Cln1‐mutations suppress Rab7‐RILP interaction and impair autophagy contributing to neuropathology in a mouse model of infantile neuronal ceroid lipofuscinosis

Abstract

Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease (LSD) caused by inactivating mutations in the CLN1 gene. CLN1 encodes palmitoyl‐protein thioesterase‐1 (PPT1), a lysosomal enzyme that catalyzes the deacylation of S‐palmitoylated proteins to facilitate their degradation and clearance by lysosomal hydrolases. Despite the discovery more than two decades ago that CLN1 mutations causing PPT1‐deficiency underlies INCL, the precise molecular mechanism(s) of pathogenesis has remained elusive. Here, we report that autophagy is dysregulated in Cln1−/− mice, which mimic INCL and in postmortem brain tissues as well as cultured fibroblasts from INCL patients. Moreover, Rab7, a small GTPase, critical for autophagosome‐lysosome fusion, requires S‐palmitoylation for trafficking to the late endosomal/lysosomal membrane where it interacts with Rab‐interacting lysosomal protein (RILP), essential for autophagosome‐lysosome fusion. Notably, PPT1‐deficiency in Cln1−/− mice, dysregulated Rab7‐RILP interaction and preventing autophagosome‐lysosome fusion, which impaired degradative functions of the autolysosome leading to INCL pathogenesis. Importantly, treatment of Cln1−/− mice with a brain‐penetrant, PPT1‐mimetic, small molecule, N‐tert (butyl)hydroxylamine (NtBuHA), ameliorated this defect. Our findings reveal a previously unrecognized role of CLN1/PPT1 in autophagy and suggest that small molecules functionally mimicking PPT1 may have therapeutic implications.

The neuronal ceroid lipofuscinosis‐related protein CLN8 regulates endo‐lysosomal dynamics and dendritic morphology

“…Accordingly, CLN8 may impact vesicular traffic in different ways: (1) by direct interaction with proteins associated with vesicular transport (Passantino et al., 2013); (2) by modulating the transport of soluble lysosomal proteins that affect lysosomal biogenesis (di Ronza et al., 2018; Bajaj et al., 2020). For example, palmitoyl protein thiosterase 1 (a lysosomal protein transported by the EGRESS complex; di Ronza et al., 2018; Bajaj et al., 2020) is necessary for the proper location of Rab7a and Rab7a/RILP interaction, proteins involved in the regulation of late endosome/lysosome movement (Sarkar et al., 2020); and (3) by modulating lipid biosynthesis and distribution within cells (Hermansson et al., 2005; Rusyn et al., 2008; Kuronen et al., 2012). Dysregulation of any of these processes can lead to incorrect distribution and recycling of proteins and, ultimately, alterations in vesicular traffic.…”

Autophagy in the Neuronal Ceroid Lipofuscinoses (Batten Disease)

“…As PE is a key lipid in autophagosome formation, Psd knockdown causes reduced levels of the LC3-PE conjugate ( Midorikawa et al, 2010 ). In a Ppt1 -deficient mouse model, LC3-II levels and autophagosome formation are increased, resulting in an increase in the numbers of autophagosomes ( Sarkar et al, 2020 ; Yun et al, 2020 ). In another study, LC3-II lipidation experiments showed lysosomal inhibition and a decrease in autophagosome accumulation in PPT1 -deficient human cells treated with autophagy inhibitors (e.g., chloroquine (CQ) or a chloroquine-derived molecule DC661), indicating that loss of PPT1 inhibits autophagy ( Rebecca et al, 2019 ).…”

“…In another study, LC3-II lipidation experiments showed lysosomal inhibition and a decrease in autophagosome accumulation in PPT1 -deficient human cells treated with autophagy inhibitors (e.g., chloroquine (CQ) or a chloroquine-derived molecule DC661), indicating that loss of PPT1 inhibits autophagy ( Rebecca et al, 2019 ). In patient-derived PPT1 -deficient fibroblasts, studies revealed that LC3-II levels did not differ with inhibition of lysosomal acidification in PPT1 -deficient cells but increased in fibroblasts derived from CLN1 disease patients, suggesting that autophagic vesicle turnover independent of lysosomal acidification is reduced ( Sarkar et al, 2020 ). Patient-derived PPT1 -deficient fibroblasts also display decreased LC3 colocalization with both LAMP1 and LAMP2, suggesting altered autophagosomal-lysosomal fusion ( Figure 3 ) ( Sarkar et al, 2020 ).…”

“…In patient-derived PPT1 -deficient fibroblasts, studies revealed that LC3-II levels did not differ with inhibition of lysosomal acidification in PPT1 -deficient cells but increased in fibroblasts derived from CLN1 disease patients, suggesting that autophagic vesicle turnover independent of lysosomal acidification is reduced ( Sarkar et al, 2020 ). Patient-derived PPT1 -deficient fibroblasts also display decreased LC3 colocalization with both LAMP1 and LAMP2, suggesting altered autophagosomal-lysosomal fusion ( Figure 3 ) ( Sarkar et al, 2020 ). In CLN1 disease models, including mice and human fibroblasts, co-localization of ras-related protein (RAB) 7 (RAB7) with LAMP2 is decreased, as well as poor interactions between RAB7 and RAB7-interacting lysosomal protein (RILP) ( Figure 3 ) ( Sarkar et al, 2020 ).…”

Interorganelle communication, aging, and neurodegeneration

“…It is an open question as to whether and how parkin regulates these contacts. Interestingly, CLN1 mutations, which cause a form of neuronal ceroid lipofuscinosis similar to CLN8, also suppress Rab7-RILP interaction, leading to impaired autophagy (Sarkar et al 2020).…”