A growing body of evidence indicates that valproic acid (VPA), a histone deacetylase inhibitor used to treat epilepsy and mood disorders, has histone deacetylase‐related and ‐unrelated neurotoxic activity, the mechanism of which is still poorly understood. We report that VPA induces neuronal cell death through an atypical calpain‐dependent necroptosis pathway that initiates with downstream activation of c‐Jun N‐terminal kinase 1 (JNK1) and increased expression of receptor‐interacting protein 1 (RIP‐1) and is accompanied by cleavage and mitochondrial release/nuclear translocation of apoptosis‐inducing factor, mitochondrial release of Smac/DIABLO, and inhibition of the anti‐apoptotic protein X‐linked inhibitor of apoptosis (XIAP). Coinciding with apoptosis‐inducing factor nuclear translocation, VPA induces phosphorylation of the necroptosis‐associated histone H2A family member H2AX, which is known to contribute to lethal DNA degradation. These signals are inhibited in neuronal cells that express constitutively activated MEK/ERK and/or PI3‐K/Akt survival pathways, allowing them to resist VPA‐induced cell death. The data indicate that VPA has neurotoxic activity and identify a novel calpain‐dependent necroptosis pathway that includes JNK1 activation and RIP‐1 expression.
Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)‐hydroxycholesterol, 24(S)OH‐C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin‐cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH‐C. Proteoliposomes containing apolipoprotein A‐I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A‐I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH‐C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH‐C esterification. When highly neurotoxic 24(S)OH‐C was treated with enzyme and proteoliposomes before incubation with differentiated SH‐SY5Y cells, the neuron survival improved. The esters of 24(S)OH‐C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH‐C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH‐C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol‐induced neurotoxic injuries to neurons in culture.
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.
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.
Insulin‐like growth factor‐1 (IGF‐1) is a neuroprotective growth factor that promotes neuronal survival by inhibition of apoptosis. To examine whether IGF‐1 exerts cytoprotective effects against extracellular inflammatory stimulation, ventral spinal cord 4.1 (VSC4.1) motoneuron cells were treated with interferon‐gamma (IFN‐γ). Our data demonstrated apoptotic changes, increased calpain:calpastatin and Bax:Bcl‐2 ratios, and expression of apoptosis‐related proteases (caspase‐3 and ‐12) in motoneurons rendered by IFN‐γ in a dose‐dependent manner. Post‐treatment with IGF‐1 attenuated these changes. In addition, IGF‐1 treatment of motoneurons exposed to IFN‐γ decreased expression of inflammatory markers (cyclooxygenase‐2 and nuclear factor‐kappa B:inhibitor of kappa B ratio). Furthermore, IGF‐1 attenuated the loss of expression of IGF‐1 receptors (IGF‐1Rα and IGF‐1Rβ) and estrogen receptors (ERα and ERβ) induced by IFN‐γ. To determine whether the protective effects of IGF‐1 are associated with ERs, ERs antagonist ICI and selective siRNA targeted against ERα and ERβ were used in VSC4.1 motoneurons. Distinctive morphological changes were observed following siRNA knockdown of ERα and ERβ. In particular, apoptotic cell death assessed by TUNEL assay was enhanced in both ERα and ERβ‐silenced VSC4.1 motoneurons following IFN‐γ and IGF‐1 exposure. These results suggest that IGF‐1 protects motoneurons from inflammatory insult by a mechanism involving pivotal interactions with ERα and ERβ.
Expression of a familial Alzheimer's disease (AD)‐linked mutant of amyloid β precursor protein (APP) or the binding of transforming growth factor β2 to wild‐type (wt)‐APP causes neuronal death by activating an intracellular death signal (a APP‐mediated intracellular death signal) in the absence of the involvement of amyloid β (Aβ) toxicity in vitro. These neuronal death models may therefore be regarded as Aβ‐independent neuronal death models related to AD. A recent study has shown that the A673T mutation in the APP isoform APP770, corresponding to the A598T mutation in the most prevalent neuronal APP isoform APP695 (an AD‐protective mutant of APP), is linked to a reduction in the incidence rate of AD. Consistent with this, cells expressing the AD‐protective mutant of APP produce less Aβ than cells expressing wt‐APP. In this study, transforming growth factor β2 caused death in cultured neuronal cells expressing wt‐APP, but not in those expressing the AD‐protective mutant of APP. This result suggests that the AD‐protective mutation of APP reduces the incidence rate of AD by attenuating the APP‐mediated intracellular death signal. In addition, a mutation that causes hereditary cerebral hemorrhage with amyloidosis‐Dutch type also attenuated the APP‐mediated intracellular death signal.
Alzheimer's disease (AD) is a neurodegenerative disorder that represents the most common type of dementia among elderly people. Amyloid beta (Aβ) peptides in extracellular Aβ plaques, produced from the amyloid precursor protein (APP) via sequential processing by β‐ and γ‐secretases, impair hippocampal synaptic plasticity, and cause cognitive dysfunction in AD patients. Here, we report that Aβ peptides also impair another form of synaptic plasticity; cerebellar long‐term depression (LTD). In the cerebellum of commonly used AD mouse model, APPswe/PS1dE9 mice, Aβ plaques were detected from 8 months and profound accumulation of Aβ plaques was observed at 18 months of age. Biochemical analysis revealed relatively high levels of APP protein and Aβ in the cerebellum of APPswe/PS1dE9 mice. At pre‐Aβ accumulation stage, LTD induction, and motor coordination are disturbed. These results indicate that soluble Aβ oligomers disturb LTD induction and cerebellar function in AD mouse model.
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.
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.
The positron emission tomography (PET) ligand 11C‐labeled Pittsburgh compound B (PIB) is used to image β‐amyloid (Aβ) deposits in the brains of living subjects with the intent of detecting early stages of Alzheimer's disease (AD). However, deposits of human‐sequence Aβ in amyloid precursor protein transgenic mice and non‐human primates bind very little PIB. The high stoichiometry of PIB:Aβ binding in human AD suggests that the PIB‐binding site may represent a particularly pathogenic entity and/or report local pathologic conditions. In this study, 3H‐PIB was employed to track purification of the PIB‐binding site in > 90% yield from frontal cortical tissue of autopsy‐diagnosed AD subjects. The purified PIB‐binding site comprises a distinct, highly insoluble subfraction of the Aβ in AD brain with low buoyant density because of the sodium dodecyl sulfate‐resistant association with a limited subset of brain proteins and lipids with physical properties similar to lipid rafts and to a ganglioside:Aβ complex in AD and Down syndrome brain. Both the protein and lipid components are required for PIB binding. Elucidation of human‐specific biological components and pathways will be important in guiding improvement of the animal models for AD and in identifying new potential therapeutic avenues.
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.
Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron‐like cell lines over‐expressing the mitochondrial GSH transporter, the 2‐oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up‐regulation of the anti‐apoptotic protein, B cell lymphoma 2 (Bcl‐2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl‐2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co‐immunoprecipitated with Bcl‐2 from rat brain lysates in a GSH‐dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl‐2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels.
The consumption of ethanol by pregnant women may cause neurological abnormalities, affecting learning and memory processes in children, and are collectively described as fetal alcohol spectrum disorders. However, the molecular mechanisms underlying these changes are still poorly understood. In our previous studies, we found that ethanol treatment of postnatal day 7 (P7) mice significantly enhances the anandamide levels but not the 2‐arachidonylglycerol (2‐AG) levels and induces widespread neurodegeneration, but the reason for the lack of significant effects of ethanol on the 2‐AG level is unknown. In this study, we examined developmental changes in diacylglycerol lipase‐α, β (DAGL‐α and β) and monoacylglycerol lipase (MAGL). We found that the levels of these proteins were significantly higher in adult brains compared to those detected early in brain development. Next, we examined the influence of P7 ethanol treatment on these enzymes, finding that it differentially altered the DAGL‐α protein and mRNA levels but consistently enhanced those of the DAGL‐β. Interestingly, the ethanol treatment enhanced MAGL protein and mRNA levels. Inhibition of MAGL with KML29 failed to induce neurodegeneration in P7 mice. Collectively, these findings suggest that ethanol significantly activates DAGL‐β and MAGL in the neonatal brain, resulting in no net change in 2‐AG levels.
Munc13‐1 is a pre‐synaptic active‐zone protein essential for neurotransmitter release and involved in pre‐synaptic plasticity in brain. Ethanol, butanol, and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13‐1 with EC50s of 52 mM, 26 mM, and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13‐1 C1 exclusively at Glu‐582, which was identified by mass spectrometry. Mutation of Glu‐582 to alanine, leucine, and histidine reduced the alcohol binding two‐ to five‐fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild‐type Munc13‐1 compared with the mutants. If Munc13‐1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss‐of‐function mutation in the conserved Dunc‐13 in Drosophila melanogaster. The Dunc‐13P84200/+ heterozygotes have 50% wild‐type levels of Dunc‐13 mRNA and display a very robust increase in ethanol self‐administration. This phenotype is reversed by the expression of the rat Munc13‐1 protein within the Drosophila nervous system. The present studies indicate that Munc13‐1 C1 has binding site(s) for alcohols and Munc13‐1 activity is sufficient to restore normal self‐administration to Drosophila mutants deficient in Dunc‐13 activity.
An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.
Trafficking of G protein‐coupled receptors plays a crucial role in controlling the precise signalling of the receptor as well as its proper regulation. Metabotropic glutamate receptor 1 (mGluR1), a G protein‐coupled receptor, is a member of the group I mGluR family. mGluR1 plays a critical role in neuronal circuit formation and also in multiple types of synaptic plasticity. This receptor has also been reported to be involved in various neuropsychiatric diseases. Other than the central nervous system, mGluR1 plays crucial roles in various non‐neuronal cells like hepatocytes, skin cells, etc. Although it has been reported that mGluR1 gets endocytosed on ligand application, the events after the internalization of the receptor has not been studied. We show here that mGluR1 internalizes on ligand application. Subsequent to endocytosis, majority of the receptors localize at the recycling compartment and no significant presence of the receptor was noticed in the lysosome. Furthermore, mGluR1 returned to the cell membrane subsequent to ligand‐mediated internalization. We also show here that the recycling of mGluR1 is dependent on the activity of protein phosphatase 2A. Thus, our data suggest that the ligand‐mediated internalized receptors recycle back to the cell surface in protein phosphatase 2A‐dependent manner.
Abnormal autophagy may contribute to neurodegeneration in Parkinson's disease (PD). However, it is largely unknown how autophagy is dysregulated by oxidative stress (OS), one of major pathogenic causes of PD. We recently discovered the potential autophagy regulator gene family including Tnfaip8/Oxi‐α, which is a mammalian target of rapamycin (mTOR) activator down‐regulated by OS in dopaminergic neurons (J. Neurochem., 112, 2010 , 366). Here, we demonstrate that the OS‐induced Tnfaip8 l1/Oxi‐β could increase autophagy by a unique mechanism that increases the stability of tuberous sclerosis complex 2 (TSC2), a critical negative regulator of mTOR. Tnfaip8 l1/Oxi‐β and Tnfaip8/Oxi‐α are the novel regulators of mTOR acting in opposition in dopaminergic (DA) neurons. Specifically, 6‐hydroxydopamine (6‐OHDA) treatment up‐regulated Tnfaip8 l1/Oxi‐β in DA neurons, thus inducing autophagy, while knockdown of Tnfaip8 l1/Oxi‐β prevented significantly activation of autophagic markers by 6‐OHDA. FBXW5 was identified as a novel binding protein for Tnfaip8 l1/Oxi‐β. FBXW5 is a TSC2 binding receptor within CUL4 E3 ligase complex, and it promotes proteasomal degradation of TSC2. Thus, Tnfaip8 l1/Oxi‐β competes with TSC2 to bind FBXW5, increasing TSC2 stability by preventing its ubiquitination. Our data show that the OS‐induced Tnfaip8 l1/Oxi‐β stabilizes TSC2 protein, decreases mTOR phosphorylation, and enhances autophagy. Therefore, altered regulation of Tnfaip8 l1/Oxi‐β may contribute significantly to dysregulated autophagy observed in dopaminergic neurons under pathogenic OS condition.
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.
Radiotherapy is the major treatment modality for primary and metastatic brain tumors which involves the exposure of brain to ionizing radiation. Ionizing radiation can induce various detrimental pathophysiological effects in the adult brain, and Alzheimer's disease and related neurodegenerative disorders are considered to be late effects of radiation. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation in cultured primary neurons similar to that in Alzheimer's disease. We demonstrated that exposure to 0.5 or 2 Gy γ rays causes increased phosphorylation of tau protein at several phosphorylation sites in a time‐ and dose‐dependent manner. Consistently, we also found ionizing radiation causes increased activation of GSK3β, c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase before radiation‐induced increase in tau phosphorylation. Specific inhibitors of these kinases almost fully blocked radiation‐induced tau phosphorylation. Our studies further revealed that oxidative stress plays an important role in ionizing radiation‐induced tau phosphorylation, likely through the activation of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase, but not GSK3β. Overall, our studies suggest that ionizing radiation may cause increased risk for development of Alzheimer's disease by promoting abnormal tau phosphorylation.
Dysregulated metabolism and consequent extracellular accumulation of amyloid‐β (Aβ) peptides in the brain underlie the pathogenesis of Alzheimer's disease. Extracellular Aβ in the brain parenchyma is mainly secreted from the pre‐synaptic terminals of neuronal cells in a synaptic activity‐dependent manner. The p24 family member p24α2 reportedly attenuates Aβ generation by inhibiting γ‐secretase processing of amyloid precursor protein; however, the pattern of expression and localization of p24α2 in the brain remains unknown. We performed immunohistochemical staining and subcellular fractionation for p24α2 in the mouse brain. Immunostaining showed that p24α2 is broadly distributed in the gray matter of the central nervous system and is predominantly localized to synapses. Subcellular fractionation revealed prominent localization of p24α2 in the pre‐synaptic terminals. Immunoisolation of synaptic vesicles (SV) indicated that p24α2 is condensed at active zone‐docked SV. During development, p24α2 expression is highest in the post‐natal period and gradually decreases with age. We also confirmed that amyloid precursor protein and γ‐secretase components are localized at active zone‐docked SV. Our results suggest a novel functional role for p24α2 in the regulation of synaptic transmission and synaptogenesis, and provide evidence for the participation of p24α2 in the regulation of Aβ generation and secretion in the brain.
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