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.
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.
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.
The effect of psychoactive drugs on depression has usually been studied in cases of prolonged drug addiction and/or withdrawal, without much emphasis on the effects of subchronic or recreational drug use. To address this issue, we exposed laboratory rats to subchronic regimens of heroin or cocaine and tested long‐term effects on (i) depressive‐like behaviors, (ii) brain‐derived neurotrophic factor (BDNF) levels in reward‐related brain regions, and (iii) depressive‐like behavior following an additional chronic mild stress procedure. The long‐term effect of subchronic cocaine exposure was a general reduction in locomotor activity whereas heroin exposure induced a more specific increase in immobility during the forced swim test. Both cocaine and heroin exposure induced alterations in BDNF levels that are similar to those observed in several animal models of depression. Finally, both cocaine and heroin exposure significantly enhanced the anhedonic effect of chronic mild stress. These results suggest that subchronic drug exposure induces depressive‐like behavior which is accompanied by modifications in BDNF expression and increases the vulnerability to develop depressive‐like behavior following chronic stress. Implications for recreational and small‐scale drug users are discussed.
Cocaine is a recreational drug of abuse that binds to the dopamine transporter, preventing reuptake of dopamine into pre‐synaptic terminals. The increased presence of synaptic dopamine results in stimulation of both pre‐ and post‐synaptic dopamine receptors, considered an important mechanism by which cocaine elicits its reinforcing properties. However, the effects of acute cocaine administration on pre‐synaptic dopamine function remain unclear. Non‐invasive imaging techniques such as positron emission tomography have revealed impaired pre‐synaptic dopamine function in chronic cocaine users. Similar impairments have been seen in animal studies, with microdialysis experiments indicating decreased basal dopamine release. Here we use micro positron emission tomography imaging techniques in mice to measure dopamine synthesis capacity and determine the effect of acute cocaine administration of pre‐synaptic dopamine function. We show that a dose of 20 mg/kg cocaine is sufficient to elicit hyperlocomotor activity, peaking 15–20 min post treatment (p < 0.001). However, dopamine synthesis capacity in the striatum was not significantly altered by acute cocaine treatment (: 0.0097 per min vs. 0.0112 per min in vehicle controls, p > 0.05). Furthermore, expression levels of two key enzymes related to dopamine synthesis, tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase, within the striatum of scanned mice were not significantly affected by acute cocaine pre‐treatment (p > 0.05). Our findings suggest that while the regulation of dopamine synthesis and release in the striatum have been shown to change with chronic cocaine use, leading to a reduced basal tone, these adaptations to pre‐synaptic dopaminergic neurons are not initiated following a single exposure to the drug.
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.
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
Parkinson's disease (PD) and diabetes belong to the most common neurodegenerative and metabolic syndromes, respectively. Epidemiological links between these two frequent disorders are controversial. The neuropathological hallmarks of PD are protein aggregates composed of amyloid‐like fibrillar and serine‐129 phosphorylated (pS129) α‐synuclein (AS). To study if diet‐induced obesity could be an environmental risk factor for PD‐related α‐synucleinopathy, transgenic (TG) mice, expressing the human mutant A30P AS in brain neurons, were subjected after weaning to a lifelong high fat diet (HFD). The TG mice became obese and glucose‐intolerant, as did the wild‐type controls. Upon aging, HFD significantly accelerated the onset of the lethal locomotor phenotype. Coinciding with the premature movement phenotype and death, HFD accelerated the age of onset of brainstem α‐synucleinopathy as detected by immunostaining with antibodies against pathology‐associated pS129. Amyloid‐like neuropathology was confirmed by thioflavin S staining. Accelerated onset of neurodegeneration was indicated by Gallyas silver‐positive neuronal dystrophy as well as astrogliosis. Phosphorylation of the activation sites of the pro‐survival signaling intermediate Akt was reduced in younger TG mice after HFD. Thus, diet‐induced obesity may be an environmental risk factor for the development of α‐synucleinopathies. The molecular and cellular mechanisms remain to be further elucidated.
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.
The orphan nuclear receptor estrogen‐related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH‐SY5Y cells. RA induced neurite outgrowth of SH‐SY5Y cells with an increase in DAergic neuron‐like properties, including up‐regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up‐regulated by RA, and participated in RA effect on SH‐SY5Y cells. ERRγ over‐expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA‐induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo‐like kinase 2 was up‐regulated in ERRγ‐over‐expressing SH‐SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation.
Lewy bodies, mainly composed of α‐synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low‐order αS oligomers are neurotoxic and critical species in the pathogenesis of α‐synucleinopathies. To develop disease modifying therapies for α‐synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo‐induced cross‐linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N‐terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long‐term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers.
Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca2+/calmodulin‐dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse‐enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state‐dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca2+/calmodulin binding and activation) loses its affinity for the receptor. Ca2+ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα‐binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca2+‐dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα‐sensitive site. Together, the long intracellular C‐terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5‐dependent Ca2+ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross‐talk between the two receptors.
Recent studies reveal that cocaine experience results in persistent neuroadaptive changes within glutamate (Glu) synapses in brain areas associated with drug reward. However, it remains unclear whether cocaine affects Glu release in drug‐naive animals and how it is altered by drug experience. Using high‐speed amperometry with enzyme‐based and enzyme‐free biosensors in freely moving rats, we show that an initial intravenous cocaine injection at a low self‐administering dose (1 mg/kg) induces rapid, small and transient Glu release in the nucleus accumbens shell (NAc), which with subsequent injections rapidly becomes a much stronger, two‐component increase. Using cocaine‐methiodide, cocaine's analog that does not cross the blood–brain barrier, we confirm that the initial cocaine‐induced Glu release in the NAc has a peripheral neural origin. Unlike cocaine, Glu responses induced by cocaine‐methiodide rapidly habituate following repeated exposure. However, after cocaine experience this drug induces cocaine‐like Glu responses. Hence, the interoceptive actions of cocaine, which essentially precede its direct actions in the brain, play a critical role in experience‐dependent alterations in Glu release, cocaine‐induced neural sensitization and may contribute to cocaine addiction.
Alzheimer β‐amyloid (Aβ) peptides can self‐organize into oligomeric ion channels with high neurotoxicity potential. Cholesterol is believed to play a key role in this process, but the molecular mechanisms linking cholesterol and amyloid channel formation have so far remained elusive. Here, we show that the short Aβ22‐35 peptide, which encompasses the cholesterol‐binding domain of Aβ, induces a specific increase of Ca2+ levels in neural cells. This effect is neither observed in calcium‐free medium nor in cholesterol‐depleted cells, and is inhibited by zinc, a blocker of amyloid channel activity. Double mutations V24G/K28G and N27R/K28R in Aβ22‐35 modify cholesterol binding and abrogate channel formation. Molecular dynamic simulations suggest that cholesterol induces a tilted α‐helical topology of Aβ22‐35. This facilitates the establishment of an inter‐peptide hydrogen bond network involving Asn‐27 and Lys‐28, a key step in the octamerization of Aβ22‐35 which proceeds gradually until the formation of a perfect annular channel in a phosphatidylcholine membrane. Overall, these data give mechanistic insights into the role of cholesterol in amyloid channel formation, opening up new therapeutic options for Alzheimer's disease.
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.
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