👤 Sabateeshan Mathavarajah

The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases that affect people of all ages and ethnicities, yet many of the associated genes/proteins are not well characterized. Mutations in MFSD8 (major facilitator superfamily domain-containing 8) cause an infantile form of NCL referred to as CLN7 disease. In this study, we revealed the localization and binding partners of an ortholog of human MFSD8 (Mfsd8) in the social amoeba Dictyostelium discoideum. Putative lysosomal targeting motifs are conserved in Dictyostelium Mfsd8, as are several residues mutated in CLN7 disease patients. Mfsd8 tagged with GFP localizes to endocytic compartments, which includes acidic intracellular vesicles and late endosomes. We pulled-down GFP-Mfsd8 and used mass spectrometry to reveal the Mfsd8 interactome during Dictyostelium growth and starvation. Among the identified hits were the Dictyostelium ortholog of human cathepsin D (CtsD), as well as proteins linked to the functions of the CLN3 (Cln3) and CLN5 (Cln5) orthologs in Dictyostelium. To study the function of Mfsd8, we validated a publically available mfsd8 Show less

Mutations in CLN3 cause a juvenile form of neuronal ceroid lipofuscinosis (NCL). This devastating neurological disorder, commonly known as Batten disease, is currently untreatable due to a lack of understanding of the physiological role of the protein. Recently, work in the social amoeba Dictyostelium discoideum has provided valuable new insight into the function of CLN3 in the cell. More specifically, research has linked the Dictyostelium homolog (gene: cln3, protein: Cln3) to protein secretion, adhesion, and aggregation during starvation, which initiates multicellular development. In this study, we used comparative transcriptomics to explore the mechanisms underlying the aberrant response of cln3 Show less

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating neurological disorders that have a global distribution and affect people of all ages. Commonly known as Batten disease, this form of neurodegeneration is linked to mutations in 13 genetically distinct genes. The precise mechanisms underlying the disease are unknown, in large part due to our poor understanding of the functions of NCL proteins. The social amoeba Show less

Mutations in CLN3 cause a juvenile form of neuronal ceroid lipofuscinosis (NCL), commonly known as Batten disease. Currently, there is no cure for NCL and the mechanisms underlying the disease are not well understood. In the social amoeba Dictyostelium discoideum, the CLN3 homolog, Cln3, localizes predominantly to the contractile vacuole (CV) system. This dynamic organelle functions in osmoregulation, and intriguingly, osmoregulatory defects have been observed in mammalian cell models of CLN3 disease. Therefore, we used Dictyostelium to further study the involvement of CLN3 in this conserved cellular process. First, we assessed the localization of GFP-Cln3 during mitosis and cytokinesis, where CV system function is essential. GFP-Cln3 localized to the CV system during mitosis and cln3 Show less

Mutations in CLN5 cause neuronal ceroid lipofuscinosis (NCL), a currently untreatable neurodegenerative disorder commonly known as Batten disease. Several genetic models have been generated to study the function of CLN5, but one limitation has been the lack of a homolog in lower eukaryotic model systems. Our previous work revealed a homolog of CLN5 in the social amoeba Dictyostelium discoideum. We used a Cln5-GFP fusion protein to show that the protein is secreted and functions as a glycoside hydrolase in Dictyostelium. Importantly, we also revealed this to be the molecular function of human CLN5. In this study, we generated an antibody against Cln5 to show that the endogenous protein is secreted during the early stages of Dictyostelium development. Like human CLN5, the Dictyostelium homolog is glycosylated and requires this post-translational modification for secretion. Cln5 secretion bypasses the Golgi complex, and instead, occurs via an unconventional pathway linked to autophagy. Interestingly, we observed co-localization of Cln5 and GFP-Cln3 as well as increased secretion of Cln5 and Cln5-GFP in cln3 Show less

Ceroid lipofuscinosis neuronal 5 (CLN5) is a member of a family of proteins that are linked to neuronal ceroid lipofuscinosis (NCL). This devastating neurological disorder, known commonly as Batten disease, affects all ages and ethnicities and is currently incurable. The precise function of CLN5, like many of the NCL proteins, remains to be elucidated. In this study, we report the localization, molecular function, and interactome of Cln5, the CLN5 homolog in the social amoeba Dictyostelium discoideum. Residues that are glycosylated in human CLN5 are conserved in the Dictyostelium homolog as are residues that are mutated in patients with CLN5 disease. Dictyostelium Cln5 contains a putative signal peptide for secretion and we show that the protein is secreted during growth and starvation. We also reveal that both Dictyostelium Cln5 and human CLN5 are glycoside hydrolases, providing the first evidence in any system linking a molecular function to CLN5. Finally, immunoprecipitation coupled with mass spectrometry identified 61 proteins that interact with Cln5 in Dictyostelium. Of the 61 proteins, 67% localize to the extracellular space, 28% to intracellular vesicles, and 20% to lysosomes. A GO term enrichment analysis revealed that a majority of the interacting proteins are involved in metabolism, catabolism, proteolysis, and hydrolysis, and include other NCL-like proteins (e.g., Tpp1/Cln2, cathepsin D/Cln10, cathepsin F/Cln13) as well as proteins linked to Cln3 function in Dictyostelium (e.g., AprA, CfaD, CadA). In total, this work reveals a CLN5 homolog in Dictyostelium and further establishes this organism as a complementary model system for studying the functions of proteins linked to NCL in humans. Show less