RESUMO
The Dlg4 gene encodes for post-synaptic density protein 95 (PSD95), a major synaptic protein that clusters glutamate receptors and is critical for plasticity. PSD95 levels are diminished in ageing and neurodegenerative disorders, including Alzheimer's disease and Huntington's disease. The epigenetic mechanisms that (dys)regulate transcription of Dlg4/PSD95, or other plasticity genes, are largely unknown, limiting the development of targeted epigenome therapy. We analysed the Dlg4/PSD95 epigenetic landscape in hippocampal tissue and designed a Dlg4/PSD95 gene-targeting strategy: a Dlg4/PSD95 zinc finger DNA-binding domain was engineered and fused to effector domains to either repress (G9a, Suvdel76, SKD) or activate (VP64) transcription, generating artificial transcription factors or epigenetic editors (methylating H3K9). These epi-editors altered critical histone marks and subsequently Dlg4/PSD95 expression, which, importantly, impacted several hippocampal neuron plasticity processes. Intriguingly, transduction of the artificial transcription factor PSD95-VP64 rescued memory deficits in aged and Alzheimer's disease mice. Conclusively, this work validates PSD95 as a key player in memory and establishes epigenetic editing as a potential therapy to treat human neurological disorders.
Assuntos
Doença de Alzheimer/genética , Comportamento Animal , Cognição , Proteína 4 Homóloga a Disks-Large/genética , Repressão Epigenética , Hipocampo/metabolismo , Memória , Ativação Transcricional , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Epigênese Genética , Código das Histonas , Humanos , Camundongos , Camundongos Transgênicos , Ratos , Dedos de ZincoRESUMO
Dendrite arbor growth, or dendritogenesis, is choreographed by a diverse set of cues, including the NMDA receptor (NMDAR) subunits NR2A and NR2B. While NR1NR2B receptors are predominantly expressed in immature neurons and promote plasticity, NR1NR2A receptors are mainly expressed in mature neurons and induce circuit stability. How the different subunits regulate these processes is unclear, but this is likely related to the presence of their distinct C-terminal sequences that couple different signaling proteins. Calcium-calmodulin-dependent protein kinase II (CaMKII) is an interesting candidate as this protein can be activated by calcium influx through NMDARs. CaMKII triggers a series of biochemical signaling cascades, involving the phosphorylation of diverse targets. Among them, the activation of cAMP response element-binding protein (CREB-P) pathway triggers a plasticity-specific transcriptional program through unknown epigenetic mechanisms. Here, we found that dendritogenesis in hippocampal neurons is impaired by several well-characterized constructs (i.e., NR2B-RS/QD) and peptides (i.e., tatCN21) that specifically interfere with the recruitment and interaction of CaMKII with the NR2B C-terminal domain. Interestingly, we found that transduction of NR2AΔIN, a mutant NR2A construct with increased interaction to CaMKII, reactivates dendritogenesis in mature hippocampal neurons in vitro and in vivo. To gain insights into the signaling and epigenetic mechanisms underlying NMDAR-mediated dendritogenesis, we used immunofluorescence staining to detect CREB-P and acetylated lysine 27 of histone H3 (H3K27ac), an activation-associated histone tail mark. In contrast to control mature neurons, our data shows that activation of the NMDAR/CaMKII/ERK-P/CREB-P signaling axis in neurons expressing NR2AΔIN is not correlated with increased nuclear H3K27ac levels.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dendritos/enzimologia , Hipocampo/enzimologia , Histonas/metabolismo , Neurogênese , Plasticidade Neuronal , Receptores de N-Metil-D-Aspartato/metabolismo , Acetilação , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Dendritos/efeitos dos fármacos , Idade Gestacional , Hipocampo/efeitos dos fármacos , Hipocampo/embriologia , Mutação , Neurogênese/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Peptídeos/farmacologia , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Interferência de RNA , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais , TransfecçãoRESUMO
N-methyl-D-aspartate receptors (NMDARs) are known to regulate axonal refinement and dendritic branching. However, because NMDARs are abundantly present as tri-heteromers (e.g., NR1/NR2A/NR2B) during development, the precise role of the individual subunits NR2A and NR2B in these processes has not been elucidated. Ventral spinal cord neurons (VSCNs) provide a unique opportunity to address this problem, because the expression of both NR2A and NR2B (but not NR1) is downregulated in culture. Exogenous NR2A or NR2B were introduced into these naturally NR2-null neurons at 4 DIV, and electrophysiological recordings at 11 DIV confirmed that synaptic NR1NR2A receptors and NR1NR2B receptors were formed, respectively. Analysis of the dendritic architecture showed that introduction of NR2B, but not NR2A, dramatically increased the number of secondary and tertiary dendritic branches of VSCNs. Whole cell patch-clamp recordings further indicated that the newly formed branches in NR2B-expressing neurons were able to establish functional synapses because the frequency of miniature AMPA-receptor synaptic currents was increased. Using previously described mutants, we also found that disruption of the interaction between NR2B and RasGRF1 dramatically impaired dendritic branch formation in VSCNs. The differential role of the NR2A and NR2B subunits and the requirement for RasGRF1 in regulating branch formation was corroborated in hippocampal cultures. We conclude that the association between NR1NR2B-receptors and RasGRF1 is needed for dendritic branch formation in VSCNs and hippocampal neurons in vitro. The dominated NR2A expression and the limited interactions of this subunit with the signaling protein RasGRF1 may contribute to the restricted dendritic arbor development in the adult CNS.