RESUMO
It is now established that the development of the CNS requires equilibrium between cell survival and apoptosis. Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts a powerful protective effect on cerebellar granule cells by inhibiting the caspase 3. In contrast, Fas ligand (FasL) plays an essential role during ontogenesis in eliminating supernumerary neurons by apoptosis. To determine if PACAP and FasL interact during cerebellar development, we characterized the effects of these factors on cerebellar morphogenesis and caspase 3 activity in PACAP+/+ and PACAP-/- mice. First, we demonstrated in vivo that PACAP is able to reverse the diminution of internal granule cell layer thickness induced by FasL in PACAP+/+ and PACAP-/- mice. Second, ex vivo and immunohistochemical studies revealed that interaction between FasL and PACAP occurs through the caspase 3 activity. Third, behavioural study showed a significant difference for the PACAP + FasL group in the righting reflex test at P8 which does not persist at P60. Finally, a time course study revealed that the pro-apoptotic effect of FasL characterized at P8 was followed by a progressive compensatory mechanism in caspase 3 activity and bromodeoxyuridine incorporation. These data suggest that PACAP and FasL interact during cerebellar development to control apoptosis of granule cells and may affect some motor cerebellar functions.
Assuntos
Comportamento Animal/fisiologia , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Proteína Ligante Fas/metabolismo , Neurônios/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Bromodesoxiuridina/metabolismo , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Proteína Ligante Fas/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Neurônios/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reflexo/efeitos dos fármacos , Estatísticas não Paramétricas , Fatores de TempoRESUMO
Previous studies have demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic effects during neurodevelopment. In particular, the occurrence of PACAP and its receptors in the cerebellum during pre- and postnatal periods suggests that it could play a crucial role in ontogenesis of this structure. To test this hypothesis, we compared the histogenesis of cerebellar cortex in wild-type and PACAP-knockout (PACAP-/-) mice at postnatal days (P)4 and 7. Morphometric analysis of PACAP-/- mice revealed a significant reduction in the thickness of the external granule cell layer at P4 and of the internal granule cell layer at P7. Expression of nestin, a neural precursor marker, and synaptophysin, a mature neuronal marker, was quantified by real-time PCR and Western blot. No modification of nestin expression was noticed between wild-type and PACAP-/- mice, but a substantial decrease in synaptophysin expression was observed in PACAP-/- mice at P4 and P7. Immunohistochemistry revealed a reduction in synaptophysin labelling in the molecular and internal granule cell layers of PACAP-/- mice at P7. Caspase-3 activation was significantly increased in PACAP-/- mice at P4 and P7. Autoradiographic studies revealed no difference in PACAP binding site distributions and PACAP was effective at stimulating cAMP production in both wild-type and PACAP-/- cultured granule cells. This study demonstrates that disruption of the PACAP gene induces alteration of the immature cerebellum. Neuronal differentiation of granule cells was delayed whereas cell death that naturally occurs during ontogeny was increased in PACAP-/- mice. These data provide the first evidence of a physiological role for PACAP during cerebellar development.
Assuntos
Cerebelo/anormalidades , Cerebelo/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Células-Tronco/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Caspase 3/metabolismo , Morte Celular/genética , Diferenciação Celular/genética , Cerebelo/metabolismo , AMP Cíclico/metabolismo , Ativação Enzimática/fisiologia , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/fisiopatologia , Nestina , Neurônios/citologia , Células-Tronco/citologia , Sinaptofisina/metabolismoRESUMO
In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are present on granule cells during both the development period and in adulthood. Treatment of granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and induces differentiation. PACAP also protects cerebellar granule cells against the deleterious effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of expression very similar to that described in rodents, suggesting that PACAP could also exert neurodevelopmental and neuroprotective functions in the cerebellum of primates including human.
