Spinal muscular atrophy (SMA) is an autosomal recessive disorder in humans which results in the loss of motor neurons. It is caused by reduced levels of the survival motor neuron (SMN) protein as a result of loss or mutation of the SMN1 gene. SMN is encoded by two genes, SMN1 and SMN2, which essentially differ by a single nucleotide in exon 7. As a result, the majority of the transcript from SMN2 lacks exon 7 (SMNDelta7). SMNDelta7 may be toxic and detrimental in SMA, which, if true, could lead to adverse effects with drugs that stimulate expression of SMN2. To determine the role of SMNDelta7 in SMA, we created transgenic mice expressing SMNDelta7 and crossed them onto a severe SMA background. We found that the SMNDelta7 is not detrimental in that it extends survival of SMA mice from 5.2 to 13.3 days. Unlike mice with selective deletion of SMN exon 7 in muscle, these mice with a small amount of full-length SMN (FL-SMN) did not show a dystrophic phenotype. This indicates that low levels of FL-SMN as found in SMA patients and absence of FL-SMN in muscle tissue have different effects and raises the question of the importance of high SMN levels in muscle in the presentation of SMA. SMN and SMNDelta7 can associate with each other and we suggest that this association stabilizes SMNDelta7 protein turnover and ameliorates the SMA phenotype by increasing the amount of oligomeric SMN. The increased survival of the SMNDelta7 SMA mice we report will facilitate testing of therapies and indicates the importance of considering co-complexes of SMN and SMNDelta7 when analyzing SMN function.

Spinal muscular atrophy (SMA) is caused by loss of the survival motor neuron gene (SMN1) and retention of the SMN2 gene. The copy number of SMN2 affects the amount of SMN protein produced and the severity of the SMA phenotype. While loss of mouse Smn is embryonic lethal, two copies of SMN2 prevents this embryonic lethality resulting in a mouse with severe SMA that dies 5 days after birth. Here we show that expression of full-length SMN under the prion promoter (PrP) rescues severe SMA mice. The PrP results in high levels of SMN in neurons at embryonic day 15. Mice homozygous for PrP-SMN with two copies of SMN2 and lacking mouse Smn survive for an average of 210 days and lumbar motor neuron root counts in these mice were normal. Expression of SMN solely in skeletal muscle using the human skeletal actin (HSA) promoter resulted in no improvement of the SMA phenotype or extension of survival. One HSA line displaying nerve expression of SMN did affect the SMA phenotype with mice living for an average of 160 days. Thus, we conclude that expression of full-length SMN in neurons can correct the severe SMA phenotype in mice. Furthermore, a small increase of SMN in neurons has a substantial impact on survival of SMA mice while high SMN levels in mature skeletal muscle alone has no impact.

5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.

Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein. Previously, cultured SMA motor neurons showed reduced growth cone size and axonal length. Furthermore, reduction of SMN in zebrafish resulted in truncation followed by branching of motor neuron axons. In this study, motor neurons labeled with green fluorescent protein (GFP) were examined in SMA mice from embryonic day 10.5 to postnatal day 2. SMA motor axons showed no defect in axonal formation or outgrowth at any stage of development. However, a significant increase in synapses lacking motor axon input was detected in embryonic SMA mice. Therefore, one of the earliest detectable morphological defects in the SMA mice is the loss of synapse occupation by motor axons. This indicates that in severe SMA mice there are no defects in motor axon formation however, we find evidence of denervation in embryogenesis.

(99m)Tc-RAFT-RGD allowed the experimental in vivo molecular imaging of myocardial angiogenesis.

No abstract

Introduction - Myocardial angiogenesis following reperfusion of an infarcted area affects postinfarct remodelling and may therefore impact on patient prognosis. The non-invasive molecular imaging of angiogenesis is thus of potential clinical relevance in this setting. Angiogenic vessels overexpress the α v β 3 integrin. Tc-RAFT-RGD is a novel 99m Tc-labelled tracer composed of four α v β 3 integrin-specific cyclo(RGDfK) sequences tethered on a cyclodecapeptide platform. Hypothesis - We hypothesized that Tc-RAFT-RGD binding to the α v β 3 integrin would allow the molecular imaging of myocardial angiogenesis in vivo. Methods - Myocardial infarction was induced by left anterior descending coronary artery occlusion for 45 min prior to reperfusion in 24 anesthetized male Wistar rats. Fourteen days later, the animals were injected intravenously with Tc-RAFT-RGD (n = 16) or Tc-RAFT-RAD (negative control, n = 8). Tracer uptake was assessed 60 min following injection in 12 and 4 animals injected with Tc-RAFT-RGD and Tc-RAFT-RAD, respectively. In those animals, the hearts were quickly excised for autoradiographic imaging, nitroblue tetrazolium (NBT) infarct staining, CD31 immunostaining of neovessels, and for gamma-well counting of myocardial tracer activity in the infarcted and normal areas. The remaining animals were dedicated to in vivo dual-isotope pinhole SPECT imaging following the simultaneous injection of Tl-201 and Tc-RAFT-RGD (n = 4) or Tc-RAFT-RAD (n = 4). Results - Neovessels were observed in the infarcted area using CD31 immunostaining. The Tc-RAFT-RGD infarcted-to-normal zone activity ratios by gamma-well counting and ex vivo imaging (2.6 ± 0.6 and 3.9 ± 1.0, respectively) were significantly higher than those of Tc-RAFT-RAD (1.7 ± 0.5 and 2.1 ± 0.4, respectively, P< 0.05). Quantification of short-axis images from SPECT acquisitions indicated similar Tl-201 defect magnitudes in animals injected with Tc-RAFT-RGD and Tc-RAFT-RAD (0.3 ± 0.1 and 0.4 ± 0.1, P =NS). The infarcted area was readily visible in vivo with Tc-RAFT-RGD but not with Tc-RAFT-RAD (infarct-to-normal zone activity ratio, 2.8 ± 0.6 and 1.6 ± 0.4, respectively, P< 0.05). Conclusions - Tc-RAFT-RGD allowed the experimental in vivo molecular imaging of myocardial angiogenesis.