Glutamate transport is a critical process in the brain that maintains low extracellular levels of glutamate to allow for efficient neurotransmission and prevent excitotoxicity. Loss of glutamate transport function is implicated in epilepsy, traumatic brain injury, and amyotrophic lateral sclerosis. It remains unclear whether or not glutamate transport can be modulated in these disease conditions to improve outcome. Here, we show that sirtuin (SIRT)4, a mitochondrial sirtuin, is up‐regulated in response to treatment with the potent excitotoxin kainic acid. Loss of SIRT4 leads to a more severe reaction to kainic acid and decreased glutamate transporter expression and function in the brain. Together, these results indicate a critical and novel stress response role for SIRT4 in promoting proper glutamate transport capacity and protecting against excitotoxicity.
Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin‐1 (ET‐1) were examined in rat cultured astrocytes. Expressions of VEGF‐R1 and ‐R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF‐R3 and Tie‐2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF‐R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF‐E, a selective VEGF‐R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF165 were reduced by selective antagonists for VEGF‐R1 and ‐R2. Treatment with ET‐1 increased VEGF‐R1 mRNA and protein levels. The effects of ET‐1 on VEGF‐R1 mRNA were mimicked by Ala1,3,11,15‐ET‐1, a selective agonist for ETB receptors, and inhibited by BQ788, an ETB antagonist. ET‐1 did not affect the mRNA levels of VEGF‐R2, ‐R3, and Tie‐2. Pre‐treatment with ET‐1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET‐1 induces up‐regulation of VEGF‐R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues.
Ischemic postconditioning is increasingly being investigated as a therapeutic approach for cerebral ischemia. However, the majority of studies are focused on the acute protection of neurons per se . Whether and how postconditioning affects multiple cells in the recovering neurovascular unit remains to be fully elucidated. Here, we asked whether postconditioning may modulate help‐me signaling between injured neurons and reactive microglia. Rats were subjected to 100 min of focal cerebral ischemia, then randomized into a control versus postconditioning group. After 3 days of reperfusion, infarct volumes were significantly reduced in animals treated with postconditioning, along with better neurologic outcomes. Immunostaining revealed that ischemic postconditioning increased expression of vascular endothelial growth factor (VEGF ) in neurons within peri‐infarct regions. Correspondingly, we confirmed that VEGFR 2 was expressed on Iba1‐positive microglia/macrophages, and confocal microscopy showed that in postconditioned rats, these cells were polarized to a ramified morphology with higher expression of M2‐like markers. Treating rats with a VEGF receptor 2 kinase inhibitor negated these effects of postconditioning on microglia/macrophage polarization. In vitro , postconditoning after oxygen‐glucose deprivation up‐regulated VEGF release in primary neuron cultures, and adding VEGF to microglial cultures partly shifted their M2‐like markers. Altogether, our findings support the idea that after postconditioning, injured neurons may release VEGF as a ‘help‐me’ signal that promotes microglia/macrophage polarization into potentially beneficial phenotypes.
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A virtual issue on Neuroinflammation in nervous system disorders, edited by Tammy Kielian, is now available: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1471-4159/homepage/virtual_issues.htm#neuroinflammation
In this study, in vitro and in vivo experiments were carried out with the high‐affinity multifunctional D2/D3 agonist D‐512 to explore its potential neuroprotective effects in models of Parkinson's disease and the potential mechanism(s) underlying such properties. Pre‐treatment with D‐512 in vitro was found to rescue rat adrenal Pheochromocytoma PC12 cells from toxicity induced by 6‐hydroxydopamine administration in a dose‐dependent manner. Neuroprotection was found to coincide with reductions in intracellular reactive oxygen species, lipid peroxidation, and DNA damage. In vivo, pre‐treatment with 0.5 mg/kg D‐512 was protective against neurodegenerative phenotypes associated with systemic administration of MPTP, including losses in striatal dopamine, reductions in numbers of DAergic neurons in the substantia nigra (SN), and locomotor dysfunction. These observations strongly suggest that the multifunctional drug D‐512 may constitute a novel viable therapy for Parkinson's disease.
It has been postulated that the accumulation of extracellular α‐synuclein (α‐syn) might alter the neuronal membrane by formation of ‘pore‐like structures’ that will lead to alterations in ionic homeostasis. However, this has never been demonstrated to occur in brain neuronal plasma membranes. In this study, we show that α‐syn oligomers rapidly associate with hippocampal membranes in a punctate fashion, resulting in increased membrane conductance (5 fold over control) and the influx of both calcium and a fluorescent glucose analogue. The enhancement in intracellular calcium (1.7 fold over control) caused a large increase in the frequency of synaptic transmission (2.5 fold over control), calcium transients (3 fold over control), and synaptic vesicle release. Both primary hippocampal and dissociated nigral neurons showed rapid increases in membrane conductance by α‐syn oligomers. In addition, we show here that α‐syn caused synaptotoxic failure associated with a decrease in SV2, a membrane protein of synaptic vesicles associated with neurotransmitter release. In conclusion, extracellular α‐syn oligomers facilitate the perforation of the neuronal plasma membrane, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation.
The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, full‐length membrane‐bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis, either via interaction of mRAGE with β‐amyloid peptide (Aβ) or inhibition of the mRAGE‐activated signaling pathway. In the present study, we showed that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and Transformer2β‐1 (Tra2β‐1) were involved in the alternative splicing of mRAGE and esRAGE. Functionally, two factors had an antagonistic effect on the regulation. Glucose deprivation induced an increased ratio of mRAGE/esRAGE via up‐regulation of hnRNP A1 and down‐regulation of Tra2β‐1. Moreover, the ratios of mRAGE/esRAGE and hnRNP A1/Tra2β‐1 were increased in peripheral blood mononuclear cells from AD patients. The results provide a molecular basis for altered splicing of mRAGE and esRAGE in AD pathogenesis.
Intracellular protein trafficking is tightly regulated, and improper trafficking might be the fundamental provocateur for human diseases including neurodegeneration. In neurons, protein trafficking to and from the plasma membrane affects synaptic plasticity. Voltage‐gated potassium channel 2.1 (Kv2.1) is a predominant delayed rectifier potassium (K+) current, and electrical activity patterns of dopamine (DA) neurons within the substantia nigra are generated and modulated by the orchestrated function of different ion channels. The pathological hallmark of Parkinson's disease (PD) is the progressive loss of these DA neurons, resulting in the degeneration of striatal dopaminergic terminals. However, whether trafficking of Kv2.1 channels contributes to PD remains unclear. In this study, we demonstrated that MPTP/MPP+ increases the surface expression of the Kv2.1 channel and causes nigrostriatal degeneration by using a subchronic MPTP mouse model. The inhibition of the Kv2.1 channel by using a specific blocker, guangxitoxin‐1E, protected nigrostriatal projections against MPTP/MPP+ insult and thus facilitated the recovery of motor coordination. These findings highlight the importance of trafficking of Kv2.1 channels in the pathogenesis of PD.
Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from non‐specific effects after cholesterol manipulation. Furthermore, it remains unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole‐cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase or methyl‐β‐cyclodextrin impaired three different forms of endocytosis, including slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca2+ channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of methyl‐β‐cyclodextrin reduced exocytosis, mainly by decreasing the readily releasable pool and the vesicle replenishment after readily releasable pool depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses.
Adropin is expressed in the CNS and plays a crucial role in the development of stroke. However, little is currently known about the effects of adropin on the blood‐brain barrier (BBB) function after intracerebral hemorrhage (ICH). In this study, the role of adropin in collagenase‐induced ICH was investigated in mice. At 1‐h post‐ICH, mice were administered with recombinant human adropin by intranasal. Brain water +content, BBB permeability, and neurological function were measured at different time intervals. Proteins were quantified using western blot analysis, and the localizations of adropin and Notch1 were visualized via immunofluorescence staining. It is shown that adropin reduced brain water content and improved neurological functions. Adropin preserved the functionality of BBB by increasing N‐cadherin expression and reducing extravasation of albumin. Moreover, in vivo knockdown of Notch1 and Hes1 both abolished the protective effects of adropin. Taken together, our data demonstrate that adropin constitutes a potential treatment value for ICH by preserving BBB and improving functional outcomes through the Notch1 signaling pathway.
Accumulating evidence indicates that increased intracellular Na+ concentration ([Na+]i) in astroglial cells is associated with the development of brain edema under ischemic conditions, but the underlying mechanisms are still elusive. Here, we report that in primary cultured rat cortical astrocytes, elevations of [Na+]i reflecting those achieved during ischemia cause a marked decrease in hypotonicity‐evoked current mediated by volume‐regulated anion channel (VRAC). Pharmacological manipulations revealed that VRAC inhibition was not due to the reverse mode of the plasma membrane sodium/calcium exchanger. The negative modulation of VRAC was also observed in an astrocytic cell line lacking the predominant astrocyte water channel aquaporin 4, indicating that [Na+]i effect was not mediated by the regulation of aquaporin 4 activity. The inward rectifier Cl? current, which is also expressed by cultured astrocytes, was not affected by [Na+]i increase. VRAC depression by high [Na+]i was confirmed in adult astrocytes, suggesting that it was not developmentally regulated. Altogether, these results provide the first evidence that intracellular Na+ dynamics can modulate astrocytic membrane conductance that controls functional processes linked to cell volume regulation and add further support to the concept that limiting astrocyte intracellular Na+ accumulation might be a favorable strategy to counteract the development of brain edema.
Drebrin an actin‐bundling key regulator of dendritic spine genesis and morphology, has been recently proposed as a regulator of hippocampal glutamatergic activity which is critical for memory formation and maintenance. Here, we examined the effects of genetic deletion of drebrin on dendritic spine and on the level of complexes containing major brain receptors. To this end, homozygous and heterozygous drebrin knockout mice generated in our laboratory and related wild‐type control animals were studied. Level of protein complexes containing dopamine receptor D1/dopamine receptor D2, 5‐hydroxytryptamine receptor 1A (5‐HT1AR), and 5‐hydroxytryptamine receptor 7 (5‐HT7R) were significantly reduced in hippocampus of drebrin knockout mice whereas no significant changes were detected for GluR1, 2, and 3 and NR1 as examined by native gel‐based immunoblotting. Drebrin depletion also altered dendritic spine formation, morphology, and reduced levels of dopamine receptor D1 in dendritic spines as evaluated using immunohistochemistry/confocal microscopy. Electrophysiological studies further showed significant reduction in memory‐related hippocampal synaptic plasticity upon drebrin depletion. These findings provide unprecedented experimental support for a role of drebrin in the regulation of memory‐related synaptic plasticity and neurotransmitter receptor signaling, offer relevant information regarding the interpretation of previous studies and help in the design of future studies on dendritic spines.
This review explores the evolving landscape of G-protein-coupled receptor (GPCR)-based genetically encoded fluorescent indicators (GEFIs), with a focus on their development, structural components, engineering strategies, and applications. We highlight the unique features of this indicator class, emphasizing the importance of both the sensing domain (GPCR structure and activation mechanism) and the reporting domain (circularly permuted fluorescent protein (cpFP) structure and fluorescence modulation). Further, we discuss indicator engineering approaches, including the selection of suitable cpFPs and expression systems. Additionally, we showcase the diversity and flexibility of their application by presenting a summary of studies where such indicators were used. Along with all the advantages, we also focus on the current limitations as well as common misconceptions that arise when using these indicators. Finally, we discuss future directions in indicator engineering, including strategies for screening with increased throughput, optimization of the ligand-binding properties, structural insights, and spectral diversity.
It is well known that sleep disorders are harmful to people's health and performance, and growing evidence suggests that sleep deprivation (SD ) can trigger neuroinflammation in the brain. The nucleotide‐binding domain and leucine‐rich repeat protein‐3 (NLRP 3) inflammasome is reported to be relevant to the neuroinflammation induced by SD , but the regulatory signaling that governs the NLRP 3 inflammasome in SD is still unknown. Meanwhile, whether the regulatory action of antidepressants in astrocytes could affect the neuroinflammation induced by SD also remains obscure. In this study, we were the first to discover that the antidepressant fluoxetine, a type of specific serotonin reuptake inhibitor widely used in clinical practice, could suppress the neuroinflammation and neuronal apoptosis induced by SD . The main findings from this study are as follows: (i) SD stimulated the expression of activated NLRP 3 inflammasomes and the maturation of IL ‐1β/18 via suppressing the phosphorylation of STAT 3 in astrocytes; (ii) SD decreased the activation of AKT and stimulated the phosphorylation of GSK ‐3β, which inhibited the phosphorylation of STAT 3; (iii) the NLRP 3 inflammasome expression stimulated by SD was partly mediated by the P2X7 receptor; (iv) an agonist of STAT 3 could significantly abolish the expression of NLRP 3 inflammasomes induced by an agonist of the P2X7 receptor in primary cultured astrocytes; (v) the administration of fluoxetine could reverse the stimulation of NLRP 3 inflammasome expression and function by SD through elevating the activation of STAT 3. In conclusion, our present research suggests the promising possibility that fluoxetine could ameliorate the neuronal impairment induced by SD .
DJ‐1 is an oxidative stress sensor that localizes to the mitochondria when the cell is exposed to oxidative stress. DJ‐1 mutations that result in gene deficiency are linked to increased risk of Parkinson's disease (PD). Activation of microglial stress conditions that are linked to PD may result in neuronal death. We postulated that DJ‐1 deficiency may increase microglial neurotoxicity. We found that down‐regulation of DJ‐1 in microglia using an shRNA approach increased cell sensitivity to dopamine as measured by secreted pro‐inflammatory cytokines such as IL‐1β and IL‐6. Furthermore, we discovered that DJ‐1‐deficient microglia had increased monoamine oxidase activity that resulted in elevation of intracellular reactive oxygen species and nitric oxide leading to increased dopaminergic neurotoxicity. Rasagaline, a monoamine oxidase inhibitor approved for treatment of PD, reduced the microglial pro‐inflammatory phenotype and significantly reduced neurotoxicity. Moreover, we discovered that DJ‐1‐deficient microglia have reduced expression of triggering receptor expressed on myeloid cells 2 (TREM2), previously suggested as a risk factor for pro‐inflammation in neurodegenerative diseases. Further studies of DJ‐1‐mediated cellular pathways in microglia may contribute useful insights into the development of PD providing future avenues for therapeutic intervention.
Synaptic impairment rather than neuronal loss may be the leading cause of cognitive dysfunction in brain aging. Certain small Rho‐GTPases are involved in synaptic plasticity, and their dysfunction is associated with brain aging and neurodegeneration. Rho‐GTPases undergo prenylation by attachment of geranylgeranylpyrophosphate (GGPP) catalyzed by GGTase‐I. We examined age‐related changes in the abundance of Rho and Rab proteins in membrane and cytosolic fractions as well as of GGTase‐I in brain tissue of 3‐ and 23‐month‐old C57BL/6 mice. We report a shift in the cellular localization of Rho‐GTPases toward reduced levels of membrane‐associated and enhanced cytosolic levels of those proteins in aged mouse brain as compared with younger mice. The age‐related reduction in membrane‐associated Rho proteins was associated with a reduction in GGTase‐Iβ levels that regulates binding of GGPP to Rho‐GTPases. Proteins prenylated by GGTase‐II were not reduced in aged brain indicating a specific targeting of GGTase‐I in the aged brain. Inhibition of GGTase‐I in vitro modeled the effects of aging we observed in vivo. We demonstrate for the first time a decrease in membrane‐associated Rho proteins in aged brain in association with down‐regulation of GGTase‐Iβ. This down‐regulation could be one of the mechanisms causing age‐related weakening of synaptic plasticity.
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