Reports of research work funded by grants prior to 2016

Victoria University of Wellington

Determining the Mechanism and Therapeutic Efficacy of the LIPIN-1 Modifier on Cerebellar Neurodegeneration in Niemann-Pick Type C Disease

AB Munkacsi
School of Biological Sciences

Niemann-Pick type C (NPC) disease is a fatal, paediatric neurodegenerative disease due to lysosomal accumulation of cholesterol and sphingolipids.  Currently, there is no FDA-approved therapy to treat NPC disease.  In 2011, our laboratory reported the amelioration of the lipid accumulation in NPC patient fibroblasts with a drug that is FDA-approved to treat cancer; however, in subsequent studies in our laboratory, we have determined that this drug does not ameliorate neurodegeneration in the Npc1-/- mouse model of NPC disease.  Thus, other candidate therapies must be identified.  The objectives of this project were to test the therapeutic efficacy of the lipin-1 modifier of NPC disease, a modifier that I have identified and have not yet published.  I have completed a significant portion of these aims with WMRF funding and anticipate publishing these results in the next six months.  Specifically, my progress with the WMRF funding is the following:

Aim 1

To identify the mechanism by which murine lipin-1 exacerbates NPC disease severity.  We crossed the Npc1+/- mice with the Lpin1+/- mice to generate Npc1-/- Lpin1-/- mice.  We recovered only 8 out of 299 Npc1-/- Lpin1-/- animals, a result that is not significantly consistent with the expected Mendelian ratio of recovering one double mutant every 16 births (χ2 test, P = 0.0107), suggesting the lipin-1 deficiency causes embryonic lethality of the Npc1-/- mice. Of the 8 Npc1-/- Lpin1-/- mice that did survive, these animals died between pre-birth and six weeks, a lifespan that is significantly shorter than the 10-12 week lifespan of the Npc1-/- mouse and the 44-48 week lifespan of the Lpin1-/- mouse.  In addition to exacerbated lethality, the Npc1-/- Lpin1-/- animals exhibited enhanced motor function decline compared to their parental single mutant animals, though this was not quantified given there were not enough Npc1-/- Lpin1-/- animals, due to the exacerbated lethality, to commit to the training time required for a balance beam assay conducted weekly starting at week three.  To examine cells regulating ataxia, we visualised the Purkinje cell layer in the cerebellum. At P44, the Purkinje cell layer is not observed in the Npc1-/- Lpin1-/- animal while it is observed in the WT, Npc1-/-, and Lpin1-/- mice, a result that is in agreement with the exacerbated motor function deficit. In summary, we demonstrate that lipin-1 deficiency exacerbates NPC disease phenotypes in the Npc1-/- mouse.

Given the recent observation that lipin-1 is required for macroautophagy in muscle cells and the previous demonstrations of macroautophagic defects in NP-C disease, we hypothesized that macroautophagy was a mechanism underlying the lipin-mediated exacerbation of NP-C disease.  Macroautophagy is a fundamental cellular process in which cytosolic proteins and organelles are engulfed by an autophagosome in the cytoplasm and degraded in the lysosome.  We quantified the induction of macroautophagy by examining levels of the autophagosome marker, microtubule-associated protein-1 light chain 3 (LC3-II).  In the liver, LC3-II levels were significantly increased in each of the Lpin1-/- and Npc1-/- animals compared to WT animals by 1.8- and 2.9-fold, respectively. LC3-II levels were further increased in the Npc1-/- Lpin1-/- animals by 31.3-, 17.2-, and 10.5-fold compared to WT, Lpin1-/-, and Npc1-/- animals, respectively (Fig. 1).  In the cerebellum, LC3-II levels were not significantly increased in the Lpin1-/- animals and were significantly increased by 119-fold in the Npc1-/- animals compared to WT animals.  Strikingly in the Npc1-/- Lpin1-/- animals, LC3-II was increased 215-fold compared to WT and 1.8-fold compared to Npc1-/- animals (Fig. 1).  These results demonstrate that lipin-1 deficiency dramatically exacerbates the macroautophagic defect in the liver and cerebellum of Npc1-/- animals.