In non‐excitatory cells, stromal interaction molecule 1 (STIM1) and STIM2 mediate store‐operated calcium entry via an interaction with ORAI1 calcium channels. However, in neurons, STIM2 over‐expression appears to play a role in calcium homeostasis that is different from STIM1 over‐expression. The aim of this study was to establish the role and localization of native STIM2 in the neuronal cell. Co‐immunoprecipitation experiments revealed that the interaction between endogenous STIM2 and ORAI1 was greater in a low‐calcium medium than in a high‐calcium medium. Using a Proximity Ligation Assay (PLA), the number of apparent complexes of endogenous STIM2 with ORAI1 was quantified. No change in the number of PLA signals was observed in the presence of thapsigargin, which depletes calcium from the endoplasmic reticulum (ER). However, the number of apparent STIM2‐ORAI1 complexes increased when intracellular and subsequently ER calcium concentrations were decreased by BAPTA‐AM or a low‐calcium medium. Both Fura‐2 acetoxymethyl ester calcium imaging and PLA in the same neuronal cell indicated that the calcium responses correlated strongly with the number of endogenous STIM2‐ORAI1 complexes. The small drop in calcium levels in the ER caused by decreased intracellular calcium levels appeared to initiate the calcium‐sensitive and thapsigargin‐insensitive interaction between STIM2 and ORAI1.
Polysialic acid (PSA) is a major regulator of cell–cell interactions in the developing nervous system and in neural plasticity in the adult. As a polyanionic molecule with high water‐binding capacity, PSA increases the intercellular space generating permissive conditions for cell motility. PSA enhances stem cell migration and axon path finding and promotes repair in the lesioned peripheral and central nervous systems, thus contributing to regeneration. As a next step in developing an improved PSA‐based approach to treat nervous system injuries, we searched for small organic compounds that mimic PSA and identified as a PSA mimetic 5‐nonyloxytryptamine oxalate, described as a selective 5‐hydroxytryptamine receptor 1B (5‐HT1B) agonist. Similar to PSA, 5‐nonyloxytryptamine binds to the PSA‐specific monoclonal antibody 735, enhances neurite outgrowth of cultured primary neurons and process formation of Schwann cells, protects neurons from oxidative stress, reduces migration of astrocytes and enhances myelination in vitro. Furthermore, nonyloxytryptamine treatment enhances expression of the neural cell adhesion molecule (NCAM) and its polysialylated form PSA‐NCAM and reduces expression of the microtubule‐associated protein MAP2 in cultured neuroblastoma cells. These results demonstrate that 5‐nonyloxytryptamine mimics PSA and triggers PSA‐mediated functions, thus contributing to the repertoire of molecules with the potential to improve recovery in acute and chronic injuries of the mammalian peripheral and central nervous systems.
The molecular mechanisms causing the loss of dopaminergic neurons containing neuromelanin in the substantia nigra and responsible for motor symptoms of Parkinson's disease are still unknown. The discovery of genes associated with Parkinson's disease (such as alpha synuclein (SNCA), E3 ubiquitin protein ligase (parkin), DJ‐1 (PARK7), ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL‐1), serine/threonine‐protein kinase (PINK‐1), leucine‐rich repeat kinase 2 (LRRK2), cation‐transporting ATPase 13A1 (ATP13A), etc.) contributed enormously to basic research towards understanding the role of these proteins in the sporadic form of the disease. However, it is generally accepted by the scientific community that mitochondria dysfunction, alpha synuclein aggregation, dysfunction of protein degradation, oxidative stress and neuroinflammation are involved in neurodegeneration. Dopamine oxidation seems to be a complex pathway in which dopamine o‐quinone, aminochrome and 5,6‐indolequinone are formed. However, both dopamine o‐quinone and 5,6‐indolequinone are so unstable that is difficult to study and separate their roles in the degenerative process occurring in Parkinson's disease. Dopamine oxidation to dopamine o‐quinone, aminochrome and 5,6‐indolequinone seems to play an important role in the neurodegenerative processes of Parkinson's disease as aminochrome induces: (i) mitochondria dysfunction, (ii) formation and stabilization of neurotoxic protofibrils of alpha synuclein, (iii) protein degradation dysfunction of both proteasomal and lysosomal systems and (iv) oxidative stress. The neurotoxic effects of aminochrome in dopaminergic neurons can be inhibited by: (i) preventing dopamine oxidation of the transporter that takes up dopamine into monoaminergic vesicles with low pH and dopamine oxidative deamination catalyzed by monoamino oxidase (ii) dopamine o‐quinone, aminochrome and 5,6‐indolequinone polymerization to neuromelanin and (iii) two‐electron reduction of aminochrome catalyzed by DT‐diaphorase. Furthermore, dopamine conversion to NM seems to have a dual role, protective and toxic, depending mostly on the cellular context.
Temporal lobe epilepsy (TLE) often becomes refractory, and patients with TLE show a high incidence of psychiatric symptoms, including anxiety and depression. Therefore, it is necessary to identify molecules that were previously unknown to contribute to epilepsy and its associated disorders. We previously found that the sialyltransferase ST3Gal IV is up‐regulated within the neural circuits through which amygdala‐kindling stimulation propagates epileptic seizures. In contrast, this study demonstrated that kindling stimulation failed to evoke epileptic seizures in ST3Gal IV‐deficient mice. Furthermore, approximately 80% of these mice failed to show tonic–clonic seizures with stimulation, whereas all littermate wild‐type mice showed tonic–clonic seizures. This indicates that the loss of ST3Gal IV does not cause TLE in mice. Meanwhile, ST3Gal IV‐deficient mice exhibited decreased acclimation in the open field test, increased immobility in the forced swim test, enhanced freezing during delay auditory fear conditioning, and sleep disturbances. Thus, the loss of ST3Gal IV modulates anxiety‐related behaviors. These findings indicate that ST3Gal IV is a key molecule in the mechanisms underlying anxiety – a side effect of TLE – and may therefore also be an effective target for treating epilepsy, acting through the same circuits.
Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β‐actin and GAP‐43 mRNAs. β‐actin 3′UTR has a defined element for interaction with ZBP1, but GAP‐43 mRNA shows no homology to this RNA sequence. Here, we show that an AU‐rich regulatory element (ARE) in GAP‐43′s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP‐43 mRNA levels increase after in vivo injury, and GAP‐43 mRNA shows an increased half‐life in regenerating axons. GAP‐43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co‐immunoprecipitate in an RNA‐dependent fashion. Reporter mRNA with the GAP‐43 ARE competes with endogenous β‐actin mRNA for axonal localization and decreases axon length and branching similar to the β‐actin 3′UTR competing with endogenous GAP‐43 mRNA. Conversely, over‐expressing GAP‐43 coding sequence with its 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP‐43′s 3′UTR.
Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2‐knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2‐knockout mice were significantly lower than those in wild‐type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons.
Spinocerebellar ataxia type 3 (SCA3) is one of at least nine inherited neurodegenerative diseases caused by an expansion of a polyglutamine tract within corresponding disease‐specific proteins. In case of SCA3, mutation of Ataxin‐3 results in aggregation of misfolded protein, formation of intranuclear as well as cytosolic inclusion bodies and cell death in distinct neuronal populations. Since cyclin‐dependent kinase‐5 (CDK5) has been shown to exert beneficial effects on aggregate formation and cell death in various polyglutamine diseases, we tested its therapeutic potential for SCA3. Our data show increased caspase‐dependent Ataxin‐3 cleavage, aggregation, and neurodegeneration in the absence of sufficient CDK5 activity. This disease‐propagating effect could be reversed by mutation of the caspase cleavage site in Ataxin‐3. Moreover, reduction of CDK5 expression levels by RNAi in vivo enhances SCA3 toxicity as assayed in a Drosophila model for SCA3. In summary, we present CDK5 as a potent neuroprotectant, regulating cleavage and thereby toxicity of Ataxin‐3 and other polyglutamine proteins.
Middle cerebral artery occlusion (MCAO) induces secondary damages in the hippocampus that is remote from primary ischemic regions. Tau hyperphosphorylation is an important risk for neurodegenerative diseases. Increased tau phosphorylation has been identified in ischemic cortex, but little is known regarding the changes in the hippocampus. We showed that unilateral transient MCAO induced accumulation of hyperphosphorylated tau and concurrent dephosphorylation of glycogen synthase kinase‐3β at Ser 9 in the ipsilateral hippocampus. These MCAO‐induced changes were not reproduced when glutamatergic inputs from the entorhinal cortex to the hippocampus were transected; however, the changes were mimicked by intrahippocampal N‐methyl‐d ‐aspartate (NMDA) administration. Inhibition of NMDA receptor (NMDAR) subunit NR2B, but not NR2A activity in the hippocampus attenuated the accumulation of hyperphosphorylated tau and spatial cognitive impairment in MCAO rats. Together, our data suggest that overactivation of NR2B‐containing NMDARs through entorhinal–hippocampal connection plays an important role in the accumulation of hyperphosphorylated tau in the hippocampus following MCAO. Glycogen synthase kinase‐3β is an important protein kinase involved in NMDARs‐mediated tau hyperphosphorylation. This study indicates that early inhibition of NR2B‐containing NMDARs may represent a potential strategy to prevent or delay the occurrence of post‐stroke dementia.
Japanese encephalitis virus (JEV), a single‐stranded RNA (ssRNA) virus, is the leading cause of encephalitis in Asia. Microglial activation is one of the key events in JEV‐induced neuroinflammation. Although the various microRNAs (miRNAs) has been shown to regulate microglia activation during pathological conditions including neuroviral infections, till date, the involvement of miRNAs in JEV infection has not been evaluated. Hence, we sought to evaluate the possible role of miRNAs in mediating JEV‐induced microglia activation. Initial screening revealed significant up‐regulation of miR‐29b in JEV‐infected mouse microglial cell line (BV‐2) and primary microglial cells. Furthermore, using bioinformatics tools, we identified tumor necrosis factor alpha‐induced protein 3, a negative regulator of nuclear factor‐kappa B signaling as a potential target of miR‐29b. Interestingly, in vitro knockdown of miR‐29b resulted in significant over‐expression of tumor necrosis factor alpha‐induced protein 3, and subsequent decrease in nuclear translocation of pNF‐κB. JEV infection in BV‐2 cell line elevated inducible nitric oxide synthase, cyclooxygenase‐2, and pro‐inflammatory cytokine expression levels, which diminished after miR‐29b knockdown. Collectively, our study demonstrates involvement of miR‐29b in regulating JEV‐ induced microglial activation.
Dysregulation of hypothalamic–pituitary–adrenal (HPA) axis activation is associated with changes in addiction‐related behaviors. In this study, we tested whether sex differences in the acute effects of methamphetamine (MA) exposure involve differential activation of the HPA axis. Male and female mice were injected with MA (1 mg/kg) or saline for comparison of plasma corticosterone and analysis of the immediate early gene c‐Fos in brain. There was a prolonged elevation in corticosterone levels in female compared to male mice. C‐Fos was elevated in both sexes following MA in HPA axis‐associated regions, including the hypothalamic paraventricular nucleus (PVN), central amygdala, cingulate, and CA3 hippocampal region. MA increased the number of c‐Fos and c‐Fos/glucocorticoid receptor (GR) dual‐labeled cells to a greater extent in males than females in the cingulate and CA3 regions. MA also increased the number of c‐fos/vasopressin dual‐labeled cells in the PVN as well as the number and percentage of c‐Fos/GR dual‐labeled cells in the PVN and central amygdala, although no sex differences in dual labeling were found in these regions. Thus, sex differences in MA‐induced plasma corticosterone levels and activation of distinct brain regions and proteins involved in HPA axis regulation may contribute to sex differences in acute effects of MA on the brain.
Human immunodeficiency virus‐1 (HIV) is a public health issue and a major complication of the disease is NeuroAIDS. In vivo, microglia/macrophages are the main cells infected. However, a low but significant number of HIV‐infected astrocytes has also been detected, but their role in the pathogenesis of NeuroAIDS is not well understood. Our previous data indicate that gap junction channels amplify toxicity from few HIV‐infected into uninfected astrocytes. Now, we demonstrated that HIV infection of astrocytes results in the opening of connexin43 hemichannels (HCs). HIV‐induced opening of connexin43 HCs resulted in dysregulated secretion of dickkopf‐1 protein (DKK1, a soluble wnt pathway inhibitor). Treatment of mixed cultures of neurons and astrocytes with DKK1, in the absence of HIV infection, resulted in the collapse of neuronal processes. HIV infection of mixed cultures of human neurons and astrocytes also resulted in the collapse of neuronal processes through a DKK1‐dependent mechanism. In addition, dysregulated DKK1 expression in astrocytes was observed in human brain tissue sections of individuals with HIV encephalitis as compared to tissue sections from uninfected individuals. Thus, we demonstrated that HIV infection of astrocytes induces dysregulation of DKK1 by a HC‐dependent mechanism that contributes to the brain pathogenesis observed in HIV‐infected individuals.
Cerebral ischaemia rapidly depletes cellular ATP. Whilst this deprives brain tissue of a valuable energy source, the concomitant production of adenosine mitigates the damaging effects of energy failure by suppressing neuronal activity. However, the production of adenosine and other metabolites, and their loss across the blood–brain barrier, deprives the brain of substrates for the purine salvage pathway, the primary means by which the brain makes ATP. Because of this, cerebral ATP levels remain depressed after brain injury. To test whether manipulating cellular ATP levels in brain tissue could affect functional neuronal outcomes in response to oxygen/glucose deprivation (OGD), we examined the effects of creatine and d ‐ribose and adenine (RibAde). In hippocampal slices creatine delayed ATP breakdown, reduced adenosine release, retarded both the depression of synaptic transmission and the anoxic depolarization caused by OGD, and improved the recovery of transmission. In contrast, RibAde increased cellular ATP, caused increased OGD‐induced adenosine release and accelerated the depression of synaptic transmission, but did not improve functional recovery. However, RibAde improved the viability of cerebellar granule cells when administered after OGD. Our data indicate that RibAde may be effective in promoting recovery of brain tissue after injury, potentially via enhancement of salvage‐mediated ATP production.
CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long‐term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody‐mediated cell adhesion, and neurite outgrowth on HIgM12‐coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates it's in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases.
Growing evidence suggests that oxidative stress, as associated with spinal cord injury (SCI), may play a critical role in both neuroinflammation and neuropathic pain conditions. The production of the endogenous aldehyde acrolein, following lipid peroxidation during the inflammatory response, may contribute to peripheral sensitization and hyperreflexia following SCI via the TRPA1‐dependent mechanism. Here, we report that there are enhanced levels of acrolein and increased neuronal sensitivity to the aldehyde for at least 14 days after SCI. Concurrent with injury‐induced increases in acrolein concentration is an increased expression of TRPA1 in the lumbar (L3–L6) sensory ganglia. As proof of the potential pronociceptive role for acrolein, intrathecal injections of acrolein revealed enhanced sensitivity to both tactile and thermal stimuli for up to 10 days, supporting the compound's pro‐nociceptive functionality. Treatment of SCI animals with the acrolein scavenger hydralazine produced moderate improvement in tactile responses as well as robust changes in thermal sensitivity for up to 49 days. Taken together, these data suggest that acrolein directly modulates SCI‐associated pain behavior, making it a novel therapeutic target for preclinical and clinical SCI as an analgesic.
Girdin, an actin‐binding protein, possesses versatile functions in a multitude of cellular processes. Although several studies have shown that Girdin is involved in the cell DNA synthesis, actin cytoskeleton rearrangement, and cell motility, the molecular mechanisms of Girdin in tumor development and progression remain elusive. In this study, through over‐expression and siRNA experiments, we found that Girdin increased migration of LN229 human glioblastoma cells. On the other hand, reducing Girdin impaired F‐actin polymerization, which is essential for cell morphogenesis and motility. Matrix metalloproteinase 2, critical in human glioma migration and invasion, was down‐regulated upon Girdin reduction and led to decreased invasion in vitro and in vivo. In addition, silencing Girdin expression impaired the phosphorylation of two important adhesion molecules, integrin β1 and focal adhesion kinase, resulting in cell adhesion defects. Our immunohistochemical study on human gliomas tissue sections indicated that Girdin expression was positively related with glioma malignancy, supporting the in vitro and in vivo results from cell lines. Collectively, our findings suggest a critical role for Girdin in glioma infiltration.
The main purpose of this study was to evaluate whether donepezil, acetylcholinesterase inhibitor, shown to play a protective role through inhibiting glycogen synthesis kinase‐3β (GSK‐3β) activity, could also exert neuroprotective effects by stimulating protein phosphatase 2A (PP2A) activity in the amyloid‐beta (Aβ)42‐induced neuronal toxicity model of Alzheimer's disease. In Aβ42‐induced toxic conditions, each PP2A and GSK‐3β activity measured at different times showed time‐dependent reverse pattern toward the direction of accelerating neuronal deaths with the passage of time. In addition, donepezil pre‐treatment showed dose‐dependent stepwise increase of neuronal viability and stimulation of PP2A activity. However, such effects on them were significantly reduced through the depletion of PP2A activity with either okadaic acid or PP2Ac siRNA. In spite of blocked PP2A activity in this Aβ42 insult, however, donepezil pretreatment showed additional significant recovering effect on neuronal viability when compared to the value without donepezil. Moreover, donepezil partially recovered its dephosphorylating effect on hyperphosphorylated tau induced by Aβ42. This observation led us to assume that additional mechanisms of donepezil, including its inhibitory effect on GSK‐3β activity and/or the activation role of nicotinic acetylcholine receptors (nAChRs), might be involved. Taken together, our results suggest that the neuroprotective effects of donepezil against Aβ42‐induced neurotoxicity are mediated through activation of PP2A, but its additional mechanisms including regulation of GSK‐3β and nAChRs activity would partially contribute to its effects.
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