Showing papers in "Molecular Brain in 2018"

Targeting NMDA receptors in stroke: new hope in neuroprotection

Abstract: NMDA (N-methyl-d-aspartate) receptors (NMDARs) play a central role in excitotoxic neuronal death caused by ischemic stroke, but NMDAR channel blockers have failed to be translated into clinical stroke treatments. However, recent research on NMDAR-associated signaling complexes has identified important death-signaling pathways linked to NMDARs. This led to the generation of inhibitors that inhibit these pathways downstream from the receptor without necessarily blocking NMDARs. This therapeutic approach may have fewer side effects and/or provide a wider therapeutic window for stroke as compared to the receptor antagonists. In this review, we highlight the key findings in the signaling cascades downstream of NMDARs and the novel promising therapeutics for ischemic stroke.

proBDNF is modified by advanced glycation end products in Alzheimer's disease and causes neuronal apoptosis by inducing p75 neurotrophin receptor processing.

Abstract: Alzheimer disease (AD) is a complex pathology related to multiple causes including oxidative stress. Brain-derived neurotrophic factor (BDNF) is a neutrotrophic factor essential for the survival and differentiation of neurons and is considered a key target in the pathophysiology of various neurodegenerative diseases, as for example AD. Contrarily to BDNF, the precursor form of BDNF (proBDNF) induces apoptosis through the specific interaction with p75 and its co-receptor, Sortilin. We used hippocampal tissue and cerebrospinal fluid from AD patients and controls. to study the localization and the levels of proBDNF, p75 and Sortilin as well as the post-traduccional modifications of proBDNF induced by Radical Oxygen Species, by immunofluorescence and Western blot. Differentiation and survival were assessed on differentiated mouse hippocampal neurons derived from postnatal neural stem cells from WT animals or from the transgenic AD animal model APP/PS1∆E9, based on mutations of familiar AD. In AD patients we observe a significative increase of proBDNF and Sortilin expression and a significative increase of the ratio proBDNF/BDNF in their cerebrospinal fluid compared to controls. In addition, the proBDNF of AD patients is modified by ROS-derived advanced glycation end products, which prevent the processing of the proBDNF to the mature BDNF, leading to an increase of pathogenicity and a decrease of trophic effects. The cerebrospinal fluid from AD patients, but not from controls, induces apoptosis in differentiated hippocampal neurons mainly by the action of AGE-modified proBDNF present in the cerebrospinal fluid of the patients. This effect is triggered by the activation and processing of p75 that stimulate the internalization of the intracellular domain (ICD) within the nucleus causing apoptosis. Induction of apoptosis and p75 ICD internalization by AD patients-derived proBDNF is further enhanced in neuron cultures from the AD model expressing the APP/PS1∆E9 transgene. Our results indicate the importance of proBDNF neurotoxic signaling in AD pathology essentially by three mechanisms: i) by an increase of proBDNF stability due to ROS-induced post-traductional modifications; ii) by the increase of expression of the p75 co-receptor, Sortilin and iii) by the increase of the basal levels of p75 processing found in AD.

Cannabidiol enhances morphine antinociception, diminishes NMDA-mediated seizures and reduces stroke damage via the sigma 1 receptor

Abstract: Cannabidiol (CBD), the major non-psychotomimetic compound present in the Cannabis sativa plant, exhibits therapeutic potential for various human diseases, including chronic neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, ischemic stroke, epilepsy and other convulsive syndromes, neuropsychiatric disorders, neuropathic allodynia and certain types of cancer. CBD does not bind directly to endocannabinoid receptors 1 and 2, and despite research efforts, its specific targets remain to be fully identified. Notably, sigma 1 receptor (σ1R) antagonists inhibit glutamate N-methyl-D-aspartate acid receptor (NMDAR) activity and display positive effects on most of the aforesaid diseases. Thus, we investigated the effects of CBD on three animal models in which NMDAR overactivity plays a critical role: opioid analgesia attenuation, NMDA-induced convulsive syndrome and ischemic stroke. In an in vitro assay, CBD disrupted the regulatory association of σ1R with the NR1 subunit of NMDAR, an effect shared by σ1R antagonists, such as BD1063 and progesterone, and prevented by σ1R agonists, such as 4-IBP, PPCC and PRE084. The in vivo administration of CBD or BD1063 enhanced morphine-evoked supraspinal antinociception, alleviated NMDA-induced convulsive syndrome, and reduced the infarct size caused by permanent unilateral middle cerebral artery occlusion. These positive effects of CBD were reduced by the σ1R agonists PRE084 and PPCC, and absent in σ1R−/− mice. Thus, CBD displays antagonist-like activity toward σ1R to reduce the negative effects of NMDAR overactivity in the abovementioned experimental situations.

Early Life Stress, Depression And Parkinson's Disease: A New Approach.

Abstract: This review aims to shed light on the relationship that involves exposure to early life stress, depression and Parkinson’s disease (PD). A systematic literature search was conducted in Pubmed, MEDLINE, EBSCOHost and Google Scholar and relevant data were submitted to a meta-analysis . Early life stress may contribute to the development of depression and patients with depression are at risk of developing PD later in life. Depression is a common non-motor symptom preceding motor symptoms in PD. Stimulation of regions contiguous to the substantia nigra as well as dopamine (DA) agonists have been shown to be able to attenuate depression. Therefore, since PD causes depletion of dopaminergic neurons in the substantia nigra, depression, rather than being just a simple mood disorder, may be part of the pathophysiological process that leads to PD. It is plausible that the mesocortical and mesolimbic dopaminergic pathways that mediate mood, emotion, and/or cognitive function may also play a key role in depression associated with PD. Here, we propose that a medication designed to address a deficiency in serotonin is more likely to influence motor symptoms of PD associated with depression. This review highlights the effects of an antidepressant, Fluvoxamine maleate, in an animal model that combines depressive-like symptoms and Parkinsonism.

Novel and de novo mutations in pediatric refractory epilepsy

Abstract: Pediatric refractory epilepsy is a broad phenotypic spectrum with great genetic heterogeneity. Next-generation sequencing (NGS) combined with Sanger sequencing could help to understand the genetic diversity and underlying disease mechanisms in pediatric epilepsy. Here, we report sequencing results from a cohort of 172 refractory epilepsy patients aged 0–14 years. The pathogenicity of identified variants was evaluated in accordance with the American College of Medical Genetics and Genomics (ACMG) criteria. We identified 43 pathogenic or likely pathogenic variants in 40 patients (23.3%). Among these variants, 74.4% mutations (32/43) were de novo and 60.5% mutations (26/43) were novel. Patients with onset age of seizures ≤12 months had higher yields of deleterious variants compared to those with onset age of seizures > 12 months (P = 0.006). Variants in ion channel genes accounted for the greatest functional gene category (55.8%), with SCN1A coming first (16/43). 81.25% (13/16) of SCN1A mutations were de novo and 68.8% (11/16) were novel in Dravet syndrome. Pathogenic or likely pathogenic variants were found in the KCNQ2, STXBP1, SCN2A genes in Ohtahara syndrome. Novel deleterious variants were also found in West syndrome, Doose syndrome and glucose transporter type 1 deficiency syndrome patients. One de novo MECP2 mutation were found in a Rett syndrome patient. TSC1/TSC2 variants were found in 60% patients with tuberous sclerosis complex patients. Other novel mutations detected in unclassified epilepsy patients involve the SCN8A, CACNA1A, GABRB3, GABRA1, IQSEC2, TSC1, VRK2, ATP1A2, PCDH19, SLC9A6 and CHD2 genes. Our study provides novel insights into the genetic origins of pediatric epilepsy and represents a starting-point for further investigations into the molecular pathophysiology of pediatric epilepsy that could eventually lead to better treatments.

Resolvin D2 protects against cerebral ischemia/reperfusion injury in rats.

Abstract: Cerebral ischemia/reperfusion (I/R) injury is a critical factor leading to a poor prognosis for ischemic stroke patients. ω-3 fatty acid supplements taken as part of a daily diet have been shown to improve the prognosis of patients with ischemic stroke. In this study, we aimed to investigate the potential effects of resolvin D2 (RvD2), a derivative of ω-3 fatty acids, and its possible advantage on cerebral I/R injury in rats. Cerebral I/R caused by middle cerebral artery occlusion and reperfusion (MCAO/R) was established in Sprague-Dawley rats. First, in rats fed a regular diet, the MCAO/R stimulus led to a significant decrease in endogenous production of RvD2. Exogenous supply of RvD2 via intraperitoneal injection reversed MCAO/R-induced brain injury, including infarction, inflammatory response, brain edema, and neurological dysfunction. Meanwhile, RvD2 reversed the MCAO/R-induced decrease in the protein level of GPR18, which has been identified as a receptor for RvD2, especially in neurons and brain microvascular endothelial cells (BMVECs). Furthermore, RvD2 exerted rescue effects on MCAO/R-induced neuron and BMVEC death. Moreover, GPR18 antagonist O-1918 could block the rescue effects of RvD2, possibly at least partially though the GPR18-ERK1/2-NOS signaling pathway. Finally, compared with ω-3 fatty acid supplements, RvD2 treatment had a better rescue effect on cerebral infarction, which may be due to the MCAO/R-induced decrease in 5-lipoxygense phosphorylation and subsequent RvD2 generation. In conclusion, compared with ω-3 fatty acids, RvD2 may be an optimal alternative and complementary treatment for ischemic stroke patients with recanalization treatment.

Warburg effect hypothesis in autism Spectrum disorders.

Abstract: Autism spectrum disorder (ASD) is a neurodevelopmental disease which is characterized by a deficit in social interactions and communication with repetitive and restrictive behavior. In altered cells, metabolic enzymes are modified by the dysregulation of the canonical WNT/β-catenin pathway. In ASD, the canonical WNT/β-catenin pathway is upregulated. We focus this review on the hypothesis of Warburg effect stimulated by the overexpression of the canonical WNT/β-catenin pathway in ASD. Upregulation of WNT/β-catenin pathway induces aerobic glycolysis, named Warburg effect, through activation of glucose transporter (Glut), pyruvate kinase M2 (PKM2), pyruvate dehydrogenase kinase 1(PDK1), monocarboxylate lactate transporter 1 (MCT-1), lactate dehydrogenase kinase-A (LDH-A) and inactivation of pyruvate dehydrogenase complex (PDH). The aerobic glycolysis consists to a supply of a large part of glucose into lactate regardless of oxygen. Aerobic glycolysis is less efficient in terms of ATP production than oxidative phosphorylation because of the shunt of the TCA cycle. Dysregulation of energetic metabolism might promote cell deregulation and progression of ASD. Warburg effect regulation could be an attractive target for developing therapeutic interventions in ASD.

Chronic restraint stress induces hippocampal memory deficits by impairing insulin signaling

Abstract: Chronic stress is a psychologically significant factor that impairs learning and memory in the hippocampus. Insulin signaling is important for the development and cognitive function of the hippocampus. However, the relation between chronic stress and insulin signaling at the molecular level is poorly understood. Here, we show that chronic stress impairs insulin signaling in vitro and in vivo, and thereby induces deficits in hippocampal spatial working memory and neurobehavior. Corticosterone treatment of mouse hippocampal neurons in vitro caused neurotoxicity with an increase in the markers of autophagy but not apoptosis. Corticosterone treatment impaired insulin signaling from early time points. As an in vivo model of stress, mice were subjected to chronic restraint stress. The chronic restraint stress group showed downregulated insulin signaling and suffered deficits in spatial working memory and nesting behavior. Intranasal insulin delivery restored insulin signaling and rescued hippocampal deficits. Our data suggest that psychological stress impairs insulin signaling and results in hippocampal deficits, and these effects can be prevented by intranasal insulin delivery.

Interleukin-1 beta promotes neuronal differentiation through the Wnt5a/RhoA/JNK pathway in cortical neural precursor cells.

Abstract: Pro-inflammatory cytokine interleukin-1 beta (IL-1β) is a key mediator of inflammation and stress in the central nervous system (CNS), and is highly expressed in the developing brain. In this study, we investigated the possible role of IL-1β in neuronal differentiation of cortical neural precursor cells (NPCs). We showed that stimulation with IL-1β increased expression levels of neurotrophin-3 (NT3) and neurogenin 1 (Ngn1) and promoted neurite outgrowth. We also found that IL-1β increased mRNA and protein levels of Wnt5a. Knockdown of Wnt5a by transfection with Wnt5a siRNA inhibited IL-1β-induced neuronal differentiation. Moreover, IL-1β-induced Wnt5a expression was regulated by nuclear factor kappa B (NF-κB) activation, which is involved in IL-1β-mediated neuronal differentiation. To examine the role of Wnt5a in neuronal differentiation of NPCs, we exogenously added Wnt5a. We found that exogenous Wnt5a promotes neuronal differentiation, and activates the RhoA/Rho-associated kinase (ROCK)/c-jun N-terminal kinase (JNK) pathway. In addition, Wnt5a-induced neuronal differentiation was blocked by RhoA siRNA, as well as by a specific Rho-kinase inhibitor (Y27632) or a SAPK/JNK inhibitor (SP600125). Furthermore, treatment with RhoA siRNA, Y27632, or SP600125 suppressed the IL-1β-induced neuronal differentiation. Therefore, these results suggest that the sequential Wnt5a/RhoA/ROCK/JNK pathway is involved in IL-1β-induced neuronal differentiation of NPCs.

Long non-coding RNA H19 and MALAT1 gene variants in patients with ischemic stroke in a northern Chinese Han population.

Abstract: Objectives Long non-coding RNAs (lncRNAs) have been identified as key regulators in the development of atherosclerosis, which is a major cause of ischemic stroke. However, to date, there are no reports on the association between lncRNA gene variation and the risk of ischemic stroke. Therefore, we assessed the association between H19 and MALAT1 gene polymorphisms and susceptibility to ischemic stroke in a northern Chinese Han population. Methods In our study, we genotyped four genetic variations in lncRNA-H19 and -MALAT1 (rs217727, rs2251375, rs619586, and rs3200401) in a case–control study of 567 ischemic stroke patients and 552 control subjects. Results We found that the TT genotype of the rs217727 polymorphism within H19 was significantly associated with increased risk of ischemic stroke in our northern Chinese Han population (odds ration (OR) = 1.519, 95% confidence interval (CI) = 1.072–2.152, p = 0.018). Stratified analysis based on stroke subtype revealed that the increased risk was more evident in small vessel ischemic stroke (OR = 1.941, 95% CI = 1.260–2.992, p = 0.02). Individuals with the TT genotype had a 1.941 times higher risk of small vessel ischemic stroke when compared with the subjects of CC + CT. These correlations remained after adjusting for confounding risk factors of stroke (OR = 1.913, 95% CI = 1.221–2.998, p = 0.005). However, there was no significant association between H19 rs2251375 or MALAT1 rs3200401 and ischemic stroke in either total population analysis or subgroup analysis. Conclusion In conclusion, our findings suggest that the H19 rs217727 gene polymorphism contributes to small vessel ischemic stroke susceptibility in the Chinese Han population and may serve as a potential indicator for ischemic stroke susceptibility.

Lentivirus-mediated expression of human secreted amyloid precursor protein-alpha prevents development of memory and plasticity deficits in a mouse model of Alzheimer's disease

Abstract: Alzheimer’s disease (AD) is a neurodegenerative disease driven in large part by accumulated deposits in the brain of the amyloid precursor protein (APP) cleavage product amyloid-β peptide (Aβ). However, AD is also characterised by reductions in secreted amyloid precursor protein-alpha (sAPPα), an alternative cleavage product of APP. In contrast to the neurotoxicity of accumulated Αβ, sAPPα has many neuroprotective and neurotrophic properties. Increasing sAPPα levels has the potential to serve as a therapeutic treatment that mitigates the effects of Aβ and rescue cognitive function. Here we tested the hypothesis that lentivirus-mediated expression of a human sAPPα construct in a mouse model of AD (APPswe/PS1dE9), begun before the onset of plaque pathology, could prevent later behavioural and electrophysiological deficits. Male mice were given bilateral intra-hippocampal injections at 4 months of age and tested 8–10 months later. Transgenic mice expressing sAPPα performed significantly better than untreated littermates in all aspects of the spatial water maze task. Expression of sAPPα also resulted in partial rescue of long-term potentiation (LTP), tested in vitro. These improvements occurred in the absence of changes in amyloid pathology. Supporting these findings on LTP, lentiviral-mediated expression of sAPPα for 3 months from 10 months of age, or acute sAPPα treatment in hippocampal slices from 18 to 20 months old transgenic mice, completely reversed the deficits in LTP. Together these findings suggest that sAPPα has wide potential to act as either a preventative or restorative therapeutic treatment in AD by mitigating the effects of Aβ toxicity and enhancing cognitive reserve.

Autophagy is increased following either pharmacological or genetic silencing of mGluR5 signaling in Alzheimer's disease mouse models.

Abstract: Alzheimer’s disease (AD) is characterized by neurotoxicity mediated by the accumulation of beta amyloid (Aβ) oligomers, causing neuronal loss and progressive cognitive decline. Genetic deletion or chronic pharmacological inhibition of mGluR5 by the negative allosteric modulator CTEP, rescues cognitive function and reduces Aβ aggregation in both APPswe/PS1ΔE9 and 3xTg-AD mouse models of AD. In late onset neurodegenerative diseases, such as AD, defects arise at different stages of the autophagy pathway. Here, we show that mGluR5 cell surface expression is elevated in APPswe/PS1ΔE9 and 3xTg-AD mice. This is accompanied by reduced autophagy (accumulation of p62) as the consequence of increased ZBTB16 expression and reduced ULK1 activity, as we have previously observed in Huntington’s disease (HD). The chronic (12 week) inhibition of mGluR5 with CTEP in APPswe/PS1ΔE9 and 3xTg-AD mice prevents the observed increase in mGluR5 surface expression. In addition, mGluR5 inactivation facilitates the loss of ZBTB16 expression and ULK1 activation as a consequence of ULK-Ser757 dephosphorylation, which promotes the loss of expression of the autophagy marker p62. Moreover, the genetic ablation of mGluR5 in APPswe/PS1ΔE9 mice activated autophagy via similar mechanisms to pharmacological blockade. This study provides further evidence that mGluR5 overactivation contributes to inhibition of autophagy and can result in impaired clearance of neurotoxic aggregates in multiple neurodegenerative diseases. Thus, it provides additional support for the potential of mGluR5 inhibition as a general therapeutic strategy for neurodegenerative diseases such as AD and HD.

Down-regulation of ghrelin receptors on dopaminergic neurons in the substantia nigra contributes to Parkinson’s disease-like motor dysfunction

Abstract: Ghrelin exerts a wide range of physiological actions throughout the body and appears to be a promising target for disease therapy. Endogenous ghrelin receptors (GHSRs) are present in extrahypothalamic sites including the substantia nigra pars compacta (SNc), which is related to phenotypic dysregulation or frank degeneration in Parkinson’s disease (PD). Here we found a dramatic decrease in the expression of GHSR in PD-specific induced pluripotent stem cell (iPSC)-derived dopaminergic (DAnergic) neurons generated from patients carrying parkin gene (PARK2) mutations compared to those from healthy controls. Consistently, a significant decrease in the expression of GHSR was found in DAnergic neurons of isogenic PARK2-iPSC lines that mimicked loss of function of the PARK2 gene through CRISPR Cas9 technology. Furthermore, either intracerebroventricular injection or microinjection into the SNc of the selective GHSR1a antagonist [D-Lys3]-GHRP6 in normal mice produced cataleptic behaviors related to dysfunction of motor coordination. These findings suggest that the down-regulation of GHSRs in SNc-DA neurons induced the initial dysfunction of DA neurons, leading to extrapyramidal disorder under PD.

Relationships between the acoustic startle response and prepulse inhibition in C57BL/6J mice: a large-scale meta-analytic study

Abstract: Prepulse inhibition (PPI) is the suppression of a startle reflex response to a startle stimulus that occurs when a weak prepulse stimulus precedes the startle stimulus PPI is measured to assess sensorimotor gating across species, including humans and rodents Reduced PPI, which is thought to reflect dysfunction of sensorimotor gating, is reported in patients with psychiatric disorders, such as schizophrenia, bipolar disorder, and post-traumatic stress disorder (PTSD), and in animal models of these disorders Individual differences in basal startle reactivity occur even in a genetically homogenous group of animals; however, there is limited information regarding whether basal levels of the startle response are associated with variations in PPI levels Here, to explore the relationship between an acoustic startle response (ASR) and PPI, we performed a meta-analysis of data obtained from more than 1300 C57BL/6J male mice on the influence of an ASR to 110- and 120-dB startle stimuli on the PPI levels of the ASR at 74- and 78-dB prepulse intensities Examination of scatter plots of the ASR amplitudes and PPI levels followed by correlation analyses indicated that there is no simple linear relationship between the two measures; when mice were divided into three groups on the basis of their startle amplitudes, there were positive correlations between the amplitude of the ASR to the 110-dB stimulus and PPI levels in a group of mice that showed lower ASR amplitudes among the genetically homogenous group, whereas no significant correlations were identified in groups of mice that showed intermediate and higher ASR amplitudes As indicated by the correlation analysis, the lowest responders to the 110-dB stimulus exhibited lower levels of PPI than the intermediate or higher responders In contrast, for the 120-dB stimulus, a negative correlation was identified between the amplitude of the ASR to the 120-dB stimulus and the PPI levels in the groups of mice that showed intermediate or higher ASR amplitudes Lower and intermediate responders showed higher levels of PPI than higher responders to the 120-dB stimulus These findings suggest that basal startle reactivity may affect PPI levels in male C57BL/6J mice, thus representing one potential confounding factor that may confuse the interpretation of PPI results These findings emphasize the importance of careful examination of startle reactivity to ensure a reliable assessment of PPI

Plasticity changes in forebrain activity and functional connectivity during neuropathic pain development in rats with sciatic spared nerve injury

Abstract: Neuropathic pain is a major worldwide health problem. Although central sensitization has been reported in well-established neuropathic conditions, information on the acute brain activation patterns in response to peripheral nerve injury is lacking. This study first mapped the brain activity in rats immediately following spared nerve injury (SNI) of the sciatic nerve. Using blood-oxygenation-level-dependent functional magnetic resonance imaging (BOLD-fMRI), we observed sustained activation in the bilateral insular cortices (ICs), primary somatosensory cortex (S1), and cingulate cortex. Second, this study sought to link this sustained activation pattern with brain sensitization. Using manganese-enhanced magnetic resonance imaging (MEMRI), we observed enhanced activity in the ipsilateral anterior IC (AIC) in free-moving SNI rats on Days 1 and 8 post-SNI. Furthermore, enhanced functional connectivity between the ipsilateral AIC, bilateral rostral AIC, and S1 was observed on Day 8 post-SNI. Chronic electrophysiological recording experiments were conducted to confirm the tonic neuronal activation in selected brain regions. Our data provide evidence of tonic activation-dependent brain sensitization during neuropathic pain development and offer evidence that the plasticity changes in the IC and S1 may contribute to neuropathic pain development.

TLR4-mediated autophagic impairment contributes to neuropathic pain in chronic constriction injury mice

Abstract: Neuropathic pain is a complex, chronic pain state characterized by hyperalgesia, allodynia, and spontaneous pain. Accumulating evidence has indicated that the microglial Toll-like receptor 4 (TLR4) and autophagy are implicated in neurodegenerative diseases, but their relationship and role in neuropathic pain remain unclear. In this study, we examined TLR4 and its association with autophagic activity using a chronic constriction injury (CCI)-induced neuropathic pain model in wild-type (WT) and TLR4-knockout (KO) mice. The mice were assigned into four groups: WT-Contralateral (Contra), WT-Ipsilateral (Ipsi), TLR4 KO-Contra, and TLR4 KO-Ipsi. Behavioral and mechanical allodynia tests and biochemical analysis of spinal cord tissue were conducted following CCI to the sciatic nerve. Compared with the Contra group, mechanical allodynia in both the WT- and TLR4 KO-Ipsi groups was significantly increased, and a marked decrease of allodynia was observed in the TLR4 KO-Ipsi group. Although glial cells were upregulated in the WT-Ipsi group, no significant change was observed in the TLR4 KO groups. Moreover, protein expression and immunoreactive cell regulation of autophagy (Beclin 1, p62) were significantly increased in the neurons, but not microglia, of WT-Ipsi group compared with the WT-Contra group. The level of PINK1, a marker for mitophagy was increased in the neurons of WT, but not in TLR4 KO mice. Together, these results show that TLR4-mediated p62 autophagic impairment plays an important role in the occurrence and development of neuropathic pain. And what is more, microglial TLR4-mediated microglial activation might be indirectly coupled to neuronal autophage.

RNAseq analysis of hippocampal microglia after kainic acid-induced seizures.

Abstract: Microglia have been shown to be of critical importance to the progression of temporal lobe epilepsy. However, the broad transcriptional changes that these cells undergo following seizure induction is not well understood. As such, we utilized RNAseq analysis upon microglia isolated from the hippocampus to determine expression pattern alterations following kainic acid induced seizure. We determined that microglia undergo dramatic changes to their expression patterns, particularly with regard to mitochondrial activity and metabolism. We also observed that microglia initiate immunological activity, specifically increasing interferon beta responsiveness. Our results provide novel insights into microglia transcriptional regulation following acute seizures and suggest potential therapeutic targets specifically in microglia for the treatment of seizures and epilepsy.

Distribution of Caskin1 protein and phenotypic characterization of its knockout mice using a comprehensive behavioral test battery.

Abstract: Calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1 (Caskin1) is a direct binding partner of the synaptic adaptor protein CASK. Because Caskin1 forms homo-multimers and binds not only CASK but also other neuronal proteins in vitro, it is anticipated to have neural functions; but its exact role in mammals remains unclear. Previously, we showed that the concentration of Caskin1 in the spinal dorsal horn increases under chronic pain. To characterize this protein, we generated Caskin1-knockout (Caskin1-KO) mice and specific anti-Caskin1 antibodies. Biochemical and immunohistochemical analyses demonstrated that Caskin1 was broadly distributed in the whole brain and spinal cord, and that it primarily localized at synapses. To elucidate the neural function of Caskin1 in vivo, we subjected Caskin1-KO mice to comprehensive behavioral analysis. The mutant mice exhibited differences in gait, enhanced nociception, and anxiety-like behavior relative to their wild-type littermates. In addition, the knockouts exhibited strong freezing responses, with or without a cue tone, in contextual and cued-fear conditioning tests as well as low memory retention in the Barnes Maze test. Taken together, these results suggest that Caskin1 contributes to a wide spectrum of behavioral phenotypes, including gait, nociception, memory, and stress response, in broad regions of the central nervous system.

Poly-arginine R18 and R18D (D-enantiomer) peptides reduce infarct volume and improves behavioural outcomes following perinatal hypoxic-ischaemic encephalopathy in the P7 rat

Abstract: We examined the neuroprotective efficacy of the poly-arginine peptide R18 and its D-enantiomer R18D in a perinatal hypoxic-ischaemic (HI) model in P7 Sprague-Dawley rats. R18 and R18D peptides were administered intraperitoneally at doses of 30, 100, 300 or 1000 nmol/kg immediately after HI (8% O2/92%N2 for 2.5 h). The previously characterised neuroprotective JNKI-1-TATD peptide at a dose of 1000 nmol/kg was used as a control. Infarct volume and behavioural outcomes were measured 48 h after HI. For the R18 and R18D doses examined, total infarct volume was reduced by 25.93% to 43.80% (P = 0.038 to < 0.001). By comparison, the JNKI-1-TATD reduced lesion volume by 25.27% (P = 0.073). Moreover, R18 and R18D treatment resulted in significant improvements in behavioural outcomes, while with JNKI-1-TATD there was a trend towards improvement. As an insight into the likely mechanism underlying the effects of R18, R18D and JNKI-1-TATD, the peptides were added to cortical neuronal cultures exposed to glutamic acid excitotoxicity, resulting in up to 89, 100 and 71% neuroprotection, respectively, and a dose dependent inhibition of neuronal calcium influx. The study further confirms the neuroprotective properties of poly-arginine peptides, and suggests a potential therapeutic role for R18 and R18D in the treatment of HIE.

Exposure to mild blast forces induces neuropathological effects, neurophysiological deficits and biochemical changes

Abstract: Direct or indirect exposure to an explosion can induce traumatic brain injury (TBI) of various severity levels. Primary TBI from blast exposure is commonly characterized by internal injuries, such as vascular damage, neuronal injury, and contusion, without external injuries. Current animal models of blast-induced TBI (bTBI) have helped to understand the deleterious effects of moderate to severe blast forces. However, the neurological effects of mild blast forces remain poorly characterized. Here, we investigated the effects caused by mild blast forces combining neuropathological, histological, biochemical and neurophysiological analysis. For this purpose, we employed a rodent blast TBI model with blast forces below the level that causes macroscopic neuropathological changes. We found that mild blast forces induced neuroinflammation in cerebral cortex, striatum and hippocampus. Moreover, mild blast triggered microvascular damage and axonal injury. Furthermore, mild blast caused deficits in hippocampal short-term plasticity and synaptic excitability, but no impairments in long-term potentiation. Finally, mild blast exposure induced proteolytic cleavage of spectrin and the cyclin-dependent kinase 5 activator, p35 in hippocampus. Together, these findings show that mild blast forces can cause aberrant neurological changes that critically impact neuronal functions. These results are consistent with the idea that mild blast forces may induce subclinical pathophysiological changes that may contribute to neurological and psychiatric disorders.

Systemic overexpression of SQSTM1/p62 accelerates disease onset in a SOD1 H46R -expressing ALS mouse model

Abstract: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a selective loss of upper and lower motor neurons. Recent studies have shown that mutations in SQSTM1 are linked to ALS. SQSTM1 encodes SQSTM1/p62 that regulates not only autophagy via the association with MAP1LC3/LC3 and ubiquitinated proteins but also the KEAP1-NFE2L2/Nrf2 anti-oxidative stress pathway by interacting with KEAP1. Previously, we have demonstrated that loss of SQSTM1 exacerbates disease phenotypes in a SOD1H46R-expressing ALS mouse model. To clarify the effects of SQSTM1 overexpression in this model, we generated SQSTM1 and SOD1 H46R double-transgenic (SQSTM1;SOD1 H46R ) mice. SQSTM1;SOD1 H46R mice exhibited earlier disease onset and shorter lifespan than did SOD1 H46R mice. Conversely, disease progression after the onset rather slightly but significantly slowed in SQSTM1;SOD1 H46R mice. However, there were observable differences neither in the number of Nissl positive neurons nor in the distribution of ubiquitin-positive and/or SQSTM1-positive aggregates between SOD1 H46R and SQSTM1;SOD1 H46R mice. It was noted that these protein aggregates were mainly observed in neuropil, and partly localized to astrocytes and/or microglia, but not to MAP2-positive neuronal cell bodies and dendrites at the end-stage of disease. Nonetheless, the biochemically-detectable insoluble SQSTM1 and poly-ubiquitinated proteins were significantly and progressively increased in the spinal cord of SQSTM1;SOD1 H46R mice compared to SOD1 H46R mice. These results suggest that overexpression of SQSTM1 in SOD1 H46R mice accelerates disease onset by compromising the protein degradation pathways.

GABAergic deficits and schizophrenia-like behaviors in a mouse model carrying patient-derived neuroligin-2 R215H mutation

Abstract: Schizophrenia (SCZ) is a severe mental disorder characterized by delusion, hallucination, and cognitive deficits. We have previously identified from schizophrenia patients a loss-of-function mutation Arg215→His215 (R215H) of neuroligin 2 (NLGN2) gene, which encodes a cell adhesion molecule critical for GABAergic synapse formation and function. Here, we generated a novel transgenic mouse line with neuroligin-2 (NL2) R215H mutation. The single point mutation caused a significant loss of NL2 protein in vivo, reduced GABAergic transmission, and impaired hippocampal activation. Importantly, R215H KI mice displayed anxiety-like behavior, impaired pre-pulse inhibition (PPI), cognition deficits and abnormal stress responses, recapitulating several key aspects of schizophrenia-like behaviors. Our results demonstrate a significant impact of a single point mutation NL2 R215H on brain functions, providing a novel animal model for the study of schizophrenia and neuropsychiatric disorders.

What does LTP tell us about the roles of CaMKII and PKMζ in memory

Abstract: In “Criteria for identifying the molecular basis of the engram (CaMKII, PKMζ),” Lisman proposes that elucidating the mechanism of LTP maintenance is key to understanding memory storage. He suggests three criteria for a maintenance mechanism to evaluate data on CaMKII and PKMζ as memory storage molecules: necessity, occlusion, and erasure. Here we show that when the criteria are tested, the results reveal important differences between the molecules. Inhibiting PKMζ reverses established, protein synthesis-dependent late-LTP, without affecting early-LTP or baseline synaptic transmission. In contrast, blocking CaMKII has two effects: 1) inhibiting CaMKII activity blocks LTP induction but not maintenance, and 2) disrupting CaMKII interactions with NMDARs in the postsynaptic density (PSD) depresses both early-LTP and basal synaptic transmission equivalently. To identify a maintenance mechanism, we propose a fourth criterion — persistence. PKMζ increases for hours during LTP maintenance in hippocampal slices, and for over a month in specific brain regions during long-term memory storage in conditioned animals. In contrast, increased CaMKII activity lasts only minutes following LTP induction, and CaMKII translocation to the PSD in late-LTP or memory has not been reported. Lastly, do the PKMζ and CaMKII models integrate the many other signaling molecules important for LTP? Activity-dependent PKMζ synthesis is regulated by many of the signaling molecules that induce LTP, including CaMKII, providing a plausible mechanism for new gene expression in the persistent phosphorylation by PKMζ maintaining late-LTP and memory. In contrast, CaMKII autophosphorylation and translocation do not appear to require new protein synthesis. Therefore, the cumulative evidence supports a core role for PKMζ in late-LTP and long-term memory maintenance, and separate roles for CaMKII in LTP induction and for the maintenance of postsynaptic structure and synaptic transmission in a mechanism distinct from late-LTP.

Biological function of Lemur tyrosine kinase 2 (LMTK2): implications in neurodegeneration.

Abstract: Neurodegenerative disorders are frequent, incurable diseases characterised by abnormal protein accumulation and progressive neuronal loss. Despite their growing prevalence, the underlying pathomechanism remains unclear. Lemur tyrosine kinase 2 (LMTK2) is a member of a transmembrane serine/threonine-protein kinase family. Although it was described more than a decade ago, our knowledge on LMTK2’s biological functions is still insufficient. Recent evidence has suggested that LMTK2 is implicated in neurodegeneration. After reviewing the literature, we identified three LMTK2-mediated mechanisms which may contribute to neurodegenerative processes: disrupted axonal transport, tau hyperphosphorylation and enhanced apoptosis. Moreover, LMTK2 gene expression is decreased in an Alzheimer’s disease mouse model. According to these features, LMTK2 might be a promising therapeutic target in near future. However, further investigations are required to clarify the exact biological functions of this unique protein.

Expression of functional inhibitory neurotransmitter transporters GlyT1, GAT-1, and GAT-3 by astrocytes of inferior colliculus and hippocampus

Abstract: Neuronal inhibition is mediated by glycine and/or GABA. Inferior colliculus (IC) neurons receive glycinergic and GABAergic inputs, whereas inhibition in hippocampus (HC) predominantly relies on GABA. Astrocytes heterogeneously express neurotransmitter transporters and are expected to adapt to the local requirements regarding neurotransmitter homeostasis. Here we analyzed the expression of inhibitory neurotransmitter transporters in IC and HC astrocytes using whole-cell patch-clamp and single-cell reverse transcription-PCR. We show that most astrocytes in both regions expressed functional glycine transporters (GlyTs). Activation of these transporters resulted in an inward current (IGly) that was sensitive to the competitive GlyT1 agonist sarcosine. Astrocytes exhibited transcripts for GlyT1 but not for GlyT2. Glycine did not alter the membrane resistance (RM) arguing for the absence of functional glycine receptors (GlyRs). Thus, IGly was mainly mediated by GlyT1. Similarly, we found expression of functional GABA transporters (GATs) in all IC astrocytes and about half of the HC astrocytes. These transporters mediated an inward current (IGABA) that was sensitive to the competitive GAT-1 and GAT-3 antagonists NO711 and SNAP5114, respectively. Accordingly, transcripts for GAT-1 and GAT-3 were found but not for GAT-2 and BGT-1. Only in hippocampal astrocytes, GABA transiently reduced RM demonstrating the presence of GABAA receptors (GABAARs). However, IGABA was mainly not contaminated by GABAAR-mediated currents as RM changes vanished shortly after GABA application. In both regions, IGABA was stronger than IGly. Furthermore, in HC the IGABA/IGly ratio was larger compared to IC. Taken together, our results demonstrate that astrocytes are heterogeneous across and within distinct brain areas. Furthermore, we could show that the capacity for glycine and GABA uptake varies between both brain regions.

Characterization of temperature-sensitive leak K + currents and expression of TRAAK, TREK-1, and TREK2 channels in dorsal root ganglion neurons of rats

Abstract: Leak K+ currents are mediated by two-pore domain K+ (K2P) channels and are involved in controlling neuronal excitability. Of 15 members of K2P channels cloned so far, TRAAK, TREK-1, and TREK-2 are temperature sensitive. In the present study, we show that strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels was present mainly in small-sized dorsal root ganglion (DRG) neurons of rats. The percentages of neurons with strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels were 27, 23, and 20%, respectively. Patch-clamp recordings were performed to examine isolated leak K+ currents on acutely dissociated small-sized rat DRG neurons at room temperature of 22 °C, cool temperature of 14 °C and warm temperature of 30 °C. In majority of small-sized DRG neurons recorded (76%), large leak K+ currents were observed at 22 °C and were inhibited at 14 °C and potentiated at 30 °C, suggesting the presence of temperature-sensitive K2P channels in these neurons. In a small population (18%) of small-sized DRG neurons, cool temperature of 14 °C evoked a conductance which was consistent with TRPM8 channel activation in cold-sensing DRG neurons. In these DRG neurons, leak K+ currents were very small at 22 °C and were not potentiated at 30 °C, suggesting that few temperature-sensitive K2P channels was present in cold-sensing DRG neurons. For DRG neurons with temperature-sensitive leak K+ currents, riluzole, norfluoxetine and prostaglandin F2α (PGE2α) inhibited the leak K+ currents at both 30 °C and 22 °C degree, and did not have inhibitory effects at 14 °C. Collectively, the observed temperature-sensitive leak K+ currents are consistent with the expression of temperature-sensitive K2P channels in small-sized DRG neurons.

Naked mole-rat cortical neurons are resistant to acid-induced cell death

Abstract: Regulation of brain pH is a critical homeostatic process and changes in brain pH modulate various ion channels and receptors and thus neuronal excitability. Tissue acidosis, resulting from hypoxia or hypercapnia, can activate various proteins and ion channels, among which acid-sensing ion channels (ASICs) a family of primarily Na+ permeable ion channels, which alongside classical excitotoxicity causes neuronal death. Naked mole-rats (NMRs, Heterocephalus glaber) are long-lived, fossorial, eusocial rodents that display remarkable behavioral/cellular hypoxia and hypercapnia resistance. In the central nervous system, ASIC subunit expression is similar between mouse and NMR with the exception of much lower expression of ASIC4 throughout the NMR brain. However, ASIC function and neuronal sensitivity to sustained acidosis has not been examined in the NMR brain. Here, we show with whole-cell patch-clamp electrophysiology of cultured NMR and mouse cortical and hippocampal neurons that NMR neurons have smaller voltage-gated Na+ channel currents and more hyperpolarized resting membrane potentials. We further demonstrate that acid-mediated currents in NMR neurons are of smaller magnitude than in mouse, and that all currents in both species are reversibly blocked by the ASIC antagonist benzamil. We further demonstrate that NMR neurons show greater resistance to acid-induced cell death than mouse neurons. In summary, NMR neurons show significant cellular resistance to acidotoxicity compared to mouse neurons, contributing factors likely to be smaller ASIC-mediated currents and reduced NaV activity.

Fibronectin type III domain-containing protein 5 interacts with APP and decreases amyloid β production in Alzheimer’s disease

Abstract: The deposition of Amyloid-beta peptides (Aβ) is detected at an earlier stage in Alzheimer’s disease (AD) pathology. Thus, the approach toward Aβ metabolism is considered to play a critical role in the onset and progression of AD. Mounting evidence suggests that lifestyle-related diseases are closely associated with AD, and exercise is especially linked to the prevention and the delayed progression of AD. We previously showed that exercise is more effective than diet control against Aβ pathology and cognitive deficit in AD mice fed a high-fat diet; however, the underlying molecular mechanisms remain poorly understood. On the other hand, a report suggested that exercise induced expression of fibronectin type III domain-containing protein 5 (FNDC5) in the hippocampus of mice through PGC1α pathway. Thus, in the current study, we investigated a possibility that FNDC5 interacts with amyloid precursor protein (APP) and affects Aβ metabolism. As a result, for the first time ever, we found the interaction between FNDC5 and APP, and forced expression of FNDC5 significantly decreased levels of both Aβ40 and Aβ42 secreted in the media. Taken together, our results indicate that FNDC5 significantly affects β-cleavage of APP via the interaction with APP, finally regulating Aβ levels. A deeper understanding of the mechanisms by which the interaction between APP and FNDC5 may affect Aβ production in an exercise-dependent manner would provide new preventive strategies against the development of AD.

Accelerated FRET-PAINT Microscopy

Abstract: Recent development of FRET-PAINT microscopy significantly improved the imaging speed of DNA-PAINT, the previously reported super-resolution fluorescence microscopy with no photobleaching problem. Here we try to achieve the ultimate speed limit of FRET-PAINT by optimizing the camera speed, dissociation rate of DNA probes, and bleed-through of the donor signal to the acceptor channel, and further increase the imaging speed of FRET-PAINT by 8-fold. Super-resolution imaging of COS-7 microtubules shows that high-quality 40-nm resolution images can be obtained in just tens of seconds.

Antidepressant-like effects of translocator protein (18 kDa) ligand ZBD-2 in mouse models of postpartum depression.

Abstract: The 18 kDa translocator protein (TSPO) is primarily localized in the outer mitochondrial membrane of steroid-synthesizing cells in the central and peripheral nervous systems. One of the protein’s main functions is transporting substrate cholesterol into the mitochondria in a prerequisite process for steroid synthesis. Clinical trials have indicated that TSPO ligands might be valuable in treating some neuropathies and psychopathies. However, limited information is known about the role of TSPO in postpartum depression (PPD). The TSPO ligand ZBD-2, a derivative of XBD173, was synthesized in our laboratory. Behavioral tests, enzyme linked immunosorbent assay, and Western blot were employed to evaluate ZBD-2’s efficacy against PPD and to elucidate the potential underlying molecular mechanism. The TSPO levels significantly decreased in the basolateral amygdala of PPD models. After treatment for 2 weeks, ZBD-2 alleviated depression-like behaviors and enhanced the TSPO level in a PPD animal model. The underlying mechanisms of ZBD-2 were related to regulate the hypothalamic-pituitary-adrenal axis, enhance 5-HT and BDNF secretion, and maintain the excitatory and inhibitory synaptic protein expression to normal levels. Our results directly confirm that ZBD-2 exerts a therapeutic effect on PPD, which provides a new target for anti-PPD drug development.