Different expression levels of spartin cause broad spectrum of cellular consequences in human neuroblastoma cells.

Hereditary spastic paraplegia describes a diverse group of neurodegenerative conditions characterised by progressive spasticity and weakness of the lower limbs. Mutations in the SPG20 gene encoding spartin cause an autosomal recessive hereditary spastic paraplegia known as Troyer syndrome. To evaluate the cellular consequences of sustained spartin depletion in neuronal cells, we established several clonal SH-SY5Y cell lines with different level of spartin knockdown. Here, we report that cells with modest spartin downregulation show signs of neuronal differentiation such as increased neuritogenesis and cytoskeleton rearrangement. Interestingly, we also indicate that permanent high level spartin depletion results in impaired cell growth and multiple mitochondrial aberrations, which we speculate, arise as a result of chronic oxidative stress. Our studies demonstrate that the scale of spartin downregulation is the major factor that determines the severity of cellular consequences observed and suggest that there is a critical level of spartin expression which must be maintained for proper cellular functions.

Identification of novel spartin-interactors shows spartin is a multifunctional protein.

Hereditary spastic paraplegia describes a group of neurodegenerative diseases characterized by lower limb progressive weakness and spasticity. Troyer syndrome is an autosomal recessive form of hereditary spastic paraplegia caused by a frameshift mutation (1110delA) in the SPG20 gene encoding spartin protein, the cellular function of which remains unknown. Knowledge about spartin-interactors is also very limited. In this study, we apply a broad spectrum of proteomics techniques to identify novel spartin-binding proteins. We used a Tandem Affinity Purification technique followed by HPLC-mass spectrometry to characterize potential spartin-binding partners. Selected putative interactions were confirmed by co-immunoprecipitation experiments. We identified 94 potential spartin-binding proteins which were grouped into functional categories. We performed co-immunoprecipitation experiments to confirm that spartin interacts with GRP78, GRP75 and nucleolin proteins. Additionally, our mass spectrometry results confirmed previously published information about spartin interaction with ubiquitin and the E3 ubiquitin-protein ligases, AIP4/Itch and AIP5/WWP1. Our studies suggest that spartin is a multifunctional protein and for the first time we suggest a role for spartin in protein folding and turnover both in mitochondria and endoplasmic reticulum. We also show for the first time interaction between spartin and a nucleolar protein, nucleolin.