Chronic brain hypoperfusion (CBH) is a common clinical feature of Alzheimer's disease and vascular dementia, but the underlying molecular mechanism is unclear. Our previous study reported that the down-regulation of promotes amyloidogenesis via regulation of amyloid precursor protein and b-site amyloid precursor protein cleaving enzyme 1 (BACE1) expression at the post-transcriptional level in CBH rats with bilateral common carotid artery occlusion (2VO). CBH owing to unilateral common carotid artery occlusion (UCCAO) increases tau phosphorylation levels at multiple phosphorylation sites in the brain, but the molecular mechanism is poorly understood. The purpose of this study was to investigate whether miR-195 could both deregulate amyloid metabolism and indirectly deregulate tau phosphorylation in CBH. We observed that 2VO leads to tau hyperphosphorylation at Ser202/Thr205, Ser262, Thr231, and Ser422 and to the conversion from cyclin-dependent kinase 5 (Cdk5)/ p35 to Cdk5/p25 in rat hippocampi. Endogenous miR-195 was knocked down using over-expression of its antisense molecule (pre-AMO-miR-195) via a lentivirus (lenti-pre-AMO-miR-195); this knockdown increased the tau phosphorylation at Ser202/ Thr205, Ser262, Thr231, Ser422, and the Cdk5/p25 activation, but over-expression of miR-195 using lenti-pre-miR-195 decreased the tau phosphorylation and Cdk5/p25 activation. Further in vitro studies demonstrated that miR-195 overexpression prevented tau hyperphosphorylation and Cdk5/p35 activity, which were increased by miR-195 inhibition. A dual luciferase reporter assay showed that miR-195 bound to the Cdk5r1 gene, which encodes p35 protein, in the 3 0 UTR and inhibited p35 expression. We concluded that tau hyperphosphorylation involves the down-regulation of miR-195, which is mediated by Cdk5/p25 activation in 2VO rats. Our findings demonstrated that down-regulation of miR-195 led to increased vulnerability via the regulation of multiple targets.

Chronic brain hypoperfusion (CBH) induces the accumulation of abnormal cellular proteins, accompanied by cognitive decline, and the autophagic-lysosomal system is abnormal in dementia. Whether CBH accounts for autophagic-lysosomal neuropathology remains unknown. Here, we show that CBH significantly increased the number of autophagic vacuoles (AVs) with high LC3-II levels, but decreased SQSTM1 and cathepsin D levels in the hippocampi of rats following bilateral common carotid artery occlusion (2VO) for 2 weeks. Further studies showed that microRNA-27a (Mir27a) was upregulated at 2 weeks compared with the sham group. Additionally, LAMP-2 proteins were downregulated by Mir27a overexpression, upregulated by Mir27a inhibition, and unchanged by binding-site mutations or miR-masks, indicating that lamp-2 is the target of Mir27a. Knockdown of endogenous Mir27a prevented the reduction of LAMP-2 protein expression as well as the accumulation of AVs in the hippocampi of 2VO rats. Overexpression of Mir27a induced, while the knockdown of Mir27a reduced, the accumulation of AVs and the LC3-II level in cultured neonatal rat neurons. The results revealed that CBH in rats at 2 weeks could induce inefficient lysosomal clearance, which is regulated by the Mir27a-mediated downregulation of LAMP-2 protein expression. These findings provide an insight into a novel molecular mechanism of autophagy at the miRNA level.

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