glycogen and Neuronal-Ceroid-Lipofuscinoses

Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: Vision loss not only is often one of the earliest symptoms of JNCL, but also has been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J background. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also reported for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.

Topics: Animals; Atrophy; Autophagy; Blindness; Cell Line; Disease Models, Animal; Gene Knock-In Techniques; Gene Knockdown Techniques; Glycogen; Humans; Lysosomes; Membrane Glycoproteins; Mice; Mice, Transgenic; Microscopy, Electron; Molecular Chaperones; Mutation; Neuronal Ceroid-Lipofuscinoses; Retinal Pigment Epithelium; RNA, Small Interfering

The neuronal ceroid lipofuscinoses (NCLs) constitute a group of autosomal recessive neurodegenerative diseases affecting children. To date, the disease pathogenesis remains unknown, although the role of lysosomal impairment is widely recognized across the different diseases. Recently, the creation of simple models of juvenile NCL (Batten disease) has provided additional insights into the disease mechanism at the molecular level. We report defects in metabolism identified in the Schizosacchromyces pombe yeast model, where btn1, the orthologue of CLN3, has been deleted, using a metabolomics approach based on high resolution 1H and 13C NMR spectroscopy. Such changes represent the first documented metabolic changes associated with deletion of btn1. A decrease in extracellular glucose and increases in the concentration of extracellular ethanol and alanine labelling demonstrate increased glycolytic flux that may arise from vacuolar impairment, whilst amino acid changes were detected which were also in accordance with defective vacuolar functionality. That these changes were detected using a metabolomic based approach advocates its use to further analyse other yeast models of human disease to better understand the function of orthologue genes.

Topics: Amino Acids; Cell Division; Child; Cytoplasmic Granules; Gene Deletion; Glucose; Glycerol; Glycogen; Glycolysis; HeLa Cells; Humans; Magnetic Resonance Spectroscopy; Membrane Glycoproteins; Membrane Proteins; Metabolomics; Microscopy, Electron, Transmission; Molecular Chaperones; Neuronal Ceroid-Lipofuscinoses; RNA Interference; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Sucrose

Ultrastructural studies of uncultured amniotic fluid cells obtained by genetic amniocentesis at 16 wk of gestation demonstrated 3 major cell types. Membrane bound curvilinear cytosomes were observed in about 30% of a subpopulation of dark, elongated cells. These are considered typical of the inclusions of the late infantile variant of neuronal ceroid-lipofuscinosis. This technique was used to monitor 6 at-risk pregnancies of which 2 were identified as affected. We have followed 6 of the 7 fetuses through to delivery with confirmation of our findings by skin biopsy in 4 and with clinical observations of a fifth child. There are major problems involved in the use of uncultured amniotic fluid cells for prenatal diagnosis. In addition to a great deal of heterogeneity of cell type, there is a considerable amount of tissue debris and a very high proportion of nonviable cells. We have examined chorionic villus tissues of 3 fetuses known to have inborn errors of lysosomal metabolism without finding any evidence of storage material. This is taken as an indication that the mutant gene(s) is not expressed in these tissues at this early stage of pregnancy. Notwithstanding these limitations, the usefulness of this technique in monitoring at-risk pregnancies has to be determined.

Topics: Amniocentesis; Amniotic Fluid; Child, Preschool; Chorionic Villi; Female; Glycogen; Humans; Male; Microscopy, Electron; Neuronal Ceroid-Lipofuscinoses; Organelles; Pregnancy; Prenatal Diagnosis