RESUMO
BACKGROUND: Genome-wide association studies have established clusterin (CLU) as a genetic modifier for late-onset Alzheimer's disease (AD). Both protective and risk alleles have been identified which may be associated with its expression levels. However, the physiological function of clusterin in the central nervous system remains largely unknown. METHODS: We examined Clu expression in mouse brains by immunohistochemistry and high-resolution imaging. We performed electrophysiological recordings and morphological analysis of dendritic spines in wild-type and Clu knockout mice. We tested synaptic function of astrocytic Clu using neuron-glia co-cultures and by AAV-mediated astroglial Clu expression in vivo. Finally, we investigated the role of astrocytic Clu on synaptic properties and amyloid pathology in 5xFAD transgenic mouse model of AD. RESULTS: We show that astrocyte secreted Clu co-localizes with presynaptic puncta of excitatory neurons. Loss of Clu led to impaired presynaptic function and reduced spine density in vivo. Neurons co-cultured with Clu-overexpressing astrocytes or treated with conditioned media from HEK293 cells transfected with Clu displayed enhanced excitatory neurotransmission. AAV-mediated astroglial Clu expression promoted excitatory neurotransmission in wild-type mice and rescued synaptic deficits in Clu knockout mice. Overexpression of Clu in the astrocytes of 5xFAD mice led to reduced Aß pathology and fully rescued the synaptic deficits. CONCLUSION: We identify Clu as an astrocyte-derived synaptogenic and anti-amyloid factor; the combination of these activities may influence the progression of late-onset AD.
Assuntos
Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Clusterina/metabolismo , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Clusterina/genética , Modelos Animais de Doenças , Estudo de Associação Genômica Ampla , Humanos , Camundongos Transgênicos , Neuropatologia/métodos , Transmissão Sináptica/genéticaRESUMO
Neuroinflammation has been increasingly recognized to play a critical role in Alzheimer's disease (AD). The epoxy fatty acids (EpFAs) are derivatives of the arachidonic acid metabolism pathway and have anti-inflammatory activities. However, their efficacy is limited because of their rapid hydrolysis by the soluble epoxide hydrolase (sEH). We report that sEH is predominantly expressed in astrocytes and is elevated in postmortem brain tissue from patients with AD and in the 5xFAD ß amyloid mouse model of AD. The amount of sEH expressed in AD mouse brains correlated with a reduction in brain EpFA concentrations. Using a specific small-molecule sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), we report that TPPU treatment protected wild-type mice against LPS-induced inflammation in vivo. Long-term administration of TPPU to the 5xFAD mouse model via drinking water reversed microglia and astrocyte reactivity and immune pathway dysregulation. This was associated with reduced ß amyloid pathology and improved synaptic integrity and cognitive function on two behavioral tests. TPPU treatment correlated with an increase in EpFA concentrations in the brains of 5xFAD mice, demonstrating brain penetration and target engagement of this small molecule. These findings support further investigation of TPPU as a potential therapeutic agent for the treatment of AD.
Assuntos
Doença de Alzheimer , Epóxido Hidrolases , Doença de Alzheimer/tratamento farmacológico , Animais , Compostos de Epóxi , Humanos , Camundongos , Compostos de Fenilureia , PiperidinasRESUMO
Strong evidence implicates the complement pathway as an important contributor to amyloid pathology in Alzheimer's disease (AD); however, the role of complement in tau modulation remains unclear. Here we show that the expression of C3 and C3a receptor (C3aR1) are positively correlated with cognitive decline and Braak staging in human AD brains. Deletion of C3ar1 in PS19 mice results in the rescue of tau pathology and attenuation of neuroinflammation, synaptic deficits, and neurodegeneration. Through RNA sequencing and cell-type-specific transcriptomic analysis, we identify a C3aR-dependent transcription factor network that regulates a reactive glial switch whose inactivation ameliorates disease-associated microglia and neurotoxic astrocyte signatures. Strikingly, this C3aR network includes multiple genes linked to late-onset AD. Mechanistically, we identify STAT3 as a direct target of C3-C3aR signaling that functionally mediates tau pathogenesis. All together our findings demonstrate a crucial role for activation of the C3-C3aR network in mediating neuroinflammation and tau pathology.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/patologia , Complemento C3a/metabolismo , Citocinas/metabolismo , Receptores de Complemento/metabolismo , Transdução de Sinais/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/complicações , Doença de Alzheimer/patologia , Animais , Encéfalo/fisiopatologia , Proteínas de Ligação ao Cálcio , Transtornos Cognitivos/etiologia , Complemento C3a/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Potenciais Evocados/fisiologia , Feminino , Redes Reguladoras de Genes/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Receptores de Complemento/genética , Transdução de Sinais/efeitos dos fármacos , Tauopatias/complicações , Regulação para Cima/fisiologia , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
The progression of tau pathology in Alzheimer's disease follows a stereotyped pattern, and recent evidence suggests a role of synaptic connections in this process. Astrocytes are well positioned at the neuronal synapse to capture and degrade extracellular tau as it transits the synapse and hence could potentially have the ability to inhibit tau spreading and delay disease progression. Our study shows increased expression and activity of Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, in response to tau pathology in both human brains with dementia and transgenic mouse models. Exogenous TFEB expression in primary astrocytes enhances tau fibril uptake and lysosomal activity, while TFEB knockout has the reverse effect. In vivo, induced TFEB expression in astrocytes reduces pathology in the hippocampus of PS19 tauopathy mice, as well as prominently attenuates tau spreading from the ipsilateral to the contralateral hippocampus in a mouse model of tau spreading. Our study suggests that astrocytic TFEB plays a functional role in modulating extracellular tau and the propagation of neuronal tau pathology in tauopathies such as Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Hipocampo/metabolismo , Sinapses/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Astrócitos/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Hipocampo/patologia , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Sinapses/genética , Sinapses/patologia , Proteínas tau/genéticaRESUMO
Neurogenic differentiation factor 2 (NeuroD2) is a highly expressed transcription factor in the developing central nervous system. In newborn neurons, NeuroD2-mediated gene expression promotes differentiation, maturation, and survival. In addition to these early, cell-intrinsic developmental processes, NeuroD2 in postmitotic neurons also regulates synapse growth and ion channel expression to control excitability. While NeuroD2 transactivation can be induced in an activity-dependent manner, little is known about how expression of NeuroD2 itself is regulated. Using genome-wide, mRNA-based microarray analysis, we found that NeuroD2 is actually one of hundreds of genes whose mRNA levels are suppressed by synaptic activity, in a manner dependent upon N-methyl d-aspartate receptor (NMDAR) activation. We confirmed this observation both in vitro and in vivo and provide evidence that this happens at the level of transcription and not mRNA stability. Our experiments further indicate that suppression of NeuroD2 message by NMDARs likely involves both CaMKII and MAPK but not voltage-gated calcium channels, in contrast to its mechanism of transactivation. We predict from these data that NMDARs may transduce information about the level of synaptic activity a developing neuron receives, to down-regulate NeuroD2 and allow proper maturation of cortical circuits by suppressing expression of neurite and synaptic growth promoting gene products.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Perfilação da Expressão Gênica , Camundongos , Análise em Microsséries , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
KEY POINTS: Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks to allow proper brain function. Disrupting this balance may lead to autism spectral disorders and epilepsy. We show the basic helix-loop-helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell-intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo. We identify two genes potentially downstream of NeuroD2-mediated transcription that regulate these parameters: gastrin-releasing peptide and the small conductance, calcium-activated potassium channel, SK2. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability. Our results offer insight into how synaptic innervation and intrinsic excitability are coordinated during cortical development. ABSTRACT: Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks for proper brain function. Disruption of this balance during development may lead to autism spectral disorders and epilepsy. Synaptic excitation is counterbalanced by synaptic inhibition but also by attenuation of cell-intrinsic neuronal excitability. To maintain proper excitation levels during development, neurons must sense activity over time and regulate the expression of genes that control these parameters. While this is a critical process, little is known about the transcription factors involved in coordinating gene expression to control excitatory/inhibitory synaptic balance. We show here that the basic helix-loop-helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell-intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo as shown by ex vivo analysis of a NeuroD2 knockout mouse. Using microarray analysis and comparing wild-type and NeuroD2 knockout cortical networks, we identified two potential gene targets of NeuroD2 that contribute to these processes: gastrin-releasing peptide (GRP) and the small conductance, calcium-activated potassium channel, SK2. We found that the GRP receptor antagonist RC-3059 and the SK2 specific blocker apamin partially reversed the effects of increased NeuroD2 expression on inhibitory synaptic drive and action potential repolarization, respectively. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability and offer insight into how these processes are coordinated during cortical development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Neuropeptídeos/fisiologia , Células Piramidais/fisiologia , Córtex Somatossensorial/fisiologia , Sinapses/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Peptídeo Liberador de Gastrina/genética , Potenciais Pós-Sinápticos Inibidores , Camundongos Knockout , Neuropeptídeos/genética , Ratos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genéticaRESUMO
In humans, atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. The genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. It was noted previously that Nkx2-5, which is expressed in the PV myocardium and reinforces a chamber-like myocardial identity in the PV, is lacking in the SAN. Here we present evidence that in mice Shox2 antagonizes the transcriptional output of Nkx2-5 in the PV myocardium and in a functional Nkx2-5(+) domain within the SAN to determine cell fate. Shox2 deletion in the Nkx2-5(+) domain of the SAN caused sick sinus syndrome, associated with the loss of the pacemaker program. Explanted Shox2(+) cells from the embryonic PV myocardium exhibited pacemaker characteristics including node-like electrophysiological properties and the capability to pace surrounding Shox2(-) cells. Shox2 deletion led to Hcn4 ablation in the developing PV myocardium. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in the PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. A similar mechanism is also adopted in differentiated embryoid bodies. We found that Shox2 interacts with Nkx2-5 directly, and discovered a substantial genome-wide co-occupancy of Shox2, Nkx2-5 and Tbx5, further supporting a pivotal role for Shox2 in the core myogenic program orchestrating venous pole and pacemaker development.
Assuntos
Proteínas de Homeodomínio/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Veias Pulmonares/metabolismo , Nó Sinoatrial/metabolismo , Fatores de Transcrição/fisiologia , Animais , Relógios Biológicos , Diferenciação Celular , Linhagem da Célula , Separação Celular , Eletrocardiografia , Corpos Embrioides/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Coração/embriologia , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Fenótipo , Estrutura Terciária de Proteína , Proteínas com Domínio T/metabolismoRESUMO
BACKGROUND: The assembly of neural circuits requires the concerted action of both genetically determined and activity-dependent mechanisms. Calcium-regulated transcription may link these processes, but the influence of specific transcription factors on the differentiation of synapse-specific properties is poorly understood. Here we characterize the influence of NeuroD2, a calcium-dependent transcription factor, in regulating the structural and functional maturation of the hippocampal mossy fiber (MF) synapse. RESULTS: Using NeuroD2 null mice and in vivo lentivirus-mediated gene knockdown, we demonstrate a critical role for NeuroD2 in the formation of CA3 dendritic spines receiving MF inputs. We also use electrophysiological recordings from CA3 neurons while stimulating MF axons to show that NeuroD2 regulates the differentiation of functional properties at the MF synapse. Finally, we find that NeuroD2 regulates PSD95 expression in hippocampal neurons and that PSD95 loss of function in vivo reproduces CA3 neuron spine defects observed in NeuroD2 null mice. CONCLUSION: These experiments identify NeuroD2 as a key transcription factor that regulates the structural and functional differentiation of MF synapses in vivo.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Espinhas Dendríticas/genética , Hipocampo/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Neuropeptídeos/genética , Sinapses/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Espinhas Dendríticas/metabolismo , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Sinapses/metabolismoRESUMO
Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) or small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of the unique DUF283 domain within dicer is largely unknown. Here we report the first structure of the DUF283 domain from the Arabidopsis thaliana DCL4. The DUF283 domain adopts an alpha-beta-beta-beta-alpha topology and resembles the structural similarity to the double-stranded RNA-binding domain. Notably, the N-terminal alpha helix of DUF283 runs cross over the C-terminal alpha helix orthogonally, therefore, N- and C-termini of DUF283 are in close proximity. Biochemical analysis shows that the DUF283 domain of DCL4 displays weak dsRNA binding affinity and specifically binds to double-stranded RNA-binding domain 1 (dsRBD1) of Arabidopsis DRB4, whereas the DUF283 domain of DCL1 specifically binds to dsRBD2 of Arabidopsis HYL1. These data suggest a potential functional role of the Arabidopsis DUF283 domain in target selection in small RNA processing.
Assuntos
Arabidopsis/química , Ribonuclease III/química , Sítios de Ligação , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/químicaRESUMO
Neurexins are highly polymorphic cell-surface adhesive molecules in neurons. In cultured mammalian cell system, they were found to be involved in synaptogenesis. Here, we report for the first time that Drosophila neurexin is required for synapse formation and associative learning in larvae. Drosophila genome encodes a single functional neurexin (CG7050; Neurexin-1 or Nrx-1), which is a homolog of vertebrate alpha-neurexin. Neurexin-1 is expressed in central nervous system and highly enriched in synaptic regions of the ventral ganglion and brain. Neurexin-1 null mutants are viable and fertile, but have shortened lifespan. The synapse number is decreased in central nervous system in Neurexin-1 null mutants. In addition, Neurexin-1 null mutants exhibit associative learning defect in larvae.