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.

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Systematic kinetic, equilibrium binding, melting, and electrophoretic studies were carried out with oligonucleotides to determine the sequence specificities of chromomycin A3 (CHR) binding to DNA at the self-complementary tetranucleotide level. Decamers of the forms d(GTA-XGCY-TAC) and d(GTA-XCGY-TAC), with X, Y = A, G, C, or T, were used for this purpose. Results indicate that the binding preferences for CHR are in the order -GGCC- > -CGCG- > -GCGC-, -CCGG- > -AGCT- > -ACGT-, -TGCA- > -TCGA-. Detergent-induced drug dissociation studies revealed that CHR dissociates very slowly from both -GGCC- and -CGCG- sequences, with the former being measurably slower than the latter which in turn is at least an order of magnitude slower than the rest of the sequences. Thermal denaturation measurements indicate that the binding of CHR stabilizes the DNA duplex, with -GGCC- and -CGCG- exhibiting the largest effects. Results of gel electrophoretic retardation experiments support our general findings on the relative binding order. Our experimental results support earlier NMR findings by other researchers implicating the preference of aureolic acid drugs at the 5'GC3' step and further reveal significant modulations by the adjacent base pairs. Attempts were made to rationalize our results with the known detailed structural information from NMR studies.

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