RESUMO
Inherited myelopathies are a small, but important subset of diseases that cause dysfunction of the spinal cord. Manifestations can include various combinations of signs and symptoms, including disturbance of gait, spasticity, paraplegia, amyotrophy, sensory loss, and urinary sphincter dysfunction. These diseases can be divided into classes that include (1) distal axonopathies-exemplified by hereditary spastic paraplegia, (2) motor neuron diseases including familial amyotrophic lateral sclerosis and spinal muscular atrophy, (3) inborn errors of metabolism such as adrenomyeloneuropathy, and (4) other inherited diseases with myelopathy as part of their spectrum of manifestations. Although the inherited myelopathies are relatively rare diseases, knowledge of them and their manifestations is important for the physician faced with a patient with myelopathy, particularly if there are similarly affected individuals in the patient's family. In addition, understanding the pathophysiologic underpinnings of these diseases provides insight into the molecular biology of the nervous system and provides a gateway toward developing treatments for these diseases.
Assuntos
Doenças da Medula Espinal/congênito , Doenças da Medula Espinal/fisiopatologia , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Predisposição Genética para Doença/genética , Humanos , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/fisiopatologia , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatologia , Paraplegia Espástica Hereditária/diagnóstico , Paraplegia Espástica Hereditária/fisiopatologia , Doenças da Medula Espinal/diagnósticoRESUMO
Adenylyl cyclase types 1 (AC1) and 8 (AC8), the two major calmodulin-stimulated adenylyl cyclases in the brain, couple NMDA receptor activation to cAMP signaling pathways. Cyclic AMP signaling pathways are important for many brain functions, such as learning and memory, drug addiction, and development. Here we show that wild-type, AC1, AC8, or AC1&8 double knockout (DKO) mice were indistinguishable in tests of acute pain, whereas behavioral responses to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant, were reduced or abolished in AC1&8 DKO mice. AC1 and AC8 are highly expressed in the anterior cingulate cortex (ACC), and contribute to inflammation-induced activation of CREB. Intra-ACC administration of forskolin rescued behavioral allodynia defective in the AC1&8 DKO mice. Our studies suggest that AC1 and AC8 in the ACC selectively contribute to behavioral allodynia.
Assuntos
Adenilil Ciclases/deficiência , Encéfalo/enzimologia , Calmodulina/metabolismo , AMP Cíclico/metabolismo , Hiperalgesia/genética , Vias Neurais/enzimologia , Dor/genética , Adenilil Ciclases/genética , Animais , Comportamento Animal/fisiologia , Encéfalo/fisiopatologia , AMP Cíclico/análogos & derivados , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Adjuvante de Freund , Hiperalgesia/enzimologia , Hiperalgesia/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Vias Neurais/fisiopatologia , Dor/enzimologia , Dor/fisiopatologia , Medição da Dor , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) produce the most common inherited form of Parkinson's disease (PD) but the function of LRRK2 remains poorly understood. The presynaptic role of multiple genes linked to PD including α-synuclein (α-syn) has suggested that LRRK2 may also influence neurotransmitter release, a possibility supported by recent work. However, the use of disease-associated mutants that cause toxicity complicates the analysis. To determine whether LRRK2 normally influences the synaptic vesicle, we have now used a combination of imaging and electrophysiology to study LRRK2 knockout (KO) mice. Surprisingly, we find that in hippocampal (generally excitatory) neurons, the loss of LRRK2 does not affect synaptic vesicle exocytosis, endocytosis or the mobility of α-syn. Double KO (DKO) mice lacking LRRK1 as well as LRRK2 also show no defect in transmitter release by hippocampal neurons. However, in striatal neurons, which express LRRK2 at higher levels, the loss of LRRK2 leads to modest acceleration of synaptic vesicle endocytosis. Thus, endogenous LRRK2 normally slows synaptic vesicle recycling at striatal terminals.
RESUMO
The importance of the cAMP signaling pathway in the modulation of ethanol sensitivity has been suggested by studies in organisms from Drosophila melanogaster to man. However, the involvement of specific isoforms of adenylyl cyclase (AC), the molecule that converts ATP to cAMP, has not been systemically determined in vivo. Because AC1 and AC8 are the only AC isoforms stimulated by calcium, and ethanol modulates calcium flux by the NMDA receptor, we hypothesized that these ACs would be important in the neural response to ethanol. AC1 knock-out (KO) mice and double knock-out (DKO) mice with genetic deletion of both AC1 and AC8 display substantially increased sensitivity to ethanol-induced sedation compared with wild-type (WT) mice, whereas AC8 KO mice are only minimally more sensitive. In contrast, AC8 KO and DKO mice, but not AC1 KO mice, demonstrate decreased voluntary ethanol consumption compared with WT mice. DKO mice do not display increased sleep time compared with WT mice after administration of ketamine or pentobarbital, indicating that the mechanism of enhanced ethanol sensitivity in these mice is likely distinct from the antagonism of ethanol of the NMDA receptor and potentiation of the GABA(A) receptor. Ethanol does not enhance calcium-stimulated AC activity, but the ethanol-induced phosphorylation of a discrete subset of protein kinase A (PKA) substrates is compromised in the brains of DKO mice. These results indicate that the unique activation of PKA signaling mediated by the calcium-stimulated ACs is an important component of the neuronal response to ethanol.
Assuntos
Adenilil Ciclases/metabolismo , Cálcio/farmacologia , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Neurônios/efeitos dos fármacos , Adenilil Ciclases/deficiência , Análise de Variância , Animais , Ataxia/fisiopatologia , Comportamento Animal , Western Blotting/métodos , Depressores do Sistema Nervoso Central/sangue , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Etanol/sangue , Antagonistas de Aminoácidos Excitatórios/farmacologia , Preferências Alimentares/efeitos dos fármacos , Agonistas GABAérgicos/farmacologia , Isoxazóis/farmacologia , Ketamina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Pentobarbital/farmacologia , Fosforilação/efeitos dos fármacos , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Quinina/farmacologia , Tempo de Reação/efeitos dos fármacos , Reflexo/efeitos dos fármacos , Sacarina/farmacologia , Sono/efeitos dos fármacos , Sono/genéticaRESUMO
Fetal alcohol exposure results in cognitive and neurobehavioral deficits, but the effects of modifying genetic loci on the severity of these sequelas have not been well characterized. Although the cAMP signaling pathway has been shown to be an important modulator of ethanol sensitivity in adult mice, its potential role in modulating ethanol-induced neurodegeneration has not been examined. Adenylyl cyclases (ACs) 1 and 8 produce cAMP in response to intracellular calcium elevation and modulate several aspects of neuronal function, including ethanol sensitivity. AC1 and AC8 are expressed widely throughout the brain of neonatal mice, and genetic deletion of both AC1 and AC8 in double-knock-out (DKO) mice enhances ethanol-induced neurodegeneration in the brains of neonatal mice. In addition, ethanol treatment induces significantly greater levels of caspase-3 activation in the brains of DKO mice compared with wild-type (WT) mice, reflecting higher numbers of apoptotic neurons. Administration of the NMDA receptor antagonist MK801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine hydrogen maleate] or the GABA(A) receptor potentiator phenobarbital, which mimics components of the effects of ethanol on neurons, results in significantly greater neurodegeneration in the brains of neonatal DKO mice than WT mice. Furthermore, loss of a single calcium-stimulated AC isoform potentiates neurodegeneration after administration of ethanol, MK801, or phenobarbital. In contrast, the levels of physiological cell death, death after hypoxia/ischemia, and excitotoxic cell death are not increased in the brains of DKO mice. Thus, AC1 and AC8 are critical modulators of neurodegeneration induced by activity blockade in the neonatal brain and represent genetic loci that may potentially modify the severity of fetal alcohol syndrome.
Assuntos
Adenilil Ciclases/metabolismo , Encéfalo/efeitos dos fármacos , Cálcio/farmacologia , Etanol , Doenças Neurodegenerativas/induzido quimicamente , Anilidas/metabolismo , Animais , Animais Recém-Nascidos , Comportamento Animal , Western Blotting/métodos , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Caspase 3 , Caspases/metabolismo , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Etanol/sangue , Moduladores GABAérgicos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Hipóxia/metabolismo , Hipóxia/patologia , Hibridização In Situ/métodos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/fisiologia , Fármacos Neuroprotetores/farmacologia , Oligopeptídeos/metabolismo , Fenobarbital/farmacologia , Coloração pela Prata/métodos , Fatores de TempoRESUMO
The regulated release of polypeptides has a central role in physiology, behavior, and development, but the mechanisms responsible for production of the large dense core vesicles (LDCVs) capable of regulated release have remained poorly understood. Recent work has implicated cytosolic adaptor protein AP-3 in the recruitment of LDCV membrane proteins that confer regulated release. However, AP-3 in mammals has been considered to function in the endolysosomal pathway and in the biosynthetic pathway only in yeast. We now find that the mammalian homolog of yeast VPS41, a member of the homotypic fusion and vacuole protein sorting (HOPS) complex that delivers biosynthetic cargo to the endocytic pathway in yeast, promotes LDCV formation through a common mechanism with AP-3, indicating a conserved role for these proteins in the biosynthetic pathway. VPS41 also self-assembles into a lattice, suggesting that it acts as a coat protein for AP-3 in formation of the regulated secretory pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/metabolismo , Exocitose/fisiologia , Biogênese de Organelas , Via Secretória/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Células COS , Chlorocebus aethiops , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Endossomos/metabolismo , Humanos , Fusão de Membrana , Proteínas de Membrana/metabolismo , Células PC12 , Transporte Proteico , Ratos , Vesículas Secretórias/metabolismo , Fatores de Transcrição/genética , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND: The Ca2+-stimulated adenylyl cyclases (ACs), AC1 and AC8, are key components of long-term memory processing. AC1 and AC8 double knockout mice (Adcy1(-/-)Adcy8(-/-); DKO) display impaired fear memory processing; the mechanism of this impairment is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesize that the Ca2+-stimulated ACs modulate long-lasting transcriptional changes essential for fear memory consolidation and maintenance. Here, we report a genome-wide study of gene expression changes associated with conditioned fear (CF) memory in wild-type and DKO mice to identify AC-dependent gene regulatory changes that occur in the amygdala and hippocampus at baseline and different time points after CF learning. We observed an overall decrease in transcriptional changes in DKO mice across all time points, but most strikingly, at periods when memory consolidation and retention should be occurring. Further, we identified a shared set of transcription factor binding sites in genes upregulated in wild-type mice that were associated with downregulated genes in DKO mice. To prove the temporal and regional importance of AC activity on different stages of memory processing, the tetracycline-off system was used to produce mice with forebrain-specific inducible expression of AC8 on a DKO background. CF behavioral results reveal that adult restoration of AC8 activity in the forebrain is sufficient for intact learning, while cessation of this expression at any time point across learning causes memory deficits. CONCLUSIONS/SIGNIFICANCE: Overall, these studies demonstrate that the Ca2+-stimulated ACs contribute to the formation and maintenance of fear memory by a network of long-term transcriptional changes.
Assuntos
Adenilil Ciclases/metabolismo , Cálcio/metabolismo , Medo , Regulação da Expressão Gênica , Memória , Animais , Western Blotting , Imuno-Histoquímica , Camundongos , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Although ethanol exerts widespread action in the brain, only recently has progress been made in understanding the specific events occurring at the synapse during ethanol exposure. Mice deficient in the calcium-stimulated adenylyl cyclases, AC1 and AC8 (DKO), demonstrate increased sedation duration and impaired phosphorylation by protein kinase A (PKA) following acute ethanol treatment. While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have used phosphoproteomic techniques and identified several presynaptic proteins that are phosphorylated in the brains of wild type mice (WT) after ethanol exposure, including synapsin, a known PKA target. Phosphorylation of synapsins I and II, as well as phosphorylation of non-PKA targets, such as, eukaryotic elongation factor-2 (eEF-2) and dynamin is significantly impaired in the brains of DKO mice. This deficit is primarily driven by AC1, as AC1-deficient, but not AC8-deficient mice also demonstrate significant reductions in phosphorylation of synapsin and eEF-2 in cortical and hippocampal tissues. DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade.