Discussion
CbCln3Δex7/8 cerebellar precursor cells represent the first genetically accurate neuron-derived culture model of JNCL. Homozygous CbCln3Δex7/8 cells express mutant battenin and JNCL-hallmark mitochondrial ATPase subunit c accumulation, upon aging of cells at confluency. Moreover, this is the first study to indicate recessive endosomal/lysosomal membrane trafficking defects and mitochondrial dysfunction that precedes subunit c deposition in an accurate JNCL model. Importantly, these defects are likely to be early events in the JNCL disease process and may particularly impact neuronal function.
Abnormal cathepsin D localization and processing in homozygous CbCln3Δex7/8 cells and Cln3Δex7/8 mice likely reflects altered vesicular trafficking and/or lysosomal pH, which is known to impact cathepsin D processing [14,16]. Indeed, CLN3 overexpression in HEK-293 cells altered lysosomal pH and cathepsin D processing [17], and lysosomal pH homeostasis is disrupted in JNCL [10,15]. It is noteworthy that cathepsin B and the CLN2-encoded enzyme, TPPI, are also altered in JNCL [18-20]. Nevertheless, despite the cathepsin D protein alterations that are observed in homozygous CbCln3Δex7/8 cells, cathepsin D enzymatic activity does not appear to be reduced. Thus, decreased cathepsin D activity is unlikely to account for subunit c accumulation in JNCL.
Aging of homozygous CbCln3Δex7/8 cells at confluency is necessary to induce significantly accumulated subunit c protein. However, membrane organelle disruptions precede subunit c accumulation in homozygous CbCln3Δex7/8 cells, since they are observed without aging at confluency. Lysosomal and endosomal size and distribution are altered, and mitochondria are abnormally elongated and functionally compromised in sub-confluent homozygous CbCln3Δex7/8 cultures. These observations argue that membrane trafficking defects do not result from excessive subunit c accumulation compromising the lysosome, but rather are early events in the disease process preceding subunit c accumulation. Mitochondrial abnormalities, which have also been reported in JNCL patients and other animal models [21-23], may result from ineffective turnover by autophagy, a lysosomal-targeted pathway [24]. Alternatively, or simultaneously, battenin deficiency may impact mitochondrial function upstream of turnover, affecting mitochondrial biogenesis and/or altered transport and processing of mitochondrial proteins.
In wild-type CbCln3 neuronal precursor cells battenin primarily co-localizes with early and late endosomes. Battenin immunostaining in homozygous CbCln3Δex7/8 neuronal precursors is significantly reduced in abundance, but mutant signal also co-localizes with endosomal markers suggesting mutant battenin protein with C-terminal epitopes is trafficked similar to wild-type protein. In other studies, CLN3/battenin protein localization has been reported to partially overlap with late endosomes and lysosomes in non-neuronal cells [7], and to lysosomes, synaptosomes [8] and endosomes [9,25] in neurons. These data jointly indicate that battenin resides in a subset of vesicular compartments linking multiple membrane trafficking pathways, perhaps functioning in vesicular transport and/or fusion. Endocytic and lysosomal-targeted pathways, including mitochondrial autophagy, are specifically implicated in this study.