Functional Characterization of Hereditary Spastic Paraplegia Proteins Spastin and ZFYVE27

Abstract

Hereditary spastic paraplegias (HSP) are a heterogeneous group of genetic disorders characterized by progressive spasticity in the lower limbs caused primarily due to dysfunction of corticospinal tract neurons. All modes of inheritance such as autosomal dominant (AD), autosomal recessive (AR) and X-linked have been reported for HSP. Mutations in the SPG4/SPAST gene are the single most common cause for AD-HSP and accounts for up to 40% of all AD-HSP cases. In the present study, we have characterized the functional and structural properties of SPAST gene product, spastin and its interacting protein ZFYVE27, another HSP related protein.In the first part of this thesis, spastin oligomeric state as hexamer has been evaluated by biochemical and cellular approaches. Furthermore, structural information on hexameric spastin was obtained through modeling of spastin AAA domain and this modeled structure was used as a framework to classify the known mutations of spastin in HSP patients into four different functional groups based on the structural information.In the second part, compound heterozygous sequence variants of SPAST causing early age onset of HSP phenotype has been reported. The modifier effect of spastin known polymorphism (S44L) in association with another mutation has been described. Our findings further strengthen the threshold effect model of spastin, with a direct correlation between functional level of spastin and early age onset of HSP phenotype.In the third part, expansion of spastin mutational spectrum in a large HSP cohort has been reported with identification of 29 novel spastin mutations. Moreover, we attempted to determine any possible deleterious effects of these novel mutations on spastin oligomeric function using spastin structural model.In the last part of this thesis, we showed that ZFYVE27, a spastin interacting protein, functions as dimer/tetramer by means of cellular and biochemical analysis. Further, we show that although the core interaction region, hydrophobic region 3 (HR3) is dispensable for self-association of ZFYVE27, it is essential for neurite formation and co-expression of the truncated ZFYVE27 (∆HR3184-208) hampers the function of wild type ZFYVE27.Moreover, to dissect the role of ZFYVE27 in HSP pathomechanism, we endeavored to generate loss of function as well as gain of function mouse models for ZFYVE27. Conceivably, the phenotype of loss of function mouse model might mimic the clinical features of HSP. By elucidating the role of ZFYVE27 in neuritogenesis/neuropathies by means of generated mouse models, we will be able to gain novel mechanistic insights into the underlying pathomechanism of HSP.