Fig 1: Down-regulation of miR-708 in AS mice brain and identification of Nnat as one of its predicted target. (A) Quantitative RT-PCR analysis of miR-708 from the total RNA extracted from the cortex sample of wild type and AS mice at postnatal day 10 and 60 (P10 and P60). Each sample was tested in triplicate and normalized against RNU6 (internal control). Values are mean ± SD with four animals/group at P10 and six animals/group at P60. *P < 0.001 as compared to respective wild type group. (B) Venn diagram depicting the number of identified target gene predicted by three different algorithms. (C) Sequence of miR-708 and the probable binding site at the 3'-UTR of Nnat that shows 100% resemblance.
Fig 2: Nnat modulates intracellular Ca2+ homeostasis and the phosphorylation of CAMKIIa at Thr286. HT22 hippocampal cells were transiently transfected with Nnat expression plasmids (Nnata and Nnatß) for 24 h and then cells were either subjected to Ca2+ imaging experiment (A) or processed for immunoblotting using antibodies against total and Thr286 phosphorylated form of CaMKIIa (B) followed by quantitation of Thr286 phosphorylated form of CaMKIIa (C). Over-expressed Nnata and Nnatß were detected by V5 antibody. Neuro 2a cells were transfected with Nnat siRNA and 24 h post-transfection, cells were either subjected to Ca2+ imaging (D) or processed for immunoblotting using antibodies against total and Thr286 phosphorylated form of CaMKIIa (E) followed by quantitation of Thr286 phosphorylated form of CaMKIIa (F). About 50–60 randomly selected cells were assessed for measuring fluorescence intensity in Ca2+ imaging experiment and the experiment was repeated twice. Immunoblotting experiment was repeated thrice. Values are mean ± SD. *P < 0.01 compared to respective control group.
Fig 3: Comparative analysis of immunofluorescence staining of Nnat in various brain regions between wild type and AS mice at postnatal day P5 and P60. (A) Typical immunofluorescence staining of Nnat in the cortical and hippocampal region of wild type and AS mice. (B) Double immunofluorescence staining of Nnat and PV in the brain section obtained from 60 days old (P60) wild type and AS mice. Note the selective localization of Nnat in PV neurons (indicated by arrow). Arrowhead points the Nnat stained neuron that is not localized with PV neuron. Brain sections obtained from both wild type and AS mice were kept on the same slide and processed for immunostaining. Scale: 20 µm.
Fig 4: Regulation of Nnat expression by miR-708. (A) The 3'-UTR of Nnat was cloned into a dual luciferase reporter vector and then transfected into neuro 2a cells in the presence of miR-708 mimic or mimic negative control (NC). Twenty four hours after transfection, cells were processed for dual-luciferase assay. (B–D) Neuro 2a cells were transiently transfected with miR-708 mimic, inhibitor and their negative controls separately and 24 h later cells were collected and subjected to either immunoblot or quantitative PCR analysis of Nnat. (B) Representative immunoblot of Nnat along with ß-actin. (C) Quantitative analysis of band intensity of Nnat shown in B using NIH Image analysis software. (D) Quantitative RT-PCR analysis of Nnat mRNA levels. Values are mean ± SD of three independent experiments. *P < 0.001 compared to respective control group.
Fig 5: Primary cultured cortical neurons (E16 + 14DIV) prepared from AS mice embryos demonstrate increased expression of Nnat along with higher basal intracellular Ca2+ and increased phosphorylation of CaMKIIa at Thr286. (A) Double immunofluorescence staining of Nnat and Ube3a in the cortical neuron prepared from wild type and AS embryos. (B) Double immunofluorescence staining of pThr286 CaMKIIa and Ube3a showing increased phosphorylation of CaMKIIa at Thr286 in the cortical neuron cultured from AS mice embryo. At least 20 immunostained neurons from three different embryos in each group were assessed and most Ube3a-deficient neurons showed increased level of Nnat and pCaMKIIa at Thr286. (C) Comparison of intracellular basal Ca2+ level in the primary cortical neuron (E16 + 14 DIV) obtained from wild type and AS embryos using Fluo-4 AM dye. About 50–60 cells prepared from three different embryos in each group were used for evaluation. (D,E) Immunoblot analysis of Ube3a, Nnat and total and Thr286 phosphorylated form of CaMKIIa in the lysate prepared from primary cultured cortical neurons. Nnat level was normalized against ß-actin while pThr286CaMKIIa was normalized against total CaMKIIa. Values are mean ± SD of three independent experiments. *P < 0.05 compared to respective wild type control group.
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