Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins and modifies location of late endosomal compartments

Abstract

CLN3 is an endosomal/lysosomal transmembrane protein mutated in classical juvenile onset neuronal ceroid lipofuscinosis, a fatal inherited neurodegenerative lysosomal storage disorder. The function of CLN3 in endosomal/lysosomal events has remained elusive due to poor understanding of its interactions in these compartments. It has previously been shown that the localisation of late endosomal/lysosomal compartments is disturbed in cells expressing the most common disease-associated CLN3 mutant, CLN3∆ex7-8 (c.462-677del). We report here that a protracted disease causing mutant, CLN3E295K, affects the properties of late endocytic compartments, since over-expression of the CLN3E295K mutant protein in HeLa cells induced relocalisation of Rab7 and a perinuclear clustering of late endosomes/lysosomes. In addition to the previously reported disturbances in the endocytic pathway, we now show that the anterograde transport of late endosomal/lysosomal compartments is affected in CLN3 deficiency. CLN3 interacted with motor components driving both plus and minus end microtubular trafficking: tubulin, dynactin, dynein and kinesin-2. Most importantly, CLN3 was found to interact directly with active, guanosine-5'-triphosphate (GTP)-bound Rab7 and with the Rab7-interacting lysosomal protein (RILP) that anchors the dynein motor. The data presented in this study provide novel insights into the role of CLN3 in late endosomal/lysosomal membrane transport.

Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function

“…Further work is needed to fully elucidate whether CLN3 functions in regulating autophagosomal maturation via a Rab7 pathway, and how it may do so. It is nevertheless notable that Uusi-Rauva et al (39) suggested that loss of CLN3 function leads to an imbalance of GTP/GDP forms of Rab7, given their observation that EGFPRab7 recruitment to the late endosome was more rapid in JNCL patient fibroblasts with the common ϳ1-kb mutation, compared with control fibroblasts. Our observation in this study that Rab7 association with autophagosomal compartments is somewhat increased in fixed CbCln3 ⌬ex7/8 cells, as compared with wild type CbCln3 cells, would seem to support their findings.…”

“…Interestingly, CLN3 has also been proposed to mediate Rab7 function, and overexpressed CLN3 protein has been reported to co-immunoprecipitate with a Rab7-interacting lysosomal protein complex (39). To extend our analyses, particularly given the possibility that CLN3 and thapsigargin share some mechanism, and to further rule out the possibility that the observed effects were not due to an up-regulation of autophagy, we next examined the effects of thapsigargin treatment and Cln3 mutation on Rab7 autophagosomal localization in our cerebellar cell system, with and without bafilomycin, a VATPase inhibitor that blocks lysosomal acidification and fusion.…”

Mechanisms and functions of lysosome positioning

“…In these diseases, mutations in lysosomal proteins lead to the accumulation of storage material in enlarged lysosomal vacuoles. Interestingly, the abnormal lysosomes are often clustered in the juxtanuclear area, as documented for neuronal ceroid lipofuscinosis type 3 (CLN3) (Uusi-Rauva et al, 2012), and mucolipidosis type IV (ML-IV) (Li et al, 2016b). Inhibition of lysosomal proteolysis has also been found to disrupt axonal transport of late endosomes, lysosomes and autolysosomes in neurons, resulting in their accumulation in dystrophic axonal swellings characteristic of Alzheimer's disease (Lee et al, 2011).…”

“…For example, cytosol acidification causes dispersal of the perinuclear lysosome population, whereas subsequent alkalinization returns them to their central location (Heuser, 1989;Parton et al, 1991). Other perturbations, such as aggresome formation (Zaarur et al, 2014), starvation (Korolchuk et al, 2011), drug-induced apoptosis (Yu et al, 2016), expression of pathogenic mutant forms of huntingtin (Erie et al, 2015) or leucine-rich repeat kinase 2 (LRRK2) (Dodson et al, 2012) and LSDs (Uusi-Rauva et al, 2012;Li et al, 2016b), result in perinuclear clustering of lysosomes. During movement, lysosomes sometimes tubulate in a process that might involve attachment to both kinesins and dynein pulling in opposite directions (Mrakovic et al, 2012;Li et al, 2016b).…”

Cholesterol-binding molecules MLN64 and ORP1L mark distinct late endosomes with transporters ABCA3 and NPC1

“…Another protein associating with the RAB7-RILP complex that controls LE transport is neuronal ceroid lipofuscinosis protein CLN3. When mutated, this protein causes classical juvenile onset neuronal ceroid lipofuscinosis, a fatal inherited neurodegenerative lysosomal storage disorder in which lipopigments accumulate in lysosomes ( 13 ). Proteins of the ATP binding cassette class A (ABCA) proteins have recently been shown to be present in LE compartments due to a conserved targeting motif ( 14 ).…”