Lipid-induced lysosomal damage after demyelination corrupts microglia protective function in lysosomal storage disorders.

Neuropathic lysosomal storage disorders (LSDs) present with activated pro-inflammatory microglia. However, anti-inflammatory treatment failed to improve disease pathology. We characterise the mechanisms underlying microglia activation in Niemann-Pick disease type A (NPA). We establish that an NPA patient and the acid sphingomyelinase knockout (ASMko) mouse model show amoeboid microglia in neurodegeneration-prone areas. In vivo microglia ablation worsens disease progression in ASMko mice. We demonstrate the coexistence of different microglia phenotypes in ASMko brains that produce cytokines or counteract neuronal death by clearing myelin debris. Overloading microglial lysosomes through myelin debris accumulation and sphingomyelin build-up induces lysosomal damage and cathepsin B extracellular release by lysosomal exocytosis. Inhibition of cathepsin B prevents neuronal death and behavioural anomalies in ASMko mice. Similar microglia phenotypes occur in a Niemann-Pick disease type C mouse model and patient. Our results show a protective function for microglia in LSDs and how this is corrupted by lipid lysosomal overload. Data indicate cathepsin B as a key molecule mediating neurodegeneration, opening research pathways for therapeutic targeting of LSDs and other demyelinating diseases.

Single-nucleus transcriptome analysis reveals disease- and regeneration-associated endothelial cells in white matter vascular dementia.

BACKGROUND: Vascular dementia (VaD) is the accumulation of vascular lesions in the subcortical white matter of the brain. These lesions progress and there is no direct medical therapy. AIMS: To determine the specific cellular responses in VaD so as to provide molecular targets for therapeutic development. MATERIALS AND METHODS: Single-nucleus transcriptome analysis was performed in human periventricular white matter (PVWM) samples of VaD and normal control (NC) subjects. RESULTS: Differential analysis shows that cell type-specific transcriptomic changes in VaD are associated with the disruption of specific biological processes, including angiogenesis, immune activation, axonal injury and myelination. Each cell type in the neurovascular unit within white matter has a specific alteration in gene expression in VaD. In a central cell type for this disease, subcluster analysis of endothelial cells (EC) indicates that VaD contains a disease-associated EC subcluster that expresses genes associated with programmed cell death and a response to protein folding. Two other subpopulations of EC in VaD express molecular systems associated with regenerative processes in angiogenesis, and in axonal sprouting and oligodendrocyte progenitor cell maturation. CONCLUSION: This comprehensive molecular profiling of brain samples from patients with VaD reveals previously unknown molecular changes in cells of the neurovascular niche, and an attempt at regeneration in injured white matter.

NPC1 enables cholesterol mobilization during long-term potentiation that can be restored in Niemann-Pick disease type C by CYP46A1 activation.

NPC is a neurodegenerative disorder characterized by cholesterol accumulation in endolysosomal compartments. It is caused by mutations in the gene encoding NPC1, an endolysosomal protein mediating intracellular cholesterol trafficking. Cognitive and psychiatric alterations are hallmarks in NPC patients pointing to synaptic defects. However, the role of NPC1 in synapses has not been explored. We show that NPC1 is present in the postsynaptic compartment and is locally translated during LTP. A mutation in a region of the NPC1 gene commonly altered in NPC patients reduces NPC1 levels at synapses due to enhanced NPC1 protein degradation. This leads to shorter postsynaptic densities, increased synaptic cholesterol and impaired LTP in NPC1nmf164 mice with cognitive deficits. NPC1 mediates cholesterol mobilization and enables surface delivery of CYP46A1 and GluA1 receptors necessary for LTP, which is defective in NPC1nmf164 mice. Pharmacological activation of CYP46A1 normalizes synaptic levels of cholesterol, LTP and cognitive abilities, and extends life span of NPC1nmf164 mice. Our results unveil NPC1 as a regulator of cholesterol dynamics in synapses contributing to synaptic plasticity, and provide a potential therapeutic strategy for NPC patients.

Oxidative Stress and Histopathological Changes in Gills and Kidneys of Cyprinus carpio following Exposure to Benzethonium Chloride, a Cationic Surfactant.

One cationic surfactant with a wide spectrum of microbiocidal activity is benzethonium chloride (BEC). Despite being widely used, the toxicity data on vertebrate organisms are limited. Therefore, we aimed to evaluate within this study the acute toxicity of BEC on the gills and kidneys of Cyprinus carpio (European carp). An alteration of the antioxidant enzymes activities (glutathione reductase, glutathione peroxidase and glutathione S-transferase) was noticed after 96 h of exposure, along with an elevation of lipid peroxidation and decreased concentration of reduced glutathione, which confirmed that BEC was able to induce toxicity to these tissues. These metabolic effects were correlated with unspecific structural changes observed in gills and kidneys, having moderate degree of severity (such as an increase of melanomacrophages aggregation incidence and cytoplasm vacuolation of goblet cells in collecting tubules) and generally being compatible with life for the exposure time studied. The most severe structural effects were observed in gills after 96 h, noticing a lamellar aneurysm, hemorrhages and lamellar epithelium disruption due to the blood vessels and pillar cells damages and increased blood flow inside the lamellae. By our research we can confirm the utility of biochemical and histological analyses in the fish organs as tools for monitoring the water quality and ecotoxicological potential of chemicals.

SGPL1 (sphingosine phosphate lyase 1) modulates neuronal autophagy via phosphatidylethanolamine production.

Macroautophagy/autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. The roles of the bioactive signaling lipid sphingosine-1-phosphate (S1P) and its catabolic enzyme SGPL1/SPL (sphingosine phosphate lyase 1) in autophagy are increasingly recognized. Here we provide in vitro and in vivo evidence for a previously unidentified route through which SGPL1 modulates autophagy in neurons. SGPL1 cleaves S1P into ethanolamine phosphate, which is directed toward the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SGPL1fl/fl/Nes mice with developmental neural specific SGPL1 ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased BECN1/Beclin-1 and SQSTM1/p62 levels were apparent. Alterations were also noticed in downstream events of the autophagic-lysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as APP (amyloid ß [A4] precursor protein) and SNCA/α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SGPL1 in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of SQSTM1, APP and SNCA. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SGPL1fl/fl/Nes brains. Experiments using EGFP-mRFP-LC3 provided further support for blockage of the autophagic flux at initiation stages upon SGPL1 deficiency due to PE paucity. These results emphasize a formerly overlooked direct role of SGPL1 in neuronal autophagy and assume significance in the context that autophagy modulators hold an enormous therapeutic potential in the treatment of neurodegenerative diseases.

Sphingosine 1-phosphate lyase ablation disrupts presynaptic architecture and function via an ubiquitin- proteasome mediated mechanism.

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.