RESUMO
Cancer immunotherapy approaches target signaling pathways that are highly synonymous between CD4 and CD8 T-cell subsets and, therefore, often stimulate nonspecific lymphocyte activation, resulting in cytotoxicity to otherwise healthy tissue. The goal of our study was to identify intrinsic modulators of basic T lymphocyte activation pathways that could discriminately bolster CD8 anti-tumor effector responses. Using a Tbc1d10c null mouse, we observed marked resistance to a range of tumor types conferred by Tbc1d10c deficiency. Moreover, tumor-bearing Tbc1d10c null mice receiving PD-1 or CTLA-4 monotherapy exhibited a 33% or 90% cure rate, respectively. While Tbc1d10c was not expressed in solid tumor cells, Tbc1d10c disruption selectively augmented CD8 T-cell activation and cytotoxic effector responses and adoptive transfer of CD8 T cells alone was sufficient to recapitulate Tbc1d10c null tumor resistance. Mechanistically, Tbc1d10c suppressed CD8 T-cell activation and anti-tumor function by intersecting canonical NF-κB pathway activation via regulation of Map3k3-mediated IKKß phosphorylation. Strikingly, none of these cellular or molecular perturbations in the NF-κB pathway were featured in Tbc1d10c null CD4 T cells. Our findings identify a Tbc1d10c-Map3k3-NF-κB signaling axis as a viable therapeutic target to promote CD8 T-cell anti-tumor immunity while circumventing CD4 T cell-associated cytotoxicity and NF-κB activation in tumor cells.
Assuntos
NF-kappa B , Neoplasias , Camundongos , Animais , NF-kappa B/metabolismo , Linfócitos T CD8-Positivos , Ativação Linfocitária , Neoplasias/terapia , Subpopulações de Linfócitos T/metabolismo , Proteínas Ativadoras de GTPase/genéticaRESUMO
The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson's disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications.
RESUMO
The regulation of protein degradation is essential for maintaining the appropriate environment to coordinate complex cell signaling events and to promote cellular remodeling. The Autophagy linked FYVE protein (Alfy), previously identified as a molecular scaffold between the ubiquitinated cargo and the autophagic machinery, is highly expressed in the developing central nervous system, indicating that this pathway may have yet unexplored roles in neurodevelopment. To examine this possibility, we used mouse genetics to eliminate Alfy expression. We report that this evolutionarily conserved protein is required for the formation of axonal tracts throughout the brain and spinal cord, including the formation of the major forebrain commissures. Consistent with a phenotype reflecting a failure in axon guidance, the loss of Alfy in mice disrupts localization of glial guidepost cells, and attenuates axon outgrowth in response to Netrin-1. These findings further support the growing indication that macroautophagy plays a key role in the developing CNS.
Assuntos
Encéfalo/embriologia , Vias Neurais/embriologia , Neurônios/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Relacionadas à Autofagia , Técnicas de Inativação de Genes , Camundongos Endogâmicos C57BLRESUMO
There are currently no therapies that provide either protection or restoration of neuronal function for adult-onset neurodegenerative diseases such as Parkinson's disease (PD). Many clinical efforts to provide such benefits by infusion of neurotrophic factors have failed, in spite of robust effects in preclinical assessments. One important reason for these failures is the difficulty, due to diffusion limits, of providing these protein molecules in sufficient amounts to the intended cellular targets in the central nervous system. This challenge suggests an alternative approach, that of viral vector transduction to directly activate the intracellular signaling pathways that mediate neurotrophic effects. To this end we have investigated the ability of a constitutively active form of the GTPase Rheb, an important activator of mammalian target of rapamycin (mTor) signaling, to mediate neurotrophic effects in dopamine neurons of the substantia nigra (SN), a population of neurons affected in PD. We find that constitutively active hRheb(S16H) induces many neurotrophic effects in mice, including abilities to both preserve and restore the nigrostriatal dopaminergic axonal projections in a highly destructive neurotoxin model. We conclude that direct viral vector transduction of vulnerable neuronal populations to activate intracellular neurotrophic signaling pathways offers promise for the treatment of neurodegenerative disease.
Assuntos
Axônios/metabolismo , Dependovirus/genética , Neurônios Dopaminérgicos/metabolismo , Vetores Genéticos/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Neuropeptídeos/genética , Doença de Parkinson/prevenção & controle , Transdução Genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Axônios/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Fatores de Iniciação em Eucariotos , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Oxidopamina/efeitos adversos , Doença de Parkinson/terapia , Fosfoproteínas/metabolismo , Fosforilação , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismoRESUMO
OBJECTIVE: A prevailing concept in neuroscience has been that the adult mammalian central nervous system is incapable of restorative axon regeneration. Recent evidence, however, has suggested that reactivation of intrinsic cellular programs regulated by protein kinase B (Akt)/mammalian target of rapamycin (mTor) signaling may restore this ability. METHODS: To assess this possibility in the brain, we have examined the ability of adenoassociated virus (AAV)-mediated transduction of dopaminergic neurons of the substantia nigra (SN) with constitutively active forms of the kinase Akt and the GTPase Ras homolog enriched in brain (Rheb) to induce regrowth of axons after they have been destroyed by neurotoxin lesion. RESULTS: Both constitutively active myristoylated Akt and hRheb(S16H) induce regrowth of axons from dopaminergic neurons to their target, the striatum. Histological analysis demonstrates that these new axons achieve morphologically accurate reinnervation. In addition, functional reintegration into target circuitry is achieved, as indicated by partial behavioral recovery. INTERPRETATION: We conclude that regrowth of axons within the adult nigrostriatal projection, a system that is prominently affected in Parkinson's disease, can be achieved by activation of Akt/mTor signaling in surviving endogenous mesencephalic dopaminergic neurons by viral vector transduction.
Assuntos
Axônios/fisiologia , Dopamina/fisiologia , Proteínas Monoméricas de Ligação ao GTP/administração & dosagem , Regeneração Nervosa/fisiologia , Neuropeptídeos/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/administração & dosagem , Transdução de Sinais , Animais , Axônios/metabolismo , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Monoméricas de Ligação ao GTP/genética , Regeneração Nervosa/genética , Neurônios/citologia , Neurônios/patologia , Neurônios/virologia , Neuropeptídeos/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais/genética , Substância Negra/metabolismo , Substância Negra/patologia , Substância Negra/virologiaRESUMO
Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.
Assuntos
Autofagia/fisiologia , Axônios/metabolismo , Dopamina/metabolismo , Neurônios/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Degeneração Retrógrada/metabolismo , Degeneração Retrógrada/patologia , Animais , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Dependovirus/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Feixe Prosencefálico Mediano/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Transmissão/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Oxidopamina/efeitos adversos , Proteínas Proto-Oncogênicas c-akt/genética , Degeneração Retrógrada/etiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Substância Negra/patologia , Serina-Treonina Quinases TOR/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Brain-derived neurotrophic factor (BDNF) was the first purified molecule identified to directly support the development of mesencephalic dopamine neurons. However, its physiologic role has remained unknown. Based on patterns of expression, it is unlikely to serve as a target-derived neurotrophic factor, but it may instead act locally in the mesencephalon, either released by afferent projections, or in autocrine fashion. To assess a possible local role, we blocked BDNF signaling in the substantia nigra (SN) of postnatal rats by injection of either neutralizing antibodies or a peptide antagonist. These treatments increased the magnitude of developmental cell death in the SN, indicating that endogenous local BDNF does play a regulatory role. However, we also find that elimination of BDNF in brain throughout postnatal development in BDNF(fl/fl):Nestin-Cre mice has no effect on the adult number of SN dopamine neurons. We postulate that other forms of trophic support may compensate for the elimination of BDNF during early development. Although the number of SN dopamine neurons is unchanged, their organization is disrupted. We conclude that BDNF plays a physiologic role in the postnatal development of SN dopamine neurons.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dopamina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/fisiologia , Substância Negra/citologia , Substância Negra/crescimento & desenvolvimento , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/imunologia , Morte Celular/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas/métodos , Proteínas de Filamentos Intermediários/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Nestina , Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Dopamine neurons of the substantia nigra (SN) undergo a natural cell death event which is biphasic, with peaks at postnatal days (PNDs) 2 and 14. There is growing evidence that GDNF functions as a striatal target-derived neurotrophic factor to regulate the first phase. It has been unknown whether the GDNF receptor, GFRalpha1, may play a role in regulating either phase. To evaluate a possible role for GFRalpha1 we have examined its expression throughout postnatal development in the SN and particularly in the striatum, where its expression has been uncertain. GFRalpha1 mRNA is highly expressed in SN, as previously shown, with highest levels at PND14-28. We find that it is also expressed in striatum with a similar time course, but with a more discrete period of maximal expression between PND10 and PND14. The cellular basis of this maximum of expression is an increased number of GFRalpha1 mRNA-positive medium-sized neurons evenly distributed within the striatum. Immunostaining reveals GFRalpha1 protein-positive neurons with a similar morphology and distribution. We conclude that GFRalpha1 is expressed in striatum maximally late in postnatal development. In this location it may act in trans to influence the viability and development of nigral dopamine neurons.