Pre-synaptic defects of NPC1-deficient hippocampal neurons are not directly related to plasma membrane cholesterol

Imbalances in brain cholesterol homeostasis have been observed in several neurodegenerative diseases. In Niemann-Pick Type C (NPC) disease, mutations in NPC1 or NPC2 lead to endosomal cholesterol accumulation, neuronal dysfunction and death. Cholesterol in synaptic plasma membranes influences membrane fluidity, curvature, and protein function, and its depletion may adversely affect synaptic vesicle cycling. We have investigated pre-synaptic function in primary hippocampal neurons with altered cholesterol distribution because of NPC1 deficiency or cyclodextrin treatment. In NPC1-deficient neurons grown in serum-free medium, plasma membrane cholesterol was reduced and total synaptic vesicle release during prolonged stimulation was attenuated. In NPC1-deficient neurons cultured in the presence of high-density lipoproteins, plasma membrane cholesterol markedly increased, but the defects in synaptic vesicle release in NPC1-deficient neurons were exacerbated. Treatment with 1 mM methyl-beta-cyclodextrin acutely depleted plasma membrane cholesterol in wild-type neurons to levels below those in NPC1 deficiency, but did not alter synaptic vesicle exo- or endocytosis. Defects only became apparent when higher methyl-beta-cyclodextrin concentrations were used. Our data indicate that synaptic vesicle release can tolerate some degree of plasma membrane cholesterol depletion and suggest that the pre-synaptic defects in NPC1-deficient neurons are not solely caused by a reduction of plasma membrane cholesterol.