A Murine Central Nervous System-Derived Cell Line Carrying a NPC1 Gene Mutation and Expressing Multipotent Neural Stem Cell-Like Properties

The true substrate of NPC1 is controversial and not fully understood at the current time. In this presentation, we provide experimental evidence to support a novel functional role for NPC1, which involves the intracellular trafficking of amine-containing molecules. Specifically, we propose that NPC1 has the ability to sense when lysosomes become markedly concentrated with these molecules, which leads to the formation of a hybrid organelle capable of facilitating their cellular release. We demonstrate that the hybrid organelle originates from a heterotypic fusion event involving lysosomes with late endosomes. The hybrid organelle is unique in that is capable of significant expansion, referred to as vacuolization, the function of which is presumed to quickly dissipate osmotic pressure associated with the highly concentrated amine. The hybrid organelle subsequently facilitates the release of the lysosomal cargo to the plasma membrane. To better understand this specific trafficking event we employed a pulse-chase technique to localize [H]dextran to lysosomes and monitored secretion to the extracellular space as a function of time. It is well known that NPC1 is required for the efficient release of multiple lysosomal cargo relative to cells with mutated NPC1. Conversely, agents have been identified that can cause a NP-C disease phenotype by apparently inhibiting these secretion events; however, to our knowledge, no one has previously shown that NPC1 function in this secretion event could be stimulated, which, in turn, may provide insight into the true substrate of the protein. Using this secretion assay we revealed that certain amines were able to significantly increase the secretion of lysosomal dextran above untreated normal cells; whereas the same amines had no influence on cells without functional NPC1, ruling out a nonspecific influence. Furthermore, the relatively short exposure time required for the aforementioned stimulation rules out the possibility of NPC1 up-regulation as a cause. The stimulation appeared to be concentration dependent and was limited to those amines that were subject to extensive accumulation into lysosomes. These results provide an important foundation for future studies aimed at the identification of the true functional role and subsrates for this intriguing protein. 1. Neufeld EB, Wastney M, Patel S, Suresh S, Cooney AM, Dwyer NK, Roff CF, Ohno K, Morris JA, Carstea ED, Incardona JP, Strauss JF, 3rd, Vanier MT, Patterson MC, Brady RO, Pentchev PG, Blanchette-Mackie EJ 1999. The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo. J Biol Chem 274(14):9627-9635. 2. Roff CF, Goldin E, Comly ME, Cooney A, Brown A, Vanier MT, Miller SP, Brady RO, Pentchev PG 1991. Type C Niemann-Pick disease: use of hydrophobic amines to study defective cholesterol transport. Dev Neurosci 13(4-5):315-319.