A second promoter provides an alternative target for therapeutic up-regulation of utrophin in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5′ end of the gene and have shown it to be synaptically regulated in vivo. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3′ to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.

Impaired metabolic modulation of α-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle

The neuronal isoform of nitric oxide synthase (nNOS) is highly expressed in mammalian skeletal muscle, but its functional role has not been defined. NO has been implicated in the local metabolic regulation of blood flow in contracting skeletal muscle in part by antagonizing sympathetic vasoconstriction. We therefore hypothesized that nNOS in skeletal muscle is the source of the NO mediating the inhibition of sympathetic vasoconstriction in contracting muscle. In the mdx mouse, a model of Duchenne muscular dystrophy in which dystrophin deficiency results in greatly reduced expression of nNOS in skeletal muscle, we found that the normal ability of skeletal muscle contraction to attenuate α-adrenergic vasoconstriction is defective. Similar results were obtained in mutant mice that lack the gene encoding nNOS. Together these data suggest a specific role for nNOS in the local metabolic inhibition of α-adrenergic vasoconstriction in active skeletal muscle.

Myogenic stem cell function is impaired in mice lacking the forkhead/winged helix protein MNF

Myocyte nuclear factor (MNF) is a winged helix transcription factor that is expressed selectively in myogenic stem cells (satellite cells) of adult animals. Using a gene knockout strategy to generate a functional null allele at the Mnf locus, we observed that mice lacking MNF are viable, but severely runted. Skeletal muscles of Mnf−/− animals are atrophic, and satellite cell function is impaired. Muscle regeneration after injury is delayed and incomplete, and the normal timing of expression of cell cycle regulators and myogenic determination genes is dysregulated. Mnf mutant mice were intercrossed with mdx mice that lack dystrophin and exhibit only a subtle myopathic phenotype. In contrast, mdx mice that also lack MNF die in the first few weeks of life with a severe myopathy. Haploinsufficiency at the Mnf locus (Mnf+/−) also exacerbates the mdx phenotype to more closely resemble Duchenne's muscular dystrophy in humans. We conclude that MNF acts to regulate genes that coordinate the proliferation and differentiation of myogenic stem cells after muscle injury. Animals deficient in MNF may prove useful for evaluation of potential therapeutic interventions to promote muscle regeneration for patients having Duchenne's muscular dystrophy.

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase.

Huntington disease stems from a mutation of the protein huntingtin and is characterized by selective loss of discrete neuronal populations in the brain. Despite a massive loss of neurons in the corpus striatum, NO-generating neurons are intact. We recently identified a brain-specific protein that associates with huntingtin and is designated huntingtin-associated protein (HAP1). We now describe selective neuronal localizations of HAP1. In situ hybridization studies reveal a resemblance of HAP1 and neuronal nitric oxide synthase (nNOS) mRNA localizations with dramatic enrichment of both in the pedunculopontine nuclei, the accessory olfactory bulb, and the supraoptic nucleus of the hypothalamus. Both nNOS and HAP1 are enriched in subcellular fractions containing synaptic vesicles. Immunocytochemical studies indicate colocalizations of HAP1 and nNOS in some neurons. The possible relationship of HAP1 and nNOS in the brain is reminiscent of the relationship of dystrophin and nNOS in skeletal muscle and suggests a role of NO in Huntington disease, analogous to its postulated role in Duchenne muscular dystrophy.

G-utrophin, the autosomal homologue of dystrophin Dp116, is expressed in sensory ganglia and brain.

The utrophin gene is closely related to the dystrophin gene in both sequence and genomic structure. The Duchenne muscular dystrophy (DMD) locus encodes three 14-kb dystrophin transcripts in addition to several smaller isoforms, one of which, Dp116, is specific to peripheral nerve. We describe here the corresponding 5.5-kb mRNA from the utrophin locus. This transcript, designated G-utrophin, is of particular interest because it is specifically expressed in the adult mouse brain and appears to be the predominant utrophin transcript in this tissue. G-utrophin is expressed in brain sites generally different from the regions expressing beta-dystroglycan. During mouse embryogenesis G-utrophin is also seen in the developing sensory ganglia. Our data confirm the close evolutionary relationships between the DMD and utrophin loci; however, the functions for the corresponding proteins probably differ.