Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a

Abstract

MicroRNAs (miRs) are short endogenous RNAs that regulate gene expression by incomplete pairing with messenger RNAs. An increasing number of studies show that mammalian microRNAs play fundamental roles in various aspects of cellular function including differentiation, proliferation, and cell death. Recent findings demonstrating the presence of microRNAs in mature neuronal dendrites suggest their possible involvement in controlling local protein translation and synaptic function. HIV-1 Encephalopathy (HIVE) is a manifestation of HIV-1 infection that often results in neuronal damage and dysfunction. While neurons are rarely, if ever, infected by HIV-1, they are exposed to cytotoxic viral and cellular factors including the HIV-1 transactivating factor Tat. In this study, we show that Tat deregulates expression levels of selected microRNAs, including the neuronal mir-128, in primary cortical neurons. We further show that mir-128a inhibits expression of the pre-synaptic protein SNAP25, whereas the anti-mir-128a partially restores Tat/mir-128a-induced downregulation of SNAP25 expression. Altogether, our data provide a novel mechanism by which HIV-Tat perturbs neuronal activity.

Fig. 1

microRNAs whose expression is modulated (P-value < 0.01) by Tat-treatment in primary neurons. A: The fold change represents microRNA expression level in Tat-treated compared to untreated cells and it is expressed as an average of two experiments shown as Exp 1 and Exp 2. B: Quantitative RT-PCR. The mean fold change represents the amount of microRNA in the Tat-treated sample after normalization with U6 and relative to the untreated sample (detailed in Materials and Methods Section). Minimum and maximum values of the 95% confidence interval are shown.

Fig. 2

Modulation of mir-128a expression by Tat in primary neurons. Quantitative RT-PCR shows a time and dose-dependent expression of mir-128a in Tat-treated neuronal cultures. The diagram represents results from three independent experiments each performed in triplicate. The fold change is calculated over the control (neurons treated with the poly-Arginine, Arg(9), peptide) previous normalization of each sample with the U6 internal control (see also Materials and Methods Section). Standard deviation is shown.

Fig. 3

Inhibitory effect of mir-128a on SNAP25 predicted gene target. Luciferase and Renilla values were determined 24 h (A) or 48 h (B) post-transfection. A: Diagram relative to the inhibitory effect of mir-128a on the control perfect match sequence. B: Inhibitory effect of mir-128a on SNAP25 3′UTR and single mir-128a sites 1 and 2. Relative units represent the ratio between Renilla values and the Luciferase internal control. The experiments were performed in duplicates and repeated at least three times.

Fig. 4

Inhibitory effect of mir-128a on SNAP25 protein. Representative Western blot showing levels of expression of SNAP25 in mouse neuronal progenitors upon transfection of pSIL-mir-128a and controls. Alpha-tubulin was used as loading control. The diagrams on the right show densitometric analysis (average of three experiments) of the SNAP25 protein levels after normalization with alpha-tubulin.

Fig. 5

Tat-induced down regulation ofSNAP25 involves the activity of mir-128a. A: Western blot analysis performed on cellular lysates obtained at 24 and 48 h from Tat-treated (at 100 and 500 nM) rat cortical neurons and control. Grb2 antibody was used to show equal loading of protein lysates. Densitometric analysis relative to SNAP25 expression after treatment with Tat101 was normalized for Grb2 levels and calculated from three independent experiments. B: Western blot analysis performed on neuronal progenitors lysates showing the contribution of mir-128a in the Tat-mediated downregulation of SNAP25. Treatment of Tat-transfected cells with antago-mir-128a partially prevents Tat-induced reduction of SNAP25 levels. Alpha-tubulin was used as loading control. The diagram on the right shows densitometric analysis (average of three independent experiments) of the SNAP25 protein levels after normalization with alpha-tubulin.

Fig. 6

Decreased expression of SNAP25 protein in HIV-Encephalopathy. A: Full montages of the brain from normal and HIVE cases demonstrate that while SNAP25 is present throughout the cortex and white matter in the normal brain, its levels are significantly decreased in both the cortex (arrow) and the white matter (arrowhead). B: Expression of SNAP25 is also significantly reduced in the sub-cortical white matter in an area of inflammation where a giant multinucleated cell can be seen (arrow), and also decreased in the cortex on an HIVE affected area.