain region in some of the mutant mice, which resulted in reduced fertility in adult males. We thus screened 250 patients for the presence of mutations in PLXNA1, and identified different nonsynonymous mutations (p.V349L, p.V437L, p.R528W, p.H684Y, p.G720E, p.R740H, p.R813H, p.R840Q, p.A854T, p.R897H, p.L1464V, p.K1618T, p.C1744F), all at heterozygous state, in 15 patients. Most of these mutations are predicted to affect plexin-A1 stability or signaling activity based on predictive algorithms and a structural model of the protein. Moreover, in vitro experiments allowed us to show the existence of deleterious effects of eight mutations (including a transcript splicing defect), none of which are expected to result in a complete loss of protein synthesis, targeting, or signaling activity, though. Our findings indicate that signaling insufficiency through plexin-A1 can contribute to the pathogenesis of Kallmann syndrome, and further substantiate the oligogenic pattern of inheritance in this developmental disorder.
Assuntos
Síndrome de Kallmann/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Adulto , Animais , Movimento Celular , Feminino , Hormônio Liberador de Gonadotropina/deficiência , Heterozigoto , Humanos , Hipogonadismo/genética , Hipotálamo/metabolismo , Masculino , Camundongos , Mutação , Proteínas do Tecido Nervoso/fisiologia , Células Neuroendócrinas/metabolismo , Neurônios/metabolismo , Bulbo Olfatório/fisiologia , Receptores de Superfície Celular/fisiologia , Reprodução , Semaforina-3A/genética , Semaforina-3A/metabolismo , Semaforinas/metabolismo , Transdução de SinaisRESUMO
Subcellular target recognition in the CNS is the culmination of a multiple-step program including axon guidance, target recognition, and synaptogenesis. In cerebellum, basket cells (BCs) innervate the soma and axon initial segment (AIS) of Purkinje cells (PCs) to form the pinceau synapse, but the underlying mechanisms remain incompletely understood. Here, we demonstrate that neuropilin-1 (NRP1), a Semaphorin receptor expressed in BCs, controls both axonal guidance and subcellular target recognition. We show that loss of Semaphorin 3A function or specific deletion of NRP1 in BCs alters the stereotyped organization of BC axon and impairs pinceau synapse formation. Further, we identified NRP1 as a trans-synaptic binding partner of the cell adhesion molecule neurofascin-186 (NF186) expressed in the PC AIS during pinceau synapse formation. These findings identify a dual function of NRP1 in both axon guidance and subcellular target recognition in the construction of GABAergic circuitry.
Assuntos
Orientação de Axônios/fisiologia , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Neurônios GABAérgicos/fisiologia , Neuropilina-1/fisiologia , Animais , Células CHO , Moléculas de Adesão Celular/metabolismo , Técnicas de Cocultura , Cricetulus , Humanos , Fatores de Crescimento Neural/metabolismo , Neurogênese/fisiologia , Células de Purkinje/fisiologia , Semaforina-3A/fisiologia , Sinapses/fisiologiaRESUMO
Episodic ataxia type-2 (EA2) is a dominantly inherited human neurological disorder caused by loss of function mutations in the CACNA1A gene, which encodes the CaV2.1 subunit of P/Q-type voltage-gated calcium channels. It remains however unknown whether the deficit of cerebellar CaV2.1 in adult is in direct link with the disease. To address this issue, we have used lentiviral based-vector RNA interference (RNAi) to knock-down CaV2.1 expression in the cerebellum of adult mice. We show that suppression of the P/Q-type channels in Purkinje neurons induced motor abnormalities, such as imbalance and ataxic gait. Interestingly, moderate channel suppression caused no basal ataxia, while ß-adrenergic activation and exercise mimicked stress induced motor disorders. Moreover, stress-induced ataxia was stable, non-progressive and totally abolished by acetazolamide, a carbonic anhydrase inhibitor used to treat EA2. Altogether, these data reveal that P/Q-type channel suppression in adult mice supports the episodic status of EA2 disease.
Assuntos
Ataxia/etiologia , Canais de Cálcio Tipo N/metabolismo , Cerebelo/patologia , Nistagmo Patológico/etiologia , Células de Purkinje/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/fisiologia , Animais , Ataxia/genética , Ataxia/patologia , Ataxia/fisiopatologia , Canais de Cálcio Tipo N/genética , Cerebelo/metabolismo , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Movimento/fisiologia , Nistagmo Patológico/genética , Nistagmo Patológico/patologia , Nistagmo Patológico/fisiopatologia , Equilíbrio Postural/genética , RNA Interferente Pequeno/genética , Transdução GenéticaRESUMO
The establishment of precise neural circuits during development involves a variety of contact-mediated and secreted guidance molecules that are expressed in a complementary fashion by different cell types. To build a functional circuit, each cell type must first trigger an intrinsic genetic program that is led by their environment at a key time point. It is therefore essential to identify the different cell-specific and stage-specific transcriptional profiles expressed by neurons. However, very few studies have been done to address this issue thus far. Herein, we have carried out a large-scale quantitative real-time PCR analysis of all classical axon guidance molecules (i.e., Semaphorins, Netrins, Ephrins, and Slits) and their receptors expressed by Purkinje cells (PCs) at specific stages of postnatal cerebellar development in vivo. Most cerebellar connections are setup in a well-characterized sequential manner during postnatal development and lead to the fine regulation of the PC, the sole output of the structure. Our analysis of the relative expression of these guidance cues has uncovered a dynamic expression pattern corresponding to specific stages of cerebellar development, thus providing a starting point for studying the role of these axon guidance molecules in cerebellar wiring.