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Many human spinal cord injuries are anatomically incomplete but exhibit complete paralysis. It is unknown why spared axons fail to mediate functional recovery in these cases. To investigate this, we undertook a small-molecule screen in mice with staggered bilateral hemisections in which the lumbar spinal cord is deprived of all direct brain-derived innervation, but dormant relay circuits remain. We discovered that a KCC2 agonist restored stepping ability, which could be mimicked by selective expression of KCC2, or hyperpolarizing DREADDs, in the inhibitory interneurons between and around the staggered spinal lesions. Mechanistically, these treatments transformed this injury-induced dysfunctional spinal circuit to a functional state, facilitating the relay of brain-derived commands toward the lumbar spinal cord. Thus, our results identify spinal inhibitory interneurons as a roadblock limiting the integration of descending inputs into relay circuits after injury and suggest KCC2 agonists as promising treatments for promoting functional recovery after spinal cord injury.
Assuntos
Traumatismos da Medula Espinal/tratamento farmacológico , Simportadores/agonistas , Simportadores/metabolismo , Animais , Axônios , Regulação da Expressão Gênica/genética , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/genética , Neurônios/metabolismo , Recuperação de Função Fisiológica/genética , Recuperação de Função Fisiológica/fisiologia , Medula Espinal , Simportadores/uso terapêutico , Cotransportadores de K e Cl-RESUMO
Dopaminergic (DA) neurons of the ventral midbrain (VM) play vital roles in the regulation of voluntary movement, emotion and reward. They are divided into the A8, A9 and A10 subgroups. The development of the A9 group of DA neurons is an area of intense investigation to aid the generation of these neurons from stem cell sources for cell transplantation approaches to Parkinson's disease (PD). This review discusses the molecular processes that are involved in the identity, specification, maturation, target innervation and survival of VM DA neurons during development. The complex molecular interactions of a number of genetic pathways are outlined, as well as recent advances in the mechanisms that regulate subset identity within the VM DA neuronal pool. A thorough understanding of the cellular and molecular mechanisms involved in the development of VM DA neurons will greatly facilitate the use of cell replacement therapy for the treatment of PD.
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Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/citologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Doença de Parkinson/terapia , Transplante de Células/métodos , Neurônios Dopaminérgicos/fisiologia , Humanos , Modelos Biológicos , Vias Neurais/fisiologia , Neurogênese/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Parkinson's disease is the second most common neurodegenerative disease, and is characterised by the progressive degeneration of the nigrostriatal dopaminergic (DA) system. Current treatments are symptomatic, and do not protect against the DA neuronal loss. One of the most promising treatment approaches is the application of neurotrophic factors to rescue the remaining population of nigrostriatal DA neurons. Therefore, the identification of new neurotrophic factors for midbrain DA neurons, and the subsequent elucidation of the molecular bases of their effects, are important. Two related members of the bone morphogenetic protein (BMP) family, BMP2 and growth differentiation factor 5 (GDF5), have been shown to have neurotrophic effects on midbrain DA neurons both in vitro and in vivo. However, the molecular (signalling pathway(s)) and cellular (direct neuronal or indirect via glial cells) mechanisms of their effects remain to be elucidated. Using the SH-SH5Y human neuronal cell line, as a model of human midbrain DA neurons, we have shown that GDF5 and BMP2 induce neurite outgrowth via a direct mechanism. Furthermore, we demonstrate that these effects are dependent on BMP type I receptor activation of canonical Smad 1/5/8 signalling.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Fator 5 de Diferenciação de Crescimento/farmacologia , Neurogênese , Proteínas Smad/metabolismo , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Humanos , Mesencéfalo/citologia , Proteínas Recombinantes/farmacologia , Proteínas Smad/genéticaRESUMO
Regulatory programs governing neuronal death and axon regeneration in neurodegenerative diseases remain poorly understood. In adult mice, optic nerve crush (ONC) injury by severing retinal ganglion cell (RGC) axons results in massive RGC death and regenerative failure. We performed an in vivo CRISPR-Cas9-based genome-wide screen of 1,893 transcription factors (TFs) to seek repressors of RGC survival and axon regeneration following ONC. In parallel, we profiled the epigenetic and transcriptional landscapes of injured RGCs by ATAC-seq and RNA-seq to identify injury-responsive TFs and their targets. These analyses converged on four TFs as critical survival regulators, of which ATF3/CHOP preferentially regulate pathways activated by cytokines and innate immunity and ATF4/C/EBPγ regulate pathways engaged by intrinsic neuronal stressors. Manipulation of these TFs protects RGCs in a glaucoma model. Our results reveal core transcription programs that transform an initial axonal insult into a degenerative process and suggest novel strategies for treating neurodegenerative diseases.
Assuntos
Traumatismos do Nervo Óptico , Células Ganglionares da Retina , Animais , Axônios/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/fisiologia , Traumatismos do Nervo Óptico/metabolismo , Células Ganglionares da Retina/fisiologiaRESUMO
The optic nerve is part of the mammalian adult central nervous system (CNS) and has limited capability to regenerate after injury. Deletion of phosphatase and tensin homolog (PTEN), a negative regulator of the PI3 kinase/Akt pathway, has been shown to promote regeneration in retinal ganglion cells (RGCs) after optic nerve injury [1]. We present the lipidome of adult PTENloxP/loxP mice subjected to intravitreal injection of adeno-associated viruses expressing Cre (AAV-Cre) as a model of CNS neuroregeneration. At 4 weeks old, PTENloxP/loxP mice were intravitreally-injected with 2-3 µl of either AAV-Cre (KO) or AAV-PLAP (control), and two weeks later optic nerve crush was performed. At indicated time-points after crush (0 days, 7 days, 14 days), mice were euthanized and optic nerves were immediately dissected out, and then flash frozen on dry ice. A modified Bligh and Dyer [2] method was used for lipid extraction from the optic nerves, followed by liquid chromatography-mass spectrometry (LC MS-MS) lipid profiling using a Q-Exactive Orbitrap instrument coupled with Accela 600 HPLC. The raw scans were analysed with LipidSearch 4.2 and the statistical analysis was conducted through Metaboanalyst 4.0. This data is available at Metabolomics Workbench, study ID ST001477.
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Characterizing neuronal cell types demands efficient strategies for specific labeling and manipulation of individual subtypes to dissect their connectivity and functions. Recombinant viral technology offers a powerful toolbox for targeted transgene expression in specific neuronal populations. In order to achieve cell type-specific targeting, exciting progress has been made to: alter viral tropisms, design rational delivery strategies, and drive selective expression patterns with engineered DNA sequences in viral genomes. For the latter case, emerging single-cell genomic analyses provide rich databases. In this review, we will summarize current status, and point out challenges, of using viral vectors for neuronal cell type-specific visualization and manipulations. With concerted efforts, progress will continue to be made toward developing viral vectors for the vast array of neuronal subtypes in the mammalian nervous system.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Animais , NeurôniosRESUMO
Neuroblastoma (NB) is a paediatric cancer that arises in the sympathetic nervous system. Patients with stage 4 tumours have poor outcomes and 20% of high-risk cases have MYCN amplification. The bone morphogenetic proteins (BMPs) play roles in sympathetic neuritogenesis, by signalling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB; it is unknown if the expression of BMPR1A or BMPR1B is altered. We report lower BMPR2 and BMPR1B, and higher BMPR1A, expression in stage 4 and in MYCN-amplified NB. Kaplan-Meier plots showed that high BMPR2 or BMPR1B expression was linked to better survival, while high BMPR1A was linked to worse survival. Gene ontology enrichment and pathway analyses revealed that BMPR2 and BMPR1B co-expressed genes were enriched in those associated with NB differentiation. BMPR1A co-expressed genes were enriched in those associated with cell proliferation. Moreover, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This suggested that differentiation should decrease BMPR1A and increase BMPR1B expression. In agreement, nerve growth factor treatment of cultured sympathetic neurons decreased Bmpr1a expression and increased Bmpr1b expression. Overexpression of dominant negative BMPR1B, treatment with a BMPR1B inhibitor and treatment with GDF5, which signals via BMPR1B, showed that BMPR1B signalling is required for optimal neuritogenesis in NB cells, suggesting that loss of BMPR1B may alter neuritogenesis. The present study shows that expression of distinct BMPRs is associated with different survival outcomes in NB.
RESUMO
Neuroblastoma is an embryonal malignancy that arises from cells of sympathoadrenal lineage during the development of the nervous system. It is the most common pediatric extracranial solid tumor and is responsible for 15% of childhood deaths from cancer. Fifty percent of cases are diagnosed as high-risk metastatic disease with a low overall 5-year survival rate. More than half of patients experience disease recurrence that can be refractory to treatment. Amplification of the MYCN gene is an important prognostic indicator that is associated with rapid disease progression and a poor prognosis, highlighting the need for new therapeutic approaches. In recent years, there has been an increasing focus on identifying anticancer properties of naturally occurring chalcones, which are secondary metabolites with variable phenolic structures. Here, we report that 4-hydroxychalcone is a potent cytotoxin for MYCN-amplified IMR-32 and SK-N-BE (2) neuroblastoma cells, when compared to non-MYCN-amplified SH-SY5Y neuroblastoma cells and to the non-neuroblastoma human embryonic kidney cell line, HEK293t. Moreover, 4-hydroxychalcone treatment significantly decreased cellular levels of the antioxidant glutathione and increased cellular reactive oxygen species. In addition, 4-hydroxychalcone treatment led to impairments in mitochondrial respiratory function, compared to controls. In support of this, the cytotoxic effect of 4-hydroxychalcone was prevented by co-treatment with either the antioxidant N-acetyl-L-cysteine, a pharmacological inhibitor of oxidative stress-induced cell death (IM-54) or the mitochondrial reactive oxygen species scavenger, Mito-TEMPO. When combined with the anticancer drugs cisplatin or doxorubicin, 4-hydroxychalcone led to greater reductions in cell viability than was induced by either anti-cancer agent alone. In summary, this study identifies a cytotoxic effect of 4-hydroxychalcone in MYCN-amplified human neuroblastoma cells, which rationalizes its further study in the development of new therapies for pediatric neuroblastoma.
Assuntos
Morte Celular/genética , Chalconas/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Linhagem Celular Tumoral , Humanos , Neuroblastoma/patologia , Estresse OxidativoRESUMO
Neuroblastoma (NB) is the most common extracranial solid tumor of early childhood; it accounts for approximately 8-10% of all childhood cancers and is the most common cancer in children in the first year of life. Patients in the high-risk group have a poor prognosis, with relapses being common and often refractory to drug treatment in those that survive. Moreover, the drug treatment itself can lead to a range of long-term sequelae. Therefore, there is a critical need to identify new therapeutics for NB. Isoliquiritigenin (ISLQ) is a naturally-occurring, dietary chalcone-type flavonoid with a range of biological effects that depend on the cell type and context. ISLQ has potential as an anticancer agent. Here we show that ISLQ has potent cytotoxic effects on SK-N-BE(2) and IMR-32 human NB cells, which carry amplification of the MYCN gene, the main prognostic marker of poor survival in NB. ISLQ was found to increase cellular reactive oxygen species (ROS). The cytotoxic effect of ISLQ was blocked by small molecule inhibitors of oxidative stress-induced cell death, and by the antioxidant N-acetyl-l-cysteine (NAC). Combined treatment of either SK-N-B-E(2) or IMR-32 cells with ISLQ and the anticancer agent cisplatin resulted in loss of cell viability that was greater than that induced by cisplatin alone. This study provides proof-of-principle that ISLQ is a potent cytotoxin for MYCN-amplified human NB cells. This is an important first step in rationalizing the further study of ISLQ as a potential adjunct therapy for high-risk NB.
Assuntos
Células Ganglionares da Retina , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Animais , Humanos , Degeneração Neural/patologia , Degeneração Neural/metabolismo , Degeneração Neural/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disease, and is characterized by the progressive degeneration of nigrostriatal dopaminergic (DA) neurons. Current PD treatments are symptomatic, wear off over time and do not protect against DA neuronal loss. Finding a way to re-grow midbrain DA (mDA) neurons is a promising disease-modifying therapeutic strategy for PD. However, reliable biomarkers are required to allow such growth-promoting approaches to be applied early in the disease progression. miR-181a has been shown to be dysregulated in PD patients, and has been identified as a potential biomarker for PD. Despite studies demonstrating the enrichment of miR-181a in the brain, specifically in neurites of postmitotic neurons, the role of miR-181a in mDA neurons remains unknown. Herein, we used cell culture models of human mDA neurons to investigate a potential role for miR-181a in mDA neurons. We used a bioninformatics analysis to identify that miR-181a targets components of the bone morphogenetic protein (BMP) signalling pathway, including the transcription factors Smad1 and Smad5, which we find are expressed by rat mDA neurons and are required for BMP-induced neurite growth. We also found that inhibition of neuronal miR-181a, resulted in increased Smad signalling, and induced neurite growth in SH-SY5Y cells. Finally, using embryonic rat cultures, we demonstrated that miR-181a inhibition induces ventral midbrain (VM) and cortical neuronal growth. These data describe a new role for miR-181a in mDA neurons, and provide proof of principle that miR-181a dysresgulation in PD may alter the activation state of signalling pathways important for neuronal growth in neurons affected in PD.
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There is growing awareness that prenatal adversity may increase the risk of autism spectrum disorder (ASD). Here, we examined the association between hypertensive disorders of pregnancy (HDP) and ASD risk at 7 years of age using the Millennium Cohort Study (MCS), a representative cohort of 13,192 children born in the UK from 2000 to 2001. We also sought to examine cytokine expression in the serum of women with pre-eclampsia, which is the most common HDP, and whether exposure of foetal neurons to this serum could change patterns of neuronal growth. HDP were reported by mothers 9 months post-delivery. ASD was parent reported at age seven, based on a doctor or health care professional's diagnosis. Weighted logistic regression was used for data analysis, adjusting for several potential confounders including maternal alcohol consumption, education, depression, age, and poverty status. Sensitivity analyses were performed excluding pre-term births, small for gestational age (SGA), and pre-pregnancy hypertension and depression. There was a significant association between HDP and a twofold increased risk of ASD (AOR = 2.10 [95% CI 1.20-3.70]). Excluding preterm births, SGA births, and offspring of women who had pre-pregnancy hypertension or over the age of 40 did not change the results materially. At the cellular level, exposure of foetal cortical neurons to 3% serum isolated from women with an established HDP increased neuronal growth and branching in vitro. These findings indicate that HDP exposure may increase the risk of ASD in the offspring.
Assuntos
Transtorno do Espectro Autista/epidemiologia , Transtorno do Espectro Autista/etiologia , Hipertensão Induzida pela Gravidez/patologia , Adolescente , Adulto , Animais , Estudos de Casos e Controles , Proliferação de Células , Quimiocinas/metabolismo , Estudos de Coortes , Feminino , Humanos , Interleucina-6/sangue , Pessoa de Meia-Idade , Neurônios/patologia , Pré-Eclâmpsia/sangue , Gravidez , Ratos Sprague-Dawley , Fatores de Risco , Adulto JovemRESUMO
Bone morphogenetic protein 2 (BMP2) is a neurotrophic factor which induces the growth of midbrain dopaminergic (DA) neurons in vitro and in vivo, and its neurotrophic effects have been shown to be dependent on activation of BMP receptors (BMPRs) and Smad 1/5/8 signalling. However, the precise intracellular cascades that regulate BMP2-BMPR-Smad-signalling-induced neurite growth remain unknown. Endocytosis has been shown to regulate Smad 1/5/8 signalling and differentiation induced by BMPs. However, these studies were carried out in non-neural cells. Indeed, there are scant reports regarding the role of endocytosis in BMP-Smad signalling in neurons. To address this, and to further characterise the mechanisms regulating the neurotrophic effects of BMP2, the present study examined the role of dynamin-dependent endocytosis in BMP2-induced Smad signalling and neurite growth in the SH-SY5Y neuronal cell line. The activation, temporal kinetics and magnitude of Smad 1/5/8 signalling induced by BMP2 were significantly attenuated by dynasore-mediated inhibition of endocytosis in SH-SY5Y cells. Furthermore, BMP2-induced increases in neurite length and neurite branching in SH-SY5Y cells were significantly reduced following inhibition of dynamin-dependent endocytosis using dynasore. This study demonstrates that BMP2-induced Smad signalling and neurite growth is regulated by dynamin-dependent endocytosis in a model of human midbrain dopaminergic neurons.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Endocitose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Neurogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Proteína Morfogenética Óssea 2/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Neurônios Dopaminérgicos/metabolismo , Endocitose/fisiologia , Humanos , Mesencéfalo/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurogênese/fisiologiaRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by the degeneration of midbrain dopaminergic (mDA) neurons and their axons, and aggregation of α-synuclein, which leads to motor and late-stage cognitive impairments. As the motor symptoms of PD are caused by the degeneration of a specific population of mDA neurons, PD lends itself to neurotrophic factor therapy. The goal of this therapy is to apply a neurotrophic factor that can slow down, halt or even reverse the progressive degeneration of mDA neurons. While the best known neurotrophic factors are members of the glial cell line-derived neurotrophic factor (GDNF) family, their lack of clinical efficacy to date means that it is important to continue to study other neurotrophic factors. Bone morphogenetic proteins (BMPs) are naturally secreted proteins that play critical roles during nervous system development and in the adult brain. In this review, we provide an overview of the BMP ligands, BMP receptors (BMPRs) and their intracellular signalling effectors, the Smad proteins. We review the available evidence that BMP-Smad signalling pathways play an endogenous role in mDA neuronal survival in vivo, before outlining how exogenous application of BMPs exerts potent effects on mDA neuron survival and axon growth in vitro and in vivo. We discuss the molecular mechanisms that mediate these effects, before highlighting the potential of targeting the downstream effectors of BMP-Smad signalling as a novel neuroprotective approach to slow or stop the degeneration of mDA neurons in PD.
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Neuroblastoma is the most common extracranial pediatric solid tumor, arising from the embryonic sympathoadrenal lineage of the neural crest, and is responsible for 15% of childhood cancer deaths. Although survival rates are good for some patients, those children diagnosed with high-risk neuroblastoma have survival rates as low as 35%. Thus, neuroblastoma remains a significant clinical challenge and the development of novel therapeutic strategies is essential. Given that there is widespread epigenetic dysregulation in neuroblastoma, epigenetic pharmacotherapy holds promise as a therapeutic approach. In recent years, histone deacetylase (HDAC) inhibitors, which cause selective activation of gene expression, have been shown to be potent chemotherapeutics for the treatment of a wide range of cancers. Here we examined the ability of the FDA-approved drug Romidepsin, a selective HDAC1/2 inhibitor, to act as a cytotoxic agent in neuroblastoma cells. Treatment with Romidepsin at concentrations in the low nanomolar range induced neuroblastoma cell death through caspase-dependent apoptosis. Romidepsin significantly increased histone acetylation, and significantly enhanced the cytotoxic effects of the cytotoxic agent 6-hydroxydopamine, which has been shown to induce cell death in neuroblastoma cells through increasing reactive oxygen species. Romidepsin was also more potent in MYCN-amplified neuroblastoma cells, which is an important prognostic marker of poor survival. This study has thus demonstrated that the FDA-approved chemotherapeutic drug Romidepsin has a potent caspase-dependent cytotoxic effect on neuroblastoma cells, whose effects enhance cell death induced by other cytotoxins, and suggests that Romidepsin may be a promising chemotherapeutic candidate for the treatment of neuroblastoma.
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Antibióticos Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Depsipeptídeos/administração & dosagem , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Caspase/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , HumanosRESUMO
INTRODUCTION: Neurotrophic factors (NTFs) have been evaluated for neuroprotective effects in Parkinson's disease (PD). However, clinical trials examining the efficacy of intracerebral administration of NTFs on motor symptoms in PD have produced mixed results, and are thus inconclusive. The objective of this systematic review and meta-analysis was to determine the effects of intracerebral NTF application on motor symptoms in people with PD. METHODS: We searched PubMed, MEDLINE, EMBASE, and Cochrane from inception through to March 31 2016 for open-label trials and randomized controlled trials (RCTs) which intracerebrally administered NTFs to PD patients, and which performed motor examination using the Unified Parkinson's Disease Rating Scale. RESULTS: Eight studies with a total of 223 participants were included. Fixed effects analysis revealed that NTF treatment did not significantly reduce motor symptoms in PD patients compared to placebo controls (P = 0.98). Combining open-label and RCT data, both treatment with NTFs (P < 0.001) and treatment with placebo (P < 0.05) significantly improved motor function in PD patients when compared to predicted symptoms in untreated PD controls. Finally, random effects analysis revealed that NTF-treated PD patients were not significantly likely to improve following intracerebral NTF administration (P = 0.25). CONCLUSION: In conclusion, intracerebral NTF administration does not improve motor symptoms in PD patients, when compared to placebo-treated controls. These findings may guide therapeutic decisions and inform future research on NTFs and their application in PD.