ABSTRACT
A disturbed autophagic pathway leads to chronically increased levels of autophagic vacuoles in Niemann Pick Type-C 1 (NPC1) deficient neurons. Since these accumulations potentially contribute to neuronal cell death associated with the disease, we investigated two pharmacological strategies which potentially reduce the number of autophagic structures under following aspects: efficiency, sustainability and effect on neuronal cell viability. The strategies comprised (i) an interruption of the autophagic flux by the class III PI3K inhibitor 3-methyladenine (3-MA) and (ii) an acceleration of the autophagic execution by 2-hydroxypropyl-ß-cyclodextrin (pCD). Our data show that the inhibition of autophagy with 3-MA only initially reduced the number of autophagic vacuoles in cultured neurons. Prolonged treatments with the PI3K-inhibitor reversed this lowering effect. The re-increase in the number of autophagic vacuoles was combined with a defect in the integrity of lysosomes which endangered further survival of cells. The treatment with pCD evoked a slow but sustained reduction of autophagic structures and had no negative effects on neuronal survival.
Subject(s)
Adenine/analogs & derivatives , Autophagy/drug effects , Enzyme Inhibitors/pharmacology , Neurons/cytology , Proteins/metabolism , Vacuoles/drug effects , beta-Cyclodextrins/pharmacology , 2-Hydroxypropyl-beta-cyclodextrin , Adenine/pharmacology , Animals , Cells, Cultured , Cerebral Cortex/cytology , Cholesterol/metabolism , Embryo, Mammalian , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Intracellular Signaling Peptides and Proteins , Lysosomal-Associated Membrane Protein 2/metabolism , Lysosomes/drug effects , Mice , Mice, Inbred BALB C , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , Neurons/drug effects , Neurons/metabolism , Niemann-Pick C1 Protein , Proteins/genetics , Time Factors , Vacuoles/metabolismABSTRACT
Niemann-Pick disease type C (NPC) is a fatal, progressive neurovisceral disorder. Several studies report that the autophagic flux is disturbed in NPC1-deficient (NPC1-/-) cells. Since it has been suggested that the autophagic defect may contribute to the neurodegeneration, we used cell cultures of NPC1-deficient and NPC1-wildtype neurons to investigate whether the disturbance influences neuronal survival. We found a genotype-dependent difference in survival, when autophagy is induced during culturing. NPC1-deficient neurons are more sensitive to rapamycin treatment and starvation than wildtype neurons. The subsequent search for defects in regulatory components of the autophagic pathway and the autophagic flux brought up results which differ from previous reports on somatic cells in one essential aspect: we exclude that an enhanced formation of autophagosomes contributes to the imbalanced autophagic flux in NPC1 deficient neurons. We found that solely the clearance of autophagosomes is delayed in these cells, which leads to an accumulation of autophagic vacuoles within the lysosomal compartment. Lowering the abnormal lipid load of the acidic organelles with cyclodextrin is sufficient to correct the autophagic flux and prevents premature death of NPC1-/- neurons under autophagic stress. From our results, we conclude that a pharmacological intervention in the neuropathology of NPC-disease should focus on the restoration of the lysosomal degradation capacity of cells.
