MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Nav?2 protein coding region in rat with chronic brain hypoperfusion.
ABSTRACT: Previous studies have demonstrated that the trafficking defects of Nav1.1/Nav1.2 are involved in the dementia pathophysiology. However, the detailed mechanisms are not fully understood. Moreover, whether the impaired miRNAs regulation linked to dementia is a key player in sodium channel trafficking disturbance remains unclear. The cognitive impairment induced by chronic cerebral ischemia through chronic brain hypoperfusion (CBH) is likely reason to precede dementia. Therefore, our goal in the present study was to examine the role of microRNA-9 (miR-9) in regulating Nav1.1/Nav1.2 trafficking under CBH generated by bilateral common carotid artery occlusion (2VO).The impairment of Nav1.1/Nav1.2 trafficking and decreased expression of Nav?2 were found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO. MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR. Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane. Further study showed that overexpression of miR-9 inhibited the Nav?2 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay. However, binding-site mutation or miR-masks failed to influence Nav?2 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Nav?2 is a potential target for miR-9. Lentivirus-mediated miR-9 overexpression also inhibited Nav?2 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats, whereas injection of lentivirus-mediated miR-9 knockdown could reverse the impaired trafficking of Nav1.1/Nav1.2 triggered by 2VO.We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Nav?2 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH.
Project description:BACKGROUND:Chronic brain hypoperfusion (CBH) is closely related to Alzheimer's disease (AD) and vascular dementia (VaD). Meanwhile, synaptic pathology plays a prominent role in the initial stage of AD and VaD. However, whether and how CBH impairs presynaptic plasticity is currently unclear. METHODS:In the present study, we performed a battery of techniques, including primary neuronal culture, patch clamp, stereotaxic injection of the lentiviral vectors, morris water maze (MWM), dual luciferase reporter assay, FM1-43 fluorescence dye evaluation, qRT-PCR and western blot, to investigate the regulatory effect of miR-153 on hippocampal synaptic vesicle release both in vivo and in vitro. The CBH rat model was generated by bilateral common carotid artery ligation (2VO). RESULTS:Compared to sham rats, 2VO rats presented decreased field excitatory postsynaptic potential (fEPSP) amplitude and increased paired-pulse ratios (PPRs) in the CA3-CA1 pathway, as well as significantly decreased expression of multiple vesicle fusion-related proteins, including SNAP-25, VAMP-2, syntaxin-1A and synaptotagmin-1, in the hippocampi. The levels of microRNA-153 (miR-153) were upregulated in the hippocampi of rats following 2VO surgery, and in the plasma of dementia patients. The expression of the vesicle fusion-related proteins affected by 2VO was inhibited by miR-153, elevated by miR-153 inhibition, and unchanged by binding-site mutation or miR masks. FM1-43 fluorescence images showed that miR-153 blunted vesicle exocytosis, but this effect was prevented by either 2'-O-methyl antisense oligoribonucleotides to miR-153 (AMO-153) and miR-masking of the miR-153 binding site in the 3' untranslated region (3'UTR) of the Snap25, Vamp2, Stx1a and Syt1 genes. Overexpression of miR-153 by lentiviral vector-mediated miR-153 mimics (lenti-pre-miR-153) decreased the fEPSP amplitude and elevated the PPR in the rat hippocampus, whereas overexpression of the antisense molecule (lenti-AMO-153) reversed these changes triggered by 2VO. Furthermore, lenti-AMO-153 attenuated the cognitive decline of 2VO rats. CONCLUSIONS:Overexpression of miR-153 controls CBH-induced presynaptic vesicle release impairment by posttranscriptionally regulating the expression of four vesicle release-related proteins by targeting the 3'UTRs of the Stx1a, Snap25, Vamp2 and Syt1 genes. These findings identify a novel mechanism of presynaptic plasticity impairment during CBH, which may be a new drug target for prevention or treatment of AD and VaD. Video Abstract.
Project description:Impaired synaptic plasticity and neuron loss are hallmarks of Alzheimer's disease and vascular dementia. Here, we found that chronic brain hypoperfusion (CBH) by bilateral common carotid artery occlusion (2VO) decreased the total length, numbers and crossings of dendrites and caused neuron death in rat hippocampi and cortices. It also led to increase in N-terminal ?-amyloid precursor protein (N-APP) and death receptor-6 (DR6) protein levels and in the activation of caspase-3 and caspase-6. Further study showed that DR6 protein was downregulated by miR-195 overexpression, upregulated by miR-195 inhibition, and unchanged by binding-site mutation and miR-masks. Knockdown of endogenous miR-195 by lentiviral vector-mediated overexpression of its antisense molecule (lenti-pre-AMO-miR-195) decreased the total length, numbers and crossings of dendrites and neuron death, upregulated N-APP and DR6 levels, and elevated cleaved caspase-3 and caspase-6 levels. Overexpression of miR-195 using lenti-pre-miR-195 prevented these changes triggered by 2VO. We conclude that miR-195 is involved in CBH-induced dendritic degeneration and neuron death through activation of the N-APP/DR6/caspase pathway.
Project description:Volatile anesthetics depress neurotransmitter release in a brain region- and neurotransmitter-selective manner by unclear mechanisms. Voltage-gated sodium channels (Navs), which are coupled to synaptic vesicle exocytosis, are inhibited by volatile anesthetics through reduction of peak current and modulation of gating. Subtype-selective effects of anesthetics on Nav might contribute to observed neurotransmitter-selective anesthetic effects on release. We analyzed anesthetic effects on Na+ currents mediated by the principal neuronal Nav subtypes Nav1.1, Nav1.2, and Nav1.6 heterologously expressed in ND7/23 neuroblastoma cells using whole-cell patch-clamp electrophysiology. Isoflurane at clinically relevant concentrations induced a hyperpolarizing shift in the voltage dependence of steady-state inactivation and slowed recovery from fast inactivation in all three Nav subtypes, with the voltage of half-maximal steady-state inactivation significantly more positive for Nav1.1 (-49.7 ± 3.9 mV) than for Nav1.2 (-57.5 ± 1.2 mV) or Nav1.6 (-58.0 ± 3.8 mV). Isoflurane significantly inhibited peak Na+ current (I Na) in a voltage-dependent manner: at a physiologically relevant holding potential of -70 mV, isoflurane inhibited peak I Na of Nav1.2 (16.5% ± 5.5%) and Nav1.6 (18.0% ± 7.8%), but not of Nav1.1 (1.2% ± 0.8%). Since Nav subtypes are differentially expressed both between neuronal types and within neurons, greater inhibition of Nav1.2 and Nav1.6 compared with Nav1.1 could contribute to neurotransmitter-selective effects of isoflurane on synaptic transmission.
Project description:BACKGROUND:Microglial polarization is a dynamic response to acute brain hypoxia induced by stroke and traumatic brain injury (TBI). However, studies on the polarization of microglia in chronic cerebral circulation insufficiency (CCCI) are limited. Our objective was to investigate the effect of CCCI on microglial polarization after chronic brain hypoperfusion (CBH) and explore the underlying molecular mechanisms. METHODS:CBH model was established by bilateral common carotid artery occlusion (2-vessel occlusion, 2VO) in rats. Using the stereotaxic injection technique, lenti-pre-miR-195 and anti-miR-195 oligonucleotide fragments (lenti-pre-AMO-miR-195) were injeted into the CA1 region of the hippocampus to construct animal models with high or low expression of miR-195. Immunofluorescence staining and flow cytometry were conducted to examine the status of microglial polarization. In vitro, Transwell co-culture system was taken to investigate the role of miR-195 on neuronal-microglial communication through CX3CL1-CX3CR1 signaling. Quantitative real-time PCR was used to detect the level of miR-195 and inflammatory factors. The protein levels of CX3CL1 and CX3CR1 were evaluated by both western blot and immunofluorescence staining. RESULTS:CBH induced by 2VO initiated microglial/macrophage activation in the rat hippocampus from 1?week to 8?weeks, as evaluated by increased ratio of (CD68+ and CD206+)/Iba-1 immunofluorescence. And the microglial/macrophage polarization was shifted towards the M1 phenotype at 8 weeks following CBH. The expression of CX3CL1 and CX3CR1 was increased in the hippocampus of 2VO rats at 8 weeks. An in vitro study in a Transwell co-culture system demonstrated that transfection of either primary-cultured neonatal rat neurons (NRNs) or microglial BV2 cells with AMO-195-induced M1 polarization of BV2 cells and increased CX3CL1 and CX3CR1 expression and that these effects were reversed by miR-195 mimics. Furthermore, the upregulation of miR-195 induced by lenti-pre-miR-195 injection prevented microglial/macrophage polarization to M1 phenotype triggered by hippocampal injection of lenti-pre-AMO-miR-195 and 2VO surgery. CONCLUSIONS:Our findings conclude that downregulation of miR-195 in the hippocampus is involved in CBH-induced microglial/macrophage polarization towards M1 phenotype by governing communication between neurons and microglia through the regulation of CX3CL1 and CX3CR1 signaling. This indicates that miR-195 may provide a new strategy for clinical prevention and treatment of CBH.
Project description:Paclitaxel, a chemotherapeutic agent, causes neuropathic pain whose supraspinal pathophysiology is not fully understood. Dysregulation of sodium channel expression, studied mainly in the periphery and spinal cord level, contributes to the pathogenesis of neuropathic pain. We examined gene expression of sodium channel (Nav) subunits by real time polymerase chain reaction (PCR) in the anterior cingulate cortex (ACC) at day 7 post first administration of paclitaxel, when mice had developed paclitaxel-induced thermal hyperalgesia. The ACC was chosen because increased activity in the ACC has been observed during neuropathic pain. In the ACC of vehicle-treated animals the threshold cycle (Ct) values for Nav1.4, Nav1.5, Nav1.7, Nav1.8 and Nav1.9 were above 30 and/or not detectable in some samples. Thus, comparison in mRNA expression between untreated control, vehicle-treated and paclitaxel treated animals was done for Nav1.1, Nav1.2, Nav1.3, Nav1.6, Nax as well as Nav?1-Nav?4. There were no differences in the transcript levels of Nav1.1-Nav1.3, Nav1.6, Nax, Nav?1-Nav?3 between untreated and vehicle-treated mice, however, vehicle treatment increased Nav?4 expression. Paclitaxel treatment significantly increased the mRNA expression of Nav1.1, Nav1.2, Nav1.6 and Nax, but not Nav1.3, sodium channel alpha subunits compared to vehicle-treated animals. Treatment with paclitaxel significantly increased the expression of Nav?1 and Nav?3, but not Nav?2 and Nav?4, sodium channel beta subunits compared to vehicle-treated animals. These findings suggest that during paclitaxel-induced neuropathic pain (PINP) there is differential upregulation of sodium channels in the ACC, which might contribute to the increased neuronal activity observed in the area during neuropathic pain.
Project description:High-density accumulation of voltage-gated sodium (Nav) channels at nodes of Ranvier ensures rapid saltatory conduction along myelinated axons. To gain insight into mechanisms of node assembly in the CNS, we focused on early steps of nodal protein clustering. We show in hippocampal cultures that prenodes (i.e., clusters of Nav channels colocalizing with the scaffold protein ankyrinG and nodal cell adhesion molecules) are detected before myelin deposition along axons. These clusters can be induced on purified neurons by addition of oligodendroglial-secreted factor(s), whereas ankyrinG silencing prevents their formation. The Nav isoforms Nav1.1, Nav1.2, and Nav1.6 are detected at prenodes, with Nav1.6 progressively replacing Nav1.2 over time in hippocampal neurons cultured with oligodendrocytes and astrocytes. However, the oligodendrocyte-secreted factor(s) can induce the clustering of Nav1.1 and Nav1.2 but not of Nav1.6 on purified neurons. We observed that prenodes are restricted to GABAergic neurons, whereas clustering of nodal proteins only occurs concomitantly with myelin ensheathment on pyramidal neurons, implying separate mechanisms of assembly among different neuronal subpopulations. To address the functional significance of these early clusters, we used single-axon electrophysiological recordings in vitro and showed that prenode formation is sufficient to accelerate the speed of axonal conduction before myelination. Finally, we provide evidence that prenodal clusters are also detected in vivo before myelination, further strengthening their physiological relevance.
Project description:Epilepsy is a disease characterized by abnormal brain activity and a predisposition to generate epileptic seizures, leading to neurobiological, cognitive, psychological, social, and economic impacts for the patient. There are several known causes for epilepsy; one of them is the malfunction of ion channels, resulting from mutations. Voltage-gated sodium channels (NaV) play an essential role in the generation and propagation of action potential, and malfunction caused by mutations can induce irregular neuronal activity. That said, several genetic variations in NaV channels have been described and associated with epilepsy. These mutations can affect channel kinetics, modifying channel activation, inactivation, recovery from inactivation, and/or the current window. Among the NaV subtypes related to epilepsy, NaV1.1 is doubtless the most relevant, with more than 1500 mutations described. Truncation and missense mutations are the most observed alterations. In addition, several studies have already related mutated NaV channels with the electrophysiological functioning of the channel, aiming to correlate with the epilepsy phenotype. The present review provides an overview of studies on epilepsy-associated mutated human NaV1.1, NaV1.2, NaV1.3, NaV1.6, and NaV1.7.
Project description:Research involving recombinant voltage-gated sodium (Nav) channels has unique challenges. Multiple factors contribute, but undoubtedly at the top of the list is these channels' DNA instability. Once introduced into bacterial hosts, Nav channel plasmid DNA will almost invariably emerge mutagenized and unusable, unless special conditions are adopted. This is particularly true for Nav1.1 (gene name SCN1A), Nav1.2 (SCN2A), and Nav1.6 (SCN8A), but less so for Nav1.4 (SCN4A) and Nav1.5 (SCN5A) while other Nav channel isoforms such as Nav1.7 (SCN9A) lie in between. The following recommendations for Nav plasmid DNA amplification and preparation address this problem. Three points are essential:•Bacterial propagation using Stbl2 cells at or below 30 °C.•Bias toward slow-growing, small bacterial colonies.•Comprehensive sequencing of the entire Nav channel coding region.
Project description:Variants implicated in childhood epilepsy have been identified in all four voltage-gated sodium channels that initiate action potentials in the central nervous system. Previous research has focused on the functional effects of particular variants within the most studied of these channels (NaV1.1, NaV1.2 and NaV1.6); however, there have been few comparative studies across channels to infer the impact of mutations in patients with epilepsy. Here we compare patterns of variation in patient and public databases to test the hypothesis that regions of known functional significance within voltage-gated sodium (NaV) channels have an increased burden of deleterious variants. We assessed mutational burden in different regions of the Nav channels by (1) performing Fisher exact tests on odds ratios to infer excess variants in domains, segments, and loops of each channel in patient databases versus public "control" databases, and (2) comparing the cumulative distribution of variant sites along DNA sequences of each gene in patient and public databases (i.e., independent of protein structure). Patient variant density was concordant among channels in regions known to play a role in channel function, with statistically significant higher patient variant density in S4-S6 and DIII-DIV and an excess of public variants in SI-S3, DI-DII, DII-DIII. On the other hand, channel-specific patterns of patient burden were found in the NaV1.6 inactivation gate and NaV1.1 S5-S6 linkers, while NaV1.2 and NaV1.6 S4-S5 linkers and S5 segments shared patient variant patterns that contrasted with those in NaV1.1. These different patterns may reflect different roles played by the NaV1.6 inactivation gate in action potential propagation, and by NaV1.1 S5-S6 linkers in loss of function and haploinsufficiency. Interestingly, NaV1.2 and NaV1.6 both lack amino acid substitutions over significantly long stretches in both the patient and public databases suggesting that new mutations in these regions may cause embryonic lethality or a non-epileptic disease phenotype.
Project description:Voltage-gated sodium channels (Nav1.1-Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein-protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.