MicroRNA-27a Promotes Inefficient Lysosomal Clearance in the Hippocampi of Rats Following Chronic Brain Hypoperfusion

Background/Aims: Previous studies have suggested that autophagy is activated in distinct cerebrovascular diseases, including stroke. However, the underlying regulatory mechanism of autophagy under stroke remained elusive. Accumulating evidence indicates that dysfunctions of microRNAs (miRNAs) are involved in the pathological process of stroke. Therefore, this study was taken to identify the effect of microRNA-9a-5p (miR-9a-5p) on autophagy in rats following stroke. Methods: The rat model of focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) surgery; The neurological outcomes were defined by neurological evaluation and infarct volume; The western blotting and immunofluorescence assays were used to detected the protein levels of microtubule-associated protein 1 light chain 3 (LC3) and autophagy related 5 (ATG5); The mRNA level of miR-9a-5p, LC3 and ATG5 were quantified by real-time RT-PCR; The luciferase activities of ATG5 and miR-9a-5p was detected by luciferase assay. Results: We showed here that the level of miR-9a-5p was decreased in the ischemic region of rats after MCAO. Overexpression of miR-9a-5p by miR-9a-5p agomir reduced infarct volume and alleviated neurological deficit. Moreover, we found that autophagy was activated by miR-9a-5p inhibition and inactivated by miR-9a-5p overexpression both in the MCAO rat and in SY-5Y cell lines, and unchanged by miR-masks as indicated by LC3 expression. Furthermore, the protein level of ATG5 was decreased by miR-9a-5p overexpression, but increased by miR-9a-5p inhibition and unchanged by miR-masks transfection. In addition, the luciferase assay data showed that miR-9a-5p suppressed the luciferase activity of 3’UTR of ATG5, whereas the repressive effect was relieved by mutation of binding sites. Conclusion: Our study demonstrated that miR-9a-5p may play a critical role in regulating the process of autophagy through targeting ATG5 expression, and overexpression of miR-9a-5p may be a potential approach in alleviating ischemia injury induced by MCAO.

Section: Discussionmentioning

confidence: 99%

MicroRNA-9a-5p Alleviates Ischemia Injury After Focal Cerebral Ischemia of the Rat by Targeting ATG5-Mediated Autophagy

Wang¹,

Lei²,

Zhang³

et al. 2017

“…The method for preparation was similar to the previous study with minor modifications [3]. The hippocampus samples from each rat were fixed in 3% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) and post-fixed with 1% osmium tetroxide in Sorensen’s phosphate buffer.…”

Section: Methodsmentioning

confidence: 99%

“…CBH has been demonstrated to induce autophagic-lysosomal neuropathology [3], reduced dendritic spine density [4], glial activation [5], inflammatory reactions and white matter damage [6]. …”

Section: Introductionmentioning

confidence: 99%

“…The mechanisms underlying this impairment include β amyloid (Aβ) overproduction, tau hyperphosphorylation [7], oxidative stress impairment [8], inflammation [9], synaptic dysfunction [10] and neuronal loss [11]. Previous studies have identified factors that regulate CBH, which include hypoxia inducible factor-1 (HIF-1) [12], β amyloid precursor protein (β-APP), cyclin dependent kinase 5 (CDK5), glycogen synthase kinase-3β (GSK-3β) [13], mitogen-activated protein kinase (MAPK) [14] and non-protein molecules such as microRNAs [1, 3, 15, 16]. However, the precise molecular mechanisms of CBH are not fully understood.…”

Section: Introductionmentioning

confidence: 99%

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MiR-9 Regulates the Expression of BACE1 in Dementia Induced by Chronic Brain Hypoperfusion in Rats

Xie¹,

Zhao

Zhou

et al. 2017

Background/Aims: MicroRNA-9 (miR-9) plays important roles in nervous system diseases such as glioblastoma and neurodegenerative disorders. However, how miR-9 contributes to dementia requires further study. In this study, we evaluated the role of miR-9 in dementia and the molecular mechanisms underlying its effects. Methods: A rat model of dementia was created by occlusion of the bilateral common carotid artery (2VO) for 8 weeks. Learning and memory were assessed using the Morris Water Maze (MWM). MicroRNA expression profiling was performed according to a protocol provided by LC Sciences, and quantitative real-time PCR (qRT-PCR) was used to detect the level of miR-9. Transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining were used to assess pathological changes in brain tissue. Western blot and immunofluorescence were employed to detect the expression of β-site APP cleaving enzyme 1 (BACE1) and c-AMP response element-binding protein (CREB). Results: Learning and memory were significantly impaired in 2VO rats, and these changes were accompanied by neuronal loss and glial activation in brain tissues. miR-9 was greatly upregulated in both the hippocampus and cortex of rats following 2VO. Knockdown of endogenous miR-9 via lentiviral vector-mediated delivery of its antisense molecule (lenti-pre-AMO-miR-9) reduced the vulnerability to dementia, reversed the increase in BACE1 expression, and ameliorated the reduction in CREB expression triggered by 2VO. BACE1 protein levels were significantly increased, but CREB protein levels were significantly decreased in the presence of miR-9 in cultured neonatal rat neurons (NRNs). AMO-miR-9 rescued the upregulation of BACE1 and downregulation of CREB elicited by miR-9 in rats. Dual luciferase assay experiments showed that overexpression of miR-9 inhibited the expression of CREB by targeting its 3’UTR domain. CREB protein was downregulated by miR-9 overexpression which was reversed by miR-9 inhibition in cultured NRNs. TEM imaging showed that miR-9 caused damage to NRNs, which was reversed by addition of AMO-miR-9. Conclusion: We conclude that miR-9 plays an important role in regulating the process of dementia induced by 2VO in rats by increasing BACE1 expression via downregulation of CREB.

“…In neurons, autophagy is responsible for the homeostatic pathway [6,7], and is also associated with CCH induced by two-vessel occlusion (2VO) surgery. For example, the accumulation of autophagic vacuoles and elevated microtubule-associated protein 1 light chain 3 (LC3) levels were found in the cortex of 2VO rats at 2 weeks after surgery [8]. Liu et al reported that the level of LC3 was significantly increased in both the cortex and hippocampal CA1 area of 2VO rats at 5 weeks after surgery, and GABA receptors reversed the neuronal damage by activating autophagy [9].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of MicroRNA-96 Ameliorates Cognitive Impairment and Inactivation Autophagy Following Chronic Cerebral Hypoperfusion in the Rat

Liu

Wang

et al. 2018

Background/Aims: Chronic cerebral hypoperfusion (CCH) is a high-risk factor for vascular dementia and Alzheimer’s disease. Autophagy plays a critical role in the initiation and progression of CCH. However, the underlying mechanisms remain unclear. In this study, we identified the effect of a microRNA (miR) on autophagy under CCH. Methods: A CCH rat model was established by two-vessel occlusion (2VO). Learning and memory abilities were assessed by the Morris water maze. The protein levels of LC3, beclin-1, and mTOR were detected by western blotting and immunofluorescence assays, miR-96 expression was assessed by real-time PCR, luciferase assays were used to determine the effect of miR-96 on the 3′ untranslated region (UTR) of mTOR, and the number of autophagosomes was examined by electron microscopy. Results: The level of miR-96 was significantly increased in 2VO rats, and inhibition of miR-96 ameliorated the cognitive impairment induced by 2VO. Furthermore, the number of LC3- and beclin-1-positive autophagosomes was increased in 2VO rats, and was decreased after miR-96 antagomir injection. However, the protein level of mTOR was reduced in 2VO rats, and it was down-regulated by miR-96 overexpression and up-regulated by miR-96 inhibition in 2VO rats and primary culture cells. Moreover, the luciferase activity of the 3′-UTR of mTOR was suppressed by miR-96, which was relieved by mutation of the miR-96 binding sites. Conclusion: Our study demonstrated that miR-96 may play a key role in autophagy under CCH by regulating mTOR; therefore, miR-96 may represent a potential therapeutic target for CCH.