RESUMO
Cortical interneurons establish inhibitory microcircuits throughout the neocortex and their dysfunction has been implicated in epilepsy and neuropsychiatric diseases. Developmentally, interneurons migrate from a distal progenitor domain in order to populate the neocortex - a process that occurs at a slower rate in humans than in mice. In this study, we sought to identify factors that regulate the rate of interneuron maturation across the two species. Using embryonic mouse development as a model system, we found that the process of initiating interneuron migration is regulated by blood vessels of the medial ganglionic eminence (MGE), an interneuron progenitor domain. We identified two endothelial cell-derived paracrine factors, SPARC and SerpinE1, that enhance interneuron migration in mouse MGE explants and organotypic cultures. Moreover, pre-treatment of human stem cell-derived interneurons (hSC-interneurons) with SPARC and SerpinE1 prior to transplantation into neonatal mouse cortex enhanced their migration and morphological elaboration in the host cortex. Further, SPARC and SerpinE1-treated hSC-interneurons also exhibited more mature electrophysiological characteristics compared to controls. Overall, our studies suggest a critical role for CNS vasculature in regulating interneuron developmental maturation in both mice and humans.
Assuntos
Movimento Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Eminência Mediana/irrigação sanguínea , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Osteonectina/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Potenciais de Ação , Animais , Córtex Cerebral/embriologia , Córtex Cerebral/cirurgia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Interneurônios/metabolismo , Interneurônios/transplante , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Eminência Mediana/embriologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Neovascularização Fisiológica , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/transplante , Osteonectina/metabolismo , Comunicação Parácrina , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Transdução de SinaisRESUMO
Cortical interneurons (cINs) are locally projecting inhibitory neurons that are distributed throughout the cortex. Due to their relatively limited range of influence, their arrangement in the cortex is critical to their function. cINs achieve this arrangement through a process of tangential and radial migration and apoptosis during development. In this study, we investigated the role of clustered protocadherins (cPcdhs) in establishing the spatial patterning of cINs through the use of genetic cPcdh knockout mice. cPcdhs are expressed in cINs and are known to play key functions in cell spacing and cell survival, but their role in cINs is poorly understood. Using spatial statistical analysis, we found that the 2 main subclasses of cINs, parvalbumin-expressing and somatostatin-expressing (SST) cINs, are nonrandomly spaced within subclass but randomly with respect to each other. We also found that the relative laminar distribution of each subclass was distinctly altered in whole α- or ß-cluster mutants. Examination of perinatal time points revealed that the mutant phenotypes emerged relatively late, suggesting that cPcdhs may be acting during cIN morphological elaboration and synaptogenesis. We then analyzed an isoform-specific knockout for pcdh-αc2 and found that it recapitulated the α-cluster knockout but only in SST cells, suggesting that subtype-specific expression of cPcdh isoforms may help govern subtype-specific spatial distribution.
RESUMO
Whereas past work indicates that cortical interneurons (cINs) can be generically produced from stem cells, generating large numbers of specific subtypes of this population has remained elusive. This reflects an information gap in our understanding of the transcriptional programs required for different interneuron subtypes. Here, we have utilized the directed differentiation of stem cells into specific subpopulations of cortical interneurons as a means to identify some of these missing factors. To establish this approach, we utilized two factors known to be required for the generation of cINs, Nkx2-1 and Dlx2. As predicted, their regulated transient expression greatly improved the differentiation efficiency and specificity over baseline. We extended upon this "cIN-primed" model in order to establish a modular system whereby a third transcription factor could be systematically introduced. Using this approach, we identified Lmo3 and Pou3f4 as genes that can augment the differentiation and/or subtype specificity of cINs in vitro.
Assuntos
Córtex Cerebral/citologia , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Interneurônios/classificação , Interneurônios/fisiologia , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Técnicas In Vitro , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Técnicas de Patch-Clamp , Transplante de Células-Tronco , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução GenéticaRESUMO
Recreating developmental structures in vitro has been a primary challenge for stem cell biologists. Recent studies in Cell Stem Cell (Eiraku et al., 2008) and Nature (Gaspard et al., 2008) demonstrate that embryonic stem cells can recapitulate early cortical development, enabling them to generate specific cortical subtypes.
Assuntos
Linhagem da Célula/fisiologia , Córtex Cerebral/crescimento & desenvolvimento , Células-Tronco Embrionárias/citologia , Neurogênese/fisiologia , Neurônios/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Padronização Corporal , Diferenciação Celular , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Células-Tronco Embrionárias/fisiologia , Fatores de Transcrição Forkhead/genética , Técnicas de Introdução de Genes , Homeostase , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/transplante , Proteínas tau/genéticaRESUMO
Olfactory ensheathing cells (OECs) transplanted into the lesioned CNS can stimulate reportedly different degrees of regeneration, remyelination, and functional recovery, but little is known about the mechanisms OECs may use to stimulate endogenous repair. Here, we used a functional proteomic approach, isotope-coded affinity tagging and mass spectrometry, to identify active components of the OEC secreteome that differentially stimulate outgrowth. SPARC (secreted protein acidic rich in cysteine) (osteonectin) was identified as an OEC-derived matricellular protein that can indirectly enhance the ability of Schwann cells to stimulate dorsal root ganglion outgrowth in vitro. SPARC stimulates Schwann cell-mediated outgrowth by cooperative signal with laminin-1 and transforming growth factor beta. Furthermore, when SPARC-null OECs were transplanted into lesioned rat spinal cord, the absence of OEC-secreted SPARC results in an attenuation of outgrowth of specific subsets of sensory and supraspinal axons and changes the pattern of macrophage infiltration in response to the transplanted cells. These data provide the first evidence for a role for SPARC in modulating different aspects of CNS repair and indicate that SPARC can change the activation state of endogenous Schwann cells, resulting in the promotion of outgrowth in vitro, and in vivo.
Assuntos
Neuritos/fisiologia , Bulbo Olfatório/metabolismo , Osteonectina/metabolismo , Células de Schwann/fisiologia , Traumatismos da Medula Espinal/terapia , Animais , Bovinos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuritos/efeitos dos fármacos , Bulbo Olfatório/citologia , Bulbo Olfatório/transplante , Osteonectina/uso terapêutico , Ratos , Ratos Sprague-Dawley , Células de Schwann/efeitos dos fármacos , Traumatismos da Medula Espinal/metabolismoRESUMO
Bridging of a lesion site and minimizing local damage to create an environment permissive for regeneration are both primary components of a successful strategy to repair spinal cord injury (SCI). Olfactory ensheathing cells (OECs) are prime candidates for autologous transplantation to bridge this gap, but little is known currently about their mechanism of action. In addition, OECs from the accessible lamina propria (LP) of the olfactory mucosa are a more viable source in humans but have yet to be tested for their ability to promote regeneration in established SCI models. Here, mouse LP-OECs expressing green fluorescent protein (GFP) transplanted directly into both rat and mouse dorsolateral spinal cord lesion sites demonstrate limited migration but interact with host astrocytes to develop a new transitional zone at the lesion border. LP-OECs also promote extensive migration of host Schwann cells into the central nervous system repair zone and stimulate angiogenesis to provide a biological scaffold for repair. This novel environment created by transplanted and host glia within the spinal cord inhibits cavity and scar formation and promotes extensive sprouting of multiple sensory and motor axons into and through the lesion site. Sixty days after rat SCI, serotonin- and tyrosine hydroxylase-positive axons sprouted across the lesion into the distal cord, although axotomized rubrospinal axons did not. Thus, even in a xenotransplant paradigm, LP-OECs work collaboratively with host glial cells to create an environment to ameliorate local damage and simultaneously promote a regenerative response in multiple axonal populations.
Assuntos
Biotina/análogos & derivados , Neuroglia/fisiologia , Mucosa Olfatória/patologia , Regeneração/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Cicatrização/fisiologia , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/transplante , Biotina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Dextranos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde , Imuno-Histoquímica/métodos , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neurofilamentos/metabolismo , Neuroglia/transplante , Proteínas Oncogênicas v-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Traumatismos da Medula Espinal/patologia , Fatores de Tempo , Transplante Autólogo/métodos , Tubulina (Proteína)/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Pituitary adenylate cyclase activating peptide (PACAP), a neuroregulatory peptide, is found in germinative regions of the CNS, including the olfactory bulb, throughout adulthood. We show that 1) PACAP immunoreactivity is also present in the neonatal mouse and adult mouse and rat olfactory epithelium, 2) PACAP expression pattern differs between neonatal and adult mice, and 3) PACAP is produced by olfactory ensheathing cells. PACAP may thus be a key factor in the uniquely supportive role of olfactory ensheathing cells in regeneration of neurons from olfactory epithelium and lesioned spinal cord. Using calcium imaging, we demonstrated physiological responses to PACAP in both neonatal and adult olfactory receptor neurons (ORNs). We propose that PACAP plays an important role in normal turnover of ORNs by providing neurotrophic support during development and regeneration and neuroprotective support of mature neurons.
Assuntos
Cálcio/metabolismo , Neuropeptídeos/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Olfato/fisiologia , Animais , Animais Recém-Nascidos , Camundongos , Neuropeptídeos/análise , Neuropeptídeos/biossíntese , Neurônios Receptores Olfatórios/química , Neurônios Receptores Olfatórios/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Ratos , Ratos Sprague-Dawley , Especificidade da EspécieRESUMO
Olfactory ensheathing cells (OECs) continuously support the regeneration of olfactory receptor neurons (ORNs). In addition, OECs promote regeneration of neurons within the CNS in a number of transplantation paradigms, but details of exactly how they support regeneration remain elusive. The majority of studies using OECs to promote regeneration have thus far focused on understanding the cell biology of OECs purified from the olfactory bulb (OB). Here we show that a population of OECs similar to those obtained from the OB is present in the lamina propria (LP) beneath the olfactory epithelium (OE). These OECs are the first glial cells encountered by the axons of developing ORNs as they exit the OE and display distinct and variable expression of p75, S100beta, GFAP, and O4, characteristic markers of bulb OECs. Once purified in vitro, they display Schwann cell-like and astrocyte-like properties and expand rapidly. In addition to resembling OB-OECs, LP-OECs also express a unique combination of developmentally important proteins-CD 44, beta1 integrin, P200, Notch 3, NG2, VEGF, and PACAP and CREB binding protein (CBP/p300)-not previously reported in OB-OECs. These data suggest that LP-OECs, like OB-OECs, are a developmentally distinct class of glia that are capable of both immature and mature function, depending on environmental stimuli, within the adult nervous system.