Autophagy Is Disrupted in a Knock-in Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis

Abstract

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 ⌬ex7/8 knock-in mice and CbCln3 ⌬ex7/8 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.Macroautophagy (herein referred to as autophagy) is a nonselective process by which cytoplasmic constituents are turned over (reviewed in Refs. 1 and 2). Elegant work in yeast has led to the genetic dissection of many of the proteins involved in this pathway (3, 4), which are mostly conserved in mammals (5, 6).The pathway is initiated at times of stress or nutritional deprivation to generate metabolites for cellular survival, but autophagy is also responsible for normal housekeeping. Although the regulatory control of autophagy remains to be fully elucidated, the general process from engulfment to degradation has been delineated (2). Initiation of the pathway involves de novo formation of an isolation membrane that expands to engulf cytoplasmic constituents, including organelles, and encloses to form a double membrane autophagic vacuole. This formed autophagic vacuole is then increasingly acidified, and proteases are delivered through fusion with late endosomes and lysosomes. The inner membrane is degraded to form a single membrane autolysosome, where constituents are finally completely degraded for recycling of metabolites to the cell.Autophagy is emerging as a major pathway involved in a number of neurodegenerative diseases, including Parkinson disease (7, 8), Huntington disease (9 -11), and Alzheimer disease (12, 13). In each case, autophagic vacuoles accumulate, suggesting that activation of autophagy is a common feature ...

Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function

“…In previous work employing a genetically accurate neuronal progenitor cell model of JNCL that bears a homozygous ϳ1-kb deletion in the murine Cln3 gene, recapitulating the most common genetic defect found in JNCL patients (7), we further demonstrated that Cln3 mutation leads to LC3-II-positive autophagosome accumulation, even preceding the onset of detectable storage material (17). To further dissect the autophagy pathway abnormalities caused by Cln3 mutation, here we have developed a high throughput, cell-based autophagy assay, employing the use of a green fluorescent protein-tagged LC3 transgene (GFP-LC3), stably expressed in our mouse Cln3 cell culture model of JNCL.…”

“…Although the established autophagy-related phenotypes in CLN3 models and the relationship of these to neurodegeneration in JNCL are incompletely understood, we further reasoned that developing a screening assay around autophagy in an accurate genetic model would serve as a useful starting point because abnormalities in autophagy are detected early in the disease process (17). Furthermore, the CLN3 protein is documented to be present in autophagosomes and autolysosomes/lysosomes (17), and defects in this pathway are hypothesized to be important in the development of mitochondrial ATPase subunit c-containing lysosomal storage material, a pathological hallmark in JNCL and other forms of NCL (17,42,50). Here, we have successfully developed a GFP-LC3-based autophagy assay in our murine cerebellar neuronal progenitor cell model of JNCL, and importantly, we have demonstrated that chemical biology studies using this model can successfully identify compounds with conserved bioactivity in human JNCL patient-derived disease relevant cell types.…”

“…Further work with the CbCln3 ⌬ex7/8/⌬ex7/8 cells identified elevated steady-state levels of LC3-II, which corresponded to an increased number of autophagosomes (17). To develop a high throughput version of this cell-based assay for further analysis of this phenotype and to identify autophagy modifiers in these cells, we subsequently established derivative wild type and homozygous CbCln3 ⌬ex7/8 cell lines stably expressing a GFP-LC3 transgene.…”

“…Although the primary function of CLN3 has not yet been fully uncovered it is proposed to play a role in vesicular trafficking because its deficiency leads to altered dis-tribution of endosomal and lysosomal proteins and phospholipids (7)(8)(9)(10)(11)(12), abnormal morphology of endocytic and lysosomal organelles (7,11), lysosomal pH dyshomeostasis (13)(14)(15), and amino acid transport defects (16). The hallmark JNCL storage material containing subunit c accumulates in both autophagosomes and lysosomes implicating further impact of CLN3 deficiency on the autophagy pathway (17).…”

Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease

“…13,14 The impaired autophagosomal-lysosomal fusion is likely to affect autophagic degradation of mitochondria. Indeed, mitochondrial dysfunction has been reported in several LSDs: GM1-gangliosidosis 15, 16 and mucolipidosis II, III, and IV, 17,18 neuronal ceroid lipofuscinosis or Batten disease, 19 Gaucher disease, 20 and multiple sulfatase deficiency. 21 However, accumulation of anomalous mitochondria in these diseases is not simply a result of autophagic block, but rather a consequence of a particular mechanism, which is disease-and even tissue-specific.…”