The function of amyloid precursor protein (APP) is unknown, although the discovery that it contributes to the regulation of surface expression of N‐methyl‐d ‐aspartate (NMDA) receptors has afforded new insights into its functional significance. Since APP is a member of a gene family that contains two other members, amyloid precursor‐like proteins 1 and 2 (APLP1 and APLP2), it is important to determine if the related APP proteins possess the same properties as APP with respect to their interactions with NMDA receptors. Following expression in mammalian cells, both APLP1 and APLP2 behaved similarly to APP in that they both co‐immunoprecipitated with the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B, via interaction with the obligatory GluN1 subunit. Immunoprecipitations from detergent extracts of adult mammalian brain showed co‐immunoprecipitation of APLP1 and APLP2 with GluN2A‐ and GluN2B‐containing NMDA receptors. Furthermore, similarly to APP, APLP1 and APLP2 both enhanced GluN1/GluN2A and GluN1/GluN2B cell surface expression. Thus, all the three members of the APP gene family behave similarly in that they each contribute to the regulation of cell surface NMDA receptor homoeostasis.
The STriatal‐Enriched protein tyrosine Phosphatase 61 (STEP61) inhibits the activity of the tyrosine kinase Fyn and dephosphorylates the GluN2B subunit of the NMDA receptor, whereas the protein kinase A phosphorylation of STEP61 inhibits the activity of the phosphatase (Pharmacol. Rev., 64, 2012 , p. 65). Previously, we found that ethanol activates Fyn in the dorsomedial striatum (DMS) leading to GluN2B phosphorylation, which, in turn, underlies the development of ethanol intake (J. Neurosci., 30, 2010 , p. 10187). Here, we tested the hypothesis that inhibition of STEP61 by ethanol is upstream of Fyn/GluN2B. We show that exposure of mice to ethanol increased STEP61 phosphorylation in the DMS, which was maintained after withdrawal and was not observed in other striatal regions. Specific knockdown of STEP61 in the DMS of mice enhanced ethanol‐mediated Fyn activation and GluN2B phosphorylation, and increased ethanol intake without altering the level of water, saccharine, quinine consumption or spontaneous locomotor activity. Together, our data suggest that blockade of STEP61 activity in response to ethanol is sufficient for the activation of the Fyn/GluN2B pathway in the DMS. Being upstream of Fyn and GluN2B, inactive STEP61 in the DMS primes the induction of ethanol intake.
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.
As an endogenous gaseous molecule, hydrogen sulfide (H2S) has attracted extensive attention because of its multiple biological effects. However, the effect of H2S on amygdala‐mediated emotional memory has not been elucidated. Here, by employing Pavlovian fear conditioning, an animal model widely used to explore the neural substrates of emotion, we determined whether H2S could regulate emotional memory. It was shown that the H2S levels in the amygdala of rats were significantly elevated after cued fear conditioning. Both intraamygdala and systemic administrations of H2S markedly enhanced amygdala‐dependent cued fear memory in rats. Moreover, it was found that H2S selectively increased the surface expression and currents of NMDA‐type glutamate receptor subunit 2B (GluN2B)‐containing NMDA receptors (NMDARs) in lateral amygdala of rats, whereas blockade of GluN2B‐containing NMDARs in lateral amygdala eliminated the effects of H2S to enhance amygdalar long‐term potentiation and cued fear memory. These results demonstrate that H2S can regulate amygdala‐dependent emotional memory by promoting the function of GluN2B‐containing NMDARs in amygdala, suggesting that H2S‐associated signaling may hold potential as a new target for the treatment of emotional disorders.
Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and β‐hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β‐hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of β‐hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β‐hydroxybutyrate was present in these neurons. In addition, the NMDA receptor‐induced calcium responses in the neurons were diminished in the presence of β‐hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of β‐hydroxybutyrate augmented transmitter release induced by the KATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of KATP channels in the effects of ketone bodies on transmitter release.
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.
Zinc (Zn2+) is believed to play a relevant role in the physiology and pathophysiology of the brain. Hence, Zn2+ homeostasis is critical and involves different classes of molecules, including Zn2+ transporters. The ubiquitous Zn2+ transporter‐1 (ZNT‐1) is a transmembrane protein that pumps cytosolic Zn2+ to the extracellular space, but its function in the central nervous system is not fully understood. Here, we show that ZNT‐1 interacts with GluN2A‐containing NMDA receptors, suggesting a role for this transporter at the excitatory glutamatergic synapse. First, we found that ZNT‐1 is highly expressed at the hippocampal postsynaptic density (PSD) where NMDA receptors are enriched. Two‐hybrid screening, coimmunoprecipitation experiments and clustering assay in COS‐7 cells demonstrated that ZNT‐1 specifically binds the GluN2A subunit of the NMDA receptor. GluN2A deletion mutants and pull‐down assays indicated GluN2A(1390–1464) domain as necessary for the binding to ZNT‐1. Most importantly, ZNT‐1/GluN2A complex was proved to be dynamic, since it was regulated by induction of synaptic plasticity. Finally, modulation of ZNT‐1 expression in hippocampal neurons determined a significant change in dendritic spine morphology, PSD‐95 clusters and GluN2A surface levels, supporting the involvement of ZNT‐1 in the dynamics of excitatory PSD.
Neuropilin tolloid‐like 1 (Neto1), is a CUB domain‐containing transmembrane protein that was recently identified as a novel component of the NMDA receptor complex. Here, we have investigated the possible association of Neto1 with the amyloid precursor protein (APP)695/GluN1/GluN2A and APP695/GluN1/GluN2B NMDA receptor trafficking complexes that we have previously identified. Neto1HA was shown to co‐immunoprecipitate with assembled NMDA receptors via GluN2A or GluN2B subunits; Neto1HA did not co‐immunoprecipitate APP695FLAG. Co‐immunoprecipitations from mammalian cells co‐transfected with APP695FLAG, Neto1HA and GluN1/GluN2A or GluN1/GluN2B revealed that all four proteins co‐exist within one macromolecular complex. Immunoprecipitations from native brain tissue similarly revealed the existence of a GluN1/GluN2A or GluN2B/APP/Neto1 complex. Neto1HA caused a reduction in the surface expression of both NMDA receptor subtypes, but had no effect on APP695FLAG‐ or PSD‐95αc‐Myc enhanced surface receptor expression. The Neto1 binding domain of GluN2A was mapped using GluN1/GluN2A chimeras and GluN2A truncation constructs. The extracellular GluN2A domain does not contribute to association with Neto1HA but deletion of the intracellular tail resulted in a loss of Neto‐1HA co‐immunoprecipitation which was paralleled by a loss of association between GluN2A and SAP102. Thus, Neto1 is concluded to be a component of APP/NMDA receptor trafficking complexes.
Cu/Zn‐superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER‐Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER‐Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER‐Golgi transport by over‐expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER‐Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.
Imprinting in chicks is a good model for elucidating the processes underlying neural plasticity changes during juvenile learning. We recently reported that neural activation of a telencephalic region, the core region of the hyperpallium densocellulare (HDCo), was critical for success of visual imprinting, and that N‐Methyl‐D‐aspartic (NMDA) receptors containing the NR2B subunit (NR2B/NR1) in this region were essential for imprinting. Using electrophysiological and multiple‐site optical imaging techniques with acute brain slices, we found that long‐term potentiation (LTP) and enhancement of NR2B/NR1 currents in HDCo neurons were induced in imprinted chicks. Enhancement of NR2B/NR1 currents as well as an increase in surface NR2B expression occurred even following a brief training that was too weak to induce LTP or imprinting behavior. This means that NR2B/NR1 activation is the initial step of learning, well before the activation of alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors which induces LTP. We also showed that knockdown of NR2B/NR1 inhibited imprinting, and inversely, increasing the surface NR2B expression by treatment with a casein kinase 2 inhibitor successfully reduced training time required for imprinting. These results suggest that imprinting stimuli activate post‐synaptic NR2B/NR1 in HDCo cells, increase NR2B/NR1 signaling through up‐regulation of its expression, and induce LTP and memory acquisition.
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.
The 19‐transmembrane, multisubunit γ‐secretase complex generates the amyloid β‐peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β‐amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen‐2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high‐resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen‐2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N‐terminal maltose binding protein tag on Pen‐2 still permits incorporation into the complex and subsequent presenilin‐1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen‐2 and the γ‐secretase complex.
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.
Individuals at risk of developing Alzheimer's disease (AD) often exhibit hippocampal hyperexcitability. A growing body of evidence suggests that perturbations in the glutamatergic tripartite synapse may underlie this hyperexcitability. Here, we used a tau mouse model of AD (rTg(TauP301L)4510) to examine the effects of tau pathology on hippocampal glutamate regulation. We found a 40% increase in hippocampal vesicular glutamate transporter, which packages glutamate into vesicles, and has previously been shown to influence glutamate release, and a 40% decrease in hippocampal glutamate transporter 1, the major glutamate transporter responsible for removing glutamate from the extracellular space. To determine whether these alterations affected glutamate regulation in vivo, we measured tonic glutamate levels, potassium‐evoked glutamate release, and glutamate uptake/clearance in the dentate gyrus, cornu ammonis 3(CA3), and cornu ammonis 1(CA1) regions of the hippocampus. P301L tau expression resulted in a 4‐ and 7‐fold increase in potassium‐evoked glutamate release in the dentate gyrus and CA3, respectively, and significantly decreased glutamate clearance in all three regions. Both release and clearance correlated with memory performance in the hippocampal‐dependent Barnes maze task. Alterations in mice expressing P301L were observed at a time when tau pathology was subtle and before readily detectable neuron loss. These data suggest novel mechanisms by which tau may mediate hyperexcitability.
Drugs of abuse modulate the function and activity of the mesolimbic dopamine circuit. To identify novel mediators of drug‐induced neuroadaptations in the ventral tegmental area (VTA), we performed RNA sequencing analysis on VTA samples from mice administered repeated saline, morphine, or cocaine injections. One gene that was similarly up‐regulated by both drugs was serum‐ and glucocorticoid‐inducible kinase 1 (SGK1). SGK1 activity, as measured by phosphorylation of its substrate N‐myc downstream regulated gene (NDRG), was also increased robustly by chronic drug treatment. Increased NDRG phosphorylation was evident 1 but not 24 h after the last drug injection. SGK1 phosphorylation itself was similarly modulated. To determine the role of increased SGK1 activity on drug‐related behaviors, we over‐expressed constitutively active (CA) SGK1 in the VTA. SGK1‐CA expression reduced locomotor sensitization elicited by repeated cocaine, but surprisingly had the opposite effect and promoted locomotor sensitization to morphine, without affecting the initial locomotor responses to either drug. SGK1‐CA expression did not significantly affect morphine or cocaine conditioned place preference, although there was a trend toward increased conditioned place preference with both drugs. Further characterizing the role of this kinase in drug‐induced changes in VTA may lead to improved understanding of neuroadaptations critical to drug dependence and addiction.
Cholinergic neurons in the CNS are involved in synaptic plasticity and cognition. Both muscarinic and nicotinic acetylcholine receptors (nAChRs) influence plasticity and cognitive function. The mechanism underlying nAChR‐induced plasticity, however, has remained elusive. Here, we demonstrate morphological changes in dendritic spines following activation of α4β2* nAChRs, which are expressed on glutamatergic pre‐synaptic termini of cultured hippocampal neurons. Exposure of the neurons to nicotine resulted in a lateral enlargement of spine heads. This was abolished by dihydro‐β‐erythroidine, an antagonist of α4β2* nAChRs, but not by α‐bungarotoxin, an antagonist of α7 nAChRs. Tetanus toxin or a mixture of 2‐amino‐5‐phosphonovaleric acid and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, antagonists of NMDA‐ and AMPA‐type glutamate receptors, blocked the nicotine‐induced spine remodeling. In addition, nicotine exerted full spine‐enlarging response in the post‐synaptic neuron whose β2 nAChR expression was knocked down. Finally, pre‐treatment with nicotine enhanced the Ca2+‐response of the neurons to glutamate. These data suggest that nicotine influences the activity of glutamatergic neurotransmission through the activation of pre‐synaptic α4β2 nAChRs, resulting in the modulation of spinal architecture and responsiveness. The present findings may represent one of the cellular mechanisms underlying cholinergic tuning of brain function.
In vitro and in vivo studies suggest that the basolateral membrane of choroid plexus cells, which is in contact with blood vessels, is involved in the uptake of the reduced form of vitamin C, ascorbic acid (AA), through the sodium‐vitamin C cotransporter, (SVCT2). Moreover, very low levels of vitamin C were observed in the brains of SVCT2‐null mice. The oxidized form of vitamin C, dehydroascorbic acid (DHA), is incorporated through the facilitative glucose transporters (GLUTs). In this study, the contribution of SVCT2 and GLUT1 to vitamin C uptake in human choroid plexus papilloma (HCPP) cells in culture was examined. Both the functional activity and the kinetic parameters of GLUT1 and SVCT2 in cells isolated from HCPP were observed. Finally, DHA uptake by GLUT1 in choroid plexus cells was assessed in the presence of phorbol‐12‐myristate‐13‐acetate (PMA)‐activated human neutrophils. A marked increase in vitamin C uptake by choroid plexus cells was observed that was associated with superoxide generation and vitamin C oxidation (bystander effect). Thus, vitamin C can be incorporated by epithelial choroid plexus papilloma cells using the basolateral polarization of SVCT2 and GLUT1. This mechanism may be amplified with neutrophil infiltration (inflammation) of choroid plexus tumors.
Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α‐synuclein (α‐Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α‐Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co‐cultures of neurons and astrocytes. We found that oligomeric but not monomeric α‐Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre‐incubation of cells with isotope‐reinforced polyunsaturated fatty acids (D‐PUFAs) completely prevented the effect of oligomeric α‐Syn on lipid peroxidation. Inhibition of lipid peroxidation with D‐PUFAs further protected cells from cell death induced by oligomeric α‐Syn. Thus, lipid peroxidation induced by misfolding of α‐Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease.
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