The role of sphingolipid metabolism in coupled lysosome and mitochondrial dysfunction in neurodegenerative disease models.

Background
Both mitochondria and lysosomes are key regulators of cellular metabolism and dysfunction of these two organelles has been associated with neurodegenerative diseases including Parkinson's disease (PD). Recent studies show the complex network and importance of membrane contact sites (MCS) in providing platforms for protein interactions, signaling events, lipid exchange and cholesterol homeostasis (Martello et al., 2020).
Niemann Pick type-C (NPC) disease (Vanier, 2010) is a progressive neurodegenerative disease characterized on a cellular level by cholesterol accumulation in the late endosomes/lysosomes (LE/Lys), that has been used to decipher the role of MCS and study sterol trafficking (Colaco et al., 2020; Höglinger et al., 2019; Meng et al., 2020; Torres et al., 2017). NPC1 mutant cells are associated with impaired mitochondrial fission and increased cholesterol in lysosomes and mitochondria (Saffari et al., 2017; Torres et al., 2017; Wos et al., 2016). Previous work by the group showed striking STARD3-dependent expansion of LE/Lys:mitochondria MCS in NPC1-deficient cells (Höglinger et al., 2019). This expansion may be linked to mitochondrial cholesterol import from lysosomes triggering mitochondrial dysfunction. Recent work from the group also showed that LE/Lys:ER MCS expansion by expression of LE/Lys tethering protein ORPP1L, reverses cholesterol accumulation in NPC cellular models (Höglinger et al., 2019).
Mutations of the glucocerebrosidase (GBA1) gene is considered the most important identified genetic vulnerability factor for PD, where the risk of developing PD is increased 20-30 fold (Sidransky et al., 2009). GBA1 encodes Beta-glucocerebrosidase (GCase) that catalyses the hydrolysis of glucosylceramide to glucose and ceramide. GBA1 mutation carrier PD patients exhibit more severe cognitive symptoms and earlier age of onset (Do et al., 2019). Recent study showed that GBA1 mutant PD patient derived neurons have prolonged LE/Lys:mitochondria MCS caused by defective modulation of the untethering protein TBC1D15, which mediates Rab7 GTP hydrolysis for contact untethering (Kim et al., 2021). This dysregulation was linked to decreased GBA1 lysosomal enzyme activity in patient derived neurons and could be rescued by increasing enzyme activity with a GCase modulator. These defects resulted in disrupted mitochondrial distribution and function and could be further rescued by TBC1D15 in PD patient derived GBA1-linked neurons. In addition, preliminary studies by our group also showed increased LE/Lys:mitochondria MCS in sphingosine kinase CRISPR KO cells, where the glucosylceramide precursor ceramide is elevated. Interestingly, in addition to transporting cholesterol, NPC1 was recently shown to mediate the egress of lysosomal sphingosine (Altuzar et al., 2021), likely accounting for the accumulation of sphingolipids in NPC1-deficient cells and possibly contributing to the expanded LE/Lys:mitochondria MCS.
Our aim for this PhD project is to characterize LE/Lys:mitochondria MCS dynamics and mitophagy in iPSC neuronal cells derived from NPC1 patients compared with healthy controls. We further aim to study the relationship between sphingolipid metabolism and LE/Lys:mitochondria MCS dynamics.
Aims
Aim 1: Characterization of LE/Lys:mitochondria positioning, dynamics and MCS in NPC1 neuronal cell model.

Aim 2: Identify compounds that affect lysosome-mitochondria MCS.

Aim 3: Validation of results using in vivo zebrafish model