The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are devastating forms of neurodegeneration that affect the global population. Mutations have been documented in 13 genetically distinct CLN genes (PPT1/CLN1, TPP1/CLN2, CLN3, DNAJC5/CLN4, CLN5, CLN6, MFSD8/CLN7, CLN8, CTSD/CLN10, PGRN/CLN11, ATP13A2/CLN12, CTSF/CLN13, KCTD7/CLN14), each of which causes a specific subtype of the disease (e.g., mutations in CLN3 cause CLN3 disease). While the NCLs affect all ages and ethnicities, the disease is recognized as the most common form of childhood neurodegeneration.
Table taken from Huber, 2016
Clinical manifestations of the NCLs include seizures, vision loss, reduced mental capacity, decline in motor function, and a reduced lifespan. At the cellular level, ceroid lipofuscin accumulates in neurons, as well as other cell types outside of the central nervous system, due to aberrant lysosomal function. Unfortunately, there is currently no cure for the NCLs, in large part due to our poor understanding of the proteins associated with the disease. CLN proteins localize to the endomembrane system, which plays an important role in intracellular digestion, trafficking, and secretion in eukaryotic cells.
Figure taken from Huber, 2020
Dictyostelium has emerged as an exceptional model system for studying the functions of proteins linked to human neurological disorders, including lissencephaly, epilepsy, Alzhemier’s disease, Parkinson’s disease, Huntington’s disease, and prion diseases. Intriguingly, the Dictyostelium genome encodes several homologs of human CLN proteins. Thus, it provides an excellent opportunity to reveal the precise functions of CLN proteins in eukaryotic cells.
Table taken from Huber et al., 2020.
Our current work is focused on studying the localizations and functions of the Dictyostelium homologs of human CLN3 (Cln3), CLN5 (Cln5), and MFSD8/CLN7 (Mfsd8).
Cln3
In Dictyostelium, Cln3 localizes primarily to the contractile vacuole (CV) system, and to a lesser extent, compartments of the endocytic pathway and the Golgi complex (Huber et al., 2014; Huber et al., 2017; Huber, 2017). The CV system is a dynamic organelle with established and putative functions such as osmoregulation, ion homeostasis, and vesicular trafficking. Evidence from other organisms supports a function for CLN3 in these processes. We are using this knowledge to fuel research on characterizing the role of Cln3 in CV system function to ultimately determine its molecular function within the cell.
In Dictyostelium, Cln3 localizes primarily to the contractile vacuole (CV) system, and to a lesser extent, compartments of the endocytic pathway and the Golgi complex (Huber et al., 2014; Huber et al., 2017; Huber, 2017). The CV system is a dynamic organelle with established and putative functions such as osmoregulation, ion homeostasis, and vesicular trafficking. Evidence from other organisms supports a function for CLN3 in these processes. We are using this knowledge to fuel research on characterizing the role of Cln3 in CV system function to ultimately determine its molecular function within the cell.
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Localization of GFP-Cln3 in Dictyostelium. Figure on the left taken from Huber et al., 2014. Figure on the right taken from Huber, 2017.
During growth, cln3-deficiency increases the rate of cell proliferation, impairs cytokinesis, and impacts osmoregulation (Huber et al., 2014; Mathavarajah et al., 2018). During the early stages of multicellular development, loss of cln3 impacts protein secretion and reduces adhesion (Huber et al., 2014; Huber, 2017; Huber et al., 2017). Loss of cln3 also affects gene expression, which includes homologs of human CLN genes (TPP1/CLN2, CLN5, CTSD/CLN10, PGRN/CLN11, and CTSF/CLN13) (Huber and Mathavarajah, 2019). Finally, cln3-deficiency affects lysosomal enzyme activity, endo-lysosomal pH, and nitric oxide homeostasis (Huber and Mathavarajah, 2019). During the mid-to-late stages of multicellular development, cln3-deficiency causes cells to develop precociously (Huber et al., 2014). Importantly, some cln3-deficiency phenotypes can be rescued by re-introducing either Dictyostelium Cln3 or human CLN3 into cln3- cells (Huber et al., 2014; Huber et al., 2017).
Cln5
Dictyostelium was used to show for the first time that Dictyostelium Cln5 and human CLN5 have glycoside hydrolase activity (Huber and Mathavarajah, 2018a). In Dictyostelium, Cln5 localizes to the ER where it is glycosylated (Huber and Mathavarajah, 2018a; Huber and Mathavarajah, 2018b). The protein is then trafficked to the cell cortex and CV system, which modulates its secretion (via Cln3 and autophagy induction) (Huber and Mathavarajah, 2018b).
Dictyostelium was used to show for the first time that Dictyostelium Cln5 and human CLN5 have glycoside hydrolase activity (Huber and Mathavarajah, 2018a). In Dictyostelium, Cln5 localizes to the ER where it is glycosylated (Huber and Mathavarajah, 2018a; Huber and Mathavarajah, 2018b). The protein is then trafficked to the cell cortex and CV system, which modulates its secretion (via Cln3 and autophagy induction) (Huber and Mathavarajah, 2018b).
Localization of Cln5 in Dictyostelium. Figure taken from Huber and Mathavarajah, 2018b.
An analysis of the Cln5 interactome revealed that the protein interacts with lysosomal enzymes, other homologs of human CLN proteins (TPP1/CLN2, CTSD/CLN10, and CTSF/CLN13), and proteins linked to Cln3 function in Dictyostelium (Huber and Mathavarajah, 2018a). Loss of cln5 reduces cell-substrate and cell-to-cell adhesion during the early stages of multicellular development, as well as cAMP-mediated chemotaxis (Huber and Mathavarajah, 2018b).
Mfsd8
Mfsd8 GFP-fusion proteins localize to compartments of the endocytic pathway (Huber et al., 2020). An analysis of the Mfsd8 interactome revealed that the protein interacts with ion transporters, proteins involved in glucose and lipid metabolism, as well as other CLN proteins (e.g., CtsD) (Huber et al., 2020). Mfsd8 also interacts with proteins linked to Cln3 function in Dictyostelium and Cln5-interactors (Huber et al., 2020). Finally, our data indicates that Mfsd8 may play an important role in the secretion of Cln5 and CtsD (Huber et al., 2020).
Mfsd8 GFP-fusion proteins localize to compartments of the endocytic pathway (Huber et al., 2020). An analysis of the Mfsd8 interactome revealed that the protein interacts with ion transporters, proteins involved in glucose and lipid metabolism, as well as other CLN proteins (e.g., CtsD) (Huber et al., 2020). Mfsd8 also interacts with proteins linked to Cln3 function in Dictyostelium and Cln5-interactors (Huber et al., 2020). Finally, our data indicates that Mfsd8 may play an important role in the secretion of Cln5 and CtsD (Huber et al., 2020).
Localization of GFP-Mfsd8 and Mfsd8-GFP in Dictyostelium. Figure taken from Huber et al., 2020.
The molecular networking of CLN proteins in Dictyostelium
Since mutations in CLN proteins cause the accumulation of ceroid lipofuscin within cells, and result in similar clinical manifestations between the different subtypes, the proteins are thought to have shared functions, regulate similar processes, and/or participate in shared or converging pathways. In support of this hypothesis, model systems used to study the NCLs show common phenotypes. At the cellular level, CLN proteins display similar localizations, have common binding partners, and regulate the expression and activities of one another. Below is a model illustrating our current understanding of the networking of CLN proteins in Dictyostelium.
Since mutations in CLN proteins cause the accumulation of ceroid lipofuscin within cells, and result in similar clinical manifestations between the different subtypes, the proteins are thought to have shared functions, regulate similar processes, and/or participate in shared or converging pathways. In support of this hypothesis, model systems used to study the NCLs show common phenotypes. At the cellular level, CLN proteins display similar localizations, have common binding partners, and regulate the expression and activities of one another. Below is a model illustrating our current understanding of the networking of CLN proteins in Dictyostelium.
Figure taken from Huber, 2020
The networking of CLN proteins in Dictyostelium. (1) Material is taken up by the cell and incorporated into an endosome, which matures into a lysosome. Tpp1A, Tpp1F, Cln3, Mfsd8, CtsD localize to the late endosome/lysosome. Tpp1F also localizes extracellularly as does Ppt1 and Tpp1B. (2) Tpp1B localizes to the Golgi complex and binds the Golgi pH regulator (in addition to extracellularly, see #1). Tpp1F localizes to the Golgi complex and endoplasmic reticulum (ER) (in addition to late endosome/lysosome and extracellularly, see #1) and binds the Golgi pH regulator. Cln3 localizes to the Golgi complex (in addition to the late endosome/lysosome, see #1). Cln5 is glycosylated in the ER and then trafficked to the cell cortex and contractile vacuole (CV) system. (3) Cln3 localizes to the CV system (in addition to the late endosome/lysosome and Golgi complex, see #1 and #2). During starvation, loss of cln3 alters the intracellular activity of alpha-mannosidase (ManA), the expression of beta-glucosidase (gluA), the intracellular activity of GluA, and the expression of N-acetylglucosaminidase (nagA). Cln5 interacts with ManA, NagA, and GluA. Loss of cln3 alters the expression of nagB and the secretion of NagB during starvation. Finally, loss of cln3 alters the extracellular activities of ManA, GluA, and Nag during starvation. (4) Cln5 is secreted during starvation. Secretion is regulated by autophagy (i.e., autophagy inhibition decreases secretion) and Cln3 (i.e., cln3-deficiency alters secretion). Inside the cell, Cln5 interacts with Tpp1B. (5) Loss of cln3 alters the intracellular and extracellular activity of Tpp1 during starvation. cln3-deficiency alters the expression of tpp1F and the secretion of Tpp1F during starvation. Loss of cln3 increases the expression of tpp1A during hypertonic stress and alters the expression of tpp1D and grn during starvation. cln3-deficiency alters the expression of ctsD, the intracellular and extracellular activity of CtsD, and the secretion of CtsD during starvation. (6) Loss of cln3 alters the expression of aprA and the intracellular amount of AprA during starvation. cln3-deficiency alters the secretion of AprA during growth and starvation. Loss of cln3 alters the secretion of CfaD during growth and the amount of CadA in conditioned starvation buffer. Cln5 interacts with AprA, CfaD, CadA, and CtsD. (7) Loss of cln3 alters the expression of cprD and cprG during starvation and the expression of bip2 (luminal-binding protein 2, DDB0233663). cln3-deficiency increases the expression of cprE during hypotonic stress. Loss of cln3 alters the secretion of CprD, CprE, CprG, and Bip2 during starvation. Cln5 interacts with CprD, CprE, CprG, and Bip2. (8) Loss of cln3 alters the expression of cprF during starvation. cln3-deficiency alters the secretion of CprA and CprB during starvation and the secretion of CprF. Loss of cln3 increases the expression of cprF during hypotonic stress. (9) Like Cln5, Mfsd8 interacts with CtsD, CadA, and CfaD (see #6). Like cln3- cells (see #4 and #5), loss of mfsd8 alters the secretion of Cln5 and CtsD during starvation.
The long-term goal of our research is to use Dictyostelium to define the functions of all CLN proteins and determine how they contribute to the normal functioning of the endomembrane system in eukaryotic cells. Hopefully the results of this work will provide insight and guide future research into developing therapy options for this devastating and currently untreatable neurological disorder.
