CLN5 and CLN3 function as a complex to regulate endolysosome function

Abstract

CLN5 is a soluble endolysosomal protein whose function is poorly understood. Mutations in this protein cause a rare neurodegenerative disease, Neuronal Ceroid Lipofuscinosis. We previously found that depletion of CLN5 leads to dysfunctional retromer, resulting in the degradation of the lysosomal sorting receptor, sortilin. However, how a soluble lysosomal protein can modulate the function of a cytosolic protein, retromer, is not known. In this work, we show that deletion of CLN5 not only results in retromer dysfunction, but also in impaired endolysosome fusion events. This results in delayed degradation of endocytic proteins and in defective autophagy. CLN5 modulates these various pathways by regulating downstream interactions between CLN3, an endolysosomal integral membrane protein whose mutations also result in Neuronal Ceroid Lipofuscinosis, RAB7A, and a subset of RAB7A effectors. Our data supports a model where CLN3 and CLN5 function as an endolysosomal complex regulating various functions.

Deficiency of the Lysosomal Protein CLN5 Alters Lysosomal Function and Movement

“…Although we did not measure lysosomal area in our iPSC-derived CLN5-deficient human neurons, we noticed an increased number of non-motile lysosomes (Figure 4B) and an increase in lysosomal intensity in the cell body (Figure 2A: second, third, and fourth panels) of CLN5-deficient neurons, with the overall lysosomal mass remaining the same (Figure 2E). The increased number of non-motile lysosomes and impaired anterograde lysosomal movement explain the accumulation of lysosomes in the cell bodies of the CLN5-deficient neurons (Figure 2A: second, third, and fourth panels, and Video S1B,C), as reported by Uusi-Rauva et al Yasa et al (2021) [14] suggested that CLN5 KO HeLa cells were not able to move lysosomes as efficiently as wild-type HeLa cells. In our study, the impaired anterograde movement in the CLN5-deficient human neurons could indicate that the neuronal projections are being exempt from having their waste cleared, which could cause neuronal toxicity at the synapses.…”

“…An effector of RAB7A, RILP, regulates the activity of V-ATPase, the key component for maintaining the acidity in the lysosomal lumen [32]. Interestingly, loss of CLN5 leads to reduced RAB7A/RILP interaction [14], which could lead to even further reduction in acidity in peripheral lysosomes, as seen in Figure 2A (second, third, and fourth panels).…”

“…Lysosomes travel to distal ends of neurons to maintain local degradative activities, therefore making lysosomal movement crucial to maintain homeostasis in the neurons [29]. Some CLN proteins have been recently shown to alter lysosomal positioning [14,30]; however, whether inhibiting CLN5 changes the lysosomal movement/trafficking in human neurons is unknown. To assess the lysosomal movement in control vs. CLN5i neurons, kymographs were generated from time-lapse videos of LysoTracker Red stained lysosomes in control vs. CLN5i neurons (Figure 4A).…”

“…Lysosomes are acidic vesicles containing hydrolytic enzymes, and loss of the lysosomal protein CLN5 shows reduced acidic organelles and impaired autophagy, as observed in ovine neural cultures from the naturally occurring CLN5-deficient sheep model [8]. Loss of CLN5 is further associated with different neuronal and non-neuronal pathologies, including accumulation of autofluorescent storage materials [4,[9][10][11], impaired retromer trafficking and endosomal sorting [12][13][14], disturbed lipid metabolism [15], metal imbalance [16], and mitochondrial dysfunction [17]. A study in Dictyostelium found that Cln5 acts as a glycoside hydrolase [18] and the human CLN5 has been predicted to be a lysosomal protease [19].…”

Autophagy in the Neuronal Ceroid Lipofuscinoses (Batten Disease)

“…Evidence in these models and in patient cells suggests the accumulation of autophagosomes/autolysosomes arises from reduced autophagic degradation ( Figure 3 ) ( Cao et al, 2006 ; Chang et al, 2011 ; Vidal-Donet et al, 2013 ; Lojewski et al, 2014 ; Chandrachud et al, 2015 ; Petcherski et al, 2019 ). The mechanisms causing reduced autophagic degradation upon CLN3 -deficiency are incompletely understood but may be linked to altered RAB7-mediated trafficking and/or altered Ca 2+ signalling ( Figure 3 ) ( Huber et al, 2014 ; Chandrachud et al, 2015 ; Petcherski et al, 2019 ; Yasa et al, 2020 ; Yasa et al, 2021 ). Thus, restoration of autophagic degradation in CLN3 -deficiency models has emerged as a promising therapeutic target ( Chang et al, 2011 ; Chandrachud et al, 2015 ; Hong et al, 2016 ; Palmieri et al, 2017 ; Petcherski et al, 2019 ; Kinarivala et al, 2020 ).…”