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.
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.
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.
Bisphenol‐A (BPA) has the capability of interfering with the effects of estrogens on modulating brain function. The purpose of this study was to investigate the effects of BPA on memory and synaptic modification in the hippocampus of female mice under different levels of cycling estrogen. BPA exposure (40, 400 μg/kg/day) for 8 weeks did not affect spatial memory and passive avoidance task of gonadally intact mice but improved ovariectomy (Ovx)‐induced memory impairment, whereas co‐exposure of BPA with estradiol benzoate (EB) diminished the rescue effect of EB on memory behavior of Ovx mice. The results of morphometric measurement showed that BPA positively modified the synaptic interface structure and increased the synaptic density of CA1 pyramidal cell in the hippocampus of Ovx females, but inhibited the enhancement of EB on synaptic modification and synaptogenesis of Ovx mice. Furthermore, BPA up‐regulated synaptic proteins synapsin I and PSD‐95 and NMDA receptor NR2B but inhibited EB‐induced increase in PSD‐95 and NR2B in the hippocampus of Ovx mice. These results suggest that BPA interfered with normal hormonal regulation in synaptic plasticity and memory of female mice as a potent estrogen mimetic and as a disruptor of estrogen under various concentrations of cycling estrogen.
Brain damage after insult and cognitive decline are related to excitotoxicity and strongly influenced by aging, yet mechanisms of aging‐dependent susceptibility to excitotoxicity are poorly known. Several non‐steroidal anti‐inflammatory drugs (NSAIDs) may prevent excitotoxicity and cognitive decline in the elderly by an unknown mechanism. Interestingly, after several weeks in vitro, hippocampal neurons display important hallmarks of neuronal aging in vivo. Accordingly, rat hippocampal neurons cultured for several weeks were used to investigate mechanisms of aging‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs. We found that NMDA increased cytosolic Ca2+ concentration in young, mature and aged neurons but only promoted apoptosis in aged neurons. Resting Ca2+ levels and responses to NMDA increased with time in culture which correlated with changes in expression of NMDA receptor subunits. In addition, NMDA promoted mitochondrial Ca2+ uptake only in aged cultures. Consistently, specific inhibition of mitochondrial Ca2+ uptake decreased apoptosis. Finally, we found that a series of NSAIDs depolarized mitochondria and inhibited mitochondrial Ca2+ overload, thus preventing NMDA‐induced apoptosis in aged cultures. We conclude that mitochondrial Ca2+ uptake is critical for age‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs.
Both dopamine and glutamate are critically involved in cognitive processes such as working memory. Astrocytes, which express dopamine receptors, are essential elements in the termination of glutamatergic signaling: the astrocytic glutamate transporter GLT‐1 is responsible for > 90% of cortical glutamate uptake. The effect of dopamine depletion on glutamate transporters in the prefrontal cortex (PFC) remains unknown. In an effort to determine if astrocytes are a locus of cortical dopamine–glutamate interactions, we examined the effects of chronic dopamine denervation on PFC protein and mRNA levels of glutamate transporters. PFC dopamine denervation elicited a marked increase in GLT‐1 protein levels, but had no effect on levels of other glutamate transporters; high‐affinity glutamate transport was positively correlated with the extent of dopamine depletion. GLT‐1 gene expression was not altered. Our data suggest that dopamine depletion may lead to post‐translational modifications that result in increased expression and activity of GLT‐1 in PFC astrocytes.
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.
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.
Cannabinoid Receptor 1 (CB1) has been initially described as the receptor for Delta‐9‐Tetrahydrocannabinol in the central nervous system (CNS), mediating retrograde synaptic signaling of the endocannabinoid system. Beside its expression in various CNS regions, CB1 is ubiquituous in peripheral tissues, where it mediates, among other activities, the cell's energy homeostasis. We sought to examine the role of CB1 in the context of the evolutionarily conserved autophagic machinery, a main constituent of the regulation of the intracellular energy status. Manipulating CB1 by siRNA knockdown in mammalian cells caused an elevated autophagic flux, while the expression of autophagy‐related genes remained unaltered. Pharmacological inhibition of CB1 activity using Rimonabant likewise caused an elevated autophagic flux, which was independent of the mammalian target of rapamycin complex 1, a major switch in the control of canonical autophagy. In addition, knocking down coiled‐coil myosin‐like BCL2‐interacting protein 1, the key‐protein of the second canonical autophagy control complex, was insufficient to reduce the elevated autophagic flux induced by Rimonabant. Interestingly, lysosomal activity is not altered, suggesting a specific effect of CB1 on the regulation of autophagic flux. We conclude that CB1 activity affects the autophagic flux independently of the two major canonic regulation complexes controlling autophagic vesicle formation.
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.
Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin‐1 (TSP‐1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5–2.5 mM) for 1–10 days, and TSP‐1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra‐ and extracellular TSP‐1 levels. Exposure of cultured neurons to conditioned media from ammonia‐treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP‐1 over‐expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP‐1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP‐1 synthesis in other cell types, also reversed the ammonia‐induced TSP‐1 reduction. Likewise, we found a significant decline in TSP‐1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP‐1 may represent an important therapeutic target for CHE.
In this study, we identified a polymorphism in the 5′‐flanking region of the chicken serotonin transporter (5‐HTT) gene. Sequencing analysis revealed that in comparison with the wild‐type variant (W), a deleted variant (D) is generated by deletion of four nucleotides (5′‐AATT‐3′) and a single nucleotide change (A→T). Using a polyacrylamide gel electrophoresis system, we found that the 360‐bp DNA fragment containing the W variant with the wild‐type sequence 5′‐AATTAATT‐3′ shows intrinsic DNA curvature while the 356‐bp fragment containing the D variant lacking the four base pairs AATT is not curved. Quantitative real‐time RT‐PCR and ELISA demonstrated that the expression of 5‐HTT in D/D chickens was higher than that in W/W and W/D chickens. In addition, transient transfection experiments with chloramphenicol acetyltransferase reporter gene constructs revealed increased 5‐HTT promoter activity mediated by the D variant and a silencer activity of the W variant. Interestingly, females and males with D/D genotype showed significant greater increase in body weight from 6 weeks and 16 weeks of age, respectively, and higher body mass index. Moreover, we found that D/D chickens of both genders were physically more active than W/W and W/D chickens.
Cell adhesion molecule L1 promotes neuritogenesis and neuronal survival through triggering MAPK pathways. Based on the findings that L1 is associated with casein kinase 2 (CK2), and that deficiency in PTEN promotes neuritogenesis in vitro and regeneration after trauma, we examined the functional relationship between L1 and PTEN. In parallel, we investigated the tumor suppressor p53, which also regulates neuritogenesis. Here, we report that the intracellular domain of L1 binds to the subunit CK2α, and that knockdown of L1 leads to CK2 dephosphorylation and an increase in PTEN and p53 levels. Overexpression of L1, but not the L1 mutants L1 (S1181N, E1184V), which reduced binding between L1 and CK2, reduced expression levels of PTEN and p53 proteins, and enhanced levels of phosphorylated CK2α and mammalian target of rapamycin, which is a downstream effector of PTEN and p53. Treatment of neurons with a CK2 inhibitor or transfection with CK2α siRNA increased levels of PTEN and p53, and inhibited neuritogenesis. The combined observations indicate that L1 downregulates expression of PTEN and p53 via direct binding to CK2α. We suggest that L1 stimulates neuritogenesis by activating CK2α leading to decreased levels of PTEN and p53 via a novel, L1‐triggered and CK2α‐mediated signal transduction pathway.
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.
Uncontrolled elongation of glycogen chains, not adequately balanced by their branching, leads to the formation of an insoluble, presumably neurotoxic, form of glycogen called polyglucosan. To test the suspected pathogenicity of polyglucosans in neurological glycogenoses, we have modeled the typical glycogenosis Adult Polyglucosan Body Disease (APBD) by suppressing glycogen branching enzyme 1 (GBE1, EC 2.4.1.18) expression using lentiviruses harboring short hairpin RNA (shRNA). GBE1 suppression in embryonic cortical neurons led to polyglucosan accumulation and associated apoptosis, which were reversible by rapamycin or starvation treatments. Further analysis revealed that rapamycin and starvation led to phosphorylation and inactivation of glycogen synthase (GS, EC 2.4.1.11), dephosphorylated and activated in the GBE1‐suppressed neurons. These protective effects of rapamycin and starvation were reversed by overexpression of phosphorylation site mutant GS only if its glycogen binding site was intact. While rapamycin and starvation induce autophagy, autophagic maturation was not required for their corrective effects, which prevailed even if autophagic flux was inhibited by vinblastine. Furthermore, polyglucosans were not observed in any compartment along the autophagic pathway. Our data suggest that glycogen branching enzyme repression in glycogenoses can cause pathogenic polyglucosan buildup, which might be corrected by GS inhibition.
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