Abstract
Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6 nclf/nclf cerebellar cells and compared them to wild-type and CbCln3 Δex7/8/Δex7/8 cerebellar cells. CbCln6 nclf/nclf cells and CbCln3 Δex7/8/Δex7/8 cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6 nclf/nclf cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6 nclf/nclf and CbCln3 Δex7/8/Δex7/8 cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3 Δex7/8 and Cln6 nclf mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival.
Highlights
The neuronal ceroid lipofuscinoses (NCLs) collectively account for most cases of childhood-onset neurodegenerative disease worldwide, with clinical features of blindness, seizures, psychosis, motor and cognitive decline, and premature death
We have shown that homozygous CbCln6nclf cerebellar cells, like homozygous CbCln3Dex7/8 cerebellar cells, can be induced to accumulate mitochondrial ATP synthase subunit c in Lamp-1 positive compartments by aging in confluent culture conditions
PDI immunostain was decreased in homozygous CbCln6nclf cells and patient lymphoblasts, suggesting altered disposition of this proteinfolding chaperone, and the expression of endoplasmic reticulum (ER)-related gene sets were among the highest ranked in homozygous CbCln6nclf cells, suggesting that CLN6p is required for ER function and protein biosynthesis, perhaps of endosomal-lysosomal proteins given the clear impact of the loss of CLN6p function on endocytosis and lysosomal turnover of the subunit c protein
Summary
The neuronal ceroid lipofuscinoses (NCLs) collectively account for most cases of childhood-onset neurodegenerative disease worldwide, with clinical features of blindness, seizures, psychosis, motor and cognitive decline, and premature death (see recent reviews [1,2]). Given the overlapping clinical symptoms and disease pathology in the different forms of NCL, it has been proposed that the NCL genes encode proteins that function together or at different points in a common pathway, which most likely involves lipid and protein trafficking pathways and/or ion homeostasis [2,9]. Consistent with this hypothesis, protein-protein interaction between several NCLlinked proteins has been implicated by studies in overexpression or pull-down assay systems [10,11], and cross-correction of growth defects in patient cells by other CLN genes has been described [12]. With this set of genetic cell culture reagents, we have performed detailed, comparative phenotyping in order to determine the degree of overlap in abnormal cellular processes resulting from distinct NCL mutations
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