NPC is a rare, pediatric, genetically recessive neurological disease. The disease is caused by mutations in either Npc1 or Npc2. Loss of function in either the NPC1 or NPC2 protein results in accumulation of cholesterol and sphingolipids within the late endosomes/lysosomes. This disease causes progressive neurodegeneration, hepatomegaly and splenomegaly, and ultimately early death. Currently, this disease has no cure. Many experts consider NPC disease as ?childhood Alzheimer?s disease?. Both NPC1 and NPC2 bind to cholesterol. These 2 proteins work in concert to transport cholesterol out of the late endosomes/lysosomes to various cellular compartments, including plasma membrane (PM), Golgi, and endoplasmic reticulum (ER). Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme located at the ER. It utilizes cholesterol arriving at the ER as substrate to produce cholesteryl esters. Lacking functional NPC1 or NPC2 considerably slows the transport rate of cholesterol from the late endosomes/lysosomes to the ER. However, others and we have shown that, significant amount of cholesterol can translocate from the PM to the ER as serve as the substrate for ACAT1 for esterification, in NPC independent manner. We hypothesize that ACAT1 blockage (A1B) causes cholesterol to accumulate at the ER; this cholesterol pool moves to other subcellular membranes. In mutant NPC cells, the A1B action leads to partial fulfillment of cholesterol needs in membranes. To test this hypothesis, we conducted a mouse genetic experiment, by breeding a new mutant mouse model for NPC disease and the Acat1 gene KO mouse. The results show that Acat1 gene KO significantly delayed the clinical onset, prolonged the lifespan of the mutant Npc1 mouse by 34%, partially prevented Purkinje neuron loss in the cerebellum, and significantly improved foam cell pathology in the liver and spleen. We also performed pilot studies at the cell culture level and showed that, in mutant NPC1 cells, A1B, either by using Acat1 KO or by using a potent, small molecule ACAT1 inhibitor, restored the mislocalization of syntaxin 6, a cholesterol binding t-SNARE, back to TGN. In addition, A1B dissimilates the cholesterol laden, buoyant density late endo/lysosomes into several subcellular structures with heavier densities. A1B also restored the lower cathepsin D enzyme activity observed in the mutant NPC1 cells. In the current proposal, we propose three specific aims to further investigate the A1B actions.
Aim 1. Identify elements involved in the A1B mediated restoration of syntaxin 6 back to TGN.
Aim 2. Identify elements involved in the A1B mediated restoration in cathepsin D activity.
Aim 3. Test efficacy of a brain permeable small molecule ACAT inhibitor in ameliorating NPC disease.
The outcome of this proposal may identify a new candidate therapeutic target to alleviate Niemann-Pick Type C disease, a disease that many experts consider as childhood Alzheimer's disease, and has no cure at present.
