Investigation of the molecular basis of a form of motor neuron degenerative disease

Previous studies have shown that loss of function mutations scattered throughout the coding region of seipin (BSCL2) are associated with an autosomal-recessive form of congenital generalised lipodystrophy. Recently we have shown that mutations within a specific region of seipin (N-glycosylation motif) are associated with dominant, apparently gain-of-function mutations resulting in a variable neurological phenotype completely independent to lipodystrophy of which the hallmark characteristic is a motor neuron disease affecting the upper motor neurons, lower motor neurons, or both 10,39. A recurrent feature of human neurodegenerative disorders, including motor neuron disease, is the accumulation of disease-related proteins as aggregates within neurons or glial cells; whether such inclusions are harmful or beneficial to cells remains to be clarified. Consistent with this our preliminary studies indicate that seipin mutations associated with motor neuron degeneration result in the aggregation of the mutant protein in cell culture.

The studies described in this proposal are intended to enhance our understanding of the likely subcellular localisation and morphology of wild type seipin, providing important clues as to its function, and the effect of seipin-related motor neuron degenerative disease-associated mutations, providing important information regarding the disease process. In addition these findings will be compared with the results of similar experiments of a common BSCL2 lipodystrophy mutation, providing valuable information relating to this condition. We have already laid strong foundations for this aspect of the project by conducting extensive preliminary studies including the generation of seipin-specific antibodies, wild type and mutant tagged constructs and a comprehensive battery of wild type and mutant stably-transfected cell lines.

Another important facet of this proposal is disease gene identification, an area in which we have a strong track record, for a second variant of the dHMN-V/CMT/SPG17 phenotypic continuum. This aspect of the project will be facilitated by our recent identification of a comparatively small critical interval on the long arm of chromosome 6, comprising just 42 transcripts. Consequently, as well as being of clear value to studies into the forever-expanding number of related inherited variants of motor neuron degeneration, the findings of this project will also be of benefit to our understanding of other aggregate-associated neurodegenerative diseases as well as to the field of congenital generalised lipodystrophy.

Inherited disorders of the motor neuron form a large clinically and genetically heterogeneous group of chronic progressive diseases which are among the most puzzling and untreatable illnesses. Recently we have shown that mutations in the Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene, encoding seipin, are associated with a group of autosomal dominantly-inherited diseases of the motor neuron (principally distal hereditary motor neuropathy type V; dHMN-V, and hereditary spastic paraplegia; SPG17), the hallmark characteristic of which is a predominant degeneration of the upper motor neurons, lower motor neurons, or both. Intriguingly, null mutations in seipin have also previously been shown to be associated with an autosomal recessively-inherited form of congenital generalised lipodystrophy (CGL), a condition which appears unrelated to motor neuron degenerative disease. We have shown that mutations associated with motor neuron degeneration are located specifically within an N-glycosylation motif of seipin and lead to aggregate formation within the endoplasmic reticulum of cultured cells, whereas mutations associated with CGL do not. In the current proposal we intend to use a wide range of techniques and resources including seipin-specific antibodies, an expansive battery of mutant and wild type-tagged constructs, stably-transfected neuronal cell lines, cell fractionation and cell viability assays. This approach will provide invaluable information relating to the nature and subcellular localisation of wild type seipin including oligomerisation and transmembrane domains, the subcellular localisation, nature and composition of mutant seipin aggregates, and provide clues as to how aggregate formation leads to neurodegeneration.

A second important aspect of this proposal involves disease gene identification in a group of families with the dHMN-V/SPG17 phenotype not linked to BSCL2. Having recently mapped and tightly defined a novel locus responsible for this condition to long arm of chromosome 6, we have also established that the critical interval contains just 42 transcripts. These genes will be evaluated for disease-associated mutations. The combination of disease gene identification and functional studies in this proposal will not only greatly enhance our understanding of the function of seipin, but also extend our knowledge of the pathogenic processes that lead to motor neuron degeneration.