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.
Vagus nerve stimulation (VNS) exerts neuroprotective effects against cerebral ischemia/reperfusion (I/R) injury and modulates redox status, potentially through the activity of miR‐210, an important microRNA that is regulated by hypoxia‐inducible factor and Akt‐dependent pathways. The aim of this study was to determine the mechanisms of VNS‐ and miR‐210‐mediated hypoxic tolerance. Male Sprague–Dawley rats were preconditioned with a miR‐210 antagomir (A) or with an antagomir control (AC), followed by middle cerebral artery occlusion and VNS treatment. The animals were divided into eight groups: sham I/R, I/R, I/R+AC, I/R+A, sham I/R+VNS, I/R+VNS, I/R+VNS+AC, and I/R+VNS+A. Activation of the endogenous cholinergic a7 nicotinic acetylcholine receptor (a7nAchR) pathway was identified using double immunofluorescence staining. miR‐210 expression was measured by PCR. Behavioral outcomes, infarct volume, and neuronal apoptosis were observed at 24 h following reperfusion. Markers of oxidative stress were detected using ELISA. Rats treated with VNS showed increased miR‐210 expression as well as decreased apoptosis and antioxidant stress responses compared with the I/R group; these rats also showed increased p‐Akt protein expression and significantly decreased levels of cleaved caspase 3 in the ischemic penumbra, as measured by western blot and immunofluorescence analyses, respectively. Strikingly, the beneficial effects of VNS were attenuated following miR‐210 knockdown. In conclusion, our results indicate that miR‐210 is a potential mediator of VNS‐induced neuroprotection against I/R injury. Our study highlights the neuroprotective potential of VNS, which, to date, has been largely unexplored.
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.
In this report, we describe the localization of diacylglycerol lipase‐α (DAGLα) in nuclei from adult cortical neurons, as assessed by double‐immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double‐labeling assays using the anti‐DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα‐signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC‐35‐ and NeuN‐signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C‐β (PLCβ) family, PLCβ1, PLCβ2, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC‐35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of 2‐arachidonoylglycerol (2‐AG) by liquid chromatography and mass spectrometry (LC‐MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for 2‐AG production, and its PLCβ/DAGLα‐dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for 2‐AG locally produced within the neuronal nucleus.
Temozolomide (TMZ) has been widely used in the treatment of glioblastoma (GBM), although inherent or acquired resistance restricts the application. This study was aimed to evaluate the efficacy of sulforaphane (SFN) to TMZ‐induced apoptosis in GBM cells and the potential mechanism. Biochemical assays and subcutaneous tumor establishment were used to characterize the function of SFN in TMZ‐induced apoptosis. Our results revealed that β‐catenin and miR‐21 were concordantly expressed in GBM cell lines, and SFN significantly reduced miR‐21 expression through inhibiting the Wnt/β‐catenin/TCF4 pathway. Furthermore, down‐regulation of miR‐21 enhanced the pro‐apoptotic efficacy of TMZ in GBM cells. Finally, we observed that SFN strengthened TMZ‐mediated apoptosis in a miR‐21‐dependent manner. In conclusion, SFN effectively enhances TMZ‐induced apoptosis by inhibiting miR‐21 via Wnt/β‐catenin signaling in GBM cells. These findings support the use of SFN for potential therapeutic approach to overcome TMZ resistance in GBM treatment.
The exact effect of glycine pre‐treatment on brain ischemic tolerance (IT) remains quite controversial. The objective of this study was to investigate the potential effects of glycine on IT. We used rat models of both in vitro ischemia (oxygen and glucose deprivation) and in vivo ischemia (transient middle cerebral artery occlusion). Low doses of glycine (L‐Gly) significantly decreased hippocampal ischemic LTP (i‐LTP), infarct volume, and neurological deficit scores which were administered before ischemia was induced in rats, whereas high doses of glycine exerted deteriorative effects under the same condition. These findings suggested that exogenous glycine may induce IT in a dose‐dependent manner. Furthermore, L‐Gly‐dependent neuronal protection was inversed by L689, a selective NMDAR glycine site antagonist both in vitro (abolished i‐LTP depression) and in vivo (increased infarct size reduction), but not glycine receptor (GlyR) inhibitor strychnine. Importantly, L‐Gly‐induced IT was achieved by NR2A‐dependent cAMP‐response element binding protein phosphorylation. These data imply that glycine pre‐treatment may represent a novel strategy for inducing IT based on synaptic NMDAR‐dependent neuronal transmission.
The gene encoding leucine‐rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon‐γ (IFN‐γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN‐γ‐mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal‐regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN‐γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP‐1 cells and human peripheral blood monocytes stimulated the ERK5‐LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5‐dependent IFN‐γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages.
Methyl‐β‐cyclodextrin (MβCD) is a reagent that depletes cholesterol and disrupts lipid rafts, a type of cholesterol‐enriched cell membrane microdomain. Lipid rafts are essential for neuronal functions such as synaptic transmission and plasticity, which are sensitive to even low doses of MβCD. However, how MβCD changes synaptic function, such as N‐methyl‐d ‐aspartate receptor (NMDA‐R) activity, remains unclear. We monitored changes in synaptic transmission and plasticity after disrupting lipid rafts with MβCD. At low concentrations (0.5 mg/mL), MβCD decreased basal synaptic transmission and miniature excitatory post‐synaptic current without changing NMDA‐R‐mediated synaptic transmission and the paired‐pulse facilitation ratio. Interestingly, low doses of MβCD failed to deplete cholesterol or affect α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA‐R) and NMDA‐R levels, while clearly reducing GluA1 levels selectively in the synaptosomal fraction. Low doses of MβCD decreased the inhibitory effects of NASPM, an inhibitor for GluA2‐lacking AMPA‐R. MβCD successfully decreased NMDA‐R‐mediated long‐term potentiation but did not affect the formation of either NMDA‐R‐mediated or group I metabotropic glutamate receptor‐dependent long‐term depression. MβCD inhibited de‐depression without affecting de‐potentiation. These results suggest that MβCD regulates GluA1‐dependent synaptic potentiation but not synaptic depression in a cholesterol‐independent manner.
We explored the interplay between the intracellular energy sensor AMP‐activated protein kinase (AMPK), extracellular signal‐regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)‐induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells. PMA‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (LC3)‐I to autophagosome‐bound LC3‐II, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference‐mediated silencing of AMPK suppressed PMA‐induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA‐induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA‐induced differentiation of SH‐SY5Y cells. Therefore, PMA‐induced neuronal differentiation of SH‐SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response.
Previous studies have shown that fastigial nucleus stimulation (FNS) reduces tissue damage resulting from focal cerebral ischemia. Although the mechanisms of neuroprotection induced by FNS are not entirely understood, important data have been presented in the past two decades. MicroRNAs (miRNAs) are a newly discovered group of non‐coding small RNA molecules that negatively regulate target gene expression and are involved in the regulation of cell proliferation and cell apoptosis. To date, no studies have demonstrated whether miRNAs can serve as mediators of the brain's response to FNS, which leads to endogenous neuroprotection. Therefore, this study investigated the profiles of FNS‐mediated miRNAs. Using a combination of deep sequencing and microarray with computational analysis, we identified a novel miRNA in the rat ischemic cortex after 1 h of FNS. This novel miRNA (PC‐3p‐3469_406), herein referred to as rno‐miR‐676‐1, was upregulated in rats with cerebral ischemia after FNS. In vivo observations indicate that this novel miRNA may have antiapoptotic effects and contribute to neuroprotection induced by FNS. Our study provides a better understanding of neuroprotection induced by FNS.
Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Celastrol, a plant‐derived triterpene, has shown neuroprotective effects in various disease models. However, little is known regarding the effect of celastrol on Cd‐induced neurotoxicity. Here, we show that celastrol protected against Cd‐induced apoptotic cell death in neuronal cells. This is supported by the findings that celastrol strikingly attenuated Cd‐induced viability reduction, morphological change, nuclear fragmentation, and condensation, as well as activation of caspase‐3 in neuronal cells. Concurrently, celastrol remarkably blocked Cd‐induced phosphorylation of c‐Jun N‐terminal kinase (JNK), but not extracellular signal‐regulated kinases 1/2 and p38, in neuronal cells. Inhibition of JNK by SP600125 or over‐expression of dominant negative c‐Jun potentiated celastrol protection against Cd‐induced cell death. Furthermore, pre‐treatment with celastrol prevented Cd down‐regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and activation of phosphoinositide 3′‐kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling in neuronal cells. Over‐expression of wild‐type PTEN enhanced celastrol inhibition of Cd‐activated Akt/mTOR signaling and cell death in neuronal cells. The findings indicate that celastrol prevents Cd‐induced neuronal cell death via targeting JNK and PTEN‐Akt/mTOR network. Our results strongly suggest that celastrol may be exploited for the prevention of Cd‐induced neurodegenerative disorders.
Suppressor of cytokine signaling‐2 (SOCS2) is a regulator of intracellular responses to growth factors and cytokines. Cultured dorsal root ganglia neurons from neonatal mice with increased or decreased SOCS2 expression were examined for altered responsiveness to nerve growth factor (NGF). In the presence of NGF, SOCS2 over‐expression increased neurite length and complexity, whereas loss of SOCS2 reduced neurite outgrowth. Neither loss nor gain of SOCS2 expression altered the relative survival of these cells, suggesting that SOCS2 can discriminate between the differentiation and survival responses to NGF. Interaction studies in 293T cells revealed that SOCS2 immunoprecipitates with TrkA and a juxtamembrane motif of TrkA was required for this interaction. SOCS2 also immunoprecipitated with endogenous TrkA in PC12 Tet‐On cells. Over‐expression of SOCS2 in PC12 Tet‐On cells increased total and surface TrkA expression. In contrast, dorsal root ganglion neurons which over‐expressed SOCS2 did not exhibit significant changes in total levels but an increase in surface TrkA was noted. SOCS2‐induced neurite outgrowth in PC12 Tet‐On cells correlated with increased and prolonged activation of pAKT and pErk1/2 and required an intact SOCS2 SH2 domain and SOCS box domain. This study highlights a novel role for SOCS2 in the regulation of TrkA signaling and biology.
X‐linked Adrenoleukodystrophy (X‐ALD), an inherited peroxisomal metabolic neurodegenerative disorder, is caused by mutations/deletions in the ATP‐binding cassette transporter (ABCD1) gene encoding peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). Metabolic dysfunction in X‐ALD is characterized by the accumulation of very long chain fatty acids ≥ C22:0) in the tissues and plasma of patients. Here, we investigated the mitochondrial status following deletion of ABCD1 in B12 oligodendrocytes and U87 astrocytes. This study provides evidence that silencing of peroxisomal protein ABCD1 produces structural and functional perturbations in mitochondria. Activities of electron transport chain‐related enzymes and of citric acid cycle (TCA cycle) were reduced; mitochondrial redox status was dysregulated and the mitochondrial membrane potential was disrupted following ABCD1 silencing. A greater reduction in ATP levels and citrate synthase activities was observed in oligodendrocytes as compared to astrocytes. Furthermore, most of the mitochondrial perturbations induced by ABCD1 silencing were corrected by treating cells with suberoylanilide hydroxamic acid, an Histone deacetylase inhibitor. These observations indicate a novel relationship between peroxisomes and mitochondria in cellular homeostasis and the importance of intact peroxisomes in relation to mitochondrial integrity and function in the cell types that participate in the pathobiology of X‐ALD. These observations suggest suberoylanilide hydroxamic acid as a potential therapy for X‐ALD.
Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose‐ and time‐dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK‐Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation‐dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat‐shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant‐induced activation of the MEK‐Erk pathway. These findings provide important insights into the mechanisms of activity‐driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation‐mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure.
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