Showing papers by "Kay E. Davies published in 2003"

Pharmacological strategies for muscular dystrophy.

Abstract: Duchenne muscular dystrophy (DMD) is a fatal, genetic disorder whose relentless progression underscores the urgency for developing a cure. Although Duchenne initiated clinical trials roughly 150 years ago, therapies for DMD remain supportive rather than curative. A paradigm shift towards developing rational therapeutic strategies occurred with identification of the DMD gene. Gene- and cell-based therapies designed to replace the missing gene and/or dystrophin protein have achieved varying degrees of success. However, pharmacological strategies not designed to replace dystrophin per se appear promising, and can circumvent many hurdles hampering gene- and cell-based therapy. Here, we will review present pharmacological strategies, in particular those dealing with functional substitution of dystrophin by utrophin and enhancing muscle progenitor commitment by myostatin blockade, with a view toward facilitating drug discovery for DMD.

Neuromuscular defects in a Drosophila survival motor neuron gene mutant

Abstract: Autosomal recessive spinal muscular atrophy (SMA) is linked to mutations in the survival motor neuron (SMN) gene. The SMN protein has been implicated at several levels of mRNA biogenesis and is expressed ubiquitously. Studies in various model organisms have shown that the loss of function of the SMN gene leads to embryonic lethality. The human contains two genes encoding for SMN protein and in patients one of these is disrupted. It is thought the remaining low levels of protein produced by the second SMN gene do not suffice and result in the observed specific loss of lower motor neurons and muscle wasting. The early lethality in the animal mutants has made it difficult to understand why primarily these tissues are affected. We have isolated a Drosophila smn mutant. The fly alleles contain point mutations in smn similar to those found in SMA patients. We find that zygotic smn mutant animals show abnormal motor behavior and that smn gene activity is required in both neurons and muscle to alleviate this phenotype. Physiological experiments on the fly smn mutants show that excitatory post-synaptic currents are reduced while synaptic motor neuron boutons are disorganized, indicating defects at the neuromuscular junction. Clustering of a neurotransmitter receptor subunit in the muscle at the neuromuscular junction is severely reduced. This new Drosophila model for SMA thus proposes a functional role for SMN at the neuromuscular junction in the generation of neuromuscular defects.

Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer.

Abstract: Utrophin is highly homologous and structurally similar to dystrophin, and in gene delivery experiments in mdx mice was able to functionally replace dystrophin. We performed mini-utrophin gene transfer in Golden Retriever dogs with canine muscular dystrophy (CXMD). Unlike the mouse model, the clinicopathological phenotype of CXMD is similar to that of Duchenne muscular dystrophy (DMD). We injected an adenoviral vector expressing a synthetic utrophin into tibialis anterior muscles of newborn dogs affected with CXMD and examined transgene expression by RNA and protein analysis at 10, 30 and 60 days postinjection in cyclosporin-treated and -untreated animals. Immunosuppression by cyclosporin was required to mitigate the immune response to viral and transgene antigens. RT-PCR analysis showed the presence of the exogenous transcript in the muscle of cyclosporin-treated and -untreated animals. The transgenic utrophin was efficiently expressed at the extrajunctional membrane in immunosuppressed dogs and this expression was stable for at least 60 days. We found reduced fibrosis and increased expression of dystrophin-associated proteins (DAPs) in association with muscle areas expressing the utrophin minigene, indicating that mini-utrophin can functionally compensate for lack of dystrophin in injected muscles. For this reason, utrophin transfer to dystrophin-deficient muscle appears as a promising therapeutic approach to DMD.

A Mutation in Af4 Is Predicted to Cause Cerebellar Ataxia and Cataracts in the Robotic Mouse

Abstract: The robotic mouse is an autosomal dominant mutant that arose from a large-scale chemical mutagenesis program. It has a jerky, ataxic gait and develops adult-onset Purkinje cell loss in the cerebellum in a striking region-specific pattern, as well as cataracts. Genetic and physical mapping of the disease locus led to the identification of a missense mutation in a highly conserved region of Af4, a putative transcription factor that has been previously implicated in leukemogenesis. We demonstrate that Af4 is specifically expressed in Purkinje cells, and we hypothesize that the expression of mutant Af4 leads to neurodegeneration. This function was not identified through knock-out studies, highlighting the power of phenotype-driven mutagenesis in the mouse to identify new pathways involved in neurological disease.

Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins.

Abstract: Mouse models for genetic diseases are among the most powerful tools available for developing and testing new treatment strategies. ADAM12 is a disintegrin and metalloprotease, previously demonstrated to significantly alleviate the pathology of mdx mice, a model for Duchenne muscular dystrophy in humans. More specifically ADAM12 appeared to prevent muscle cell necrosis in the mdx mice as evidenced by morphological analysis and by the reduced levels of serum creatine kinase. In the present study we demonstrated that ADAM12 may compensate for the dystrophin deficiency in mdx mice by increasing the expression and redistribution of several components of the muscle cell-adhesion complexes. First, we analyzed transgenic mice that overexpress ADAM12 and found mild myopathic changes and accelerated regeneration following acute injury. We then analyzed changes in gene-expression profiles in mdx/ADAM12 transgenic mice compared with their littermate controls and found only a few genes with an expression change greater than 2-fold between mdx/ADAM12 and mdx. The small changes in gene expression were unexpected, considering the marked improvement of the mdx pathology when ADAM12 is overexpressed, and suggested that significant changes in mdx/ADAM12 muscle might occur post-transcriptionally. Indeed, by immunostaining and immunoblotting we found an approximately 2-fold increase in expression, and distinct extrasynaptic localization, of a7B integrin and utrophin, the functional homolog of dystrophin. The expression of the dystrophin-associated glycoproteins was also increased. In conclusion, these results demonstrate a novel way to alleviate dystrophin deficiency in mice, and may stimulate the development of new approaches to compensate for dystrophin deficiency in animals and humans.

Protein expression changes in spinal muscular atrophy revealed with a novel antibody array technology.

Abstract: Autosomal recessive proximal spinal muscular atrophy (SMA) is a severe neurodegenerative disease of childhood causing weakness and wasting secondary to motor neuron dysfunction. Over 97% of cases are caused by deletions or mutations within the survival motor neuron (SMN) gene. The SMN protein is highly expressed within brain, spinal cord and muscle, and is decreased in SMA patients. It has been shown to have an important role in RNA metabolism, but the reason for the specific motor neuron loss is still unclear. We have used a novel antibody array technology to look for differences in the expression patterns of primary muscle cultures from a type II SMA patient and a normal control. A relatively small number of differences were found within a group of proteins that function as both RNA binding proteins and transcription factors. Interactions between a number of these proteins are well established, and three of them bind in turn to p53 which interacts with SMN. A number of the changes were confirmed with western blot analysis both in the primary muscle cultures and in skeletal muscle samples from SMA patients and controls. Changes at the mRNA level were also confirmed with oligonucleotide arrays. These results suggest that a common transcription pathway may be altered in the disease state, and suggests that down-regulation of transcription factors contributes to SMA pathogenesis.

Identification of a New Pmp22 Mouse Mutant and Trafficking Analysis of a Pmp22 Allelic Series Suggesting Protein Aggregates May Be Protective in Pmp22-Associated Peripheral Neuropathy

Abstract: Microarrays are increasingly used to look at the gene expression profiles of cells or tissues and in this study we have used two separate microarray models to study the disease. Firstly nylon cDNA arrays were used on both a mouse and human model of the disease and then a new proteomic technology, the antibody array was used on the human model for comparison. Dissociated motor neurons were established from the spinal cord of the mouse models and total RNA was hybridised onto a nylon filter containing 24,000 cDNAs and representing over 90% of the mouse genome. Primary muscle cultures were established from patient and normal controls and validated as a model in which to study SMA. Total RNA was hybridised to a human filter also representing the majority of the sequenced human genome. Data was analysed and cluster analysis was performed. A number of expression changes were confirmed with Real Time PCR which occurred in both human and mouse samples. These included SMN itself, p53, a known binding partner and a recently identified splicing factor which has not previously been associated with SMA. Finally a novel technology, the antibody array, was used to compare the protein profiles of SMA and normal primary muscle cultures. This found up regulation of a number of transcription factors within the same pathway of action as p53. These results offer interesting insights into the molecular pathogenesis of SMA.

A study of short utrophin isoforms in mice deficient for full-length utrophin.

Abstract: Utrophin can functionally replace dystrophin in dystrophin-deficient muscle and may have a role in a therapeutic strategy for Duchenne muscular dystrophy. This has resulted in many investigations of the full-length muscle form of utrophin; however, the short utrophins and non-muscle forms have been relatively neglected, partly because they are difficult to analyze in the presence of the full-length form. Our study circumvents this problem by using mice deficient for the full-length form (UKOex6 mice) to study the translation and distribution of short utrophins. Four tissues were examined-kidney, testis, fetal hands/feet, and brain-and three novel short isoforms were identified, including Up120, which appears to be specific to kidney glomeruli, and Up 109, expressed in the fetal dermis. A third form, Up103, was found in testis but at extremely low levels. A cDNA for Up109 has been isolated and shown to have a unique NH2-terminal sequence. In addition, the first exons of Up109 and another short form, G-utrophin, have both been located within intron 55, 56 kb apart. Our immunological studies show that G-utrophin protein accumulates only in neural tissue, in line with its similarly restricted RNA distribution. Our study of testis expression shows, for the first time, that full-length utrophin is expressed at high levels in Leydig cells, raising the possibility that this protein is involved in testosterone secretion. We note that translation of the short utrophins, especially Up140 and Up71, is relatively inefficient and discuss the significance of this observation.

Characterisation of catalytic nucleic acids targeting the survival of motor neuron messenger RNA

Abstract: Catalytic nucleic acids have been used in a reverse genetics approach to study gene function. We are interested in applying this technology to study the autosomal recessive disease Spinal Muscular Atrophy (SMA) which is caused by loss of survival motor neuron gene (SMN) product leading to progressive motor neuron loss and muscular atrophy. Although the SMN gene is ubiquitously expressed, the cause for selective motor neuron loss is unknown. Embryonal lethality in mice has made it extremely difficult to generate animal models of SMA. We describe a procedure for selecting effective DNAzymes (DZ) and ribozymes (RZ) based on their ability to cleave the full length Smn mRNA at low magnesium concentrations, after a short time period, and at a low catalytic nucleic acid to target ratio. Using these criteria three effective RZ and DZ were generated. These results indicate that catalytic nucleic acids can effectively cleave Smn target RNA in an environment closely resembling that present in the cell and thus have potential for interference with Smn gene expression in cells and in vivo.

Chapter 16 Spinal Muscular Atrophy

Abstract: Publisher Summary This chapter discusses the clinical forms of spinal muscular atrophy (SMA), both recessive and dominant, using genetic linkage where it has been identified as the main basis for classification. The molecular pathogenesis of two types of the recessive SMA of childhood is discussed, because these diseases have revealed defects in molecular pathways, which may be of fundamental relevance to motor neurons. The possible overlap between dominant SMA and both hereditary spastic paraparesis (HSP) and hereditary motor and sensory neuropathy (HMSN) is also mentioned. The chapter describes the implications of the disorders for motor neuron biology. Most cases of SMA appear to be due to mutations in a single gene segregating in a dominant, recessive, or X-linked fashion. The SMAs represent a large number of separate single-gene disorders. The elucidation of the molecular pathogenesis will collectively tell about the nature of lower motor neuron (LMN) development and survival.