New paradigms for the treatment of lysosomal storage diseases: targeting the endocannabinoid system as a therapeutic strategy.

Over the past three decades the lysosomal storage diseases have served as model for rare disease treatment development. While these efforts have led to considerable success, important challenges remain. For example, no treatments are currently approved for nearly two thirds of all lysosomal diseases, and there is limited impact of the existing drugs on the central nervous system. In addition, the costs of these therapies are extremely high, in part due to the fact that drug development has focused on a "single hit" approach - i.e., one drug for one disease. To overcome these obstacles researchers have begun to focus on defining common disease mechanisms in the lysosomal diseases, particularly in the central nervous system, with the hope of identifying drugs that might be used in several lysosomal diseases rather than an individual disease. With this concept in mind, herein we review a new potential treatment approach for the lysosomal storage diseases that focuses on modulation of the endocannabinoid system. We provide a short introduction to lysosomal storage diseases and the endocannabinoid system, followed by a brief review of data supporting this concept.

Apolipoprotein D-mediated preservation of lysosomal function promotes cell survival and delays motor impairment in Niemann-Pick type A disease.

Lysosomal Storage Diseases (LSD) are genetic diseases causing systemic and nervous system dysfunction. The glia-derived lipid binding protein Apolipoprotein D (ApoD) is required for lysosomal functional integrity in glial and neuronal cells, ensuring cell survival upon oxidative stress or injury. Here we test whether ApoD counteracts the pathogenic consequences of a LSD, Niemann Pick-type-A disease (NPA), where mutations in the acid sphingomyelinase gene result in sphingomyelin accumulation, lysosomal permeabilization and early-onset neurodegeneration. We performed a multivariable analysis of behavioral, cellular and molecular outputs in 12 and 24 week-old male and female NPA model mice, combined with ApoD loss-of-function mutation. Lack of ApoD in NPA mice accelerates cerebellar-dependent motor deficits, enhancing loss of Purkinje neurons. We studied ApoD expression in brain sections from a NPA patient and age-matched control, and the functional consequences of ApoD supplementation in primary human fibroblasts from two independent NPA patients and two control subjects. Cell viability, lipid peroxidation, and lysosomal functional integrity (pH, Cathepsin B activity, Galectin-3 exclusion) were examined. ApoD is endogenously overexpressed in NPA patients and NPA mouse brains and targeted to lysosomes of NPA patient cells, including Purkinje neurons and cultured fibroblasts. The accelerated lysosomal targeting of ApoD by oxidative stress is hindered in NPA fibroblasts, contributing to NPA lysosomes vulnerability. Exogenously added ApoD reduces NPA-prompted lysosomal permeabilization and alkalinization, reverts lipid peroxides accumulation, and significantly increases NPA cell survival. ApoD administered simultaneously to sphingomyelin overload results in complete rescue of cell survival. Our results reveal that ApoD protection of lysosomal integrity counteracts NPA pathology. ApoD supplementation could significantly delay not only the progression of NPA disease, but also of other LSDs through its beneficial effects in lysosomal functional maintenance.

Seladin-1/DHCR24 Is Neuroprotective by Associating EAAT2 Glutamate Transporter to Lipid Rafts in Experimental Stroke.

BACKGROUND AND PURPOSE: 3ß-Hydroxysteroid-Δ24 reductase (DHCR24) or selective alzheimer disease indicator 1 (seladin-1), an enzyme of cholesterol biosynthetic pathway, has been implicated in neuroprotection, oxidative stress, and inflammation. However, its role in ischemic stroke remains unexplored. The aim of this study was to characterize the effect of seladin-1/DHCR24 using an experimental stroke model in mice. METHODS: Dhcr24(+/-) and wild-type (WT) mice were subjected to permanent middle cerebral artery occlusion. In another set of experiments, WT mice were treated intraperitoneally either with vehicle or U18666A (seladin-1/DHCR24 inhibitor, 10 mg/kg) 30 minutes after middle cerebral artery occlusion. Brains were removed 48 h after middle cerebral artery occlusion for infarct volume determination. For protein expression determination, peri-infarct region was obtained 24 h after ischemia, and Western blot or cytometric bead array was performed. RESULTS: Dhcr24(+/-) mice displayed larger infarct volumes after middle cerebral artery occlusion than their WT littermates. Treatment of WT mice with the seladin-1/DHCR24 inhibitor U18666A also increased ischemic lesion. Inflammation-related mediators were increased after ischemia in Dhcr24(+/-) mice compared with WT counterparts. Consistent with a role of cholesterol in proper function of glutamate transporter EAAT2 in membrane lipid rafts, we found a decreased association of EAAT2 with lipid rafts after ischemia when DHCR24 is genetically deleted or pharmacologically inhibited. Accordingly, treatment with U18666A decreases [(3)H]-glutamate uptake in cultured astrocytes. CONCLUSIONS: These results support the idea that lipid raft integrity, ensured by seladin-1/DHCR24, plays a crucial protective role in the ischemic brain by guaranteeing EAAT2-mediated uptake of glutamate excess.

Pharmacological reversion of sphingomyelin-induced dendritic spine anomalies in a Niemann Pick disease type A mouse model.

Understanding the role of lipids in synapses and the aberrant molecular mechanisms causing the cognitive deficits that characterize most lipidosis is necessary to develop therapies for these diseases. Here we describe sphingomyelin (SM) as a key modulator of the dendritic spine actin cytoskeleton. We show that increased SM levels in neurons of acid sphingomyelinase knock out mice (ASMko), which mimic Niemann Pick disease type A (NPA), result in reduced spine number and size and low levels of filamentous actin. Mechanistically, SM accumulation decreases the levels of metabotropic glutamate receptors type I (mGluR1/5) at the synaptic membrane impairing membrane attachment and activity of RhoA and its effectors ROCK and ProfilinIIa. Pharmacological enhancement of the neutral sphingomyelinase rescues the aberrant molecular and morphological phenotypes in vitro and in vivo and improves motor and memory deficits in ASMko mice. Altogether, these data demonstrate the influence of SM and its catabolic enzymes in dendritic spine physiology and contribute to our understanding of the cognitive deficits of NPA patients, opening new perspectives for therapeutic interventions.

Age-dependent increase in desmosterol restores DRM formation and membrane-related functions in cholesterol-free DHCR24-/- mice.

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24(-/-) mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased beta-secretase activity and diminished Abeta generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24(-/-) mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24(-/-) mouse.

Detergent resistant membrane-associated IDE in brain tissue and cultured cells: Relevance to Abeta and insulin degradation.

BACKGROUND: Insulin degrading enzyme (IDE) is implicated in the regulation of amyloid beta (Abeta) steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD). Although IDE sub-cellular localization has been well studied, the compartments relevant to Abeta degradation remain to be determined. RESULTS: Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM)-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-beta-cyclodextrin (MbetaCD), endogenous Abeta accumulated in the extracellular space and exogenous Abeta proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Abeta degradation in vitro. CONCLUSION: Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Abeta degrading proteases away from DRMs may impair the physiological turn-over of Abeta in vivo deserves further investigation in light of therapeutic strategies based on enhancing Abeta proteolysis in which DRM protease-targeting may need to be taken into account.