miR-196a Enhances Neuronal Morphology through Suppressing RANBP10 to Provide Neuroprotection in Huntington's Disease

Abstract

MicroRNAs (miRNAs) play important roles in several neurobiological processes, including the development and progression of diseases. Previously, we identified that one specific miRNA, miR-196a, provides neuroprotective effects on Huntington's disease (HD), although the detailed mechanism is still unclear. Based on our bioinformatic analyses, we hypothesize miR-196a might offer neuroprotective functions through improving cytoskeletons of brain cells. Here, we show that miR-196a could enhance neuronal morphology, further ameliorating intracellular transport, synaptic plasticity, neuronal activity, and learning and memory abilities. Additionally, we found that miR-196a could suppress the expression of RAN binding protein 10 (RANBP10) through binding to its 3' untranslated region, and higher expression of RANBP10 exacerbates neuronal morphology and intracellular transport. Furthermore, miR-196a enhances neuronal morphology through suppressing RANBP10 and increasing the ability of β-tubulin polymerization. Most importantly, we observed higher expression of RANBP10 in the brains of HD transgenic mice, and higher expression of RANBP10 might exacerbate the pathological aggregates in HD. Taken together, we provide evidence that enhancement of neuronal morphology through RANBP10 is one of the neuroprotective mechanisms for miR-196a. Since miR-196a has also been reported in other neuronal diseases, this study might offer insights with regard to the therapeutic use of miR-196a in other neuronal diseases.

Keywords: Huntington's disease; Neuronal morphology; Neuroprotection.; RANBP10; miR-196a; β-tubulin polymerization.

Figure 1

miR-196a enhances neuronal morphology, intracellular transport, synaptic plasticity, neuronal activity and learning and memory. (A) Primary cortical neurons were transfected with Ds-Red or miR-196a-DsRed, and Quantitation results show increased branches (left) and total neurite length (right) after miR-196a treatment. Primary neurons from three batches were examined. (B) The brains of non-transgenic (NTG) and miR-196a transgenic mice were subjected to Golgi staining, and quantitation results show the increased branches (left) and total neurite length (right) in the brains of miR-196a transgenic mice. Three mice from each group were examined. The number of examined neurons is indicated. (C) N2a cells cotransfected with Ds-Red & APP-YFP or miR-196a-Ds-Red & APP-YFP were used for examining intracellular transport, and quantitated results show the significantly faster velocity of APP-YFP movement during anterograde but not retrograde transport in miR-196a-Ds-Red cells. The number of examined neurons is indicated. (D-J) Brains of miR-196a transgenic mice were used for Western blotting, immunohistochemical staining and electrophysiological examination. Non-transgenic (NTG) and miR-196a transgenic mice were subjected to behavioral tests. (D) Western blotting results show the expression levels of PSD95, Synaptophysin and VAMP1 in miR-196a transgenic and NTG mice. γ-tubulin was used as an internal control. (E) Quantitation results show the increase of VAMP1/γ-tubulin in miR-196a transgenic mice. (F) Immunohistochemical staining shows the expression profiling of c-Fos in hippocampal regions of miR-196a transgenic and NTG mice. The top panel shows lower magnification images, and the bottom panel shows higher magnification of CA2-CA3 regions. (G) Quantitation results show the significant increase in c-Fos+ cells, which carry a higher intensity of c-Fos signal, in miR-196a transgenic mice. (H) Western blotting shows the expression of c-Fos in the brains of wild-type (WT) and miR-196a transgenic mice. γ-tubulin was used as an internal control. (I) Quantitation results from (H) show the significant increase of c-Fos/γ-tubulin expression in miR-196a transgenic mice. (J) fEPSP in CA1 regions was determined at different time points, and the long-term potentiation (LTP) at 40 minutes after high-frequency stimulation (HFS) is also shown. The top two graphs show the representative fEPSP. Black lines indicate traces before HFS, and red lines indicate traces at 40 minutes after HFS. (K) Quantitation results of the T-maze test show a significant difference between the two groups. * indicates a significant difference with P<0.05; ** indicates a significant difference with P <0.01. ***indicates a significant difference with P<0.001.

Figure 2

miR-196a suppresses the expression of RANBP10 through binding to 3'UTR of RANBP10. N2a cells were co-transfected with miR-196a and a reporter construct to determine the direct binding of miR-196a on 3'UTR of RANBP10. N2a cells transfected with miR-196a and the brains of miR-196a transgenic mice were used to determine the suppression of endogenous RANBP10. (A) Wild-type or mutant (mut.) 3'UTRs of RANBP10 was constructed into the 3' end of luciferase gene for the reporter assay. (B) Luciferase reporter assay shows miR-196a binds to 3'UTR of RANBP10 to suppress the expression of luciferase activity compared to those in the miR-196a non-relative control (NC) or mutant 3'UTR of RANBP10 (Mut. RANBP10 3'UTR). N=3. (C) Western blotting shows the expression of RANBP10 after treatment of miR-196a mimics and NC in N2a cells. (D) Quantitation results show the suppression of RANBP10 after treatment of miR-196a mimics in N2a cells. N=8. (E) Western blotting shows the expression of RANBP10 in the brains of miR-196a transgenic and non-transgenic (NTG) mice. (F) Quantitation results show the lower expression of RANBP10 in miR-196a transgenic mice. *indicates a significant difference with P<0.05. **indicates a significant difference with P<0.01.

Figure 3

miR-196a enhances neuronal morphology through suppressing RANBP10 to enhance β-tubulin polymerization. N2a cells were transfected 196a-GFP and RANBP10-dsRed to determine neurite outgrowth in (A) and (B). N2a cells contransfected with DsRed, miR-196a-dsRed or RANBP10-dsRed, cultured at different temperature conditions and subjected to immunostaining using RANBP10 and β-tubulin antibodies in (C)-(G). (A) Overexpression of RANBP10 blocks the function of miR-196a on total neurite outgrowth, not number of branches in N2a cells (B). (C) Representative images show the expression profiles of RANBP10 and β-tubulin in N2a cells transfected with DsRed (top panel) or miR-196a-dsRed (bottom panel). Hoechst (blue) staining shows the location of the nuclei. (D) Quantitative results show the significantly lower expression level of RANBP10 in miR-196a treatment cells. (E) Quantitative results show the significantly higher expression level of β-tubulin in miR-196a treatment cells. (F) Immunostaining results show the expression profiles of β-tubulin (green) in N2a cells transfected with DsRed (left panel), miR-196a-dsRed (middle panel) or RANBP10-dsRed (right panel) at different temperature conditions. Hoechst staining (blue) shows the location of nuclei, and transfected cells are indicated by red fluorescence. (G) Quantitative results show miR-196a significantly enhances repolymerization of β-tubulin when cultured from 37℃ to 4℃, whereas RANBP10 disrupts the neuronal morphology at different temperatures. Three batches of experiments were performed in this study. * indicates a significant difference with P <0.05. *** indicates a significant difference with P <0.001. Different letters on different bars in (e) indicate a significant difference with P <0.05.

Figure 4

RANBP10 exacerbates pathological aggregates in vitro and in vivo in HD. For the in vitro study, N2a cells were cotransfected G84Q with shRANBP10 or RANBP10 for 48 hours, and cell samples were collected for Western blotting (A and B). For the in vivo study, the brains of non-transgenic (NTG), R6/2 HD or D-Tg transgenic mice at 1.5 months of age were subjected to Western blotting. (A) Western blotting shows the expression of mutant HTT using a mEM48 antibody after cotransfection of G84Q with shRANBP10(suppression), sh-lacZ (negative control) or sh-luc (negative control). γ-tubulin was used as an internal control. (B) Western blotting shows the expression of mutant HTT using a mEM48 antibody after cotransfection of G84Q with RANBP10 (overexpression) and FUW (empty vector control). γ-tubulin was used as an internal control. (C) Western blotting shows the expression of mutant HTT in brain tissues of NTG and R6/2 mice. mEM48 and RANBP10 antibodies were used to detect mutant HTT and endogenous RANBP10, respectively. (D) Quantitation results show lower expression of endogenous RANBP10 in NTG mice compared to those of R6/2 mice. (E) Western blotting shows the expression of mutant HTT in brain tissues of D-Tg and R6/2 mice. mEM48, Flag and MAB2166 antibodies were used to detect mutant HTT, exogenous RANBP10 and endogenous mouse HTT, respectively. (F) Quantitation results show higher expression of mutant HTT in separating gel in D-Tg compared to those of R6/2 mice. (G) Quantitation results show higher expression of mutant HTT in stacking gel in D-Tg compared to those of R6/2 mice. The number of examined mice is indicated inside different bars. (H) The brains of D-Tg and R6/2 mice were subjected to Golgi staining, and quantitation results show the decreased total neurite length in the brains of D-Tg transgenic mice. Three mice from each group were examined. The number of examined neurons is indicated. *** indicates a significant difference with P <0.001.