The amnesic potential of scopolamine is well manifested through synaptic plasticity gene expression changes and behavioral paradigms of memory impairment. However, the underlying mechanism remains obscure and consequently ideal therapeutic target is lacking. In this context, chromatin‐modifying enzymes, which regulate memory gene expression changes, deserve major attention. Therefore, we analyzed the expression of chromatin‐modifying enzymes and recovery potential of enzyme modulators in scopolamine‐induced amnesia. Scopolamine administration drastically up‐regulated DNA methyltransferases (DNMT1) and HDAC2 expression while CREB‐binding protein (CBP), DNMT3a and DNMT3b remained unaffected. HDAC inhibitor sodium butyrate and DNMT inhibitor Aza‐2′deoxycytidine recovered scopolamine‐impaired hippocampal‐dependent memory consolidation with concomitant increase in the expression of synaptic plasticity genes Brain‐derived neurotrophic factor (BDNF) and Arc and level of histone H3K9 and H3K14 acetylation and decrease in DNA methylation level. Sodium butyrate showed more pronounced effect than Aza‐2′deoxycytidine and their co‐administration did not exhibit synergistic effect on gene expression. Taken together, we showed for the first time that scopolamine‐induced up‐regulation of chromatin‐modifying enzymes, HDAC2 and DNMT1, leads to gene expression changes and consequent decline in memory consolidation. Our findings on the action of scopolamine as an epigenetic modulator can pave a path for ideal therapeutic targets.
The administration of pan histone deacetylase (HDAC) inhibitors reduces ischemic damage to the CNS, both in vitro and in animal models of stroke, via mechanisms which we are beginning to understand. The acetylation of p53 is regulated by Class I HDACs and, because p53 appears to play a role in ischemic pathology, the purpose of this study was to discover, using an in vitro white matter ischemia model and an in vivo cerebral ischemia model, if neuroprotection mediated by HDAC inhibition depended on p53 expression. Optic nerves were excised from wild‐type and p53‐deficient mice, and then subjected to oxygen–glucose deprivation in the presence and absence of a specific inhibitor of Class I HDACs (MS‐275, entinostat) while compound action potentials were recorded. Furthermore, transient focal ischemia was imposed on wild‐type and p53‐deficient mice, which were subsequently treated with MS‐275. Interestingly, and in both scenarios, the beneficial effects of MS‐275 were most pronounced when p53 was absent. These results suggest that modulation of p53 activity is not responsible for MS‐275‐mediated neuroprotection, and further illustrate how HDAC inhibitors variably influence p53 and associated apoptotic pathways.
It has been suggested that propofol can modulate microglial activity and hence may have potential roles against neuroinflammation following brain ischemic insult. However, whether and how propofol can inhibit post‐cardiac arrest brain injury via inhibition of microglia activation remains unclear. A rat model of asphyxia cardiac arrest (CA) was created followed by cardiopulmonary resuscitation. CA induced marked microglial activation in the hippocampal CA1 region, revealed by increased OX42 and P2 class of purinoceptor 7 (P2X7R) expression, as well as p38 MAPK phosphorylation. Morris water maze showed that learning and memory deficits following CA could be inhibited or alleviated by pre‐treatment with the microglial inhibitor minocycline or propofol. Microglial activation was significantly suppressed likely via the P2X7R/p‐p38 pathway by propofol. Moreover, hippocampal neuronal injuries after CA were remarkably attenuated by propofol. In vitro experiment showed that propofol pre‐treatment inhibited ATP‐induced microglial activation and release of tumor necrosis factor‐α and interleukin‐1β. In addition, propofol protected neurons from injury when co‐culturing with ATP‐treated microglia. Our data suggest that propofol pre‐treatment inhibits CA‐induced microglial activation and neuronal injury in the hippocampus and ultimately improves cognitive function.
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.
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 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.
Processing of amyloid precursor protein (APP) into amyloid‐β peptide (Aβ) is crucial for the development of Alzheimer's disease (AD). Because this processing is highly dependent on its intracellular itinerary, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The sorting receptor SorCS1 has been genetically linked to AD, but the underlying molecular mechanisms are poorly understood. We analyze two SorCS1 variants; one, SorCS1c, conveys internalization of surface‐bound ligands whereas the other, SorCS1b, does not. In agreement with previous studies, we demonstrate co‐immunoprecipitation and co‐localization of both SorCS1 variants with APP. Our results suggest that SorCS1c and APP are internalized independently, although they mostly share a common post‐endocytic pathway. We introduce functional Venus‐tagged constructs to study SorCS1b and SorCS1c in living cells. Both variants are transported by fast anterograde axonal transport machinery and about 30% of anterograde APP‐positive transport vesicles contain SorCS1. Co‐expression of SorCS1b caused no change of APP transport kinetics, but SorCS1c reduced the anterograde transport rate of APP and increased the number of APP‐positive stationary vesicles. These data suggest that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles.
The significant consequences of ethanol use during pregnancy are neurobehavioral abnormalities involving hippocampal and neocortex malfunctions that cause learning and memory deficits collectively named fetal alcohol spectrum disorder. However, the molecular mechanisms underlying these abnormalities are still poorly understood and therefore warrant systematic research. Here, we document novel epigenetic abnormalities in the mouse model of fetal alcohol spectrum disorder. Ethanol treatment of P7 mice, which induces activation of caspase 3, impaired DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) levels. Inhibition of caspase 3 activity, before ethanol treatment, rescued DNMT1, DNMT3A proteins as well as DNA methylation levels. Blockade of histone methyltransferase (G9a) activity or cannabinoid receptor type‐1 (CB1R), prior to ethanol treatment, which, respectively, inhibits or prevents activation of caspase 3, rescued the DNMT1 and DNMT3A proteins and DNA methylation. No reduction of DNMT1 and DNMT3A proteins and DNA methylation was found in P7 CB1R null mice, which exhibit no ethanol‐induced activation of caspase 3. Together, these data demonstrate that ethanol‐induced activation of caspase 3 impairs DNA methylation through DNMT1 and DNMT3A in the neonatal mouse brain, and such impairments are absent in CB1R null mice. Epigenetic events mediated by DNA methylation may be one of the essential mechanisms of ethanol teratogenesis.
Sevoflurane is the most widely used anaesthetic administered by inhalation. Exposure to sevoflurane in neonatal mice can induce learning deficits and abnormal social behaviours. MicroRNA (miR)‐27a‐3p, a short, non‐coding RNA that functions as a tumour suppressor, is up‐regulated after inhalation of anaesthetic, and peroxisome proliferator‐activated receptor γ (PPAR‐γ) is one of its target genes. The objective of this study was to investigate how the miR‐27a‐3p–PPAR‐γ interaction affects sevoflurane‐induced neurotoxicity. A luciferase reporter assay was employed to identify the interaction between miR‐27a‐3p and PPAR‐γ. Primary hippocampal neuron cultures prepared from embryonic day 0 C57BL/6 mice were treated with miR‐27a‐3p inhibitor or a PPAR‐γ agonist to determine the effect of miR‐27a‐3p and PPAR‐γ on sevoflurane‐induced cellular damage. Cellular damage was assessed by a flow cytometry assay to detect apoptotic cells, immunofluorescence to detect reactive oxygen species, western blotting to detect NADPH oxidase 1/4 and ELISA to measure inflammatory cytokine levels. In vivo experiments were performed using a sevoflurane‐induced anaesthetic mouse model to analyse the effects of miR‐27a‐3p on neurotoxicity by measuring the number of apoptotic neurons using the Terminal‐deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) method and learning and memory function by employing the Morris water maze test. Our results revealed that PPAR‐γ expression was down‐regulated by miR‐27a‐3p following sevoflurane treatment in hippocampal neurons. Down‐regulation of miR‐27a‐3p expression decreased sevoflurane‐induced hippocampal neuron apoptosis by decreasing inflammation and oxidative stress‐related protein expression through the up‐regulation of PPAR‐γ. In vivo tests further confirmed that inhibition of miR‐27a‐3p expression attenuated sevoflurane‐induced neuronal apoptosis and learning and memory impairment. Our findings suggest that down‐regulation of miR‐27a‐3p expression ameliorated sevoflurane‐induced neurotoxicity and learning and memory impairment through the PPAR‐γ signalling pathway. MicroRNA‐27a‐3p may, therefore, be a potential therapeutic target for preventing or treating sevoflurane‐induced neurotoxicity.
Mutations of parkin are associated with the occurrence of autosomal recessive familial Parkinson's disease (PD). Parkin acts an E3 ubiquitin ligase, which ubiquitinates target proteins and subsequently regulates either their steady‐state levels through the ubiquitin–proteasome system or biochemical properties. In this study, we identify a novel regulatory mechanism of parkin by searching for new regulatory factors. After screening human fetal brain using a yeast two hybrid assay, we found dual‐specificity tyrosine‐(Y)‐phosphorylation‐regulated kinase 1A (Dyrk1A) as a novel binding partner of parkin. We also observed that parkin interacts and co‐localizes with Dyrk1A in mammalian cells. In addition, Dyrk1A directly phosphorylated parkin at Ser‐131, causing the inhibition of its E3 ubiquitin ligase activity. Moreover, Dyrk1A‐mediated phosphorylation reduced the binding affinity of parkin to its ubiquitin‐conjugating E2 enzyme and substrate, which could be the underlying inhibitory mechanism of parkin activity. Furthermore, Dyrk1A‐mediated phosphorylation inhibited the neuroprotective action of parkin against 6‐hydroxydopamine toxicity in dopaminergic SH‐SY5Y cells. These findings suggest that Dyrk1A acts as a novel functional modulator of parkin. Parkin phosphorylation by Dyrk1A suppresses its E3 ubiquitin ligase activity potentially contributing to the pathogenesis of PD under PD‐inducing pathological conditions.
The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6‐specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin‐1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two‐hybrid screening. The interaction was mediated by the C‐terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull‐down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post‐synaptic density and endosomes in dendritic spines. In Neuro‐2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co‐expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6‐mediated neurite formation through the interaction with SNX1.
Because the cholinergic system is down‐regulated in the brain of Alzheimer's disease patients, cognitive deficits in Alzheimer's disease patients are significantly improved by rivastigmine treatment. To address the mechanism underlying rivastigmine‐induced memory improvements, we chronically treated olfactory bulbectomized (OBX) mice with rivastigmine. The chronic rivastigmine treatments for 12–13 days starting at 10 days after OBX operation significantly improved memory‐related behaviors assessed by Y‐maze task, novel object recognition task, passive avoidance task, and Barnes maze task, whereas the single rivastigmine treatment failed to improve the memory. Consistent with the improved memory‐related behaviors, long‐term potentiation in the hippocampal CA1 region was markedly restored by rivastigmine treatments. In immunoblotting analyses, the reductions of calcium/calmodulin‐dependent protein kinase II (CaMKII) autophosphorylation and calcium/calmodulin‐dependent protein kinase IV (CaMKIV) phosphorylation in the CA1 region in OBX mice were significantly restored by rivastigmine treatments. In addition, phosphorylation of AMPAR subunit glutamate receptor 1 (GluA1) (Ser‐831) and cAMP‐responsive element‐binding protein (Ser‐133) as downstream targets of CaMKII and CaMKIV, respectively, in the CA1 region was also significantly restored by chronic rivastigmine treatments. Finally, we confirmed that rivastigmine‐induced improvements of memory‐related behaviors and long‐term potentiation were not obtained in CaMKIIα+/? mice. On the other hand, CaMKIV?/? mice did not exhibit the cognitive impairments. Taken together, the stimulation of CaMKII activity in the hippocampus is essential for rivastigmine‐induced memory improvement in OBX mice.
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.
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.
Characterization of the molecular signaling pathways underlying protein synthesis‐dependent forms of synaptic plasticity, such as late long‐term potentiation (L‐LTP ), can provide insights not only into memory expression/maintenance under physiological conditions but also potential mechanisms associated with the pathogenesis of memory disorders. Here, we report in mice that L‐LTP failure induced by the mammalian (mechanistic) target of rapamycin complex 1 (mTORC 1) inhibitor rapamycin is reversed by brain‐specific genetic deletion of PKR ‐like ER kinase, PERK (PERK KO ), a kinase for eukaryotic initiation factor 2α (eIF 2α). In contrast, genetic removal of general control non‐derepressible‐2, GCN 2 (GCN 2 KO ), another eIF 2α kinase, or treatment of hippocampal slices with the PERK inhibitor GSK 2606414, does not rescue rapamycin‐induced L‐LTP failure, suggesting mechanisms independent of eIF 2α phosphorylation. Moreover, we demonstrate that phosphorylation of eukaryotic elongation factor 2 (eEF 2) is significantly decreased in PERK KO mice but unaltered in GCN 2 KO mice or slices treated with the PERK inhibitor. Reduction in eEF 2 phosphorylation results in increased general protein synthesis, and thus could contribute to the mTORC 1‐independent L‐LTP in PERK KO mice. We further performed experiments on mutant mice with genetic removal of eEF 2K (eEF 2K KO ), the only known kinase for eEF 2, and found that L‐LTP in eEF 2K KO mice is insensitive to rapamycin. These data, for the first time, connect reduction in PERK activity with the regulation of translation elongation in enabling L‐LTP independent of mTORC 1. Thus, our findings indicate previously unrecognized levels of complexity in the regulation of protein synthesis‐dependent synaptic plasticity.
Read the Editorial Highlight for this article on page 119 . Cover Image for this issue: doi: 10.1111/jnc.14185 . 相似文献
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