Neuromuscular and Regenerative Medicine Unit

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Research

The Neuromuscular and Regenerative Medicine Unit (NRMU) is engaged in a range of research activities aimed at developing a stem cell-based therapy for solid tissues, understanding the causes of muscle diseases and ageing, trialing therapies, characterizing novel cytoskeletal structures that we identified in muscle and defining their roles in muscle disease and type II diabetes, and understanding how one gene, discovered by us, GTF2IRD1, can profoundly affect skeletal muscle and yet also play a role in human cognition and behaviour. Our approaches span from bench to bedside.

Projects

1) Novel muscle actin cytoskeleton and glucose homeostasis: implications for Type II diabetes
Skeletal muscle and adipose tissue are key tissues in the management of glucose homeostasis. Defects in glucose uptake in skeletal muscle and fat are major contributing factors in the development of Type II diabetes. We have discovered a new actin-based cytoskeleton in muscle and adipose tissue that when perturbed in transgenic and knock-out mice results in altered glucose uptake. This cytoskeleton is characterised by the presence of a specific isoform of the actin-associated protein tropomyosin, Tm5NM1. Our data indicates that this actin cytoskeleton regulates the movement of the GLUT-4 glucose transporter from an intracellular site into the plasma membrane, a process required for insulin-stimulated glucose uptake. Elucidation of the function of this novel cytoskeletal system will represent a major advance in our understanding of glucose transport in skeletal muscle and fat and potentially provide new targets for treatment of diseases of altered glucose clearance, including Type II diabetes and obesity.



2) GTF2IRD1 – Insights into muscle and the mind
We have identified a novel family of proteins encoded by the gene GTF2IRD1 that are providing new insights into the regulation of important aspects of muscle and brain function.

2a) Skeletal muscle development and adult plasticity
Adult muscle cells or myofibres are inherently plastic since they can convert from one type to another in clinical conditions such as congenital myopathies, nerve injury, in response to specific stimuli such as exercise and during the normal course of ageing. Muscle fibre plasticity is a major feature of skeletal muscle diseases. We have discovered that GTF2IRD1 can influence several aspects of the formation of mature myofibres and subsequent plasticity. Using genetically modified mice we have shown that forced expression of GTF2IRD1 in the muscles of mice can profoundly alter fibre type. We now aim to use these mice to understand the signaling events and transcriptional regulatory networks that underpin muscle fibre plasticity. Forced expression of these proteins appear to provide a means to alter fibre type composition in muscles and potentially reverse adverse changes to fibre type that occur following nerve damage and in muscle disease.


2b) Genes controlling brain function and behaviour
The function(s) of GTF2IRD1 extends beyond skeletal muscle. GTF2IRD1 lies within a block of genes on human chromosome 7q11 commonly deleted in a hemizygous manner in the neurodevelopmental disorder Williams Syndrome (WS). WS is characterised by specific neurological and cognitive defects with a unique personality profile called the Williams Syndrome Cognitive Profile (WSCP). There is much interest in WS because the neurological and cognitive defects reveal a genetic basis for specific aspects of human cognition and behaviour.
Haploinsufficiency of GTF2IRD1 and an evolutionarily related gene - GTF2I - currently constitutes the most likely explanation for the causation of the WSCP. We have made Gtf2ird1 knock-out/LacZ knock-in mouse lines to model the consequences on brain function and to study the molecular and cellular mechanisms that underpin specific aspects of human behaviour.

GTF2IRD1 is a nuclear DNA binding protein. We have identified its mechanism of DNA binding and established the criteria for any potential candidate target genes that may be under its control. In complimentary studies we are examining the functional interactions of the protein utilizing yeast 2-hybrid and biochemical studies that are informed by careful analysis of conserved domains. As well, we are using a range of model organisms from sea squirts, zebrafish to mice, to define its mechanism of function.


3) Muscle stem cells and regeneration
Stem cell based therapies hold promise for the treatment of muscle diseases, such as muscular dystrophy, and conditions resulting in muscle loss and wasting, such as sarcopenia and cachexia. In the field of muscle stem cell therapy, poor survival of transplanted donor cells has been a major hurdle for efficient engraftment. We are exploring a unique method that provides transplanted muscle stem cells with a selective survival advantage which has applications for genetically based muscle diseases and muscle wasting that occurs with cancers.


3a) Pre-clinical model for enhanced muscle stem cell transplantation
The strategy for selective engraftment of muscle stem cells is based on the forced expression of a mutant form of the drug resistance gene methylguanine methyltransferase P140K [MGMT(P140K)], originally developed for bone-marrow transplantation. Donor cells that are genetically modified to express MGMT(P140K) are resistant to the cytotoxic effects of the chemotherapeutic drugs carmustine (BCNU) plus O6benzylguanine (O6BG). Following transplantation, and multiple rounds of chemo-selection, 75% to 100% donor cell engraftment in the host hematopoietic stem cell compartment can be achieved.
We recently applied this strategy to skeletal muscle. We showed that MGMT(P140K) expressing CD34+ve donor stem cells isolated from regenerating skeletal muscles can be transplanted into injured muscles, survive and form muscle fibres, following treatment with BCNU and O6BG (Lee et al. 2009). In the absence of the drugs, donor cells fail to survive. Therefore, this strategy looks promising for the selective survival of donor cells in a solid tissue such as muscle.

We are currently testing the efficacy of this strategy in mouse models of human muscle disease using the dystrophin-null mdx mice, a model for Duchenne muscular dystrophy, and α-sarcoglycan null mice.

3b) Recruitment of muscle stem cells during tissue regeneration
We observed that transplantation of CD34+ MGMT(P140K) donor stem cells into a chemo-ablated muscle bed elicits a significant recruitment of host myogenic progenitor cells. This indicates that the donor cells generate a pro-regenerative signal and we have evidence of systemic involvement. We are employing various methods to induce muscle regeneration together with parabiosis to investigate (i) the signaling mechanisms involved in the activation and/or recruitment of endogenous muscle progenitor/stem cells and (ii) the origins and identity of the cells that are recruited.