Studies on hereditary spastic paraplegia proteins

Abstract

The hereditary spastic paraplegias (HSPs) are a clinically and genetically diverse group of inherited neurological disorders that primarily cause progressive spasticity and weakness in the lower limbs due to a length-dependent, retrograde degradation of the corticospinal motor neurons. Purely spastic paraplegia is also known as uncomplicated HSP, but complicated forms of HSP exist as well, with symptoms such as mental retardation, dementia, seizures, and optic, cortical, and cerebellar atrophy. Twenty gene products have been identified from over 40 different known SPG spastic gait loci (SPG1-46), which may be inherited in autosomal dominant, autosomal recessive, or X-linked manners.

The work presented in this thesis focuses on two different HSP proteins: atlastin-1, a member of the dynamin superfamily of large GTPases through sequence similarity, and maspardin Mast syndrome, spastic paraplegia, autosomal recessive with dementia. Mutations in the atlastin-1 gene, SPG3A, are the second most common cause of autosomal dominant HSP around 10% of all cases many of which are quite early in onset (in childhood) when compared to other forms of HSP. Diseasecausing point mutations are distributed throughout the coding region, although most are clustered in known domains GTP binding/GTPase functional areas, a coiled coil region in the middle of the protein product, and in the transmembrane areas at the Cterminal end. On the other hand, the only causative mutation in SPG21/MAST (maspardin) that has been found is a frameshift-producing alteration after the second third of the gene, which induces a premature truncation of the gene product and loss of the last 95 amino acids of the wild type protein. This mutation is only inherited in an autosomal recessive manner, and causes a complicated HSP with additional symptoms such as dementia, white matter abnormalities, and cerebellar and extrapyramidal signs.

Atlastin-1 is localized to the ER and cis-Golgi apparatus in the adult brain, and appears to exist natively as oligomers, most likely tetramers. Wild-type atlastin-1 is a functional GTPase, but in paper I we found that several missense atlastin-1 mutations have impaired GTPase activity. We also found that atlastin-1 is highly enriched in vesicular structures within growth cones, varicosities, and axonal branch points. Knockdown of atlastin-1 using small hairpin RNAs impairs axon formation and elongation during neuronal development and reduces the total number of neuronal processes. In paper II we examined a novel SPG3A mutation causative for HSP that did not affect GTPase activity or interactions between atlastin and spastin, the gene most mutated in HSP. However, immunoblots from patient lymphoblasts showed a reduction in atlastin-1 protein levels, indicating that mutant atlastin-1 may cause disease pathogenesis through a dominant-negative, loss-of-function manner through protein destabilization.

Mast syndrome is likely caused by a loss of protein function. In paper IV we generated SPG21-/- transgenic mice as a possible model for SPG21. Though SPG21-/- mice appeared normal at birth, within several months they developed a mild but progressive hind limb dysfunction. Cultured cerebral cortical neurons from SPG21-/- mice exhibited significantly more axonal branching than neurons cultured from wildtype animals, although a comprehensive neuropathological analysis did not reveal any abnormalities consistent with those observed in human HSP. While a unifying mechanism for all the genes and proteins known to be involved in HSP has yet to be found, our data support the idea that axonal trafficking and proper neurite branching may represent a common cellular pathogenic theme.