👤 Janice A Espinola

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. Show less

Juvenile neuronal ceroid lipofuscinosis is caused by mutation of a novel, endosomal/lysosomal membrane protein encoded by CLN3. The observation that the mitochondrial ATPase subunit c protein accumulates in this disease suggests that autophagy, a pathway that regulates mitochondrial turnover, may be disrupted. To test this hypothesis, we examined the autophagic pathway in Cln3(Deltaex7/8) knock-in mice and CbCln3(Deltaex7/8) cerebellar cells, accurate genetic models of juvenile neuronal ceroid lipofuscinosis. In homozygous knock-in mice, we found that the autophagy marker LC3-II was increased, and mammalian target of rapamycin was down-regulated. Moreover, isolated autophagic vacuoles and lysosomes from homozygous knock-in mice were less mature in their ultrastructural morphology than the wild-type organelles, and subunit c accumulated in autophagic vacuoles. Intriguingly, we also observed subunit c accumulation in autophagic vacuoles in normal aging mice. Upon further investigation of the autophagic pathway in homozygous knock-in cerebellar cells, we found that LC3-positive vesicles were altered and overlap of endocytic and lysosomal dyes was reduced when autophagy was stimulated, compared with wildtype cells. Surprisingly, however, stimulation of autophagy did not significantly impact cell survival, but inhibition of autophagy led to cell death. Together these observations suggest that autophagy is disrupted in juvenile neuronal ceroid lipofuscinosis, likely at the level of autophagic vacuolar maturation, and that activation of autophagy may be a prosurvival feedback response in the disease process. Show less

JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a approximately 1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Deltaex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Deltaex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival. Show less