Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathological signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacological approach to restore Mcoln1-/- astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1-/- mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1-/- astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways, and restores the lysosomal compartment in Mcoln1-/- astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.

Background: The type 1 interferon (IFN) response is part of the innate immune response and best known for its role in viral and bacterial infection. However, this pathway is also induced in sterile inflammation such as that which occurs in a number of neurodegenerative diseases, including neuronopathic Gaucher disease (nGD), a lysosomal storage disorder (LSD) caused by mutations in GBA. Methods: Mice were injected with conduritol B-epoxide, an irreversible inhibitor of acid beta-glucosidase, the enzyme defective in nGD. MyTrMaSt null mice, where four adaptors of pathogen recognition receptors (PRRs) are deficient, were used to determine the role of the IFN pathway in nGD pathology. Activation of inflammatory and other pathways was analyzed by a variety of methods including RNAseq. Results: Elevation in the expression of PRRs associated with the IFN response was observed in CBE-injected mice. Ablation of upstream pathways leading to IFN production had no therapeutic benefit on the lifespan of nGD mice but attenuated neuroinflammation. Primary and secondary pathological pathways (i.e., those associated or not with mouse survival) were distinguished, and a set of~210 genes including those related to sphingolipid, cholesterol, and lipoprotein metabolism, along with a number of inflammatory pathways related to chemokines, TNF, TGF, complement, IL6, and damage-associated microglia were classified as primary pathological pathways, along with some lysosomal and neuronal genes. Conclusions: Although IFN signaling is the top elevated pathway in nGD, we demonstrate that this pathway is not related to mouse viability and is consequently defined as a secondary pathology pathway. By elimination, we defined a number of critical pathways that are directly related to brain pathology in nGD, which in addition to its usefulness in understanding pathophysiological mechanisms, may also pave the way for the development of novel therapeutic paradigms by targeting such pathways.

Approximately 70 lysosomal storage diseases are currently known, resulting from mutations in genes encoding lysosomal enzymes and membrane proteins. Defects in lysosomal enzymes that hydrolyze sphingolipids have been relatively well studied. Gaucher disease is caused by the loss of activity of glucocerebrosidase, leading to accumulation of glucosylceramide. Gaucher disease exhibits a number of subtypes, with types 2 and 3 showing significant neuropathology. Sandhoff disease results from the defective activity of β-hexosaminidase, leading to accumulation of ganglioside GM2. Niemann-Pick type C disease is primarily caused by the loss of activity of the lysosomal membrane protein, NPC1, leading to storage of cholesterol and sphingosine. All three disorders display significant neuropathology, accompanied by neuroinflammation. It is commonly assumed that neuroinflammation is the result of infiltration of monocyte-derived macrophages into the brain; for instance, cells resembling lipid-engorged macrophages ('Gaucher cells') have been observed in the brain of Gaucher disease patients. We now review the evidence that inflammatory macrophages are recruited into the brain in these diseases and then go on to provide some experimental data that, at least in the three mouse models tested, monocyte-derived macrophages do not appear to infiltrate the brain. Resident microglia, which are phenotypically distinct from infiltrating macrophages, are the only myeloid population present in significant numbers within the brain parenchyma in these authentic mouse models, even during the late symptomatic stages of disease when there is substantial neuroinflammation. This article is part of the Special Issue "Lysosomal Storage Disorders".

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OBJECTIVEA key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins in lysosomes. This event culminates with the loss of cell homeostasis, inflammation and cell death. In this study we propose to identify the chemical etiological factors and understanding the molecular and cellular mechanisms responsible for the impairment of lysosome function in macrophages.APPROACH AND RESULTSUsing shotgun lipidomics we have discovered that a family of oxidized lipids (cholesteryl hemiesters, ChE), end products of oxidation of polyunsaturated cholesteryl esters, occurs at higher concentrations in the plasma of two cohorts of cardiovascular disease patients than in the plasma of a control cohort. Macrophages exposed to the most prevalent ChE, cholesteryl hemiazelate (ChA) exhibit lysosome enlargement, peripheral lysosomal positioning, lysosome dysfunction and lipidosis which are irreversible. The transcriptomic profile of macrophages exposed to ChA indicates that the lysosome pathway is deeply affected and is well correlated with lysosome phenotypic and functional changes. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences to the pathology.CONCLUSIONWe identify ChA not only as one of the molecules involved in the etiology of irreversible lysosome dysfunction culminating with lipidosis but also as a promoter of exocytosis of the dysfunctional lysosomes. The latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.