Genome-Wide Expression Analysis of a Spinal Muscular Atrophy Model: Towards Discovery of New Drug Targets

Spinal Muscular Atrophy is a recessive genetic disease and affects lower motor neurones and muscle tissue. A single gene is disrupted in SMA: SMN1 activity is abolished but a second copy of the gene (SMN2) provides limited activity. While the SMN protein has been shown to function in the assembly of RNA-protein complexes, it is unclear how the overall reduction in SMN activity specifically results in the neuromuscular phenotypes. Similar to humans, reduced smn activity in the fly causes earliest phenotypes in neuromuscular tissues. To uncover the effects of reduced SMN activity, we have studied gene expression in control and diseased fly tissues using whole genome micro-arrays. A number of gene expression changes are recovered and independently validated. Identified genes show trends in their predicted function: several are consistent with the function of SMN, in addition some uncover novel pathways. This and subsequent genetic analysis in the fly indicates some of the identified genes could be taken for further studies as potential drug targets for SMA and other neuromuscular disorders

MicroRNA Controlled Adenovirus Mediates Anti-Cancer Efficacy without Affecting Endogenous MicroRNA Activity

MicroRNAs are small non-coding RNA molecules that regulate mRNA translation and stability by binding to complementary sequences usually within the 3′ un-translated region (UTR). We have previously shown that the hepatic toxicity caused by wild-type Adenovirus 5 (Ad5WT) in mice can be prevented by incorporating 4 binding sites for the liver-specific microRNA, mir122, into the 3′ UTR of E1A mRNA. This virus, termed Ad5mir122, is a promising virotherapy candidate and causes no obvious liver pathology. Herein we show that Ad5mir122 maintains wild-type lytic activity in cancer cells not expressing mir122 and assess any effects of possible mir122 depletion in host cells. Repeat administration of 2×1010 viral particles of Admir122 to HepG2 tumour bearing mice showed significant anti-cancer efficacy. RT-QPCR showed that E1A mRNA was down-regulated 29-fold in liver when compared to Ad5WT. Western blot for E1A confirmed that all protein variants were knocked down. RT-QPCR for mature mir122 in infected livers showed that quantity of mir122 remained unaffected. Genome wide mRNA microarray profiling of infected livers showed that although the transcript level of >3900 different mRNAs changed more than 2-fold following Ad5WT infection, less than 600 were changed by Ad5mir122. These were then filtered to select mRNAs that were only altered by Ad5mir122 and the remaining 21 mRNAs were compared to predicted mir122 targets. No mir122 target mRNAs were affected by Ad5 mir122. These results demonstrate that the exploitation of microRNA regulation to control virus replication does not necessarily affect the level of the microRNA or the endogenous mRNA targets

Monocytes induce STAT3 activation in human mesenchymal stem cells to promote osteoblast formation

A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair

Foxp2 Regulates Gene Networks Implicated in Neurite Outgrowth in the Developing Brain

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections

MicroRNA mediated knockdown of both E1A mRNA and protein measured by RT-QPCR and western blot after intravenous injection of 2×10¹⁰ vp of Ad5mir122.

<p>A) RT QPCR for the 13S E1A mRNA transcript in the livers of mice 48 hrs after intravenous injection with 2×10¹⁰ vp of Ad5mir122, Ad5WT, Ad5Luc or PBS. Ad5mir122 shows significantly reduced E1A mRNA during liver infection when compared to Ad5WT (N = 3). Statistical analysis was performed using a two tailed student T-Test (*P = <0.05). B) Western blot to confirm that all E1A proteins variants are knocked down. Each lane represents protein extracted from an individual mouse. Control lanes containing liver from mice treated with either an E1A deleted Ad5Luc vector or PBS show no E1A signal. Ad5WT treatment shows 3 clearly defined bands corresponding to proteins produced from the 13S (36 kDa), 12S (26 kDa) and a smaller fainter band which may represent the 11S or the 10S E1A transcript product. Treatment with Admir122 shows significant knockdown in E1A protein levels for all splice variants. The blot was exposed for 1, 5 and 10 minutes with the 5 minute exposure presented here. Molecular weights were calculated against a dual colour molecular weight ladder (Bio-Rad).</p

Pharmacodynamics led dose escalation study to determine the optimal treatment dose with Ad5mir122 and Ad5WT in tumour bearing mice.

<p>A) Serum Alanine Transaminase (ALT) levels from mice receiving Ad5mir122, Ad5WT or PBS. ALT values for each group are shown at days 2, 5 and 8 (n = 3) after the first injection. Data is presented as ALT units per litre using the equation in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016152#s2" target="_blank">materials and methods</a>. B) Luciferase imaging 2 days after the first injection of Ad5mir122 (left panel) or Ad5WT (right panel). C) Luciferase imaging eight days after the first injection. Mice which received a single injection of Ad5WT are shown in the right panel and mice receiving three injections of Ad5mir122 are shown in the left panel. Images are all presented on the same scale. D) Dosage and treatment schedule for Ad5mir122 and Ad5WT. Viral doses are indicated above each line and include 10% of a Luciferase reporter virus (Ad5mir122luc). All mice were treated with bisphosphonate liposomes at day -1.</p

Ad5mir122 shows reduced E1A activity in primary hepatocytes.

<p>Primary human hepatocytes were infected with either Ad5mir122luc or Ad5WTluc at 1 vp/cell. Luciferase levels are shown 3 days post infection (n = 3) as relative light units per mg total protein. Error bars represent standard deviation.</p

The level and activity of mature mir122 <i>in vivo</i> is unaffected by Ad5mir122.

<p>Mice were injected with 2×10¹⁰ vp of either Ad5mir122, Ad5WT, an E1A deleted Ad5luc vector or PBS. Quantification of mir122 mature RNA levels was performed using a Taqman microRNA assay specific for mir122. CT values were corrected against the levels of the microRNA, let7a, as a reference gene using the method published by Pfaffl M <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016152#pone.0016152-Pfaffl1" target="_blank">[25]</a>. A) RT-QPCR for mir122 showing nine superimposed amplification curves (from three mice) in each treatment group, before correction against let7a. Samples were reverse transcribed using equal amounts of total RNA (5 ng) and RT-PCR was performed using equal amounts of cDNA. CT values shown here represent the average of the reactions from three mice plus or minus standard deviation. B) The total number of mRNA changes recorded by genome wide mRNA profiling of extracted murine hepatic RNA. Positive signals are those in which the median mRNA level changed by ≧2-fold from all mice in each group in comparison to median mRNA level in mice treated with PBS (n = 3 for all groups). The total number of genes altered is calculated using the average of the three independent replicates and therefore no error bars are shown. C) Western blot analysis of the mir122 regulated protein Aldolase A in mice treated as above. Liver protein extracts were subjected to a BCA protein assay and equal loading was confirmed by Ponceau stain (data not shown).</p

Ad5mir122 kills mir122 negative cells with Ad5WT potency.

<p>Comparison of Ad5mir122 and Ad5WT in cancer cell lines incubated at a multiplicity of infection of 100 viral particles per cell. The percentage cell survival is shown 6 days post infection (N = 5) using an MTS cell survival assay. Statistical analysis was performed using one way ANOVA (* = p<0.05, NS  =  not significant).</p