RESUMO
AMPA receptors (AMPARs) play a critical role in synaptic plasticity and learning and memory, and dysfunction or dysregulation of AMPARs could lead to various neurological and psychiatric disorders, such as Alzheimer's disease (AD). However, the dynamics and/or longitudinal changes of AMPARs in vivo during AD pathogenesis remain elusive. Here, employing 5xFAD SEP-GluA1 KI mice, we investigated endogenous AMPA receptor dynamics in a whisker deflection-associated Go/No-go learning paradigm. We found a significant increase in synaptosomal AMPA receptor subunits GluA1 in WT mice after learning, while no such changes were detected in 7-mo-old 5xFAD mice. Daily training led to an increase in endogenous spine surface GluA1 in Control mice, while this increase was absent in 5xFAD-KI mice which correlates with its learning defects in Go/No-go paradigm. Furthermore, we demonstrated that the onset of abnormal AMPAR dynamics corresponds temporally with microglia and astrocyte overactivation. Our results have shown that impairments in endogenous AMPA receptor dynamics play an important role in learning deficits in 5xFAD mice and AD pathogenesis.
Assuntos
Doença de Alzheimer , Receptores de AMPA , Humanos , Animais , Camundongos , Aprendizagem , Astrócitos , MicrogliaRESUMO
Long-term potentiation (LTP) has long been considered as an important cellular mechanism for learning and memory. LTP expression involves NMDA receptor-dependent synaptic insertion of AMPA receptors (AMPARs). However, how AMPARs are recruited and anchored at the postsynaptic membrane during LTP remains largely unknown. In this study, using CRISPR/Cas9 to delete the endogenous AMPARs and replace them with the mutant forms in single neurons, we have found that the amino-terminal domain (ATD) of GluA1 is required for LTP maintenance. Moreover, we show that GluA1 ATD directly interacts with the cell adhesion molecule neuroplastin-65 (Np65). Neurons lacking Np65 exhibit severely impaired LTP maintenance, and Np65 deletion prevents GluA1 from rescuing LTP in AMPARs-deleted neurons. Thus, our study reveals an essential role for GluA1/Np65 binding in anchoring AMPARs at the postsynaptic membrane during LTP.
Assuntos
Potenciais Pós-Sinápticos Excitadores/genética , Potenciação de Longa Duração/genética , Glicoproteínas de Membrana/genética , Células Piramidais/metabolismo , Receptores de AMPA/genética , Animais , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Cultura Primária de Células , Domínios Proteicos , Células Piramidais/citologia , Receptores de AMPA/metabolismo , Análise de Célula Única , Sinapses , Proteína Vermelha FluorescenteRESUMO
In the brain, AMPA-type glutamate receptors are major postsynaptic receptors at excitatory synapses that mediate fast neurotransmission and synaptic plasticity. α/ß-Hydrolase domain-containing 6 (ABHD6), a monoacylglycerol lipase, was previously found to be a component of AMPA receptor macromolecular complexes, but its physiological significance in the function of AMPA receptors (AMPARs) has remained unclear. The present study shows that overexpression of ABHD6 in neurons drastically reduced excitatory neurotransmission mediated by AMPA but not by NMDA receptors at excitatory synapses. Inactivation of ABHD6 expression in neurons by either CRISPR/Cas9 or shRNA knockdown methods significantly increased excitatory neurotransmission at excitatory synapses. Interestingly, overexpression of ABHD6 reduced glutamate-induced currents and the surface expression of GluA1 in HEK293T cells expressing GluA1 and stargazin, suggesting a direct functional interaction between these two proteins. The C-terminal tail of GluA1 was required for the binding between of ABHD6 and GluA1. Mutagenesis analysis revealed a GFCLIPQ sequence in the GluA1 C terminus that was essential for the inhibitory effect of ABHD6. The hydrolase activity of ABHD6 was not required for the effects of ABHD6 on AMPAR function in either neurons or transfected HEK293T cells. Thus, these findings reveal a novel and unexpected mechanism governing AMPAR trafficking at synapses through ABHD6.
Assuntos
Potenciais de Ação/fisiologia , Hipocampo/fisiologia , Monoacilglicerol Lipases/metabolismo , Neurônios/fisiologia , Receptores de AMPA/metabolismo , Transmissão Sináptica/fisiologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células HEK293 , Hipocampo/citologia , Humanos , Camundongos , Domínios Proteicos/fisiologiaRESUMO
The proper formation of synapses-specialized unitary structures formed between two neurons-is critical to mediating information flow in the brain. Synaptic cell adhesion molecules (CAMs) are thought to participate in the initiation of the synapse formation process. However, in vivo functional analysis demonstrates that most well known synaptic CAMs regulate synaptic maturation and plasticity rather than synapse formation, suggesting that either CAMs work synergistically in the process of forming synapses or more CAMs remain to be found. By screening for unknown CAMs using a co-culture system, we revealed that protein tyrosine phosphatase receptor type O (PTPRO) is a potent CAM that induces the formation of artificial synapse clusters in co-cultures of human embryonic kidney 293 cells and hippocampal neurons cultured from newborn mice regardless of gender. PTPRO was enriched in the mouse brain and localized to postsynaptic sites at excitatory synapses. The overexpression of PTPRO in cultured hippocampal neurons increased the number of synapses and the frequency of miniature EPSCs (mEPSCs). The knock-down (KD) of PTPRO expression in cultured neurons by short hairpin RNA (shRNA) reduced the number of synapses and the frequencies of the mEPSCs. The effects of shRNA KD were rescued by expressing either full-length PTPRO or a truncated PTPRO lacking the cytoplasmic domain. Consistent with these results, the N-terminal extracellular domain of PTPRO was required for its synaptogenic activity in the co-culture assay. Our data show that PTPRO is a synaptic CAM that serves as a potent initiator of the formation of excitatory synapses.SIGNIFICANCE STATEMENT The formation of synapses is critical for the brain to execute its function and synaptic cell adhesion molecules (CAMs) play essential roles in initiating the formation of synapses. By screening for unknown CAMs using a co-culture system, we revealed that protein tyrosine phosphatase receptor type O (PTPRO) is a potent CAM that induces the formation of artificial synapse clusters. Using loss-of-function and gain-of-function approaches, we show that PTPRO promotes the formation of excitatory synapses. The N-terminal extracellular domain of PTPRO was required for its synaptogenic activity in cultured hippocampal neurons and the co-culture assay. Together, our data show that PTPRO is a synaptic CAM that serves as a potent initiator of synapse formation.
Assuntos
Moléculas de Adesão de Célula Nervosa/fisiologia , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/fisiologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Técnicas de Cocultura , Potenciais Pós-Sinápticos Excitadores/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Moléculas de Adesão de Célula Nervosa/genética , Técnicas de Patch-Clamp , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genéticaRESUMO
Three neuronal pentraxins are expressed in brain, the membrane-bound "neuronal pentraxin receptor" (NPR) and the secreted proteins NP1 and NARP (i.e., NP2). Neuronal pentraxins bind to AMPARs at excitatory synapses and play important, well-documented roles in the activity-dependent regulation of neural circuits via this binding activity. However, it is unknown whether neuronal pentraxins perform roles in synapses beyond modulating postsynaptic AMPAR-dependent plasticity, and whether they may even act in inhibitory synapses. Here, we show that NPR expressed in non-neuronal cells potently induces formation of both excitatory and inhibitory postsynaptic specializations in cocultured hippocampal neurons. Knockdown of NPR in hippocampal neurons, conversely, dramatically decreased assembly and function of both excitatory and inhibitory postsynaptic specializations. Overexpression of NPR rescued the NPR knockdown phenotype but did not in itself change synapse numbers or properties. However, the NPR knockdown decreased the levels of NARP, whereas NPR overexpression produced a dramatic increase in the levels of NP1 and NARP, suggesting that NPR recruits and stabilizes NP1 and NARP on the presynaptic plasma membrane. Mechanistically, NPR acted in excitatory synapse assembly by binding to the N-terminal domain of AMPARs; antagonists of AMPA and GABA receptors selectively inhibited NPR-induced heterologous excitatory and inhibitory synapse assembly, respectively, but did not affect neurexin-1ß-induced synapse assembly as a control. Our data suggest that neuronal pentraxins act as signaling complexes that function as general trans-synaptic organizers of both excitatory and inhibitory synapses by a mechanism that depends, at least in part, on the activity of the neurotransmitter receptors at these synapses. SIGNIFICANCE STATEMENT: Neuronal pentraxins comprise three neuronal proteins, neuronal pentraxin receptor (NPR) which is a type-II transmembrane protein on the neuronal surface, and secreted neuronal pentraxin-1 and NARP. The general functions of neuronal pentraxins at synapses have not been explored, except for their basic AMPAR binding properties. Here, we examined the functional role of NPR at synapses because it is the only neuronal pentraxin that is anchored to the neuronal cell-surface membrane. We find that NPR is a potent inducer of both excitatory and inhibitory heterologous synapses, and that knockdown of NPR in cultured neurons decreases the density of both excitatory and inhibitory synapses. Our data suggest that NPR performs a general, previously unrecognized function as a universal organizer of synapses.
Assuntos
Proteína C-Reativa/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sinapses/fisiologia , Animais , Proteína C-Reativa/antagonistas & inibidores , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Técnicas de Cocultura , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Antagonistas GABAérgicos/farmacologia , Técnicas de Silenciamento de Genes , Células HEK293 , Hipocampo/fisiologia , Humanos , Camundongos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios , Técnicas de Patch-Clamp , RNA Interferente Pequeno/genética , Receptores de AMPA/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Due to the diversity of synapses in different brain areas, circuits and neurons, although many synaptic CAMs, such as Neurexins (NRXNs), Neuroligins (NLGNs), Synaptic cell adhesion molecules (SynCAMs), Leucine-rich-repeat transmembrane neuronal proteins (LRRTMs), and SLIT and NTRK-like protein (SLITRKs) have been identified as synaptogenic molecules, how these molecules determine specific synapse formation and whether other molecules driving synapse formation remain undiscovered are unclear. Here, to provide a tool for synapse labeling and synaptic CAMs screening by artificial synapse formation (ASF) assay, we generated synaptotagmin-1-tdTomato (Syt1-tdTomato) transgenic mice by inserting the tdTomato-fused synaptotagmin-1 coding sequence into the genome of C57BL/6J mice. In the brain of Syt1-tdTomato transgenic mice, the tdTomato-fused synaptotagmin-1 (SYT1-tdTomato) signals were widely observed in different areas and overlapped with synapsin-1, a widely-used synaptic marker. In the olfactory bulb, the SYT1-tdTomato signals are highly enriched in the glomerulus. In the cultured hippocampal neurons, the SYT1-tdTomato signals showed colocalization with several synaptic markers. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from Syt1-tdTomato transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from Syt1-tdTomato transgenic mice could form synaptic connections with HEK293T cells expressing NLGN2, LRRTM2, and SLITRK2 without immunostaining. Therefore, our work suggested that the Syt1-tdTomato transgenic mice with the ability to label synapses by tdTomato, and it will be a convenient tool for screening synaptogenic molecules.
Assuntos
Moléculas de Adesão Celular , Proteína Vermelha Fluorescente , Sinapses , Humanos , Camundongos , Animais , Camundongos Transgênicos , Células HEK293 , Camundongos Endogâmicos C57BL , Moléculas de Adesão Celular/metabolismo , Sinapses/fisiologia , Sinaptotagminas/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Venous hypertensive myelopathy (VHM), mainly induced by the spinal dural arteriovenous fistula, is a congestive spinal cord injury that currently has no appropriate animal model available in preclinical research. METHODS: Sprague Dawley rats (280-320 g) were used. The rats were divided into 3 groups: (1) Group 1, which underwent renal artery-dorsal spinal venous bypass (AVB group); (2) Group 2, which underwent renal artery-dorsal spinal venous bypass and drainage vein stenosis (AVB/VS group); and (3) Control group, with T13 dorsal vein ligation. The success of the model was assessed using Doppler ultrasound and 7.0-T magnetic resonance imaging. Transmission electron microscopy, histochemistry, proteomics, and western blot analysis were used to evaluate ultrastructural, pathological, and molecular features in the spinal cord and cerebrospinal fluid (CSF). RESULTS: The success rate of the arteriovenous bypass was 100% at 5 days and 83% at 2 weeks. The locomotor assessment showed decreased lower extremity strength in the AVB/VS group ( P = .0067), whereas unremarkable changes were found in the AVB and Control groups. Histochemical staining suggested a 2-fold expansion of the dorsal spinal vein in the AVB/VS group, which was lower than that in the AVB group ( P < .05); however, the former displayed greater myelin and neuronal damage ( P < .05) and slight dilatation of the central canal ( P > .05). Proteomics analysis revealed that the complement and coagulation cascade pathways were upregulated in the CSF of AVB/VS rats, whereas the C3 level was elevated both in the CSF and bilateral spinal cord. Furthermore, overexpression of C3, ITGB2, and CD9 in the spinal cord was confirmed by immunoblotting. CONCLUSION: These findings suggest that the AVB/VS model can effectively mimic the clinical and molecular characteristics of VHM. Furthermore, they suggest that impaired deep intramedullary venous drainage is the key reason for the VHM.
Assuntos
Modelos Animais de Doenças , Ratos Sprague-Dawley , Animais , Ratos , Masculino , Doenças da Medula Espinal/cirurgia , Doenças da Medula Espinal/etiologia , Doenças da Medula Espinal/diagnóstico por imagem , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/diagnóstico por imagem , Constrição Patológica , Derivação Arteriovenosa Cirúrgica , HipertensãoRESUMO
The hippocampus is essential for learning and memory, but it also plays an important role in regulating emotional behavior, as hippocampal excitability and plasticity affect anxiety and fear. Brain synaptic plasticity may be regulated by tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a known protein inhibitor of extracellular matrix (ECM), and the expression of TIMP1 in the hippocampus can be induced by neuronal excitation and various stimuli. However, the involvement of Timp1 in fear learning, anxiety, and hippocampal synaptic function remains to be established. Our study of Timp1 function in vivo revealed that Timp1 knockout mice exhibit anxiety-like behavior but normal fear learning. Electrophysiological results suggested that Timp1 knockout mice showed hyperactivity in the ventral CA1 region, but the basic synaptic transmission and plasticity were normal in the Schaffer collateral pathway. Taken together, our results suggest that deletion of Timp1 in vivo leads to the occurrence of anxiety behaviors, but that Timp1 is not crucial for fear learning.
Assuntos
Ansiedade , Medo , Camundongos Knockout , Inibidor Tecidual de Metaloproteinase-1 , Animais , Ansiedade/genética , Ansiedade/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Medo/fisiologia , Camundongos Endogâmicos C57BL , Camundongos , Masculino , Hipocampo/metabolismoRESUMO
Trafficking of α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs), mediated by AMPAR interacting proteins, enabled neurons to maintain tuning capabilities at rest or active state. α/ß-Hydrolase domain-containing 6 (ABHD6), an endocannabinoid hydrolase, was an AMPAR auxiliary subunit found to negatively regulate the surface delivery of AMPARs. While ABHD6 was found to prevent AMPAR tetramerization in endoplasmic reticulum, ABHD6 was also reported to localize at postsynaptic site. Yet, the role of ABHD6 interacting with AMPAR at postsynaptic site, and the physiological significance of ABHD6 regulating AMPAR trafficking remains elusive. Here, we generated the ABHD6 knockout (ABHD6KO) mice and found that deletion of ABHD6 selectively enhanced AMPAR-mediated basal synaptic responses and the surface expression of postsynaptic AMPARs. Furthermore, we found that loss of ABHD6 impaired hippocampal long-term depression (LTD) and synaptic downscaling in hippocampal synapses. AMPAR internalization assays revealed that ABHD6 was essential for neuronal activity-dependent endocytosis of surface AMPARs, which is independent of ABHD6's hydrolase activity. The defects of AMPAR endocytosis and LTD are expressed as deficits in learning flexibility in ABHD6KO mice. Collectively, we demonstrated that ABHD6 is an endocytic accessory protein promoting AMPAR endocytosis, thereby contributes to the formation of LTD, synaptic downscaling and reversal learning.
Assuntos
Hidrolases , Receptores de AMPA , Camundongos , Animais , Receptores de AMPA/metabolismo , Hidrolases/metabolismo , Plasticidade Neuronal/fisiologia , Aprendizagem , Sinapses/metabolismo , Endocitose , Hipocampo/metabolismo , Monoacilglicerol Lipases/metabolismoRESUMO
The extracellular matrix (ECM) is a complex molecular network distributed throughout the extracellular space of different tissues as well as the neuronal system. Previous studies have identified various ECM components that play important roles in neuronal maturation and signal transduction. ECM components are reported to be involved in neurogenesis, neuronal migration, and axonal growth by interacting or binding to specific receptors. In addition, the ECM is found to regulate synapse formation, the stability of the synaptic structure, and synaptic plasticity. Here, we mainly reviewed the effects of various ECM components on synapse formation and briefly described the related diseases caused by the abnormality of several ECM components.
Assuntos
Neurônios , Sinapses , Sinapses/fisiologia , Neurônios/metabolismo , Neurogênese , Matriz Extracelular/metabolismo , Transdução de Sinais , Plasticidade NeuronalRESUMO
BACKGROUND: Synapses are the connections between neurons in the central nervous system (CNS) or between neurons and other excitable cells in the peripheral nervous system (PNS), where electrical or chemical signals rapidly travel through one cell to another with high spatial precision. Synaptic analysis, based on synapse numbers and fine morphology, is the basis for understanding neurological functions and diseases. Manual analysis of synaptic structures in electron microscopy (EM) images is often limited by low efficiency and subjective bias. NEW METHOD: We developed a multifunctional synaptic analysis system based on several advanced deep learning (DL) models. The system achieved synapse counting in low-magnification EM images and synaptic ultrastructure analysis in high-magnification EM images. RESULTS: The synapse counting system based on ResNet18 and a Faster R-CNN model had a mean average precision (mAP) of 92.55%. For synaptic ultrastructure analysis, the Faster R-CNN model based on ResNet50 achieved a mAP of 91.60%, the DeepLab v3 + model based on ResNet50 enabled high performance in presynaptic and postsynaptic membrane segmentation with a global accuracy of 0.9811, and the Faster R-CNN model based on ResNet18 achieved a mAP of 91.41% for synaptic vesicle detection. CONCLUSIONS: The proposed multifunctional synaptic analysis system may help to overcome the experimental bias inherent in manual analysis, thereby facilitating EM image-based synaptic function studies.
Assuntos
Aprendizado Profundo , Sinapses/fisiologia , Microscopia Eletrônica , Vesículas Sinápticas , Neurônios/fisiologiaRESUMO
Accurate and efficient methods for identifying and tracking each animal in a group are needed to study complex behaviors and social interactions. Traditional tracking methods (e.g., marking each animal with dye or surgically implanting microchips) can be invasive and may have an impact on the social behavior being measured. To overcome these shortcomings, video-based methods for tracking unmarked animals, such as fruit flies and zebrafish, have been developed. However, tracking individual mice in a group remains a challenging problem because of their flexible body and complicated interaction patterns. In this study, we report the development of a multi-object tracker for mice that uses the Faster region-based convolutional neural network (R-CNN) deep learning algorithm with geometric transformations in combination with multi-camera/multi-image fusion technology. The system successfully tracked every individual in groups of unmarked mice and was applied to investigate chasing behavior. The proposed system constitutes a step forward in the noninvasive tracking of individual mice engaged in social behavior.
Assuntos
Aprendizado Profundo , Animais , Camundongos , Peixe-Zebra , Algoritmos , Redes Neurais de Computação , Comportamento SocialRESUMO
N-methyl-D-aspartate receptors (NMDARs) are essential for excitatory neurotransmission and synaptic plasticity. GluN2A and GluN2B, two predominant Glu2N subunits of NMDARs in the hippocampus and the cortex, display distinct clustered distribution patterns and mobility at synaptic and extrasynaptic sites. However, how GluN2A clusters are specifically organized and stabilized remains poorly understood. Here, we found that the previously reported GluN2A-specific binding partner Rabphilin-3A (Rph3A) has the ability to undergo phase separation, which relies on arginine residues in its N-terminal domain. Rph3A phase separation promotes GluN2A clustering by binding GluN2A's C-terminal domain. A complex formed by Rph3A, GluN2A, and the scaffolding protein PSD95 promoted Rph3A phase separation. Disrupting Rph3A's phase separation suppressed the synaptic and extrasynaptic surface clustering, synaptic localization, stability, and synaptic response of GluN2A in hippocampal neurons. Together, our results reveal the critical role of Rph3A phase separation in determining the organization and stability of GluN2A in the neuronal surface.
Assuntos
Hipocampo , Neurônios , Receptores de N-Metil-D-Aspartato , Sinapses , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/genética , Sinapses/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Neurônios/metabolismo , Rabfilina-3ARESUMO
SCOPE: Peanut stem and leaf (PSL), a traditional Chinese medicine, is widely used as a dietary supplement to improve sleep quality; however, the underlying mechanism is unclear. Here, the study aims to determine whether active compounds in PSL extract exert their effects by mediating neuronal excitability. METHODS AND RESULTS: Aqueous PSL extract (500 mg kg-1 BW) increases the duration of total sleep (TS), slow wave sleep (SWS) and rapid eye movement sleep (REMS) in BALB/c mice after 7 and 14 continuous days of intragastric administration. Two PSL extract components with flavonoid-like structures: 4',7-di-O-methylnaringenin (DMN, 61 µg kg-1 BW) and 2'-O-methylisoliquiritigenin (MIL, 12 µg kg-1 BW), show similar effects on sleep in BALB/c mice. Moreover, incubation with DMN (50 µM) and MIL (50 µM) acutely reduces voltage-gated sodium and potassium currents and suppresses the firing of evoked action potential in mouse cortical neurons, indicating the inhibition on neuronal excitability. Meanwhile, RNA-seq analysis predicts the potential regulation of voltage-gated channels, which is according with the molecular docking simulation that both MIL and DMN can bind to voltage gated sodium channels 1.2 (Nav 1.2). CONCLUSIONS: DMN and MIL are the active ingredients of PSL that improve sleep quality, suggesting that PSL promotes sleep by regulating the excitability of neurons.
Assuntos
Arachis , Flavonoides , Animais , Flavonoides/farmacologia , Camundongos , Simulação de Acoplamento Molecular , Neurônios , Extratos Vegetais/farmacologia , SonoRESUMO
Macroautophagy/autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggest that autophagy is impaired during cerebral ischemia, contributing to neuronal dysfunction and neurodegeneration. However, the outcomes after transient modification in autophagy machinery are not fully understood. This study investigated the effects of ischemic stress on autophagy and synaptic structures using a rat model of oxygen-glucose deprivation (OGD) in hippocampal neurons and a mouse model of middle cerebral artery occlusion (MCAO). Upon acute ischemia, an initial autophagy modification occurred in an upregulation manner. Following, the number of lysosomes increased, as well as lysosomal volume, indicating dysfunctional lysosomal storage. These changes were prevented by inhibiting autophagy via 3-methyladenine (3-MA) treatment or ATG7 (autophagy related 7) knockdown, or were mimicked by rapamycin (RAPA), a known activator of autophagy. This suggests that dysfunctional lysosomal storage is associated with the early burst of autophagy. Dysfunctional lysosomal storage contributed to autophagy dysfunction because the basal level of MTOR-dependent lysosomal biogenesis in the reperfusion was not sufficient to clear undegraded cargoes after transient autophagy upregulation. Further investigation revealed that impairment of synaptic ultra-structures, accompanied by dysfunctional lysosomal storage, may result from a failure in dynamic turnover of synaptic proteins. This indicates a vital role of autophagy-lysosomal machinery in the maintenance of synaptic structures. This study supports previous evidence that dysfunctional lysosomal storage may occur following the upregulation of autophagy in neurons. Appropriate autophagosome-lysosomal functioning is vital for maintenance of neuronal synaptic function and impacts more than the few known synaptic proteins.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALR: autophagic lysosome reformation; ATG7: autophagy related 7; CTSB: cathepsin B; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DEGs: differentially expressed genes; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; GO: Gene Ontology; HBSS: Hanks' balanced salt solution; HPCA: hippocalcin; i.c.v: intracerebroventricular; KEGG: kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; LSDs: lysosomal storage disorders; MAP2: microtubule-associated protein 2; MCAO: middle cerebral artery occlusion; mCTSB: mature CTSB; mCTSD: mature CTSD; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; OGD/R: oxygen-glucose deprivation/reoxygenation; PBS: phosphate-buffered saline; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; proCTSD: pro-cathepsin D; RAPA: rapamycin; RNA-seq: RNA sequencing; RPS6KB/p70S6K: ribosomal protein S6 kinase; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIM: Structured Illumination Microscopy; SNAP25: synaptosomal-associated protein 25; SQSTM1/p62: sequestosome 1; SYN1: synapsin I; SYT1: synaptotagmin I; TBST: tris-buffered saline Tween-20; TEM: transmission electron microscopy; TFEB: transcription factor EB; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride; TUBB3: tubulin, beta 3 class III.
Assuntos
Autofagia/fisiologia , Isquemia/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Regulação para Cima/fisiologia , Animais , Autofagossomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Ratos WistarRESUMO
Most fast excitatory synaptic transmissions in the mammalian brain are mediated by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs), which are ligand-gated cation channels. The membrane expression level of AMPARs is largely determined by auxiliary subunits in AMPAR macromolecules, including porcupine O-acyltransferase (PORCN), which negatively regulates AMPAR trafficking to the plasma membrane. However, whether PORCN-mediated regulation depends on AMPAR subunit composition or particular regions of a subunit has not been determined. We systematically examined the effects of PORCN on the ligand-gated current and surface expression level of GluA1, GluA2, and GluA3 AMPAR subunits, alone and in combination, as well as the PORCN-GluA interaction in heterologous HEK293T cells. PORCN inhibited glutamate-induced currents and the surface expression of investigated GluA AMPAR subunits in a subunit-independent manner. These inhibitory effects required neither the amino-terminal domain (ATD) nor the carboxy-terminal domain (CTD) of GluA subunits. In addition, PORCN interacted with AMPARs independently of their ATD or CTD. Thus, the functional inhibition of AMPARs by PORCN in transfected heterologous cells was independent of the ATD, CTD, and subunit composition of AMPARs.
RESUMO
Purkinje cells (PCs) in the cerebellum receive two excitatory afferents including granule cells-derived parallel fiber (PF) and the climbing fiber. Scaffolding protein Rack1 is highly expressed in the cerebellar PCs. Here, we found delayed formation of specific cerebellar vermis lobule and impaired motor coordination in PC-specific Rack1 conditional knockout mice. Our studies further revealed that Rack1 is essential for PF-PC synapse formation. In addition, Rack1 plays a critical role in regulating synaptic plasticity and long-term depression (LTD) induction of PF-PC synapses without changing the expression of postsynaptic proteins. Together, we have discovered Rack1 as the crucial molecule that controls PF-PC synaptogenesis and synaptic plasticity. Our studies provide a novel molecular insight into the mechanisms underlying the neural development and neuroplasticity in the cerebellum.
RESUMO
Mutations within the Shank3 gene, which encodes a key postsynaptic density (PSD) protein at glutamatergic synapses, contribute to the genetic etiology of defined autism spectrum disorders (ASDs), including Phelan-McDermid syndrome (PMS) and intellectual disabilities (ID). Although there are a series of genetic mouse models to study Shank3 gene in ASDs, there are few rat models with species-specific advantages. In this study, we established and characterized a novel rat model with a deletion spanning exons 11-21 of Shank3, leading to a complete loss of the major SHANK3 isoforms. Synaptic function and plasticity of Shank3-deficient rats were impaired detected by biochemical and electrophysiological analyses. Shank3-depleted rats showed impaired social memory but not impaired social interaction behaviors. In addition, impaired learning and memory, increased anxiety-like behavior, increased mechanical pain threshold and decreased thermal sensation were observed in Shank3-deficient rats. It is worth to note that Shank3-deficient rats had nearly normal levels of the endogenous social neurohormones oxytocin (OXT) and arginine-vasopressin (AVP). This new rat model will help to further investigate the etiology and assess potential therapeutic target and strategy for Shank3-related neurodevelopmental disorders.
RESUMO
Parylene-C is a popular polymer material in biomedical applications, with excellent physicochemical properties and microfabrication capability. Like many aromatic polymers, parylene-C also has autofluorescence, which was usually taken as a negative background noise in biomedical detection studies. However, the fluorescence intensity of thin-film (<1 µm) parylene-C was relatively weak, which may be a big limitation in visualization. In this work, we reported a simple annealing method to significantly enhance the fluorescence and achieve sufficient intensity as a visual marker. We studied the behaviors and mechanisms of the enhanced parylene-C fluorescence, then verified the feasibility and reliability of parylene-C for preparing fluorescent pipettes in targeted neuronal electrophysiology, where fluorescent guidance was strongly needed. The powerful parylene-C fabrication technique enables a precisely-controlled conformal coating along with a mass production capability, which further resulted in high-quality electrophysiological recordings of both cultured hippocampal neurons and acute hippocampal brain slices. Moreover, the enhanced parylene-C fluorescence can also be applied in more general biological operations, such as designable fluorescent micro-patterns for visualization in broader biomedical fields.
Assuntos
Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Polímeros/metabolismo , Xilenos/metabolismo , Animais , Fenômenos Eletrofisiológicos , Células HEK293 , Hipocampo/citologia , Humanos , CamundongosRESUMO
Recent genome-wide association studies (GWAS) have identified multiple risk loci that show strong associations with schizophrenia. However, pinpointing the potential causal genes at the reported loci remains a major challenge. Here we identify candidate causal genes for schizophrenia using an integrative genomic approach. Sherlock integrative analysis shows that ALMS1, GLT8D1, and CSNK2B are schizophrenia risk genes, which are validated using independent brain expression quantitative trait loci (eQTL) data and integrative analysis method (SMR). Consistently, gene expression analysis in schizophrenia cases and controls further supports the potential role of these three genes in the pathogenesis of schizophrenia. Finally, we show that GLT8D1 and CSNK2B knockdown promote the proliferation and inhibit the differentiation abilities of neural stem cells, and alter morphology and synaptic transmission of neurons. These convergent lines of evidence suggest that the ALMS1, CSNK2B, and GLT8D1 genes may be involved in pathophysiology of schizophrenia.