RESUMO
Peripheral nerve injuries result in motor and sensory dysfunction which can be recovered by compensatory or regenerative processes. In situations where axonal regeneration of injured neurons is hampered, compensation by collateral sprouting from uninjured neurons contributes to target reinnervation and functional recovery. Interestingly, this process of collateral sprouting from uninjured neurons has been associated with the activation of growth-associated programs triggered by Wallerian degeneration. Nevertheless, the molecular alterations at the transcriptomic level associated with these compensatory growth mechanisms remain to be fully elucidated. We generated a surgical model of partial sciatic nerve injury in mice to mechanistically study degeneration-induced collateral sprouting from spared fibers in the peripheral nervous system. Using next-generation sequencing and Ingenuity Pathway Analysis, we described the sprouting-associated transcriptome of uninjured sensory neurons and compare it with the activated by regenerating neurons. In vitro approaches were used to functionally assess sprouting gene candidates in the mechanisms of axonal growth. Using a novel animal model, we provide the first description of the sprouting transcriptome observed in uninjured sensory neurons after nerve injury. This collateral sprouting-associated transcriptome differs from that seen in regenerating neurons, suggesting a molecular program distinct from axonal growth. We further demonstrate that genetic upregulation of novel sprouting-associated genes activates a specific growth program in vitro, leading to increased neuronal branching. These results contribute to our understanding of the molecular mechanisms associated with collateral sprouting in vivo. The data provided here will therefore be instrumental in developing therapeutic strategies aimed at promoting functional recovery after injury to the nervous system.
Assuntos
Perfilação da Expressão Gênica , Neurogênese/genética , Nervos Periféricos/fisiologia , Células Receptoras Sensoriais/fisiologia , Transcriptoma/genética , Animais , Proliferação de Células , Feminino , Gânglios Espinais/patologia , Regulação da Expressão Gênica , Vértebras Lombares/patologia , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/patologia , Nervos Periféricos/ultraestrutura , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Células Receptoras Sensoriais/ultraestrutura , Degeneração Walleriana/genética , Degeneração Walleriana/patologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Parkinson's disease (PD) is the second most common neurodegenerative condition, characterized by motor impairment due to the progressive degeneration of dopaminergic neurons in the substantia nigra and depletion of dopamine release in the striatum. Accumulating evidence suggest that degeneration of axons is an early event in the disease, involving destruction programs that are independent of the survival of the cell soma. Necroptosis, a programmed cell death process, is emerging as a mediator of neuronal loss in models of neurodegenerative diseases. Here, we demonstrate activation of necroptosis in postmortem brain tissue from PD patients and in a toxin-based mouse model of the disease. Inhibition of key components of the necroptotic pathway resulted in a significant delay of 6-hydroxydopamine-dependent axonal degeneration of dopaminergic and cortical neurons in vitro. Genetic ablation of necroptosis mediators MLKL and RIPK3, as well as pharmacological inhibition of RIPK1 in preclinical models of PD, decreased dopaminergic neuron degeneration, improving motor performance. Together, these findings suggest that axonal degeneration in PD is mediated by the necroptosis machinery, a process here referred to as necroaxoptosis, a druggable pathway to target dopaminergic neuronal loss.
Assuntos
Axônios/patologia , Necroptose , Degeneração Neural/patologia , Doença de Parkinson/patologia , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Neuritos/patologia , Oxidopamina , Doença de Parkinson/fisiopatologia , Fosforilação , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury.
Assuntos
Regeneração Nervosa , Traumatismos dos Nervos Periféricos/fisiopatologia , Nervo Isquiático/fisiopatologia , Resposta a Proteínas não Dobradas , Fator 4 Ativador da Transcrição/metabolismo , Animais , Axônios/fisiologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Estresse do Retículo Endoplasmático , Expressão Gênica , Locomoção , Macrófagos/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recuperação de Função Fisiológica , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson's disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.
Assuntos
Axônios/fisiologia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Regeneração , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Corpo Estriado/metabolismo , Denervação , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Atividade Motora/genética , Degeneração Neural/genética , Degeneração Neural/patologia , Fenômenos Fisiológicos do Sistema Nervoso , Oxidopamina/farmacologia , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/fisiopatologia , Traumatismos dos Nervos Periféricos/reabilitaçãoRESUMO
BACKGROUND: The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh) pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1) and novel Hh-regulated genes in zebrafish embryos. RESULTS: The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo. CONCLUSION: A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in different contexts of vertebrate embryonic development.
Assuntos
Técnicas Genéticas , Proteínas Hedgehog/metabolismo , Proteínas Oncogênicas/metabolismo , Saccharomyces cerevisiae , Transativadores/metabolismo , Animais , Linhagem Celular , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Técnicas Genéticas/normas , Proteínas Hedgehog/agonistas , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transativadores/antagonistas & inibidores , Transativadores/genética , Alcaloides de Veratrum/farmacologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
Despite considerable progress, the mechanisms that control neural progenitor differentiation and behavior, as well as their functional integration into adult neural circuitry, are far from being understood. Given the complexity of the mammalian brain, non-mammalian models provide an excellent model to study neurogenesis, including both the cellular composition of the neurogenic microenvironment, and the factors required for precursor growth and maintenance. In particular, we chose to address the question of the control of progenitor proliferation by Sonic hedgehog (Shh) using the zebrafish dorsal mesencephalon, known as the optic tectum (OT), as a model system. Here we show that either inhibiting pharmacologically or eliminating hedgehog (Hh) signaling by using mutants that lack essential components of the Hh pathway reduces neural progenitor cell proliferation affecting neurogenesis in the OT. On the contrary, pharmacological gain-of-function experiments result in significant increase in proliferation. Importantly, Shh-dependent function controls neural progenitor cell behavior as sox2-positive cell populations were lost in the OT in the absence of Hh signaling, as evidenced in slow-muscle-omitted (smu) mutants and with timed cyclopamine inhibition. Expressions of essential components of the Hh pathway reveal for the first time a late dorsal expression in the embryonic OT. Our observations argue strongly for a role of Shh in neural progenitor biology in the OT and provide comparative data to our current understanding of progenitor/stem cell mechanisms that place Shh as a key niche factor in the dorsal brain.