SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre-messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3' splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model

CB₁ receptor stimulation enhances maturation of the <i>in vitro</i> differentiated neurons.

<p>(<b>A</b>) Representative images of the immunofluorescence analysis of β-III tubulin in cells differentiated from NSCs after three days of culture in 1% FBS in the presence or absence of 1 µM ACEA. (<b>B–D</b>) Bar graphs represent quantitative data of morphometric analyses of number (<b>B</b>), length (<b>C</b>) and branching level of neurites (<b>D</b>) in cells treated as described in (<b>A</b>). Data represent the mean ± SD of 4 experiments. Statistical analysis was performed using the paired t-test; the p value is indicated above the individual samples.</p

Reduced activity of ERK1/2 kinases promotes neuronal differentiation of NSCs.

<p>(<b>A</b>) Western blot analysis of the phosphorylation levels of ERK1/2 (p-ERK1/2) and rpS6 (p-rpS6), a downstream target of the mTOR pathway. Cell extracts (30 µg) from NSCs treated with or without ACEA (1 µM) and the CB₁ antagonist AM251 (1 µM) in the presence of 1% FBS for the indicated time were analysed. (<b>B</b>) Representative images of the immunofluorescence analysis of β-III tubulin in cells differentiated from NSCs after three days of culture in 1% FBS in the presence or absence of 10 µM U0126, a selective inhibitor of the ERK1/2 pathway. (<b>C</b>) The bar graph represents quantitative data (expressed in % of β-III tubulin-positive cells) of the immunofluorescence analysis of neuronal differentiation of NSCs cultured in differentiating condition in the presence or absence of 10 µM U0126. (<b>D–F</b>) Bar graphs represent quantitative data of morphometric analyses of the number (<b>D</b>), the length (<b>E</b>), and branching level of neurites (<b>F</b>) in cells treated as described in (<b>C</b>). Data represent the mean ± SD of 6 experiments. Statistical analysis was performed using the paired t-test; the p value is indicated above the individual samples.</p

Description of selected genes modulated by ACEA.

<p>Description of selected genes modulated by ACEA.</p

CB₁ receptor activation during neuronal differentiation of NSCs modulates gene expression.

<p>(<b>A–B</b>) Results of the Neurogenesis and Neural Stem Cell PCR array. List of the genes up-regulated (<b>A</b>) and down-regulated (<b>B</b>) in NSCs undergoing differentiation for 24 hours in the presence of 1% FBS and 1 µM ACEA, obtained from the PCR Array analysis. (<b>C</b>) Validation of the results of the PCR array by qPCR analysis of seven genes (<i>Drd2</i>, <i>Pax5</i>, <i>Slit2</i>, <i>Gdnf</i>, <i>Shh</i>, <i>Dll1</i> and <i>Pax6</i>) selected for their relevance for neuronal differentiation or NSC stemness. Data (mean ± SD of 5 experiments) are expressed as fold increase of ACEA-treated versus control samples, using <i>Gapdh</i> expression as standard control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054271#s2" target="_blank">Materials and Methods</a>). Statistical analysis was performed using the paired t-test; the p value is indicated above the individual samples.</p

Selective activation of CB₁ receptor is sufficient to promote neuronal differentiation of NSCs.

<p>(<b>A</b>) Dose response of the effect of ACEA (0.1–1 µM) on the neuronal differentiation of NSCs <i>in vitro</i>. (<b>B</b>) Quantitative analysis of the effect of 1 µM ACEA on the differentiation of NSCs in neurons (β-III tubulin-positive cells), oligodendrocytes (O4-positive cells), astrocytes (GFAP-positive cells) and progenitor cells (Nestin-positive cells) in control (black bars) and AEA-treated (grey bars) cells. (<b>C</b>) Time course analysis (1–6 days) of the effect of ACEA on the neuronal differentiation of NSCs (expressed as % of β-III tubulin-positive cells). (<b>D</b>) Quantitative analysis of the effect of ACEA on apoptosis of NSCs undergoing differentiation. Apoptosis was measured by immunofluorescence analysis of DNA fragmentation (TUNEL assay) in control (black bars) and AEA-treated (grey bars) cells. Data of all the experiments represent the mean ± SD of at least 3 independent experiments. Statistical analysis was performed using the paired t-test; the p value is indicated above the individual samples and the bars grouped by brackets.</p

Neural stem cells (NSCs) express high levels of CB₁ receptor.

<p>(<b>A</b>) Bright field photographs of murine NSCs forming neurospheres from single cell suspensions after one, two and three days in EGF and b-FGF. (<b>B</b>) Bar graph representing the clonogenic capacity of NSCs at the 2^nd and 10^th passage (n = 6). (<b>C</b>) Immunofluorescence analysis showing NSCs cultured in differentiating conditions (devoid of EGF and b-FGF, and supplemented with 1% FCS) and stained with the neuronal marker β-III tubulin (left panel), the oligodendrocyte marker O4 (middle panel), and co-stained with the astrocyte marker GFAP (red in the right panel) and with the neural progenitor marker Nestin (green in the right panel). Cells positive for both GFAP and Nestin were considered neural progenitors that are differentiating toward astrocytes (<i>i.e.</i> immature astrocytes). (<b>D</b>) qPCR analysis showing the levels of CB₁ and CB₂ receptor transcripts, normalized for levels of the housekeeping gene β-<i>actin</i>. (<b>E</b>) Western blot analysis showing the expression levels of CB₁ and CB₂ receptor proteins in extracts obtained from NSCs, brain and spleen.</p

List of the primers used for qPCR analyses.

<p>List of the primers used for qPCR analyses.</p