Recent investigations into the mechanisms mediating itch transmission have focused on spinal mechanisms, whereas few studies have investigated the role of the cerebral cortex in itch‐related behaviors. Human imaging studies show that several cortical regions are active in correspondence with itch, including the anterior cingulate cortex (ACC). We present here evidence of cortical modulation of pruritogen‐induced scratching behavior. We combine pharmacological, genetic, and electrophysiological approaches to show that cortical GluK1‐containing kainate (KA) receptors are involved in scratching induced by histamine and non‐histamine‐dependent itching stimuli. We further show that scratching corresponds with enhanced excitatory transmission in the ACC through KA receptor modulation of inhibitory circuitry. In addition, we found that inhibiting GluK1‐containing KA receptors in the ACC also reduced behavioral nociceptive responses induced by formalin. Our results reveal a new role of the cortex in pruritogen‐induced scratching.
Beta amyloid (Aβ) oligomers are thought to contribute to the pathogenesis of Alzheimer's disease. However, clinical trials using Aβ immunization were unsuccessful due to strong brain inflammation, the mechanisms of which are poorly understood. In this study we tested whether monoclonal antibodies to oligomeric Aβ would prevent the neurotoxicity of Aβ oligomers in primary neuronal‐glial cultures. However, surprisingly, the antibodies dramatically increased the neurotoxicity of Aβ. Antibodies bound to monomeric Aβ fragments were non‐toxic to cultured neurons, while antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein, human metapneumovirus nucleocapsid protein, and measles virus nucleocapsid protein, strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody‐oligomeric antigen complexes was abolished by removal of the Fc region from the antibodies or by removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aβ oligomers or other oligomeric/multimeric antigens and their specific antibodies can cause death and loss of neurons in primary neuronal‐glial cultures via Fc‐dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aβ or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.
Although the aberrant assembly of mutant superoxide dismutase 1 (mSOD1) is implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS), the molecular basis of superoxide dismutase 1 (SOD1) oligomerization remains undetermined. We investigated the roles of transglutaminase 2 (TG2), an endogenous cross‐linker in mSOD1‐linked ALS. TG2 interacted preferentially with mSOD1 and promoted its oligomerization in transfected cells. Purified TG2 directly oligomerized recombinant mutant SOD1 and the apo‐form of the wild‐type SOD1 proteins in a calcium‐dependent manner, indicating that misfolded SOD1 is a substrate of TG2. Moreover, the non‐cell‐autonomous effect of extracellular TG2 on the neuroinflammation was suggested, since the TG2‐mediated soluble SOD1 oligomers induced tumor necrosis factor‐α, interleukin‐1β, and nitric oxide in microglial BV2 cells. TG2 was up‐regulated in the spinal cord of pre‐symptomatic G93A SOD1 transgenic mice and in the hypoglossal nuclei of mice suffering nerve ligation. Furthermore, inhibition of spinal TG2 by cystamine significantly delayed the progression and reduced SOD1 oligomers and microglial activation. These results indicate a novel role of TG2 in SOD1 oligomer‐mediated neuroinflammation, as well as in the involvement in the intracellular aggregation of misfolded SOD1 in ALS.
The biological functions of the neuregulin 1 (NRG1) and ERBB4 genes have received much recent attention due to several studies showing associations between these genes and schizophrenia. Moreover, reduced forebrain dendritic spine density is a consistent feature of schizophrenia. It is thus important to understand the mechanisms whereby NRG1 and erbB4 modulate spine morphogenesis. Here, we show that long‐term incubation with NRG1 increases both spine size and density in cortical pyramidal neurons. NRG1 also enhances the content of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors in spines. Knockdown of ERBB4 expression prevented the effects of NRG1 on spine size, but not on spine density. The effects of NRG1 and erbB4 on spines were mediated by the RacGEF kalirin, a well‐characterized regulator of dendritic spines. Finally, we show that environmental enrichment, known to promote spine growth, robustly enhances the levels of erbB4 protein in the forebrain. These findings provide a mechanistic link between NRG1 signaling and spine morphogenesis.
HIV‐1 invades CNS in the early course of infection, which can lead to the cascade of neuroinflammation. NADPH oxidases (NOXs) are the major producers of reactive oxygen species (ROS), which play important roles during pathogenic insults. The molecular mechanism of ROS generation via microRNA‐mediated pathway in human microglial cells in response to HIV‐1 Tat protein has been demonstrated in this study. Over‐expression and knockdown of microRNAs, luciferase reporter assay, and site‐directed mutagenesis are main molecular techniques used in this study. A significant reduction in miR‐17 levels and increased NOX2, NOX4 expression levels along with ROS production were observed in human microglial cells upon HIV‐1 Tat C exposure. The validation of NOX2 and NOX4 as direct targets of miR‐17 was done by luciferase reporter assay. The over‐expression and knockdown of miR‐17 in human microglial cells showed the direct role of miR‐17 in regulation of NOX2, NOX4 expression and intracellular ROS generation. We demonstrated the regulatory role of cellular miR‐17 in ROS generation through over‐expression and knockdown of miR‐17 in human microglial cells exposed to HIV‐1 Tat C protein.
Histone deacetylase (HDAC) inhibitors prevent neural cell death in in vivo models of cerebral ischaemia, brain injury and neurodegenerative disease. One mechanism by which HDAC inhibitors may do this is by suppressing the excessive inflammatory response of chronically activated microglia. However, the molecular mechanisms underlying this anti‐inflammatory effect and the specific HDAC responsible are not fully understood. Recent data from in vivo rodent studies have shown that inhibition of class I HDACs suppresses neuroinflammation and is neuroprotective. In our study, we have identified that selective HDAC inhibition with inhibitors apicidin, MS‐275 or MI‐192, or specific knockdown of HDAC1 or 2 using siRNA, suppresses the expression of cytokines interleukin‐6 (IL‐6) and tumour necrosis factor‐alpha (TNF‐α) in BV‐2 murine microglia activated with lipopolysaccharide (LPS). Furthermore, we found that in the absence of HDAC1, HDAC2 is up‐regulated and these increased levels are compensatory, suggesting that these two HDACs have redundancy in regulating the inflammatory response of microglia. Investigating the possible underlying anti‐inflammatory mechanisms suggests an increase in protein expression is not important. Taken together, this study supports the idea that inhibitors selective towards HDAC1 or HDAC2, may be therapeutically useful for targeting neuroinflammation in brain injuries and neurodegenerative disease.
The E3 ubiquitin ligase Parkin plays a central role in the pathogenesis of many neurodegenerative diseases. Parkin promotes specific ubiquitination and affects the localization of transactivation response DNA‐binding protein 43 (TDP‐43), which controls the translation of thousands of mRNAs. Here we tested the effects of lentiviral Parkin and TDP‐43 expression on amino acid metabolism in the rat motor cortex using high frequency 13C NMR spectroscopy. TDP‐43 expression increased glutamate levels, decreased the levels of other amino acids, including glutamine, aspartate, leucine and isoleucine, and impaired mitochondrial tricarboxylic acid cycle. TDP‐43 induced lactate accumulation and altered the balance between excitatory (glutamate) and inhibitory (GABA) neurotransmitters. Parkin restored amino acid levels, neurotransmitter balance and tricarboxylic acid cycle metabolism, rescuing neurons from TDP‐43‐induced apoptotic death. Furthermore, TDP‐43 expression led to an increase in 4E‐BP levels, perhaps altering translational control and deregulating amino acid synthesis; while Parkin reversed the effects of TDP‐43 on the 4E‐BP signaling pathway. Taken together, these data suggest that Parkin may affect TDP‐43 localization and mitigate its effects on 4E‐BP signaling and loss of amino acid homeostasis.
Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein‐regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth‐associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.
Spreading depression (SD), the most likely cause of migraine aura and perhaps migraine, occurs with increased oxidative stress (OS). SD increases reactive oxygen species (ROS), and ROS, in turn, can signal to increase neuronal excitability, which includes increased SD susceptibility. SD also elevates tumor necrosis factor‐α (TNF‐α), which increases neuronal excitability. Accordingly, we probed for the cellular origin of OS from SD and its relationship to TNF‐α, which might promote SD, using rat hippocampal slice cultures. We observed significantly increased OS from SD in astrocytes and microglia but not in neurons or oligodendrocytes. Since insulin‐like growth factor‐1 (IGF‐1) mitigates OS from SD, we determined the cell types responsible for this effect. We found that IGF‐1 significantly decreased microglial but not astrocytic OS from SD. We also show that IGF‐1 abrogated the SD‐induced TNF‐α increase. Furthermore, TNF‐α application increased microglial but not astrocytic OS, an effect abrogated by IGF‐1. Next, we showed that SD increased SD susceptibility, and does so via TNF‐α. This work suggests that microglia promote SD via increased and interrelated ROS and TNF‐α signaling. Thus, IGF‐1 mitigation of microglial ROS and TNF‐α responses may be targets for novel therapeutics development to prevent SD, and perhaps migraine.
The purpose of this study was to clarify the expression of Na+‐dependent multivitamin transporter (SLC5A6/SMVT) and its contribution to the supply of biotin and pantothenic acid to the human brain via the blood–brain barrier. DNA microarray and immunohistochemical analyses confirmed that SLC5A6 is expressed in microvessels of human brain. The absolute expression levels of SLC5A6 protein in isolated human and monkey brain microvessels were 1.19 and 0.597 fmol/μg protein, respectively, as determined by a quantitative targeted absolute proteomics technique. Using an antibody‐free method established by Kubo et al. (2015), we found that SLC5A6 was preferentially localized at the luminal membrane of brain capillary endothelium. Knock‐down analysis using SLC5A6 siRNA showed that SLC5A6 accounts for 88.7% and 98.6% of total [3H]biotin and [3H]pantothenic acid uptakes, respectively, by human cerebral microvascular endothelial cell line hCMEC/D3. SLC5A6‐mediated transport in hCMEC/D3 was markedly inhibited not only by biotin and pantothenic acid, but also by prostaglandin E2, lipoic acid, docosahexaenoic acid, indomethacin, ketoprofen, diclofenac, ibuprofen, phenylbutazone, and flurbiprofen. This study is the first to confirm expression of SLC5A6 in human brain microvessels and to provide evidence that SLC5A6 is a major contributor to luminal uptake of biotin and pantothenic acid at the human blood–brain barrier.
Toll‐like receptor 4 (TLR4) activation and signalling in glial cells play critical roles in neurological disorders and in alcohol‐induced brain damage. TLR4 endocytosis upon lipopolysaccharide (LPS) stimulation regulates which signalling pathway is activated, the MyD88‐dependent or the TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF)‐dependent pathway. However, it remains elusive whether ethanol‐induced TLR4 signalling is associated with receptor internalization and trafficking, and which endocytic pathway(s) are used in cortical astrocytes. Using the adenoviral over‐expression of TLR4GFP, confocal microscopy and the imagestream technique, we show that upon ethanol or LPS stimulation, TLR4 co‐localizes with markers of the clathrin and caveolin endocytic pathways, and that this endocytosis is dependent on dynamin. Using chlorpromazin and filipin as inhibitors of the clathrin and rafts/caveolae endocytic pathways, respectively, we demostrate that TRIF‐dependent signalling relies on an intact clathrin pathway, whereas disruption of rafts/caveolae inhibits the MyD88‐ and TRIF‐dependent signalling pathways. Immunofluorescence studies also suggest that lipid rafts and clathrin cooperate for appropriate TLR4 internalization. We also show that ethanol can trigger similar endocytic pathways as LPS does, although ethanol delays clathrin internalization and alters TLR4 vesicular trafficking. Our results provide new insights into the effects of ethanol or LPS on TLR4 signalling in cortical astrocytes, events that may underlie neuroinflammation and brain damage.
Chronic glial activation and neuroinflammation induced by the amyloid‐β peptide (Aβ) contribute to Alzheimer's disease (AD) pathology. APOE4 is the greatest AD‐genetic risk factor; increasing risk up to 12‐fold compared to APOE3, with APOE4‐specific neuroinflammation an important component of this risk. This editorial review discusses the role of APOE in inflammation and AD, via a literature review, presentation of novel data on Aβ‐induced neuroinflammation, and discussion of future research directions. The complexity of chronic neuroinflammation, including multiple detrimental and beneficial effects occurring in a temporal and cell‐specific manner, has resulted in conflicting functional data for virtually every inflammatory mediator. Defining a neuroinflammatory phenotype (NIP) is one way to address this issue, focusing on profiling the changes in inflammatory mediator expression during disease progression. Although many studies have shown that APOE4 induces a detrimental NIP in peripheral inflammation and Aβ‐independent neuroinflammation, data for APOE‐modulated Aβ‐induced neuroinflammation are surprisingly limited. We present data supporting the hypothesis that impaired apoE4 function modulates Aβ‐induced effects on inflammatory receptor signaling, including amplification of detrimental (toll‐like receptor 4‐p38α) and suppression of beneficial (IL‐4R‐nuclear receptor) pathways. To ultimately develop APOE genotype‐specific therapeutics, it is critical that future studies define the dynamic NIP profile and pathways that underlie APOE‐modulated chronic neuroinflammation.
The Ketogenic diet (KD) is an effective treatment with regards to treating pharmaco‐resistant epilepsy. However, there are difficulties around compliance and tolerability. Consequently, there is a need for refined/simpler formulations that could replicate the efficacy of the KD. One of the proposed hypotheses is that the KD increases cellular mitochondrial content which results in elevation of the seizure threshold. Here, we have focussed on the medium‐chain triglyceride form of the diet and the observation that plasma octanoic acid (C8) and decanoic acid (C10) levels are elevated in patients on the medium‐chain triglyceride KD. Using a neuronal cell line (SH‐SY5Y), we demonstrated that 250‐μM C10, but not C8, caused, over a 6‐day period, a marked increase in the mitochondrial enzyme, citrate synthase along with complex I activity and catalase activity. Increased mitochondrial number was also indicated by electron microscopy. C10 is a reported peroxisome proliferator activator receptor γ agonist, and the use of a peroxisome proliferator activator receptor γ antagonist was shown to prevent the C10‐mediated increase in mitochondrial content and catalase. C10 may mimic the mitochondrial proliferation associated with the KD and raises the possibility that formulations based on this fatty acid could replace a more complex diet.
Cellular interactions mediated by the neural cell adhesion molecule (NCAM) are critical in cell migration, differentiation and plasticity. Switching of the NCAM‐interaction mode, from adhesion to signalling, is determined by NCAM carrying a particular post‐translational modification, polysialic acid (PSA). Regulation of cell‐surface PSA‐NCAM is traditionally viewed as a direct consequence of polysialyltransferase activity. Taking advantage of the polysialyltransferase Ca2+‐dependent activity, we demonstrate in TE671 cells that downregulation of PSA‐NCAM synthesis constitutes a necessary but not sufficient condition to reduce cell‐surface PSA‐NCAM; instead, PSA‐NCAM turnover required internalization of the molecule into the cytosol. PSA‐NCAM internalization was specifically triggered by collagen in the extracellular matrix (ECM) and prevented by insulin‐like growth factor (IGF1) and insulin. Our results pose a novel role for IGF1 and insulin in controlling cell migration through modulation of PSA‐NCAM turnover at the cell surface.
This Editorial highlights a study by Zimmermann and coworkers in the current issue of Journal of Neurochemistry. The authors' link suppression of PKR‐like endoplasmatic reticulum kinase (PERK) activity to eukaryotic elongation factor 2 (eEF2) dephosphorylation and mTORC1‐independent high‐frequency stimulation (HFS)‐induced long‐term potentiation (LTP) in acute hippocampal slices from PERK forebrain conditional knockout mice. The results suggest that functional interaction between the signaling pathways controlling different phases of the mRNA translation process is necessary for long‐term plasticity in the hippocampus.
A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno‐associated virus (AAV)‐mediated over‐expression of BAG1 and ROCK2‐shRNA in the red nucleus to trace [by co‐expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th8 in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV‐mediated protein over‐expression versus AAV.shRNA‐mediated down‐regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2‐shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion.
下载免费PDF全文