Smn induction in adults and embryos from a single injection of tamoxifen.

<p>(<b>A</b>) DNA analysis of adult mice i.p. injected with vehicle (corn oil) or TM (9 mg/40 g body weight) using the same 3-plex PCR reaction as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015887#pone-0015887-g004" target="_blank">Figures 4A and B</a>. Wild type mice (WT;Cre-) only amplified the wild type allele (lane 1). Doubly transgenic mice (<i>Smn^C-T-Neo/WT;Cre^Esr1</i>) in the absence of TM (-TM) displayed a low basal level of <i>pgk-neo</i> excision as has been previously reported for this <i>Cre</i> line <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015887#pone.0015887-Hayashi1" target="_blank">[37]</a>. In the absence of the <i>Cre^Esr1</i> transgene, <i>Smn^C-T-Neo/WT</i> mice injected with TM could not excise <i>pgk-neo</i> (lane 3), in all tissues analyzed, <i>pgk-neo</i> excision was only possible and efficient in the presence of <i>Cre^Esr1</i> and TM (lane 4). (<b>B</b>) PCR analysis of E18.5 embryos that received a single i.p. dose of TM (3 mg/40 g body weight) to the pregnant dam at E7.5 or E13.5 DNA was genotyped as above to differentiate <i>Smn^WT</i>, <i>Smn^C-T-Neo</i> and <i>Smn^C-T</i> alleles. Arrows identify the appropriate amplicons. (<b>C</b>) Photomicrograph of E18.5 embryos induced with TM at E7.5 or E13.5. Lines in photograph show where images were tiled together in Photoshop. (<b>D</b>) Western blot and semi-quantitative densitometry of protein extracted from brain tissue of induced and control E18.5 embryos. A small amount of protein was able to be extracted from severely deformed <i>Smn^{C-T-Neo/C-T-Neo}</i> embryos identified as “escapers” for comparison to induced <i>Smn^{C-T-Neo/C-T-Neo};Cre^Esr1</i> rescued embryos. Semi-quantitative densitometry was performed on a separate blot using the same samples shown and normalized to β-tubulin, without the uninduced mutant. Protein levels from induced homozygous embryos, <i>Smn^{C-T-Neo/C-T-Neo};Cre^ESR1</i>, (0.7±0.10) was greater than <i>Smn^WT/-</i> (0.5±0.2). Abbreviations: (WT) <i>Smn</i> wild type allele, (<i>Cre+</i> and <i>Cre-</i>) presence or absence of <i>Cre^Esr1</i>, (C-T-N/WT) <i>Smn^C-T-Neo/WT</i>, (C-T-N/C-T-N) <i>Smn^{C-T-Neo/C-T-Neo}</i>, (C-T) <i>Smn^C-T</i> allele, (C-T-Neo) <i>Smn^C-T-Neo</i> allele, (TM) tamoxifen.</p

Genotypes of <i>Smn</i> embryos at E18.5 exposed to tamoxifen at E7.5 or E13.5.

<p>(χ², E7.5 injection p = 0.051; E13.5 injection p = 0.84).</p

Generation of mutant <i>Smn</i> alleles.

<p>(<b>A</b>) Gene targeting strategy to introduce the C-T and 2B mutation into <i>Smn</i> exon 7 using the gene targeting vectors pSmnC-T-Neo and pSmn2B-Neo. (<b>B</b>) Southern blot analysis of BamH I and Pst I digested DNA from <i>neo</i> resistant ES cell clones identified homologous recombinants. Two clones from each were used to perform blastocyst injections. (<b>C</b>) Germline transmission of <i>Smn^C-T-Neo</i> and <i>Smn^2B-Neo</i> alleles were determined by direct sequencing of <i>Smn</i> exon 7 PCR products from heterozygous mice. The C-T mutation corresponds to the nucleotide transition within exon 7 of the <i>SMN2</i> gene. The 2B mutation corresponds to a mutation within the splice enhancer region 2B, changing GGA to TTT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015887#pone.0015887-DiDonato2" target="_blank">[32]</a>.</p

Smn expression is efficiently induced from the <i>Smn^C-T-Neo</i> allele <i>in vitro</i>.

<p>Two independent primary MEF cells lines were derived from double transgenic embryos (<i>Smn^C-T-Neo/WT;Cre^Esr1</i>). (<b>A</b>) Schematic of the <i>Smn^C-T-Neo</i> allele from exon 6 to exon 8. Arrows represent forward and reverse primers used in the 3-plex PCR reaction to identify <i>Smn^WT</i> (640 & 637), <i>Smn^C-T-Neo</i> (638 & 637), and <i>Smn^C-T</i> (640 & 637) alleles. Primers 640 and 637 do not amplify a product in the presence of <i>pgk-neo</i> as the amplicon exceeds the time of elongation. (<b>B</b>) 3-plex PCR amplification of DNA from MEF lines 1 and 2 treated for 1 hr with 1 mM tamoxifen (+TM). MEF lines 1 and 2 left untreated (-TM) showed a slight amount of background excision (lanes 2 & 3); however, in the presence of tamoxifen (+TM), they readily amplify the <i>Smn^C-T</i> allele (lanes 4&5). Controls in lanes 6, 7, and 8 were E10.5 embryos harvested to show the indicated genotypes from crosses using germline transmitting <i>Smn^C-T-Neo</i> and <i>Smn^C-T</i> alleles. (<b>C</b>) RNA from untreated (-TM) and induced (+TM) MEF cells were amplified by RT-PCR and <i>FL-Smn</i> transcripts directly sequenced. Induced MEFs (+TM) produced enough <i>FL-Smn</i> transcripts from the mutant C-T allele that could be detected by direct sequencing. The arrow points out the C-T mutation in +TM treated cultures. (<b>D</b>) qRT-PCR of <i>FL-Smn</i> and <i>Δ7Smn</i> from uninduced (-TM) and induced (+TM) cultures. Abbreviations: (TM) tamoxifen (C-T-N/WT;<i>Cre</i>+) <i>Smn^C-T-Neo/+;Cre^Esr1</i> (C-T-N/WT) <i>Smn^C-T-Neo/+</i> (C-T/WT) <i>Smn^C-T/+</i> (C-T-N/C-T) <i>Smn^C-T-Neo/C-T</i>.</p

Whole mount analysis of embryos.

<p><i>Smn^2B-Neo</i> or <i>Smn^C-T-Neo</i> heterozygotes were intercrossed and embryos obtained at either E9.5 or E12.5 for whole-mount analysis and genotyping. (<b><i>a–h</i></b>) E9.5 <i>Smn^C-T-Neo</i> embryos. Heterozygotes (C-T-N/WT) are identical to wild type (WT/WT) littermates. Homozygotes (C-T-N/C-T-N) are small but alive and larger than the <i>Smn^{2B-Neo/2B-Neo}</i> (2B-N/2B-N) homozygotes. (<b><i>i–p</i></b>) E12.5 <i>Smn^C-T-Neo</i> embryos. Homozygotes are extremely small compared to controls and many are being reabsorbed as shown in panel (p). (<b>a'–h'</b>) E9.5 <i>Smn^2B-Neo</i> embryos. Heterozygotes (2B-N/WT) are identical to wild type (WT/WT) littermates. Homozygotes (2B-N/2B-N) are developmentally retarded though still alive with signs of lethality clearly present before this period in some embryos that did not allow for genotyping (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015887#pone-0015887-t001" target="_blank">Table 1</a>). (<b><i>i'–p'</i></b>) E12.5 <i>Smn^2B-Neo</i> embryos. All homozygous mutant embryos are undergoing resorption. Insets in o' and p' are magnified images of embryos in panel. Scale for all E9.5 embryos is 100 µM and for E12.5 200 µM.</p

Genotype of <i>Smn^C-T-Neo</i> and <i>Smn^2B-Neo</i> intercross progeny.

<p>(+) denotes wild type, (−) denotes either C-T-Neo or 2B-Neo.</p

In vitro and in vivo effects of 2,4 diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS: Context-specific modulation of SMN transcript levels

<div><p>C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as <i>SMN2</i> transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on <i>SMN2</i> are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells <i>in vitro</i> utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of <i>Smn</i> transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient <i>SMN2</i> mRNA increases with either no change or a decrease in <i>SMNΔ7</i> and no change in <i>SMN1</i> transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings <i>in vitro</i>, <i>Smn</i> transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects <i>in vitro</i> and <i>in vivo</i>, but these are complex, context specific, and not the result of simple <i>SMN2</i> transcriptional activation.</p></div

Effect of DAQ-DcpSi treatment on <i>Smn</i> transcript levels in tissues from 2B/- SMA mice and healthy 2B/+ littermate controls.

<p>Animals were dosed with either vehicle, RG3039 (6 mg/kg) or PF-06738066 (10 mg/kg) BID via intraperitoneal injection from P4-P16 and were sacrificed 12 hours following the last dose for collection of tissues. RNA was prepared and analyzed using ddPCR as described in materials and methods. All gene expression was normalized to <i>PSMD14</i> expression and expressed relative to that in vehicle-treated 2B/- mice. All data shown as mean ± s.e.m. Numbers of animals in each data set were: 2B/- Vehicle (22); 2B/- RG3039(9); 2B/- PF-06738066 (13); 2B/+ Vehicle (7); 2B/+ RG3039(9); 2B/+ PF-06738066 (10). Significance using Student’s <i>t</i>-test: <i>P</i><0.05 (*), <i>P</i><0.01(**), <i>P</i><0.001(***), <i>P</i><0.0001(****).</p

Effect of DAQ-DcpSi on the performance of 2B/- SMA mice in a combined 55° negative geotaxis/ climb test.

<p>Data presented represent combined sexes for RG3039 (<i>n</i> = 20); vehicle (<i>n</i> = 21), PF-06738066 (<i>n</i> = 21) and vehicle treated healthy 2B/+ littermates (<i>n</i> = 24). The percent of mice able to pass the 55° negative geotaxis and climb tests simultaneously is shown. At P16, while a predominant number of vehicle treated SMA were alive, PF-06738066 or RG3039 showed improvement over vehicle SMA mice that did not reach statistical significance (Fisher’s exact test, <i>P</i>-value = 0.0708; 43% power to detect a benefit amongst SMA groups).</p

Effect of RG3039 on cellular SMN protein levels determined by Western blot.

<p>A. Neural progenitor cells treated for 48 hours (example) and B. quantification of data from 4 independent experiments; C. Fibroblasts treated for 72 hours (example) and D. quantification of data from 3 independent experiments; E. Fibroblasts treated for 6 days (example); F. SMA lymphoblasts treated for 72 hours (example). DMSO final concentration for lymphoblast cells was 0.1% and 0.2% for neural progenitors and fibroblast cell lines. All aggregate data is expressed as mean ± s.e.m. and statistical significance vs. SMA DMSO (B.) or 3813 DMSO controls (D) was assessed using Student’s <i>t</i>-test: <i>P</i><0.05 (*), <i>P</i><0.01(**) or otherwise non-significant where not indicated.</p