It has been a while since you have heard from me. We are deep in post screen characterization of our NPC hits so I thought I would take a step back to the beginning to discuss NPC disease biology.
We focused a bit on the cholesterol and lipid metabolism aspects of Niemann Pick C disease biology but not so much on any other factors that might be contributing to disease progression at the cellular level. One study that stands out, performed by Sovan Sarkar et al., showed impairment of autophagic flux leads to disease pathology. Let’s get into the nitty gritty.
So what is autophagy? Autophagy is a cellular process activated to degrade proteins, organelles and cytosolic components and regulate lipid and cholesterol metabolism, ultimately to maintain cellular homeostasis. This pathway is engaged during cell starvation, hypoxia and other stresses. There are different types of autophagy that you can read an overview about here. Endosomes, lysosomes, autophagosomes, amphisomes and autolysosomes will be referenced in this post, and the diagram below will be helpful while walking through the data.
http://www.the-scientist.com/?articles.view/articleNo/31626/title/The-Enigmatic-Membrane/
Impairment of the autophagy pathway is implicated in the pathology of other neurodegenerative and liver diseases, however, up until this point, it was not known if there is a defect in autophagic flux in NPC mutant cells.
In order to confirm this, the steady-state number of autophagosomes was confirmed by looking at accumulation of LC3-II. LC3-II is a microtubule-associated protein used as a marker to identify autophagosomes. Npc1 +/+ and Npc1 -/- cells were treated with bafilomycin A (bafA1) preventing acidification of the lysosomes and subsequent fusion of lysosomes to autophagosomes, leading to an increase in LC3-II. NPC1 mutant cells display an increase in LC3-II as compared to controls, but when you add bafA1, the autophagy inhibitor, there is an equal amount of LC3-II detected in both control and Npc1-/- cells.
These data suggest that there is not an increase in synthesis of autophagosomes because blockade of degradation in control cells has the same amount of LC3-II as bafA1 inhibited Npc1-/- cells. If there was an increase in autophagosome synthesis, then blockade of autophagy should lead to an increase in LC3-II over control cells.
In order to dive into the autophagy mechanism difference, they performed mass spec to determine which genes are expressed differently between WT and Npc1-/- cells. It was noted that Rab7, a GTPase regulating endosomal/autophagosomal maturation as well as other endosomal markers, has increased expression levels in Npc1-/- cells. LC3-II and Rab7 colocalization can be used to mark amphisomes, the fusion of late endosomes with autophagosomes. There is a significant reduction in amphisomes in Npc1-/- cells as compared to control cells. This was confirmed with FITC-Dextran, which marks the endocytic pathway.
To understand why there is a decrease in amphisomes, researchers took a look at recruitment of SNARE machinery, which mediates vesicle fusion. They determined that there is a defect in the recruitment of the SNARE machinery components, VAMP8 and VAMP3, to the late endosomes in mutant cells, leading to the inability to form amphisomes.
If amphisomes do not form properly then autophagosomes will not mature and there will not be flux through the autophagic pathway. To monitor flux, they used EGFP-mRFP-LC3II reporter fusion protein. The EGFP tag will not fluoresce in acidic environments. So if the fusion protein localizes to autolysosomes then you will only see mRFP, but if in autophagosomes, there will be fluorescence from both mRFP and EGFP. Based on reporter fluorescence, control cells have autolysosomes, but the vesicles in mutant cells are all autophagosomes. In addition, the clearance of p62, a receptor that directs degradation of ubiquitinated proteins through the autophagy pathway, is decreased in Npc1-/- cells. This means there is decreased flux through the autophagy pathway in diseased cells.
To confirm the defects in autophagy, researchers transiently expressed NPC1-EGFP in Npc1-/- cells and found that expression rescues the autophagy impairment. This was observed by a reduction of p62 and LC3-II levels and a clearance of p62 aggregates and LC3 and lysotracker positive vesicles. Interestingly, treatment with HP-β-cyclodextrin, currently in clinical trial for NPC1, does not rescue the autophagy defect but does clear cholesterol accumulation.
To take this beyond the cellular level, investigators analyzed autophagic flux in liver and cerebellar tissue of Npc1-/- mice. In both organs, LC3-II and p62 levels are elevated. It is known that these organs degenerate in the NPC disease state so it is possible that impairment of autophagic flux is contributing to the pathogenesis. In neuronal culture knockdown of Npc1 leads to an increase in p62 levels, which are then cleared upon treatment with the autophagy inducer rapamycin.
All together, these data (plus more if you go right to the literature source!) show that NPC1 mutation leads to a defect in autophagic flux. It is this impairment that may explain the disease pathology observed in both the liver and brain of Niemann Pick C patients. And development of a therapeutic that rescues this impairment may be a viable treatment for NPC. Even though induction of autophagy does not clear the cholesterol accumulations, it does induce autophagosome-lysosome fusion allowing for the clearance of cargo trapped in the defective autophagy pathway.
