Defects in the MFSD8 gene encoding the lysosomal membrane protein CLN7 lead to CLN7 disease, a neurodegenerative lysosomal storage disorder belonging to the group of neuronal ceroid lipofuscinoses. Here, we have performed a SILAC-based quantitative analysis of the lysosomal proteome using Cln7-deficient mouse embryonic fibroblasts (MEFs) from a Cln7 knockout (ko) mouse model. From 3335 different proteins identified, we detected 56 soluble lysosomal proteins and 29 highly abundant lysosomal membrane proteins. Quantification revealed that the amounts of 12 different soluble lysosomal proteins were significantly reduced in Cln7 ko MEFs compared with wild-type controls. One of the most significantly depleted lysosomal proteins was Cln5 protein that underlies another distinct neuronal ceroid lipofuscinosis disorder. Expression analyses showed that the mRNA expression, biosynthesis, intracellular sorting and proteolytic processing of Cln5 were not affected, whereas the depletion of mature Cln5 protein was due to increased proteolytic degradation by cysteine proteases in Cln7 ko lysosomes. Considering the similar phenotypes of CLN5 and CLN7 patients, our data suggest that depletion of CLN5 may play an important part in the pathogenesis of CLN7 disease. In addition, we found a defect in the ability of Cln7 ko MEFs to adapt to starvation conditions as shown by impaired mammalian target of rapamycin complex 1 reactivation, reduced autolysosome tubulation and increased perinuclear accumulation of autolysosomes compared with controls. In summary, depletion of multiple soluble lysosomal proteins suggest a critical role of CLN7 for lysosomal function, which may contribute to the pathogenesis and progression of CLN7 disease.

/pdf/loss-of-cln7-results-in-depletion-of-soluble-lysosomal-qldpe5swh2.pdf

Loss of CLN7 results in depletion of soluble lysosomal proteins and impaired mTOR reactivation.

Pathomechanisms in the neuronal ceroid lipofuscinoses.

Moving towards a new era of genomics in the neuronal ceroid lipofuscinoses.

We describe the ninth variant of neuronal ceroid lipofuscinosis (NCL) or Batten disease, due to defects in a putative new gene, CLN9. We therefore refer to the new variant as CLN9-deficient. Two Serbian sisters and two German brothers are described. Their clinical history is characteristic for juvenile NCL. They show similar gene expression patterns. The existence of this variant is supported by the presence of curvilinear inclusions, fingerprint profiles, and granular osmiophilic deposits in neurons, lymphocytes, and conjunctival cells. Enzyme screening and sequencing of the coding regions of other NCL genes was negative. CLN9-deficient cells have a distinctive phenotype. They have rounded cell bodies, have prominent nucleoli, attach poorly to the culture dish, and are sensitive to apoptosis but have increased growth rates. Gene expression of proteins involved in cell adhesion and apoptosis is altered in these cells. Sphingolipid metabolism is also perturbed. They have decreased levels of ceramide, sphingomyelin, lactosylceramide, ceramide trihexoside, and globoside and increased activity of serine palmitoyl transferase.

Impaired cell adhesion and apoptosis in the novel CLN9 Batten disease variant

The social amoeba Dictyostelium discoideum has been a preferred model organism during the last 50 years, particularly for the study of cell motility and chemotaxis, phagocytosis and macropinocytosis, intercellular adhesion, pattern formation, caspase-independent cell death and more recently autophagy and social evolution. Being a soil amoeba and professional phagocyte, thus exposed to a variety of potential pathogens, D. discoideum has also proven to be a powerful genetic and cellular model for investigating host-pathogen interactions and microbial infections. The finding that the Dictyostelium genome harbours several homologs of human genes responsible for a variety of diseases has stimulated their analysis, providing new insights into the mechanism of action of the encoded proteins and in some cases into the defect underlying the disease. Recent technological developments have covered the genetic gap between mammals and non-mammalian model organisms, challenging the modelling role of the latter. Is there a future for Dictyostelium discoideum as a model organism?

/pdf/the-past-present-and-future-of-dictyostelium-as-a-model-3j1cljiwpo.pdf

The past, present and future of Dictyostelium as a model system.

Cln3 function is linked to osmoregulation in a Dictyostelium model of Batten disease.

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 Dictyostelium discoideum has proven to be an exceptional model organism for studying a wide range of neurological disorders, including the NCLs. The Dictyostelium genome contains homologs of 11 of the 13 NCL genes. Its life cycle, comprised of both single-cell and multicellular phases, provides an excellent system for studying the effects of NCL gene deficiency on conserved cellular and developmental processes. In this review, we highlight recent advances in NCL research using Dictyostelium as a biomedical model.

https://www.mdpi.com/2073-4409/8/2/115/pdf

Recent Insights into NCL Protein Function Using the Model Organism Dictyostelium discoideum.

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. While the precise role of CLN5 in NCL pathogenesis is not known, recent work revealed that the protein has glycoside hydrolase activity. Previous work on the Dictyostelium discoideum homolog of human CLN5, Cln5, revealed its secretion during the early stages of development and its role in regulating cell adhesion and cAMP-mediated chemotaxis. Here, we used Dictyostelium to examine the effect of cln5-deficiency on various growth and developmental processes during the life cycle. During growth, cln5 - cells displayed reduced cell proliferation, cytokinesis, viability, and folic acid-mediated chemotaxis. In addition, the growth of cln5 - cells was severely impaired in nutrient-limiting media. Based on these findings, we assessed autophagic flux in growth-phase cells and observed that loss of cln5 increased the number of autophagosomes suggesting that the basal level of autophagy was increased in cln5 - cells. Similarly, loss of cln5 increased the amounts of ubiquitin-positive proteins. During the early stages of multicellular development, the aggregation of cln5 - cells was delayed and loss of the autophagy genes, atg1 and atg9, reduced the extracellular amount of Cln5. We also observed an increased amount of intracellular Cln5 in cells lacking the Dictyostelium homolog of the human glycoside hydrolase, hexosaminidase A (HEXA), further supporting the glycoside hydrolase activity of Cln5. This observation was also supported by our finding that CLN5 and HEXA expression are highly correlated in human tissues. Following mound formation, cln5 - development was precocious and loss of cln5 affected spore morphology, germination, and viability. When cln5 - cells were developed in the presence of the autophagy inhibitor ammonium chloride, the formation of multicellular structures was impaired, and the size of cln5 - slugs was reduced relative to WT slugs. These results, coupled with the aberrant autophagic flux observed in cln5 - cells during growth, support a role for Cln5 in autophagy during the Dictyostelium life cycle. In total, this study highlights the multifaceted role of Cln5 in Dictyostelium and provides insight into the pathological mechanisms that may underlie CLN5 disease.

/pdf/aberrant-autophagy-impacts-growth-and-multicellular-2jmjm4rl3e.pdf

Meagan D McLaren

Papers

Cln3 function is linked to osmoregulation in a Dictyostelium model of Batten disease.

Recent Insights into NCL Protein Function Using the Model Organism Dictyostelium discoideum.

Aberrant Autophagy Impacts Growth and Multicellular Development in a Dictyostelium Knockout Model of CLN5 Disease.