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

Abstract

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.

Altered protein secretion in Batten disease

“…While the mechanisms regulating autophagy-dependent secretion are not fully understood, autophagy (ATG) genes ( Box 1 ), autophagosomes ( Box 1 ) and multivesicular bodies ( Box 1 ) are thought to play important roles ( Duran et al, 2010 ; Manjithaya et al, 2010 ; Chen et al, 2017 ). Consistent with these findings, pharmacological treatment (ammonium chloride or chloroquine) or deletion of ATG genes ( atg1 − or atg9 − ) reduce Cln5 secretion in D. discoideum ( Huber and Mathavarajah, 2018b ; McLaren et al, 2021 ). In addition, the functions of D. discoideum Cln5 ( McLaren et al, 2021 ), mouse CLN5 ( Leinonen et al, 2017 ), sheep CLN5 ( Best et al, 2017 ) and human CLN5 ( Adams et al, 2019 ; Doccini et al, 2020 ) have all been linked to autophagy.…”

“…Mfsd8 is the D. discoideum homolog of human MFSD8 ( Table 1 ) and mutations in MFSD8 cause CLN7 disease ( Box 1 ) ( Table 1 ). In addition, recent work suggests that D. discoideum Cln5 is secreted following induction of autophagy via an unconventional pathway involving the CV system ( Huber and Mathavarajah, 2018b ; McLaren et al, 2021 ) ( Fig. 2 ).…”

“…Consistent with these findings, pharmacological treatment (ammonium chloride or chloroquine) or deletion of ATG genes (atg1 − or atg9 − ) reduce Cln5 secretion in D. discoideum (Huber and Wisniewski et al, 1994;Wisniewski et al, 1995 CSF (CLN2 disease) Reduced levels of pterins Barisićet al, 2003 CSF (CLN3 disease) Increased levels of neuron-specific enolase and tau protein Kay et al, 2009 CSF, cerebrospinal fluid. McLaren et al, 2021). In addition, the functions of D. discoideum Cln5 (McLaren et al, 2021), mouse CLN5 (Leinonen et al, 2017), sheep CLN5 (Best et al, 2017) and human CLN5 (Adams et al, 2019;Doccini et al, 2020) have all been linked to autophagy.…”

Dictyostelium discoideum as a Model for Investigating Neurodegenerative Diseases

“…At a cellular level deletion of cln5 results in an increased number of autophagosomes and ubiquitinated proteins, consistent with increased autophagic activity (Figure 4B). Development in the presence of an autophagy inhibitor impaired the formation of developmental structures, including reduction in slug size (McLaren et al, 2021). These data suggest that Cln5 plays a similar role in both Dictyostelium and human biology and may play a role in autophagy.…”

“…Further investigation of Cln5 was found that cln5 − -cells have reduced cell proliferation, cytokinesis, viability, folic acid-mediated endocytosis, and growth in nutrient-limited media. cln5 − cells develop more rapidly than wild-type cells (McLaren et al, 2021). cln5 − cells also exhibit impaired spore morphology, germination, and viability.…”