Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non‐steroidal anti‐inflammatory drugs: role of mitochondrial calcium

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.

     

HIV‐1 Tat C modulates NOX2 and NOX4 expressions through miR‐17 in a human microglial cell line

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.

     

Astrocytes mediate HIV‐1 Tat‐induced neuronal damage via ligand‐gated ion channel P2X7R

During human immunodeficiency virus (HIV)‐1 infection, perturbations in neuron–glia interactions may culminate in neuronal damage. Recently, purinergic receptors have been implicated in the promotion of virus‐induced neurotoxicity and supporting the viral life cycle at multiple stages. The astrocytes robustly express purinergic receptors. We therefore sought to examine if P2X7R, a P2X receptor subtype, can mediate HIV‐1 Tat‐induced neuronal apoptosis. Tat augmented the expression of P2X7R in astrocytes. Our data reveal the involvement of P2X7R in Tat‐mediated release of monocyte chemoattractant protein (MCP‐1) /chemokine (C‐C motif) ligand 2 (CCL2) from the astrocytes. P2X7R antagonists, such as the oxidized ATP, A438079, brilliant blue G, and broad spectrum P2 receptor antagonist suramin, attenuated Tat‐induced CCL2 release in a calcium‐ and extracellular signal‐regulated kinase (ERK)1/2‐dependent manner. Calcium chelators, (1,2‐bis(o‐aminophenoxy) ethane‐N,N,N',N'‐tetraacetic acid) acetoxymethyl ester and EGTA, and ERK1/2 inhibitor U0126 abolished chemokine (C‐C motif) ligand 2 release from astrocytes. Furthermore, in human neuronal cultures, we demonstrated P2X7R involvement in Tat‐mediated neuronal death. Importantly, in the TUNEL assay, the application of P2X7R‐specific antagonists or the knockdown of P2X7R in human astrocytes reduced HIV‐Tat‐induced neuronal death significantly, underlining the critical role of P2X7R in Tat‐mediated neurotoxicity. Our study provides novel insights into astrocyte‐mediated neuropathogenesis in HIV‐1 infection and a novel target for therapeutic management of neuroAIDS.

     

The high‐affinity D2/D3 agonist D512 protects PC12 cells from 6‐OHDA‐induced apoptotic cell death and rescues dopaminergic neurons in the MPTP mouse model of Parkinson's disease

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.

     

Different effects of bisphenol‐A on memory behavior and synaptic modification in intact and estrogen‐deprived female mice

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.

     

Regulation of the high‐affinity choline transporter activity and trafficking by its association with cholesterol‐rich lipid rafts

The sodium‐coupled, hemicholinium‐3‐sensitive, high‐affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol‐rich lipid rafts in both SH‐SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH‐SY5Y cells expressing rat CHT with filipin, methyl‐β‐cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol‐saturated MβC. Kinetic analysis of binding of [³H]hemicholinium‐3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (K_D) and maximum number of binding sites (B_max); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol‐rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.

     

CaMKIIβ is localized in dendritic spines as both drebrin‐dependent and drebrin‐independent pools

Drebrin is a major F‐actin binding protein in dendritic spines that is critically involved in the regulation of dendritic spine morphogenesis, pathology, and plasticity. In this study, we aimed to identify a novel drebrin‐binding protein involved in spine morphogenesis and synaptic plasticity. We confirmed the beta subunit of Ca²⁺/calmodulin‐dependent protein kinase II (CaMKIIβ) as a drebrin‐binding protein using a yeast two‐hybrid system, and investigated the drebrin–CaMKIIβ relationship in dendritic spines using rat hippocampal neurons. Drebrin knockdown resulted in diffuse localization of CaMKIIβ in dendrites during the resting state, suggesting that drebrin is involved in the accumulation of CaMKIIβ in dendritic spines. Fluorescence recovery after photobleaching analysis showed that drebrin knockdown increased the stable fraction of CaMKIIβ, indicating the presence of drebrin‐independent, more stable CaMKIIβ. NMDA receptor activation also increased the stable fraction in parallel with drebrin exodus from dendritic spines. These findings suggest that CaMKIIβ can be classified into distinct pools: CaMKIIβ associated with drebrin, CaMKIIβ associated with post‐synaptic density (PSD), and CaMKIIβ free from PSD and drebrin. CaMKIIβ appears to be anchored to a protein complex composed of drebrin‐binding F‐actin during the resting state. NMDA receptor activation releases CaMKIIβ from drebrin resulting in CaMKIIβ association with PSD.

     

Endothelin‐1 increases the expression of VEGF‐R1/Flt‐1 receptors in rat cultured astrocytes through ETB receptors

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 Ala^1,3,11,15‐ET‐1, a selective agonist for ET_B receptors, and inhibited by BQ788, an ET_B 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.

     

Synaptodendritic recovery following HIV Tat exposure: Neurorestoration by phytoestrogens

HIV‐1 infects the brain and, despite antiretroviral therapy, many infected individuals suffer from HIV‐1‐associated neurocognitive disorders (HAND). HAND is associated with dendritic simplification and synaptic loss. Prevention of synaptodendritic damage may ameliorate or forestall neurocognitive decline in latent HIV‐1 infections. The HIV‐1 transactivating protein (Tat) is produced during viral latency in the brain and may cause synaptodendritic damage. This study examined the integrity of the dendritic network after exposure to HIV‐1 Tat by labeling filamentous actin (F‐actin)‐rich structures (puncta) in primary neuronal cultures. After 24 h of treatment, HIV‐1 Tat was associated with the dendritic arbor and produced a significant reduction of F‐actin‐labeled dendritic puncta as well as loss of dendrites. Pre‐treatment with either of two plant‐derived phytoestrogen compounds (daidzein and liquiritigenin), significantly reduced synaptodendritic damage following HIV‐1 Tat treatment. In addition, 6 days after HIV‐1 Tat treatment, treatment with either daidzein, or liquiritigenin enhanced recovery, via the estrogen receptor, from HIV‐1 Tat‐induced synaptodendritic damage. These results suggest that either liquiritigenin or daidzein may not only attenuate acute synaptodendritic injury in HIV‐1 but may also promote recovery from synaptodendritic damage.

     

G protein‐coupled estrogen receptor‐mediated effects on cytosolic calcium and nanomechanics in brain microvascular endothelial cells

G protein‐coupled estrogen receptor (GPER) is a relatively recently identified non‐nuclear estrogen receptor, expressed in several tissues, including brain and blood vessels. The mechanisms elicited by GPER activation in brain microvascular endothelial cells are incompletely understood. The purpose of this work was to assess the effects of GPER activation on cytosolic Ca²⁺ concentration, [Ca²⁺]_i, nitric oxide production, membrane potential and cell nanomechanics in rat brain microvascular endothelial cells (RBMVEC). Extracellular but not intracellular administration of G‐1, a selective GPER agonist, or extracellular administration of 17‐β‐estradiol and tamoxifen, increased [Ca²⁺]_i in RBMVEC. The effect of G‐1 on [Ca²⁺]_i was abolished in Ca²⁺‐free saline or in the presence of a L‐type Ca²⁺ channel blocker. G‐1 increased nitric oxide production in RBMVEC; the effect was prevented by NG‐nitro‐l ‐arginine methyl ester. G‐1 elicited membrane hyperpolarization that was abolished by the antagonists of small and intermediate‐conductance Ca²⁺‐activated K⁺ channels, apamin, and charibdotoxin. GPER‐mediated responses were sensitive to G‐36, a GPER antagonist. In addition, atomic force microscopy studies revealed that G‐1 increased the modulus of elasticity, indicative of cytoskeletal changes and increase in RBMVEC stiffness. Our results unravel the mechanisms underlying GPER‐mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature.

     

LPS or ethanol triggers clathrin‐ and rafts/caveolae‐dependent endocytosis of TLR4 in cortical astrocytes

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 TLR4^GFP, 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.

     

Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell‐derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co‐localize, indicating these two proteins may function in Fe³⁺ reduction prior to Fe²⁺ permeation. Zip8, DMT1, and Steap2 co‐localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin‐mediated iron assimilation. In brain interstitial fluid, transferring‐bound iron (TBI) and non‐transferrin‐bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin‐⁵⁹Fe³⁺) and NTBI, whether presented as ⁵⁹Fe²⁺‐citrate or ⁵⁹Fe³⁺‐citrate; reductase‐independent ⁵⁹Fe²⁺ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn²⁺ inhibition of Fe²⁺ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of ⁵⁹Fe from TBI relies at least in part on an endocytosis‐independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons.

     

Positive allosteric modulation of alpha‐7 nicotinic receptors promotes cell death by inducing Ca2+ release from the endoplasmic reticulum

Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7‐nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca²⁺ signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca²⁺ signaling and cell viability. We used human SH‐SY5Y neuroblastoma cells overexpressing α7‐nAChRs (α7‐SH) and their control (C‐SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca²⁺ with Fura‐2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch‐clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7‐SH but not of C‐SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca²⁺ concentration; (iii) released Ca²⁺ from the ER by a Ca²⁺‐induced Ca²⁺ release mechanism only in α7‐SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7‐nAChRs, ryanodine receptors, or IP₃ receptors. In conclusion, positive allosteric modulation of α7‐nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca²⁺, overloading of intracellular Ca²⁺, and neuronal cell death.

     

Urotensin II promotes vagal‐mediated bradycardia by activating cardiac‐projecting parasympathetic neurons of nucleus ambiguus

Urotensin II (U‐II) is a cyclic undecapeptide that regulates cardiovascular function at central and peripheral sites. The functional role of U‐II nucleus ambiguus, a key site controlling cardiac tone, has not been established, despite the identification of U‐II and its receptor at this level. We report here that U‐II produces an increase in cytosolic Ca²⁺ concentration in retrogradely labeled cardiac vagal neurons of nucleus ambiguus via two pathways: (i) Ca²⁺ release from the endoplasmic reticulum via inositol 1,4,5‐trisphosphate receptor; and (ii) Ca²⁺ influx through P/Q‐type Ca²⁺ channels. In addition, U‐II depolarizes cultured cardiac parasympathetic neurons. Microinjection of increasing concentrations of U‐II into nucleus ambiguus elicits dose‐dependent bradycardia in conscious rats, indicating the in vivo activation of the cholinergic pathway controlling the heart rate. Both the in vitro and in vivo effects were abolished by the urotensin receptor antagonist, urantide. Our findings suggest that, in addition, to the previously reported increase in sympathetic outflow, U‐II activates cardiac vagal neurons of nucleus ambiguus, which may contribute to cardioprotection.

     

Pre‐synaptic kainate receptor‐mediated facilitation of glutamate release involves PKA and Ca2+‐calmodulin at thalamocortical synapses

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4‐aminopyridine (4‐AP)‐evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post‐synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and persisted after pre‐treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G‐protein‐independent regulation further in thalamocortical slices, the KAR‐mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca²⁺ permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca²⁺ stores by thapsigargin, or inhibition of Ca²⁺‐induced Ca²⁺‐release by ryanodine. Thus, the KA‐mediated modulation was contingent on both Ca²⁺ entry through Ca²⁺‐permeable KARs and liberation of intracellular Ca²⁺ stores. Finally, sensitivity to W‐7 indicated that the increased cytosolic [Ca²⁺] underpinning KAR‐mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre‐synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca²⁺‐calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G‐protein involvement.

     

Amyloid β‐abrogated TrkA ubiquitination in PC12 cells analogous to Alzheimer's disease

Amyloid beta (Aβ) protein is the primary proteinaceous deposit found in the brains of patients with Alzheimer's disease (AD). Evidence suggests that Aβ plays a central role in the development of AD pathology. Here, we show in PC12 cells, Aβ impairs tropomyosin receptor kinase A (TrkA) ubiquitination, phosphorylation, and its association with p75^NTR, p62, and TRAF6 induced by nerve growth factor. The ubiquitination and tyrosine phosphorylation of TrkA was also found to be impaired in postmortem human AD hippocampus compared to control. Interestingly, the nitrotyrosylation of TrkA was increased in AD hippocampus and this explains why the phosphotyrosylation and ubiquitination of TrkA was impaired. In AD brain, the production of matrix metalloproteinase‐7 (MMP‐7), which cleaves proNGF, was reduced, thereby leading to the accumulation of pro‐NGF and a decrease in the level of active NGF. TrkA signaling events, including Ras/MAPK and phosphatidylinositol 3‐kinase (PI3K)/Akt pathways, are deactivated with Aβ and in the human AD hippocampus. Findings show that Aβ blocks the TrkA ubiquitination and downstream signaling similar to AD hippocampus.