Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy

Impairment of peripheral nerve function is frequent in neurometabolic diseases, but mechanistically not well understood. Here, we report a novel disease mechanism and the finding that glial lipid metabolism is critical for axon function, independent of myelin itself. Surprisingly, nerves of Schwann cell-specific Pex5 mutant mice were unaltered regarding axon numbers, axonal calibers, and myelin sheath thickness by electron microscopy. In search for a molecular mechanism, we revealed enhanced abundance and internodal expression of axonal membrane proteins normally restricted to juxtaparanodal lipid-rafts. Gangliosides were altered and enriched within an expanded lysosomal compartment of paranodal loops. We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy, preceding disease-onset by one year. Thus, peroxisomal dysfunction causes secondary failure of local lysosomes, thereby impairing the turnover of gangliosides in myelin. This reveals a new aspect of axon-glia interactions, with Schwann cell lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels

The UbiB family member Cqd1 forms a novel membrane contact site in mitochondria

Mitochondria are essential organelles of eukaryotic cells and are characterized by their unique and complex membrane system. They are confined from the cytosol by an envelope consisting of two membranes. Signals, metabolites, proteins and lipids have to be transferred across these membranes via proteinaceous contact sites to keep mitochondria functional. In the present study, we identified a novel mitochondrial contact site in Saccharomyces cerevisiae that is formed by the inner membrane protein Cqd1 and the outer membrane proteins Por1 and Om14. Similar to what is found for the mitochondrial porin Por1, Cqd1 is highly conserved, suggesting that this complex is conserved in form and function from yeast to human. Cqd1 is a member of the UbiB protein kinase-like family (also called aarF domain-containing kinases). It was recently shown that Cqd1, in cooperation with Cqd2, controls the cellular distribution of coenzyme Q by a yet unknown mechanism. Our data suggest that Cqd1 is additionally involved in phospholipid homeostasis. Moreover, overexpression of CQD1 and CQD2 causes tethering of mitochondria to the endoplasmic reticulum, which might explain the ability of Cqd2 to rescue ERMES deletion phenotypes

Quantification of Signaling Lipids by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI MS/MS)

Lipids, such as phosphoinositides (PIPs) and diacylglycerol (DAG), are important signaling intermediates involved in cellular processes such as T cell receptor (TCR)-mediated signal transduction. Here we report identification and quantification of PIP, PIP2 and DAG from crude lipid extracts. Capitalizing on the different extraction properties of PIPs and DAGs allowed us to efficiently recover both lipid classes from one sample. Rapid analysis of endogenous signaling molecules was performed by nano-electrospray ionization tandem mass spectrometry (nano-ESI MS/MS), employing lipid class-specific neutral loss and multiple precursor ion scanning for their identification and quantification. Profiling of DAG, PIP and PIP2 molecular species in primary human T cells before and after TCR stimulation resulted in a two-fold increase in DAG levels with a shift towards 1-stearoyl-2-arachidonoyl-DAG in stimulated cells. PIP2 levels were slightly reduced, while PIP levels remained unchanged

Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites

Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. Here we reveal a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or receptor signaling triggers depletion of cholesterol and associated Gb3 from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites

Analysis of Transmembrane Domains and Lipid Modified Peptides with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

Protein–lipid interactions within the membrane are difficult to detect with mass spectrometry because of the hydrophobicity of tryptic cleavage peptides on the one hand and the noncovalent nature of the protein–lipid interaction on the other hand. Here we describe a proof-of-principle method capable of resolving hydrophobic and acylated (e.g., myristoylated) peptides by optimizing the steps in a mass spectrometric workflow. We then use this optimized workflow to detect a protein–lipid interaction <i>in vitro</i> within the hydrophobic phase of the membrane that is preserved via a covalent cross-link using a photoactivatable lipid. This approach can also be used to map the site of a protein–lipid interaction as we identify the peptide in contact with the fatty acid part of ceramide in the START domain of the CERT protein

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions

Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy

Impairment of peripheral nerve function is frequent in neurometabolic diseases, but mechanistically not well understood. Here, we report a novel disease mechanism and the finding that glial lipid metabolism is critical for axon function, independent of myelin itself. Surprisingly, nerves of Schwann cell-specific Pex5 mutant mice were unaltered regarding axon numbers, axonal calibers, and myelin sheath thickness by electron microscopy. In search for a molecular mechanism, we revealed enhanced abundance and internodal expression of axonal membrane proteins normally restricted to juxtaparanodal lipid-rafts. Gangliosides were altered and enriched within an expanded lysosomal compartment of paranodal loops. We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy, preceding disease-onset by one year. Thus, peroxisomal dysfunction causes secondary failure of local lysosomes, thereby impairing the turnover of gangliosides in myelin. This reveals a new aspect of axon-glia interactions, with Schwann cell lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels.status: publishe