2. Integrin beta 1 and CNS myelination

Multiple sclerosis (MS) is an inflammatory demyelinating disease that affects over 2,500,000 people around the world during the prime of their life. MS is an autoimmune disease triggered either by a virus, genetic susceptibility or the environment. Normally, axons in the central nervous system (CNS) are surrounded by a myelin sheath, which allows for proper electrical conductance in nerves. However, individuals who suffer from MS have areas within the CNS where the myelin sheath is demyelinated and the majority of patients are unable to reform the myelin that is lost. Understanding the process of myelination and remyelination will lead to advances for clinically inducing remyelination and improving the symptoms of MS patients. Recent work has highlighted the importance of various molecules such as integrin ´ß1 and integrin-linked kinase (ILK) in the process of myelination. Our research aims are to provide further understanding of integrin ´ß1 and ILK, and their role in myelination and remyelination in vivo. Therefore, we have generated transgenic mice expressing a dominant-negative integrin ´ß1 lacking the cytoplasmic tail in an oligodendrocyte (OL)-specific manner. As well, we have generated a conditional knock-out of ILK for depletion specifically in OLs. Currently, the dominant-negative integrin ´ß1 mouse model has allowed us to demonstrate the importance of integrin ´ß1 in myelination and remyelination. Further analysis of this model will give us a better indication of the role of integrin ´ß1 in OL development. In combination with the ILK mouse model, we will expand our knowledge of how these two proteins impact on the process of myelination and remyelination, an important prelude to the design of effective treatments for MS.

3. Integrin-linked kinase (ILK)/Kinomics/Computational Biology

Unusual features in the amino-acid sequence of ILK have led us to believe it possesses a novel catalytic mechanism and we are currently solving its crystal structure to determine if this is so. This would be a valuable finding as it would distinguish ILK from the over 500 other human protein kinases, making it a suitable therapeutic target for drug design. Our desire to fully elucidate the function of ILK in oligodendrocytes has led us into the field of kinomics, a field concerned with developing and applying methods to study the protein-kinase complement of the human genome. In the lab we have developed an in vitro assay that allows for the identification of kinase-substrates in a cell-type specific manner. Computationally we have used shape superimposition techniques to design an algorithm for structure alignment, through which conservation and variability in protein kinases can be accurately studied. This algorithm has allowed us to discover a collection of conserved water molecules that play important roles in stabilizing the protein-kinase domain. These studies on ILK and the kinome are motivated by our desire to better understand the role of the integrin-signalling pathway in oligodendrocyte behaviour but will ultimately be of much wider applicability.

Figure. Conserved water molecules and active-site connectivity are shown in the death-associated protein kinase.

4. Pathogenesis and therapeutics of Spinal Muscular Atrophy (SMA)
Spinal muscular atrophy (SMA) is the second most common autosomal recessive childhood disease after cystic fibrosis. This lethal neuropathy affects 1/10,000 live born children. It is characterized by degeneration of the alpha-motor neurons in the spinal cord, which causes proximal, symmetrical limb and trunk muscle weakness that progresses to paralysis and ultimately death. SMA is a clinically heterogeneous disease and has been classified into three groups (types I to III) based on the decreasing severity of symptoms and age of onset. Type I SMA is the most severe form and shows onset of symptoms before 6 months. Affected babies are never able to sit up and succumb to the disease before 2 years of age. Type II and Type III SMA are milder forms of the disease, and show onset of symptoms between 6 months and 17 years.

All three forms of SMA are caused by deletions or mutations in the survival motor neuron (SMN) gene. There are two almost identical copies of SMN, SMN1 and SMN2. SMN1 produces primarily full length transcript (FL-SMN), whereas SMN2 produces primarily an exon 7 alternatively spliced transcript (?7SMN) and some full length transcripts. Deletion of the SMN1 gene results in the disease, whereas similar mutations in the SMN2 gene do not show any phenotypic effects. However, it is clear from patient studies that SMN2 determines SMA disease severity in a dose dependent manner.

The mouse Smn gene is present as a single copy and does not undergo alternative splicing. Smn-/- mice are pre-implantation lethal and underscore the importance of the Smn protein for cellular survival. To gain a better understanding of SMN in terms of structure/function relationships and disease pathogenesis, it would be ideal if a panel of animals with intermediate/mild phenotypes of SMA existed. We have developed two transgenic mouse lines with recombined alleles harbouring mutations within exon 7 of the Smn gene. Our experiments show that these transgenic mice have the ability to alternatively splice the Smn gene, producing the ?7Smn transcript. A reduction of the Smn protein level is also observed in all tissues.  The ensuing mouse model for SMA has a significantly shorter lifespan than its littermates and has a reduction in the number of motor neurons in the brain stem and spinal cord.  
As we continue to analyze the above transgenic lines, we have developed cell culture models with reduced Smn levels to gain a better understanding of Smn effects on cellular properties. We have used the short interference RNA (siRNA) approach to selectively reduce Smn expression in muscle-like (C2C12) and neuronal-like (PC12) cell lines. The knockdown of Smn has effects on cell culture properties. In the muscle-like like cells, we have observed decreased number of gems (Gemini of coiled bodies), slower proliferation rates, and malformed myotubes. In the neuronal-like cells, Smn knockdown alters the expression pattern of profilin II, resulting in an increase in the neuronal-specific profilin IIa isoform. Moreover, the depletion of Smn, a known interacting partner of profilin IIa, further contributes to the increased profilin IIa availability. Altogether, this leads to an increased formation of ROCK/profilin IIa complex and an inappropriate activation of the RhoA/ROCK pathway, resulting in altered cytoskeletal integrity and a subsequent defect in neuritogenesis. We’ve also observed defects in neuritogenesis in primary neural stem cells cultured from the Smn-/-;SMN2 mouse model.

The mouse and cellular models mentioned above should help elucidate the various steps in the pathogenesis of SMA and ultimately in the design of therapeutic strategies to combat this devastating disorder.