Maturation and detoxification of synphilin-1 inclusion bodies regulated by sphingolipids

Reviewer #1 (Public Review):

The authors have shown the following:
1. SY1 aggregation enhances (in terms of number of aggregates) when Sphingolipid biosynthesis is blocked.
2. In a normal cell (where sphingolipid biosynthesis is not hampered), the aggregate of SY1 (primarily the Class I aggregate) is localized only on the mitochondrial endomembrane system.
3. The localization is due to the association of SY1 (aggregates) with mitochondrial proteins like Tom70, Tim44, etc. (Is the localization completely lost? What happens to the toxicity when the aggregates are not localized on mitochondria?)
4. This fuels the loss of mitochondrial function.
5. Mitochondrial function is further abrogated when there is a block in sphingolipid biosynthesis.
6. A similar phenomenon is conserved in mammalian cell lines.

However, my major concern is that the role of sphingolipid in the mitochondrial association of the aggregates is not proven beyond doubt. I am also missing the importance of mitochondrial association in the context of IB maturation and cellular toxicity.

Reviewer #2 (Public Review):

Summary:
The authors used a yeast model for analyzing Parkinson's disease-associated synphilin-1 inclusion bodies (SY1 IBs). In this model system, large SY1 IBs are efficiently formed from smaller potentially more toxic SY1 aggregates. Using a genome-wide approach (synthetic genetic array, SGA, combined with a high-content imaging approach), the authors identified the sphingolipid metabolic pathway as pivotal for SY1 IBs formation. Disturbances of this pathway increased SY1-triggered growth deficits, loss of mitochondrial membrane potential, increased production of reactive oxygen species (ROS), and decreased cellular ATP levels pointing to an increased energy crisis within affected cells. Notably, SY1 IBs were found to be surrounded by mitochondrial membranes using state-of-the-art super-resolution microscopy. Finally, the effects observed in the yeast for SY1 IBs turned out to be evolutionarily conserved in mammalian cells. Thus, sphingolipid metabolism might play an important role in the detoxification of misfolded proteins by large IBs formation at the mitochondrial membrane.

Strengths:
• The SY1 IB yeast model is very suitable for the analysis of genes involved in IB formation.
• The genome-wide approach combining a synthetic genetic array (SGA) with a high-content imaging approach is a compelling approach and enables the reliable identification of novel genes. The authors tightly checked the output of the screen.
• The authors clearly showed, including a couple of control experiments, that the sphingolipid metabolic pathway is crucial for SY1 IB formation and cytotoxicity.
• The localization of SY1 IBs at mitochondrial membranes has been clearly demonstrated with state-of-the-art super-resolution microscopy and biochemical methods.
• Pharmacological manipulation of the sphingolipid pathway influenced mitochondrial function and cell survival.

Weaknesses:
• It remains unclear how sphingolipids are involved in SY1 IB formation.
• It remains undefined whether failure of sphingolipid-dependent SY1 IB formation from smaller potentially more toxic aggregates occurs at the mitochondrial membrane.
• It remains open whether mitochondrial activity (e.g., respiratory activity) is needed for sphingolipid-dependent SY1 IB formation.