RESUMO
The molecular mechanisms discriminating between regenerative failure and success remain elusive. While a regeneration-competent peripheral nerve injury mounts a regenerative gene expression response in bipolar dorsal root ganglia (DRG) sensory neurons, a regeneration-incompetent central spinal cord injury does not. This dichotomic response offers a unique opportunity to investigate the fundamental biological mechanisms underpinning regenerative ability. Following a pharmacological screen with small-molecule inhibitors targeting key epigenetic enzymes in DRG neurons, we identified HDAC3 signalling as a novel candidate brake to axonal regenerative growth. In vivo, we determined that only a regenerative peripheral but not a central spinal injury induces an increase in calcium, which activates protein phosphatase 4 that in turn dephosphorylates HDAC3, thus impairing its activity and enhancing histone acetylation. Bioinformatics analysis of ex vivo H3K9ac ChIPseq and RNAseq from DRG followed by promoter acetylation and protein expression studies implicated HDAC3 in the regulation of multiple regenerative pathways. Finally, genetic or pharmacological HDAC3 inhibition overcame regenerative failure of sensory axons following spinal cord injury. Together, these data indicate that PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.
Assuntos
Gânglios Espinais/fisiologia , Histona Desacetilases/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Axônios , Células Cultivadas , Modelos Animais de Doenças , Epigênese Genética/efeitos dos fármacos , Feminino , Masculino , Camundongos , Regeneração Nervosa , Fosforilação/efeitos dos fármacos , Transdução de SinaisRESUMO
Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.
Assuntos
Axônios/enzimologia , Axônios/patologia , Sistema Nervoso Central/patologia , Quinases do Centro Germinativo/metabolismo , Regeneração Nervosa , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dependovirus/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Organoides/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Axon regeneration is a necessary step toward functional recovery after spinal cord injury. The AP-1 transcription factor c-Jun has long been known to play an important role in directing the transcriptional response of Dorsal Root Ganglion (DRG) neurons to peripheral axotomy that results in successful axon regeneration. Here we performed ChIPseq for Jun in mouse DRG neurons after a sciatic nerve crush or sham surgery in order to measure the changes in Jun's DNA binding in response to peripheral axotomy. We found that the majority of Jun's injury-responsive changes in DNA binding occur at putative enhancer elements, rather than proximal to transcription start sites. We also used a series of single polypeptide chain tandem transcription factors to test the effects of different Jun-containing dimers on neurite outgrowth in DRG, cortical and hippocampal neurons. These experiments demonstrated that dimers composed of Jun and Atf3 promoted neurite outgrowth in rat CNS neurons as well as mouse DRG neurons. Our work provides new insight into the mechanisms underlying Jun's role in axon regeneration.
Assuntos
Crescimento Neuronal , Multimerização Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Elementos Facilitadores Genéticos , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
The mammalian target of rapamycin (mTOR) positively regulates axon growth in the mammalian central nervous system (CNS). Although axon regeneration and functional recovery from CNS injuries are typically limited, knockdown or deletion of PTEN, a negative regulator of mTOR, increases mTOR activity and induces robust axon growth and regeneration. It has been suggested that inhibition of S6 kinase 1 (S6K1, gene symbol: RPS6KB1), a prominent mTOR target, would blunt mTOR's positive effect on axon growth. In contrast to this expectation, we demonstrate that inhibition of S6K1 in CNS neurons promotes neurite outgrowth in vitro by twofold to threefold. Biochemical analysis revealed that an mTOR-dependent induction of PI3K signaling is involved in mediating this effect of S6K1 inhibition. Importantly, treating female mice in vivo with PF-4708671, a selective S6K1 inhibitor, stimulated corticospinal tract regeneration across a dorsal spinal hemisection between the cervical 5 and 6 cord segments (C5/C6), increasing axon counts for at least 3 mm beyond the injury site at 8 weeks after injury. Concomitantly, treatment with PF-4708671 produced significant locomotor recovery. Pharmacological targeting of S6K1 may therefore constitute an attractive strategy for promoting axon regeneration following CNS injury, especially given that S6K1 inhibitors are being assessed in clinical trials for nononcological indications.SIGNIFICANCE STATEMENT Despite mTOR's well-established function in promoting axon regeneration, the role of its downstream target, S6 kinase 1 (S6K1), has been unclear. We used cellular assays with primary neurons to demonstrate that S6K1 is a negative regulator of neurite outgrowth, and a spinal cord injury model to show that it is a viable pharmacological target for inducing axon regeneration. We provide mechanistic evidence that S6K1's negative feedback to PI3K signaling is involved in axon growth inhibition, and show that phosphorylation of S6K1 is a more appropriate regeneration indicator than is S6 phosphorylation.
Assuntos
Axônios/metabolismo , Imidazóis/administração & dosagem , Piperazinas/administração & dosagem , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/enzimologia , Regeneração da Medula Espinal/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Sistemas de Liberação de Medicamentos , Regulação Enzimológica da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Crescimento Neuronal/efeitos dos fármacos , Ligação Proteica , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Especificidade por Substrato , Resultado do TratamentoRESUMO
High-throughput, target-based screening techniques have been utilized extensively for drug discovery in the past several decades. However, the need for more predictive in vitro models of in vivo disease states has generated a shift in strategy towards phenotype-based screens. Phenotype based screens are particularly valuable in studying complex conditions such as CNS injury and degenerative disease, as many factors can contribute to a specific cellular response. In this review, we will discuss different screening frameworks and their relative utility in examining mechanisms of neurodegeneration and axon regrowth, particularly in cell-based in vitro disease models.
Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Degeneração Neural/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Animais , Humanos , FenótipoRESUMO
A number of genes regulate regeneration of peripheral axons, but their ability to drive axon growth and regeneration in the central nervous system (CNS) remains largely untested. To address this question we overexpressed eight transcription factors and one small GTPase alone and in pairwise combinations to test whether combinatorial overexpression would have a synergistic impact on CNS neuron neurite growth. The Jun oncogene/signal transducer and activator of transcription 6 (JUN/STAT6) combination increased neurite growth in dissociated cortical neurons and in injured cortical slices. In injured cortical slices, JUN overexpression increased axon growth to a similar extent as JUN and STAT6 together. Interestingly, JUN overexpression was not associated with increased growth associated protein 43 (GAP43) or integrin alpha 7 (ITGA7) expression, though these are predicted transcriptional targets. This study demonstrates that JUN overexpression in cortical neurons stimulates axon growth, but does so independently of changes in expression of genes thought to be critical for JUNs effects on axon growth. We conclude that JUN activity underlies this CNS axonal growth response, and that it is mechanistically distinct from peripheral regeneration responses, in which increases in JUN expression coincide with increases in GAP43 expression.
Assuntos
Axônios/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Proteína Oncogênica p65(gag-jun)/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Axônios/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Regeneração Nervosa , Neurogênese , Proteína Oncogênica p65(gag-jun)/genética , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismoRESUMO
Axon regeneration in the central nervous system normally fails, in part because of a developmental decline in the intrinsic ability of CNS projection neurons to extend axons. Members of the KLF family of transcription factors regulate regenerative potential in developing CNS neurons. Expression of one family member, KLF7, is down-regulated developmentally, and overexpression of KLF7 in cortical neurons in vitro promotes axonal growth. To circumvent difficulties in achieving high neuronal expression of exogenous KLF7, we created a chimera with the VP16 transactivation domain, which displayed enhanced neuronal expression compared with the native protein while maintaining transcriptional activation and growth promotion in vitro. Overexpression of VP16-KLF7 overcame the developmental loss of regenerative ability in cortical slice cultures. Adult corticospinal tract (CST) neurons failed to up-regulate KLF7 in response to axon injury, and overexpression of VP16-KLF7 in vivo promoted both sprouting and regenerative axon growth in the CST of adult mice. These findings identify a unique means of promoting CST axon regeneration in vivo by reengineering a developmentally down-regulated, growth-promoting transcription factor.
Assuntos
Axônios/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Regeneração Nervosa/fisiologia , Tratos Piramidais/fisiologia , Animais , Axônios/metabolismo , Células Cultivadas , Etoposídeo , Feminino , Expressão Gênica , Engenharia Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Proteína Vmw65 do Vírus do Herpes Simples/genética , Humanos , Imuno-Histoquímica , Fatores de Transcrição Kruppel-Like/genética , Medições Luminescentes/métodos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Regeneração Nervosa/genética , Neuritos/metabolismo , Neuritos/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Tratos Piramidais/citologia , Tratos Piramidais/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Ativação TranscricionalRESUMO
Axonal branches of the trigeminal ganglion (TG) display characteristic growth and arborization patterns during development. Subsets of TG neurons express different receptors for growth factors, but these are unlikely to explain the unique patterns of axonal arborizations. Intrinsic modulators may restrict or enhance cellular responses to specific ligands and thereby contribute to the development of axon growth patterns. Protein tyrosine phosphatase receptor type O (PTPRO), which is required for Eph receptor-dependent retinotectal development in chick and for development of subsets of trunk sensory neurons in mouse, may be such an intrinsic modulator of TG neuron development. PTPRO is expressed mainly in TrkB-expressing (TrkB(+)) and Ret(+) mechanoreceptors within the TG during embryogenesis. In PTPRO mutant mice, subsets of TG neurons grow longer and more elaborate axonal branches. Cultured PTPRO(-/-) TG neurons display enhanced axonal outgrowth and branching in response to BDNF and GDNF compared with control neurons, indicating that PTPRO negatively controls the activity of BDNF/TrkB and GDNF/Ret signaling. Mouse PTPRO fails to regulate Eph signaling in retinocollicular development and in hindlimb motor axon guidance, suggesting that chick and mouse PTPRO have different substrate specificities. PTPRO has evolved to fine tune growth factor signaling in a cell-type-specific manner and to thereby increase the diversity of signaling output of a limited number of receptor tyrosine kinases to control the branch morphology of developing sensory neurons. The regulation of Eph receptor-mediated developmental processes by protein tyrosine phosphatases has diverged between chick and mouse.
Assuntos
Axônios/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Gânglio Trigeminal/citologia , Gânglio Trigeminal/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Gravidez , Receptor EphA1/metabolismo , Receptor trkA/metabolismo , Receptor trkC/metabolismo , Transdução de Sinais/fisiologia , Gânglio Trigeminal/embriologia , Nervo Trigêmeo/citologia , Nervo Trigêmeo/embriologia , Nervo Trigêmeo/metabolismoRESUMO
In neurons, the type 3 deiodinase (D3) inactivates thyroid hormone and reduces oxygen consumption, thus creating a state of cell-specific hypothyroidism. Here we show that hypoxia leads to nuclear import of D3 in neurons, without which thyroid hormone signaling and metabolism cannot be reduced. After unilateral hypoxia in the rat brain, D3 protein level is increased predominantly in the nucleus of the neurons in the pyramidal and granular ipsilateral layers, as well as in the hilus of the dentate gyrus of the hippocampal formation. In hippocampal neurons in culture as well as in a human neuroblastoma cell line (SK-N-AS), a 24 h hypoxia period redirects active D3 from the endoplasmic reticulum to the nucleus via the cochaperone Hsp40 pathway. Preventing nuclear D3 import by Hsp40 knockdown resulted an almost doubling in the thyroid hormone-dependent glycolytic rate and quadrupling the transcription of thyroid hormone target gene ENPP2. In contrast, Hsp40 overexpression increased nuclear import of D3 and minimized thyroid hormone effects in cell metabolism. In conclusion, ischemia/hypoxia induces an Hsp40-mediated translocation of D3 to the nucleus, facilitating thyroid hormone inactivation proximal to the thyroid hormone receptors. This adaptation decreases thyroid hormone signaling and may function to reduce ischemia-induced hypoxic brain damage.
Assuntos
Hipóxia Celular/fisiologia , Núcleo Celular/metabolismo , Proteínas de Choque Térmico HSP40/fisiologia , Iodeto Peroxidase/metabolismo , Neurônios/metabolismo , Animais , Isquemia Encefálica/metabolismo , Núcleo Celular/enzimologia , Células Cultivadas , DNA/genética , Retículo Endoplasmático/metabolismo , Glicosilação , Hipocampo/citologia , Hipocampo/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Masculino , Microscopia Eletrônica , Artéria Cerebral Média/fisiologia , Consumo de Oxigênio/fisiologia , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Receptores dos Hormônios Tireóideos/metabolismo , Transdução de Sinais/fisiologia , Hormônios Tireóideos/fisiologiaRESUMO
Injury to the central nervous system (CNS) can result in lifelong loss of function due in part to the regenerative failure of CNS neurons. Inhibitory proteins derived from myelin and the astroglial scar are major barriers for the successful regeneration of injured CNS neurons. Previously, we described the identification of a novel compound, F05, which promotes neurite growth from neurons challenged with inhibitory substrates in vitro, and promotes axonal regeneration in vivo (Usher et al., 2010). To identify additional regeneration-promoting compounds, we used F05-induced gene expression profiles to query the Broad Institute Connectivity Map, a gene expression database of cells treated with >1300 compounds. Despite no shared chemical similarity, F05-induced changes in gene expression were remarkably similar to those seen with a group of piperazine phenothiazine antipsychotics (PhAPs). In contrast to antipsychotics of other structural classes, PhAPs promoted neurite growth of CNS neurons challenged with two different glial derived inhibitory substrates. Our pharmacological studies suggest a mechanism whereby PhAPs promote growth through antagonism of calmodulin signaling, independent of dopamine receptor antagonism. These findings shed light on mechanisms underlying neurite-inhibitory signaling, and suggest that clinically approved antipsychotic compounds may be repurposed for use in CNS injured patients.
Assuntos
Antipsicóticos/farmacologia , Neuritos/efeitos dos fármacos , Fenotiazinas/farmacologia , Piperazinas/farmacologia , Regeneração/efeitos dos fármacos , Animais , Antipsicóticos/química , Encéfalo/fisiologia , Células CHO , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/farmacologia , Cricetinae , Cricetulus , Perfilação da Expressão Gênica , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenotiazinas/química , Piperazinas/química , RatosRESUMO
Axon regeneration is a fundamental problem facing neuroscientists and clinicians. Failure of axon regeneration is caused by both extrinsic and intrinsic mechanisms. New techniques to examine gene expression such as Next Generation Sequencing of the Transcriptome (RNA-Seq) drastically increase our knowledge of both gene expression complexity (RNA isoforms) and gene expression regulation. By utilizing RNA-Seq, gene expression can now be defined at the level of isoforms, an essential step for understanding the mechanisms governing cell identity, growth and ultimately cellular responses to injury and disease.
Assuntos
Axônios/fisiologia , Regeneração Nervosa/fisiologia , Animais , Humanos , Isoformas de Proteínas/fisiologiaRESUMO
Neuronal pentraxin with chromo domain (NPCD) comprises a group of neuronally expressed pentraxins with both membrane and cytosolic isoforms; the functions of cytosolic NPCD isoforms are not clear. Here, we demonstrate that a cytosolic NPCD isoform selectively interacts with the BTB-Kelch protein Mayven/Kelch-like 2 (KLHL2), an actin-binding protein implicated in process outgrowth in oligodendrocytes. The KLHL2-NPCD interaction was identified by a yeast two-hybrid screen and confirmed through colocalization and co-immunoprecipitation studies. Truncation analysis indicates that the Kelch domains of KLHL2 interact with the pentraxin domain of NPCD. NPCD forms protein inclusion bodies (aggresomes) when overexpressed in tissue culture cells, KLHL2 localizes to these aggresomes, and overexpression of KLHL2 increases NPCD aggresome formation. Since other members of the BTB-Kelch family can act as Cullin-RING type E3 ubiquitin ligases, we tested the potential role of KLHL2 as a ubiquitin ligase for NPCD. We found that KLHL2 interacts selectively with Cullin 3, a key component of BTB-Kelch ubiquitin ligase complexes. Further, overexpression of KLHL2 promotes NPCD ubiquitylation. Together, these results suggest a novel E3 ubiquitin ligase function of KLHL2, with NPCD as a substrate. As the formation of aggresomes is often associated with protein aggregation in neurodegenerative diseases, we tested the effects of NPCD overexpression and KLHL2 coexpression on neuronal viability. Overexpression of NPCD in hippocampal neurons led to cell death and apoptosis; this effect was exacerbated by KLHL2 co-expression. Our findings implicate KLHL2 in ubiquitin ligase activity, and suggest potential roles of NPCD and KLHL2 in neurodegeneration.
Assuntos
Apoptose/fisiologia , Proteína C-Reativa/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Proteína C-Reativa/genética , Células Cultivadas , Proteínas Culina/genética , Proteínas Culina/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Técnicas do Sistema de Duplo-Híbrido , UbiquitinaçãoRESUMO
Neurons in the peripheral nervous system (PNS) display a higher capacity to regenerate after injury than those in the central nervous system, suggesting cell specific transcriptional modules underlying axon growth and inhibition. We report a systems biology based search for PNS specific transcription factors (TFs). Messenger RNAs enriched in dorsal root ganglion (DRG) neurons compared to cerebellar granule neurons (CGNs) were identified using subtractive hybridization and DNA microarray approaches. Network and transcription factor binding site enrichment analyses were used to further identify TFs that may be differentially active. Combining these techniques, we identified 32 TFs likely to be enriched and/or active in the PNS. Twenty-five of these TFs were then tested for an ability to promote CNS neurite outgrowth in an overexpression screen. Real-time PCR and immunohistochemical studies confirmed that one representative TF, STAT3, is intrinsic to PNS neurons, and that constitutively active STAT3 is sufficient to promote CGN neurite outgrowth.
Assuntos
Neurônios/fisiologia , Sistema Nervoso Periférico/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Cerebelo/citologia , Gânglios Espinais/citologia , Perfilação da Expressão Gênica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries/métodos , Neurônios/citologia , Hibridização de Ácido Nucleico/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genéticaRESUMO
Injuries to the cervical spinal cord represent around 60% of all spinal cord injuries (SCIs). A major priority for patients with cervical SCIs is the recovery of any hand or arm function. The similarities between human and rodent "reach-to-eat movements" indicate that analyzing mouse forelimb reaching behavior may be a method of identifying clinically relevant treatments for people with cervical SCIs. One popular behavioral measure of forelimb functional recovery comprises the Single Pellet Retrieval Task (SPRT). The most common outcome measure for this task, however (percentage of pellets successfully retrieved), cannot readily distinguish between recovery of pre-injury motor patterns and strategic compensation. Our objective was to establish outcome measures for the SPRT that are readily adopted by different investigators and capable of measuring recovery of limb function after SCI. We used a simple semi-automated approach to high-speed tracking of mouse forepaw movements during pellet retrieval. DeepLabCut™, a machine learning based computer vision software package, was used to track individual features of the mouse forepaw, allowing a more detailed assessment of reaching behavior after SCI. Interestingly, kinematic analysis of movements pre- and post-injury illuminated persistent deficits in specific features of the reaching motor patterns, namely pronation and paw trajectory, that were poorly correlated with recovery of the ability to successfully retrieve pellets. Thus, we have developed an inexpensive method for detailed analysis of mouse reach-to-eat behavior following SCI. Further, our results suggest that binary success/fail outcome measures primarily assess an animal's ability to compensate rather than a restoration of normal function in the injured pathways and networks.
Assuntos
Medula Cervical , Traumatismos da Medula Espinal , Animais , Medula Cervical/lesões , Modelos Animais de Doenças , Membro Anterior , Humanos , Camundongos , Destreza Motora , Recuperação de Função Fisiológica , Medula EspinalRESUMO
Recovery from spinal cord injury (SCI) and other central nervous system (CNS) trauma is hampered by limits on axonal regeneration in the CNS. Regeneration is restricted by the lack of neuron-intrinsic regenerative capacity and by the repressive microenvironment confronting damaged axons. To address this challenge, we have developed a therapeutic strategy that co-targets kinases involved in both extrinsic and intrinsic regulatory pathways. Prior work identified a kinase inhibitor (RO48) with advantageous polypharmacology (co-inhibition of targets including ROCK2 and S6K1), which promoted CNS axon growth in vitro and corticospinal tract (CST) sprouting in a mouse pyramidotomy model. We now show that RO48 promotes neurite growth from sensory neurons and a variety of CNS neurons in vitro, and promotes CST sprouting and/or regeneration in multiple mouse models of spinal cord injury. Notably, these in vivo effects of RO48 were seen in several independent experimental series performed in distinct laboratories at different times. Finally, in a cervical dorsal hemisection model, RO48 not only promoted growth of CST axons beyond the lesion, but also improved behavioral recovery in the rotarod, gridwalk, and pellet retrieval tasks. Our results provide strong evidence for RO48 as an effective compound to promote axon growth and regeneration. Further, they point to strategies for increasing robustness of interventions in pre-clinical models.
Assuntos
Axônios , Traumatismos da Medula Espinal , Animais , Axônios/patologia , Modelos Animais de Doenças , Camundongos , Regeneração Nervosa/fisiologia , Neurônios/metabolismo , Tratos Piramidais/patologia , Recuperação de Função Fisiológica/fisiologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologiaRESUMO
Aging is associated with increased prevalence of axonal injuries characterized by poor regeneration and disability. However, the underlying mechanisms remain unclear. In our experiments, RNA sequencing of sciatic dorsal root ganglia (DRG) revealed significant aging-dependent enrichment in T cell signaling both before and after sciatic nerve injury (SNI) in mice. Lymphotoxin activated the transcription factor NF-κB, which induced expression of the chemokine CXCL13 by neurons. This in turn recruited CXCR5+CD8+ T cells to injured DRG neurons overexpressing major histocompatibility complex class I. CD8+ T cells repressed the axonal regeneration of DRG neurons via caspase 3 activation. CXCL13 neutralization prevented CXCR5+CD8+ T cell recruitment to the DRG and reversed aging-dependent regenerative decline, thereby promoting neurological recovery after SNI. Thus, axonal regeneration can be facilitated by antagonizing cross-talk between immune cells and neurons.
Assuntos
Envelhecimento , Axônios , Linfócitos T CD8-Positivos , Gânglios Espinais , Regeneração Nervosa , Neurônios , Nervo Isquiático , Envelhecimento/metabolismo , Animais , Axônios/fisiologia , Linfócitos T CD8-Positivos/metabolismo , Gânglios Espinais/metabolismo , Camundongos , Neurônios/metabolismo , Nervo Isquiático/lesões , Nervo Isquiático/fisiologiaRESUMO
A major barrier to regeneration of CNS axons is the presence of growth-inhibitory proteins associated with myelin and the glial scar. To identify chemical compounds with the ability to overcome the inhibition of regeneration, we screened a novel triazine library, based on the ability of compounds to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. The screen produced four "hit compounds," which act with nanomolar potency on several different neuronal types and on several distinct substrates relevant to glial inhibition. Moreover, the compounds selectively overcome inhibition rather than promote growth in general. The compounds do not affect neuronal cAMP levels, PKC activity, or EGFR (epidermal growth factor receptor) activation. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions and potentiates regeneration of optic nerve axons after nerve crush in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury.
Assuntos
Neuroglia/fisiologia , Neurônios/efeitos dos fármacos , Triazinas/farmacologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Células Cultivadas , Cerebelo/citologia , Córtex Cerebral/citologia , AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Ensaios de Triagem em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/fisiologia , Compressão Nervosa , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Nervo Óptico/citologia , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração , Medula Espinal/citologia , Triazinas/químicaRESUMO
Development and regeneration of the nervous system requires the precise formation of axons and dendrites. Kinases and phosphatases are pervasive regulators of cellular function and have been implicated in controlling axodendritic development and regeneration. We undertook a gain-of-function analysis to determine the functions of kinases and phosphatases in the regulation of neuron morphology. Over 300 kinases and 124 esterases and phosphatases were studied by high-content analysis of rat hippocampal neurons. Proteins previously implicated in neurite growth, such as ERK1, GSK3, EphA8, FGFR, PI3K, PKC, p38, and PP1a, were confirmed to have effects in our functional assays. We also identified novel positive and negative neurite growth regulators. These include neuronal-developmentally regulated kinases such as the activin receptor, interferon regulatory factor 6 (IRF6) and neural leucine-rich repeat 1 (LRRN1). The protein kinase N2 (PKN2) and choline kinase alpha (CHKA) kinases, and the phosphatases PPEF2 and SMPD1, have little or no established functions in neuronal function, but were sufficient to promote neurite growth. In addition, pathway analysis revealed that members of signaling pathways involved in cancer progression and axis formation enhanced neurite outgrowth, whereas cytokine-related pathways significantly inhibited neurite formation.
Assuntos
Forma Celular , Hipocampo/enzimologia , Regeneração Nervosa , Neurônios/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Genótipo , Hipocampo/embriologia , Humanos , Camundongos , Regeneração Nervosa/genética , Fenótipo , Fosfoproteínas Fosfatases/genética , Proteínas Quinases/genética , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Transfecção , Regulação para CimaRESUMO
Neurons in the central nervous system lose their intrinsic capacity for axon regeneration as they mature, and it is widely hypothesized that changes in gene expression are responsible. Testing this hypothesis and identifying the relevant genes has been challenging because hundreds to thousands of genes are developmentally regulated in CNS neurons, but only a small subset are likely relevant to axon growth. Here we used automated high content analysis (HCA) methods to functionally test 743 plasmids encoding developmentally regulated genes in neurite outgrowth assays using postnatal cortical neurons. We identified both growth inhibitors (Ephexin, Aldolase A, Solute Carrier 2A3, and Chimerin), and growth enhancers (Doublecortin, Doublecortin-like, Kruppel-like Factor 6, and CaM-Kinase II gamma), some of which regulate established growth mechanisms like microtubule dynamics and small GTPase signaling. Interestingly, with only one exception the growth-suppressing genes were developmentally upregulated, and the growth-enhancing genes downregulated. These data provide important support for the hypothesis that developmental changes in gene expression control neurite outgrowth, and identify potential new gene targets to promote neurite outgrowth.
Assuntos
Diferenciação Celular/fisiologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa/fisiologia , Animais , Bioensaio , Células Cultivadas , Córtex Cerebral/citologia , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Frutose-Bifosfato Aldolase/análise , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Cones de Crescimento/ultraestrutura , Inibidores do Crescimento/análise , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/análise , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/genética , Neuritos/metabolismo , Neuritos/ultraestrutura , Neurogênese/fisiologia , Plasticidade Neuronal/fisiologia , Neuropeptídeos/análise , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Tratos Piramidais/citologia , Tratos Piramidais/crescimento & desenvolvimento , Tratos Piramidais/metabolismo , RatosRESUMO
Adeno-associated virus (AAV) is an essential instrument in the neuroscientist's toolkit, which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression. In the field of neural regeneration, the transduction of neurons enables the observation and regulation of axon growth and regeneration, and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury. Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions, but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes. We studied AAV transduction in the context of prior exposure of AAV serotype 8 (AAV8) to environmental pH within the range encountered during endosomal endocytosis (pH 7.4 to pH 4.4), during which low pH-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating. Due to the fundamental nature of these processes, we hypothesized that premature exposure of AAV8 particles to acidic pH would decrease viral transduction of HT1080 cells in vitro, as measured by fluorescent reporter gene expression using high-content imaging analysis. We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency, and that quantitative levels of reporter gene expression in transduced cells were similarly decreased. The biggest decrease in transduction occurred between pH 7.4 and pH 6.4, suggesting the possible co-occurrence of a pH-associated event and viral inactivation within that range. Taken together, these findings indicate that exposure of AAV8 to acidic pH for as little as 1 hour is deleterious to transduction ability. Future studies are necessary to understand the pH-associated causative mechanisms involved. This study was approved by the University of Miami Institutional Animal Care and Use Committee, USA (Protocol #18-108-LF) on July 12, 2018.