The effect of Torin1, an mTORC1 kinase inhibitor, on Niemann-Pick type C disease cellular phenotype.
Niemann-Pick type C disease (NPC), one of more than 50 lysosomal storage diseases, is
characterized by the lysosomal accumulation of unesterified cholesterol. Most lysosomal
storage diseases do not have curative therapies available and NPC is no exception. NPC
is a fatal neurovisceral disease and the majority of patients succumb to death by
late-teens to early adult-hood years. ... read moreAlthough individual lysosomal storage diseases are
rare, collectively, the prevalence is 1 in every 5,000 live births. Recently, a
potential novel therapeutic target for lysosomal storage diseases as a group has
garnered attention. Transcription factor EB (TFEB) is a master regulator of lysosomal
biogenesis that, when overexpressed, induced lysosomal exocytosis and clearance of
pathogenic accumulant in several lysosomal storage diseases in vitro and in vivo. Thus,
my doctoral project aim was to determine whether TFEB activation is a potential
therapeutic target for NPC. Here, I tested the hypothesis that pharmacological
activation of endogenous TFEB will clear lysosomally stored cholesterol from NPC cells.
In order to promote TFEB activation, a small molecule called Torin1 was used to inhibit
mTORC1 kinase, a negative regulator of TFEB activity. In NPC cells, Torin1 treatment
promoted TFEB nuclear translocation and induction of lysosomal genes. Intriguingly,
Torin1 induced the expression of lysosomal genes even with RNAi-mediated TFEB silencing,
suggesting another transcriptional regulator is responsible. Remarkably, NPC cells
treated with Torin1 showed a reduction in lysosomal cholesterol, which was measured by
fluorescent cholesterol marker. Assays were conducted to determine the fate of
cholesterol upon release from Torin1-treated lysosomes; however, no changes were
observed in trafficking of cholesterol to other intracellular membranes. To our
surprise, measurement of cholesterol content in isolated NPC lysosomes showed Torin1
treatment did not alter the amount of this lipid. Taken together, the findings suggest
Torin1 is not a therapeutic option for NPC disease and further experiments should
include testing TFEB overexpression in NPC. In addition, this work suggests the
existence of transcriptional regulator(s) other than TFEB that regulate lysosomal gene
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Cellular & Molecular Physiology.
Advisors: Laura Liscum, and Jerry Faust.
Committee: Gary Sahagian, Maribel Rios, Eric Frank, and Barbara Schreiber.
Keyword: Physiology.read less