RESUMO
BACKGROUND: Inhibitory factors have been implicated in the failure of remyelination in demyelinating diseases. Myelin associated inhibitors act through a common receptor called Nogo receptor (NgR) that plays critical inhibitory roles in CNS plasticity. Here we investigated the effects of abrogating NgR inhibition in a non-immune model of focal demyelination in adult mouse optic chiasm. METHODOLOGY/PRINCIPAL FINDINGS: A focal area of demyelination was induced in adult mouse optic chiasm by microinjection of lysolecithin. To knock down NgR levels, siRNAs against NgR were intracerebroventricularly administered via a permanent cannula over 14 days, Functional changes were monitored by electrophysiological recording of latency of visual evoked potentials (VEPs). Histological analysis was carried out 3, 7 and 14 days post demyelination lesion. To assess the effect of NgR inhibition on precursor cell repopulation, BrdU was administered to the animals prior to the demyelination induction. Inhibition of NgR significantly restored VEPs responses following optic chiasm demyelination. These findings were confirmed histologically by myelin specific staining. siNgR application resulted in a smaller lesion size compared to control. NgR inhibition significantly increased the numbers of BrdU+/Olig2+ progenitor cells in the lesioned area and in the neurogenic zone of the third ventricle. These progenitor cells (Olig2+ or GFAP+) migrated away from this area as a function of time. CONCLUSIONS/SIGNIFICANCE: Our results show that inhibition of NgR facilitate myelin repair in the demyelinated chiasm, with enhanced recruitment of proliferating cells to the lesion site. Thus, antagonizing NgR function could have therapeutic potential for demyelinating disorders such as Multiple Sclerosis.
Assuntos
Doenças Desmielinizantes/terapia , Proteínas da Mielina/antagonistas & inibidores , Bainha de Mielina/metabolismo , Quiasma Óptico/metabolismo , Receptores de Superfície Celular/genética , Recuperação de Função Fisiológica , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bromodesoxiuridina/administração & dosagem , Movimento Celular , Proliferação de Células , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Potenciais Evocados Visuais , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica , Injeções Intraventriculares , Lisofosfatidilcolinas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Bainha de Mielina/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptor Nogo 1 , Fator de Transcrição 2 de Oligodendrócitos , Quiasma Óptico/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de TempoRESUMO
RNA interference (RNAi) is a major tool for basic and applied investigations. However, obtaining RNAi data that have physiological significance requires investigation of regulations and therapeutic strategies in appropriate in vivo settings. To examine in vivo gene regulation and protein function in the adult neural stem cell (NSC) niche, we optimized a new non-viral vector for delivery of siRNA into the subventricular zone (SVZ). This brain region contains the neural stem and progenitor cells populations that express the stem cell marker, SOX2. Temporally and spatially controlled Sox2 knockdown was achieved using the monocationic lipid vector, IC10. siRNA/IC10 complexes were stable over time and smaller (<40 nm) than jetSi complexes (≈400 nm). Immunocytochemistry showed that siRNA/IC10 complexes efficiently target both the progenitor and stem cell populations in the adult SVZ. Injection of the complexes into the lateral brain ventricle resulted in specific knockdown of Sox2 in the SVZ. Furthermore, IC10-mediated transient in vivo knockdown of Sox2-modulated expression of several genes implicated in NSC maintenance. Taken together, these data show that IC10 cationic lipid formulation can efficiently vectorize siRNA in a specific area of the adult mouse brain, achieving spatially and temporally defined loss of function.Molecular Therapy-Nucleic Acids (2013) 2, e89; doi:10.1038/mtna.2013.8; published online 23 April 2013.
RESUMO
The subventricular zone (SVZ) neural stem cell niche contains mixed populations of stem cells, transit-amplifying cells, and migrating neuroblasts. Deciphering how endogenous signals, such as hormones, affect the balance between these cell types is essential for understanding the physiology of niche plasticity and homeostasis. We show that Thyroid Hormone (T(3)) and its receptor, TRα1, are directly involved in maintaining this balance. TRα1 is expressed in amplifying and migrating cells. In vivo gain- and loss-of-function experiments demonstrate first, that T(3)/TRα1 directly repress Sox2 expression, and second, that TRα1 overexpression in the niche favors the appearance of DCX+ migrating neuroblasts. Lack of TRα increases numbers of SOX2+ cells in the SVZ. Hypothyroidism increases proportions of cells in interphase. Thus, in the adult SVZ, T(3)/TRα1 together favor neural stem cell commitment and progression toward a migrating neuroblast phenotype; this transition correlates with T(3)/TRα1-dependent transcriptional repression of Sox2.
Assuntos
Células-Tronco Adultas/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/genética , Fatores de Transcrição SOXB1/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Movimento Celular/genética , Proteína Duplacortina , Repressão Enzimática/genética , Camundongos , Camundongos Mutantes , RNA Interferente Pequeno/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais , Nicho de Células-Tronco/genética , Receptores alfa dos Hormônios Tireóideos/genética , Hormônios Tireóideos/genética , Transgenes/genéticaRESUMO
The development of the catecholaminergic system of the brain of the lamprey (Lampetra fluviatilis) was studied with immunocytochemistry in a series of larvae of different sizes by using two different antibodies directed against tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. In group 1 larvae (length: 29-54 mm, ages: 8 months to 1.5 years), the only TH-immunoreactive somata observed were located in the caudal wall of the recessus praeopticus (RP) and in the nucleus tuberculi posterioris (NTP). In group 2 larvae (length: 55-80 mm, ages: 1.5-2.5 years), the somata of immunolabeled cells of the NTP give rise to fibers, most of which are ascending and terminate in the corpus striatum. Additional immunoreactive cells are observed in the nucleus praeopticus (NP), which has differentiated, and in the spinal cord. In group 3 larvae (length: 81-110 mm, ages: 2.5-4 years), the spatial distribution of TH-immunoreactive elements (somata, fibers, and terminals) bears many resemblances to that seen in the adult. Immunolabeled cells may be observed in the olfactory bulb, in the nucleus commissurae postopticae (NCP), and in the nucleus dorsalis hypothalami (NDH). Nevertheless, some groups of TH-immunoreactive cells found in the adult are not observed in group 3 larvae; these may appear during the metamorphic phase. By comparative analysis, we show that, in spite of several differences, the spatiotemporal sequence of appearance of TH-immunoreactive cell bodies and fibers in the lamprey presents many similarities to that described in gnathostomes.
