RESUMO
Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer's disease. We demonstrate that DLK/c-Jun N-terminal kinase signaling was increased in mouse models and human patients with these disorders and that genetic deletion of DLK protected against axon degeneration, neuronal loss, and functional decline in vivo. Furthermore, pharmacological inhibition of DLK activity was sufficient to attenuate the neuronal stress response and to provide functional benefit even in the presence of ongoing disease. These findings demonstrate that pathological activation of DLK is a conserved mechanism that regulates neurodegeneration and suggest that DLK inhibition may be a potential approach to treat multiple neurodegenerative diseases.
Assuntos
Zíper de Leucina , MAP Quinase Quinase Quinases/metabolismo , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/patologia , Transdução de Sinais , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos Transgênicos , Neuroproteção , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Medula Espinal/enzimologia , Medula Espinal/patologia , Superóxido Dismutase/metabolismoRESUMO
Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules.
Assuntos
Caspase 6/metabolismo , Bibliotecas de Moléculas Pequenas/química , Sítio Alostérico , Sítios de Ligação , Caspase 6/química , Cristalografia por Raios X , Dimerização , Desenho de Fármacos , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/metabolismo , Temperatura de TransiçãoRESUMO
Excessive glutamate signaling is thought to underlie neurodegeneration in multiple contexts, yet the pro-degenerative signaling pathways downstream of glutamate receptor activation are not well defined. We show that dual leucine zipper kinase (DLK) is essential for excitotoxicity-induced degeneration of neurons in vivo. In mature neurons, DLK is present in the synapse and interacts with multiple known postsynaptic density proteins including the scaffolding protein PSD-95. To examine DLK function in the adult, DLK-inducible knockout mice were generated through Tamoxifen-induced activation of Cre-ERT in mice containing a floxed DLK allele, which circumvents the neonatal lethality associated with germline deletion. DLK-inducible knockouts displayed a modest increase in basal synaptic transmission but had an attenuation of the JNK/c-Jun stress response pathway activation and significantly reduced neuronal degeneration after kainic acid-induced seizures. Together, these data demonstrate that DLK is a critical upstream regulator of JNK-mediated neurodegeneration downstream of glutamate receptor hyper-activation and represents an attractive target for the treatment of indications where excitotoxicity is a primary driver of neuronal loss.
Assuntos
MAP Quinase Quinase Quinases/fisiologia , Degeneração Neural/fisiopatologia , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Proteína 4 Homóloga a Disks-Large , Ácido Glutâmico/fisiologia , Guanilato Quinases/fisiologia , Ácido Caínico/toxicidade , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , N-Metilaspartato/fisiologia , Degeneração Neural/genética , Degeneração Neural/patologia , Proteínas do Tecido Nervoso/fisiologia , Sinapses/fisiologiaRESUMO
Accumulating evidence from mice expressing ALS-causing mutations in superoxide dismutase (SOD1) has implicated pathological immune responses in motor neuron degeneration. This includes microglial activation, lymphocyte infiltration, and the induction of C1q, the initiating component of the classic complement system that is the protein-based arm of the innate immune response, in motor neurons of multiple ALS mouse models expressing dismutase active or inactive SOD1 mutants. Robust induction early in disease course is now identified for multiple complement components (including C1q, C4, and C3) in spinal cords of SOD1 mutant-expressing mice, consistent with initial intraneuronal C1q induction, followed by global activation of the complement pathway. We now test if this activation is a mechanistic contributor to disease. Deletion of the C1q gene in mice expressing an ALS-causing mutant in SOD1 to eliminate C1q induction, and complement cascade activation that follows from it, is demonstrated to produce changes in microglial morphology accompanied by enhanced loss, not retention, of synaptic densities during disease. C1q-dependent synaptic loss is shown to be especially prominent for cholinergic C-bouton nerve terminal input onto motor neurons in affected C1q-deleted SOD1 mutant mice. Nevertheless, overall onset and progression of disease are unaffected in C1q- and C3-deleted ALS mice, thus establishing that C1q induction and classic or alternative complement pathway activation do not contribute significantly to SOD1 mutant-mediated ALS pathogenesis in mice.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/imunologia , Complemento C1q/metabolismo , Via Clássica do Complemento/imunologia , Neurônios Motores/metabolismo , Superóxido Dismutase/genética , Animais , Complemento C1q/genética , Deleção de Genes , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microglia/citologia , Neurônios Motores/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Análise de SobrevidaRESUMO
Neurons are highly polarized cells that often project axons a considerable distance. To respond to axonal damage, neurons must transmit a retrograde signal to the nucleus to enable a transcriptional stress response. Here we describe a mechanism by which this signal is propagated through injury-induced stabilization of dual leucine zipper-bearing kinase (DLK/MAP3K12). After neuronal insult, specific sites throughout the length of DLK underwent phosphorylation by c-Jun N-terminal kinases (JNKs), which have been shown to be downstream targets of DLK pathway activity. These phosphorylation events resulted in increased DLK abundance via reduction of DLK ubiquitination, which was mediated by the E3 ubiquitin ligase PHR1 and the de-ubiquitinating enzyme USP9X. Abundance of DLK in turn controlled the levels of downstream JNK signaling and apoptosis. Through this feedback mechanism, the ubiquitin-proteasome system is able to provide an additional layer of regulation of retrograde stress signaling to generate a global cellular response to localized external insults.
Assuntos
Apoptose , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Células Receptoras Sensoriais/enzimologia , Células Receptoras Sensoriais/patologia , Ubiquitinação , Animais , Apoptose/efeitos dos fármacos , Axônios/efeitos dos fármacos , Axônios/patologia , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Peso Molecular , Compressão Nervosa , Fator de Crescimento Neural/farmacologia , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/patologia , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The caspases are a family of cytosolic proteases with essential roles in inflammation and apoptosis. Drug discovery efforts have focused on developing molecules directed against the active sites of caspases, but this approach has proved challenging and has not yielded any approved therapeutics. Here we describe a new strategy for generating inhibitors of caspase-6, a potential therapeutic target in neurodegenerative disorders, by screening against its zymogen form. Using phage display to discover molecules that bind the zymogen, we report the identification of a peptide that specifically impairs the function of caspase-6 in vitro and in neuronal cells. Remarkably, the peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site, and acts via a new allosteric mechanism that promotes caspase tetramerization. Our data illustrate that screening against the zymogen holds promise as an approach for targeting caspases in drug discovery.
Assuntos
Biopolímeros/metabolismo , Caspase 6/metabolismo , Precursores Enzimáticos/metabolismo , Peptídeos/metabolismo , Regulação Alostérica , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Peptídeos/química , Ligação ProteicaRESUMO
Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context.
Assuntos
Bioensaio/métodos , Caspase 6/metabolismo , Células/enzimologia , Ensaios Enzimáticos/métodos , Lamina Tipo A/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Inibidores de Caspase , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Inibidores de Proteases/farmacologia , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Estaurosporina/farmacologia , Especificidade por Substrato/efeitos dos fármacosRESUMO
The c-Jun N-terminal kinase (JNK) signaling pathway is essential for neuronal degeneration in multiple contexts but also regulates neuronal homeostasis. It remains unclear how neurons are able to dissociate proapoptotic JNK signaling from physiological JNK activity. In this paper, we show that the mixed lineage kinase dual leucine zipper kinase (DLK) selectively regulates the JNK-based stress response pathway to mediate axon degeneration and neuronal apoptosis without influencing other aspects of JNK signaling. This specificity is dependent on interaction of DLK with the scaffolding protein JIP3 to form a specialized JNK signaling complex. Local activation of DLK-based signaling in the axon results in phosphorylation of c-Jun and apoptosis after redistribution of JNK to the cell body. In contrast, regulation of axon degeneration by DLK is c-Jun independent and mediated by distinct JNK substrates. DLK-null mice displayed reduced apoptosis in multiple neuronal populations during development, demonstrating that prodegenerative DLK signaling is required in vivo.
Assuntos
Apoptose/fisiologia , Sistema Nervoso Central/embriologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Axônios/fisiologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Gânglios Espinais/patologia , Proteínas de Homeodomínio/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Fator de Crescimento Neural/deficiência , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/farmacocinética , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Interferente Pequeno/genética , Medula Espinal/embriologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Oligodendrocyte precursor cells (OPCs) are lineage-restricted progenitors generally limited in vivo to producing oligodendrocytes. Mechanisms controlling genesis of OPCs are of interest because of their importance in myelin development and their potential for regenerative therapies in multiple sclerosis and dysmyelinating syndromes. We show here that the SoxE transcription factors (comprising Sox8, 9, and 10) induce multipotent neural precursor cells (NPCs) from the early postnatal subventricular zone (SVZ) to become OPCs in an autonomous manner. We performed a chromatin immunoprecipitation-based bioinformatic screen and identified Suppressor of Fused (Sufu) as a direct target of repression by Sox10. In vitro, overexpression of Sufu blocked OPC production, whereas RNAi-mediated inhibition augmented OPC production. Furthermore, mice heterozygous for Sufu have increased numbers of OPCs in the telencephalon during development. We conclude that Sox10 acts to restrict the potential of NPCs toward the oligodendrocyte lineage in part by regulating the expression of Sufu.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Oligodendroglia/fisiologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXE/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Proteínas Repressoras/genética , Fatores de Transcrição SOXE/genéticaRESUMO
Oligodendrocyte precursor cells (OPCs) are generated from multiple progenitor domains in the telencephalon in developmental succession from ventral to dorsal. Previous studies showed that Wnt signaling inhibits the differentiation of OPCs into mature oligodendrocytes. Here we explored the hypothesis that Wnt signaling limits the generation of OPCs from neural progenitors during forebrain development. We manipulated Wnt signaling in mouse neural progenitor cultures and found that Wnt signaling influences progenitors cell autonomously to alter the production of OPCs, and that endogenous Wnt signaling in these cultures limits the efficiency of generating OPCs from neural progenitors. To examine these events in vivo, we electroporated a soluble Wnt inhibitor or a dominant-negative transcriptional regulator into embryonic mouse neocortical ventricular zone before the usual onset of OPC production and showed that decreasing Wnt signaling in cortical progenitors results in early production of OPCs. Our studies indicate that Wnt signaling influences the timing and extent of OPC production in the developing telencephalon.
Assuntos
Diferenciação Celular/fisiologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Telencéfalo/embriologia , Proteínas Wnt/fisiologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Eletroporação , Feminino , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neocórtex/citologia , Neocórtex/embriologia , Transdução de Sinais/genética , Telencéfalo/citologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt3RESUMO
Adult neurogenesis in the hippocampus is under complex genetic control. A recent comparative study of two inbred mouse strains using quantitative trait locus analysis has revealed that cell survival is most highly correlated with neurogenesis and identified candidate genes for further investigation.
Assuntos
Hipocampo/crescimento & desenvolvimento , Camundongos Endogâmicos BALB C/genética , Camundongos Endogâmicos C57BL/genética , Neurônios/fisiologia , Locos de Características Quantitativas , Animais , Humanos , CamundongosRESUMO
It is now widely accepted that discrete regions of the adult brain contain stem cells that continue to generate new neurons. However, it remains unclear what molecular signals define the neurogenic niche and how such signals act on the heterogeneous cell populations within these regions. Here we discuss two recent studies that demonstrate the role of Wnt and Sonic Hedgehog signaling in neurogenic zones. Wnts act on neuronal precursors that mature and contribute to the dentate gyrus (DG), whereas Sonic Hedgehog affects the bona fide stem cells and transit amplifying cells (the partially committed progeny of stem cells). These studies further define how discrete populations of cells react to specific extracellular signals provided within the neurogenic niche to survive, proliferate, and form functional mature cell types.
Assuntos
Giro Denteado/citologia , Neurônios/citologia , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Transativadores/fisiologia , Proteínas Wnt/fisiologia , Animais , Astrócitos/citologia , Diferenciação Celular/fisiologia , Linhagem da Célula , Proteínas Hedgehog , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Modelos Neurológicos , Morfogênese/fisiologia , Oligodendroglia/citologia , Ratos , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/fisiologia , Tamoxifeno/farmacologia , Proteína GLI1 em Dedos de ZincoRESUMO
Here, we show that the p53 family member, p73, is necessary for survival and long-term maintenance of CNS neurons, including postnatal cortical neurons. In p73-/- animals, cortical neuron number is normal at birth but decreases significantly by postnatal day 14 (P14)-P16 because of enhanced apoptosis. This decrease continues into adulthood, when p73-/- animals have approximately one-half as many cortical cells as their wild-type littermates. Cortical neurons express the DeltaNp73alpha protein, and overexpression of DeltaNp73 isoforms rescues cortical neurons from diverse apoptotic stimuli. Thus, DeltaNp73 isoforms are survival proteins in cortical neurons, and their deletion causes a gradual loss of cortical neurons in the weeks and months after birth. This decrease in CNS neuron number in p73-/- animals is not limited to the cortex; facial motor neuron number is decreased, and postnatal development of the olfactory bulb is greatly perturbed. These findings, together with our previous work showing that DeltaNp73 is essential for survival of peripheral sympathetic neurons (Pozniak et al., 2000), indicate that p73 isoforms are essential survival proteins in CNS as well as PNS neurons, and that they likely play a role not only during developmental cell death but also in the long-term maintenance of at least some adult neurons.