Glycosphingolipid (GSL) accumulation is implicated in the neuropathology of several lysosomal conditions, such as Krabbe disease, and may also contribute to neuronal and glial dysfunction in adult-onset conditions such as Parkinson's disease, Alzheimer's disease and multiple sclerosis. GSLs accumulate in cellular membranes and disrupt their structure; however, how membrane disruption leads to cellular dysfunction remains unknown. Using authentic cellular and animal models for Krabbe disease, we provide a mechanism explaining the inactivation of lipid raft (LR)-associated IGF-1–PI3K–Akt–mTORC2, a pathway of crucial importance for neuronal function and survival. We show that psychosine, the GSL that accumulates in Krabbe disease, leads to a dose-dependent LR-mediated inhibition of this pathway by uncoupling IGF-1 receptor phosphorylation from downstream Akt activation. This occurs by interfering with the recruitment of PI3K and mTORC2 to LRs. Akt inhibition can be reversed by sustained IGF-1 stimulation, but only during a time window before psychosine accumulation reaches a threshold level. Our study shows a previously unknown connection between LR-dependent regulation of mTORC2 activity at the cell surface and a genetic neurodegenerative disease. Our results show that LR disruption by psychosine desensitizes cells to extracellular growth factors by inhibiting signal transmission from the plasma membrane to intracellular compartments. This mechanism serves also as a mechanistic model to understand how alterations of the membrane architecture by the progressive accumulation of lipids undermines cell function, with potential implications in other genetic sphingolipidoses and adult neurodegenerative conditions.

Highlights d SRC activation causes temporally distinct effects on the endothelial cell barrier d Initially, SRC causes endothelial barrier enhancement and VE cadherin rearrangement d VE cadherin phosphorylation on Y731 is required for SRCmediated barrier enhancement d Prolonged SRC activity cause barrier disruption

Lysosomal storage diseases (LSDs) with neurological involvement are inherited genetic diseases of the metabolism characterized by lysosomal dysfunction and the accumulation of undegraded substrates altering glial and neuronal function. Often, patients with neurological manifestations present with damage to the gray and white matter and irreversible neuronal decline. The use of animal models of LSDs has greatly facilitated studying and identifying potential mechanisms of neuronal dysfunction, including alterations in availability and function of synaptic proteins, modifications of membrane structure, deficits in docking, exocytosis, recycling of synaptic vesicles, and inflammation-mediated remodeling of synapses. Although some extrapolations from findings in adult-onset conditions such as Alzheimer’s disease or Parkinson’s disease have been reported, the pathogenetic mechanisms underpinning cognitive deficits in LSDs are still largely unclear. Without being fully inclusive, the goal of this mini-review is to present a discussion on possible mechanisms leading to synaptic dysfunction in LSDs.

Krabbe Disease (KD) is a lysosomal storage disorder characterized by the genetic deficiency of the lysosomal enzyme β-galactosyl-ceramidase (GALC). Deficit or a reduction in the activity of the GALC enzyme has been correlated with the progressive accumulation of the sphingolipid metabolite psychosine, which leads to local disruption in lipid raft architecture, diffuse demyelination, astrogliosis, and globoid cell formation. The twitcher mouse, the most used animal model, has a nonsense mutation, which limits the study of how different mutations impact the processing and activity of GALC enzyme. To partially address this, we generated two new transgenic mouse models carrying point mutations frequently found in infantile and adult forms of KD. Using CRISPR-Cas9 gene editing, point mutations T513M (infantile) and G41S (adult) were introduced in the murine GALC gene and stable founders were generated. We show that GALCT513M/T513M mice are short lived, have the greatest decrease in GALC activity, have sharp increases of psychosine, and rapidly progress into a severe and lethal neurological phenotype. In contrast, GALCG41S/G41S mice have normal lifespan, modest decreases of GALC, and minimal psychosine accumulation, but develop adult mild inflammatory demyelination and slight declines in coordination, motor skills, and memory. These two novel transgenic lines offer the possibility to study the mechanisms by which two distinct GALC mutations affect the trafficking of mutated GALC and modify phenotypic manifestations in early- vs adult-onset KD.

Niemann-Pick disease type C1 (NPC1) is a lipid storage disorder in which cholesterol and glycosphingolipids accumulate in late endosomal/lysosomal compartments because of mutations in the NPC1 gene. A hallmark of NPC1 is progressive neurodegeneration of the cerebellum as well as visceral organ damage; however, the mechanisms driving this disease pathology are not fully understood. Phosphoinositides are phospholipids that play distinct roles in signal transduction and vesicle trafficking. Here, we utilized a consensus spectra analysis of MS imaging data sets and orthogonal LC/MS analyses to evaluate the spatial distribution of phosphoinositides and quantify them in cerebellar tissue from Npc1-null mice. Our results suggest significant depletion of multiple phosphoinositide species, including PI, PIP, and PIP2, in the cerebellum of the Npc1-null mice in both whole-tissue lysates and myelin-enriched fractions. Additionally, we observed altered levels of the regulatory enzyme phosphatidylinositol 4-kinase type 2α in Npc1-null mice. In contrast, the levels of related kinases, phosphatases, and transfer proteins were unaltered in the Npc1-null mouse model, as observed by Western blot analysis. Our discovery of phosphoinositide lipid biomarkers for NPC1 opens new perspectives on the pathophysiology underlying this fatal neurodegenerative disease.­

Psychosine exerts most of its toxic effects by altering membrane dynamics with increased shedding of extracellular vesicles (EVs). In this study, we discovered that a fraction of psychosine produced in the brain of the Twitcher mouse, a model for Krabbe disease, is associated with secreted EVs. We evaluated the effects of attenuating EV secretion in the Twitcher brain by depleting ceramide production with an inhibitor of neutral sphingomyelinase 2, GW4869. Twitcher mice treated with GW4869 had decreased overall EV levels, reduced EV-associated psychosine and unexpectedly, correlated with increased disease severity. Notably, characterization of well-established, neuroanatomic hallmarks of disease pathology, such as demyelination and inflammatory gliosis, remained essentially unaltered in the brains of GW4869-treated Twitcher mice compared to vehicle-treated Twitcher controls. Further analysis of Twitcher brain pathophysiology is required to understand the mechanism behind early-onset disease severity in GW4869-treated mice. The results herein demonstrate that some pathogenic lipids like psychosine may be secreted using EV pathways. Our results highlight the relevance of this secretory mechanism as a possible contributor to spreading pathogenic lipids in neurological lipidoses.

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