A function retained by the common mutant CLN3 protein is responsible for the late onset of juvenile neuronal ceroid lipofuscinosis

Abstract

The neuronal ceroid lipofuscinoses (NCLs) are common neurodegenerative disorders of childhood and are classified as lysosomal storage diseases since affected cells exhibit lysosomes containing ceroid and lipofuscin-like material. CLN3 is the most widely conserved NCL gene, suggesting that it has a basic eukaryotic cell function; its loss might be expected to cause the earliest onset and/or most severe disease. However, mutations in CLN3 are linked to juvenile NCL (JNCL), the latest onset and mildest form of NCL in children. We sought to explain this paradox. Almost all patients with JNCL are homozygous or heterozygous for an intragenic 1 kb deletion within CLN3, hitherto presumed to be a null mutation. We hypothesized that the 1 kb mutation may allow CLN3 residual function. We confirmed the presence of CLN3 transcripts in JNCL patient cells. When RNA silencing was used to deplete these transcripts in cells from JNCL patients, the lysosomes significantly increased in size, confirming the presence of functional protein in these cells. Consistently, overexpression of mutant CLN3 transcript caused lysosomes to decrease in size. We modelled the JNCL mutant transcripts and those corresponding to mouse models for Cln3 in Schizosaccharomyces pombe and confirmed that most transcripts retained significant function as we predicted. Therefore, we concluded that the common mutant CLN3 protein does indeed retain significant function and that JNCL is a mutation-specific disease phenotype. This finding has important consequences for recognition and diagnosis of disease caused by mutations in CLN3 and for the development of therapy for JNCL.

S. pombe btn1, the orthologue of the Batten disease geneCLN3, is required for vacuole protein sorting of Cpy1p and Golgi exit of Vps10p

“…Indeed, a location of CLN3 in the Golgi has been noted previously (Haskell et al, 1999;Kremmidiotis et al, 1999). Importantly, yeast genetics have allowed us to study the effects of complete absence of btn1, which is particularly important in the light of our recent finding that cells from JNCL patients retain partial CLN3 function (Kitzmüller et al, 2008).…”

“…A thorough study of the lysosome proteome has not been performed in cells completely lacking CLN3 function. However, in JNCL cells, or in cells in which CLN3 function is substantially or partially reduced, alterations in trafficking or processing of specific lysosomal enzymes have been reported (Fossale et al, 2004;Junaid and Pullarkat, 1999;Metcalfe et al, 2008;Pohl et al, 2007;Sleat et al, 1998), and lysosomal homeostasis is affected (Holopainen et al, 2001;Kitzmüller et al, 2008;Ramirez-Montealegre and Pearce, 2005). In one instance, exit of a reporter version of the cation-independent M6PR from the TGN was significantly reduced when CLN3 was depleted (Metcalfe et al, 2008).…”

Detailed Clinical Phenotype and Molecular Genetic Findings in CLN3-Associated Isolated Retinal Degeneration

“…5,47 The major predicted protein product from this deletion consists of the first 153 amino acids from exons 1 to 6 plus an additional 28 novel amino acids. 70 Although this mutation results in decreased mRNA transcript levels (eFigure 4 in the Supplement), 71 it remains controversial whether alternatively spliced products are expressed or degraded. Detailed in silico analysis predicts nonsense-mediated decay leading to loss of function with homozygous 1.02-kb deletions.…”

btn1affects cytokinesis and cell-wall deposition by independent mechanisms, one of which is linked to dysregulation of vacuole pH

“…It is tempting to speculate that pH-related defects underlie the characteristic storage of JNCL, and that neuronal cell death arises from the particular sensitivity of neuronal cells to defects in a second pathway affecting polarized growth, possibly in response to external stress. Given that we have recently shown that JNCL is a mutation-specific phenotype arising from partial loss of function of CLN3 (Kitzmüller et al, 2008), it will be necessary to use a total-loss-of-function system such as ours to further define the mechanism of action of CLN3 and correlate this with the JNCL disease phenotype.…”