Role of sulfatide-reactive vNKT cells in promoting lung Treg cells via dendritic cell modulation in asthma models.

Our previous studies have showed that sulfatide-reactive type II NKT (i.e. variant NKT, vNKT) cells inhibit the immunogenic maturation during the development of mature lung dendritic cells (LDCs), leading todeclined allergic airway inflammation in asthma. Nonetheless, the specific immunoregulatory roles of vNKT cells in LDC-mediated Th2 cell responses remain incompletely understood. Herein, we found that administration of sulfatide facilitated the generation of CD4+FoxP3+ regulatory T (Treg) cells in the lungs of wild-type mice, but not in CD1d-/- and Jα18-/- mice, after ovalbumin or house dust mite exposure. This finding implies that the enhancement of lung Treg cells by sulfatide requires vNKT cells, which dependent on invariant NKT (iNKT) cells. Furthermore, the CD4+FoxP3+ Treg cells induced by sulfatide-reactive vNKT cells were found to be associated with PD-L1 molecules expressed on LDCs, and this association was dependent on iNKT cells. Collectively, our findings suggest that in asthma-mimicking murine models, sulfatide-reactive vNKT cells facilitate the generation of lung Treg cells through inducing tolerogenic properties in LDCs, and this process is dependent on the presence of lung iNKT cells. These results may provide a potential therapeutic approach to treat allergic asthma.

NKT cells in the antitumor response: the ß version?

NKT cells recognize glycolipids presented by CD1d-expressing antigen-presenting cells (APCs) and include type I NKT cells with antitumor function and type II NKT cells, which have been reported to suppress the antitumor response. Some type II NKT cells recognize sulfatide, a glycosphingolipid with a sulfate modification of the sugar. Type I NKT cells recognize different glycosphingolipids. In this issue of the JCI, Nishio and colleagues showed that APCs could process sulfatide antigens, analogous to protein processing for peptide-reactive T cells. Antigen processing in lysosomes removed sulfate to generate a glycosphingolipid that stimulated type I NKT cells and thereby turned an antigen with no antitumor activity into one that not only stimulated type I NKT cells but also stimulated antitumor responses. These findings may extend to the development of glycolipid antigens that could stimulate anticancer responses via antigen processing by APCs.

Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer.

In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding ß-galactosylceramide (ßGalCer) without sulfate. C24:2 induced IFN-γ-dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid's function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.

Adult-Onset CNS Sulfatide Deficiency Causes Sex-Dependent Metabolic Disruption in Aging.

The interconnection between obesity and central nervous system (CNS) neurological dysfunction has been widely appreciated. Accumulating evidence demonstrates that obesity is a risk factor for CNS neuroinflammation and cognitive impairment. However, the extent to which CNS disruption influences peripheral metabolism remains to be elucidated. We previously reported that myelin-enriched sulfatide loss leads to CNS neuroinflammation and cognitive decline. In this study, we further investigated the impact of CNS sulfatide deficiency on peripheral metabolism while considering sex- and age-specific effects. We found that female sulfatide-deficient mice gained significantly more body weight, exhibited higher basal glucose levels, and were glucose-intolerant during glucose-tolerance test (GTT) compared to age-matched controls under a normal diet, whereas male sulfatide-deficient mice only displayed glucose intolerance at a much older age compared to female sulfatide-deficient mice. Mechanistically, we found that increased body weight was associated with increased food intake and elevated neuroinflammation, especially in the hypothalamus, in a sex-specific manner. Our results suggest that CNS sulfatide deficiency leads to sex-specific alterations in energy homeostasis via dysregulated hypothalamic control of food intake.

Olaparib Attenuates Demyelination and Neuroinflammation in an Organotypic Slice Culture Model of Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of the arylsulfatase A lysosomal enzyme and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Using an ex vivo murine-derived organotypic cerebellar slice culture model, we demonstrate that sulfatide induces demyelination in a concentration-dependent manner. Interestingly, our novel data demonstrate that sulfatide-induced demyelination is underpinned by PARP-1 activation, oligodendrocyte loss, pro-inflammatory cytokine expression, astrogliosis, and microgliosis. Moreover, such sulfatide-induced effects can be attenuated by the treatment with the poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor Olaparib (IC50∼100 nM) suggesting that this small molecule may be neuroprotective and limit toxin-induced demyelination. Our data support the idea that sulfatide is a key driver of demyelination and neuroinflammation in MLD and suggest that PARP-1 inhibitors have therapeutic utility in the sphere of rare demyelinating disease.

Sulfatide inhibits fibroblast growth, activation and oxidative stress induced by ectopic insulin.

AIM: To study the effect of sulfatide on gene expression and proliferation of human primary fibroblasts induced by insulin, insulin-like growth factor-1 and human growth hormone. MATERIALS AND METHODS: Human primary fibroblasts were exposed to 1, 3 and 30 µM of sulfatide or its precursor galactosylceramide (GalCer). Proliferation was determined by 3 H-thymidine incorporation and gene expression via microarray analysis. RESULTS: Sulfatide and GalCer reduced the growth rate of fibroblasts by 32%-82% when exposed to 0.5 nM insulin. After challenge with 120 µM of H2 O2 , sulfatide reduced membrane leakage. Fibroblast gene expression was altered by sulfatide in gene pathways associated with cell cycle/growth, transforming growth factor-ß function, and encoding of proteins involved in intracellular signalling. NFKBIA, a key control element in NF-кB regulation, was decreased 2-fold by sulfatide. CONCLUSIONS: Sulfatide strongly inhibits fibroblast growth. We therefore suggest the addition of sulfatide to injectable commercial insulin formulations, which would reduce adverse fibroblast growth and improve well-being in patients with diabetes.

Reduced sulfatide content in deferoxamine-induced senescent HepG2 cells.

Iron chelators, such as deferoxamine, exert an anticancer effect by altering the activity of biomolecules critical for regulation of the cell cycle, cell metabolism, and apoptotic processes. Thus, iron chelators are sometimes used in combination with radio- and/or chemotherapy in the treatment of cancer. The possibility that deferoxamine could induce a program of senescence similar to radio- and/or chemotherapy, fostering adaptation in the treatment of cancer cells, is not fully understood. Using established biochemical techniques, biomarkers linked to lipid composition, and coherent anti-Stokes Raman scattering microscopy, we demonstrated that hepatocellular carcinoma-derived HepG2 cells survive after deferoxamine treatment, acquiring phenotypic traits and representative hallmarks of senescent cells. The results support the view that deferoxamine acts in HepG2 cells to produce oxidative stress-induced senescence by triggering sequential mitochondrial and lysosomal dysfunction accompanied by autophagy blockade. We also focused on the lipidome of senescent cells after deferoxamine treatment. Using mass spectrometry, we found that the deferoxamine-induced senescent cells presented marked remodeling of the phosphoinositol, sulfatide, and cardiolipin profiles, which all play a central role in cell signaling cascades, intracellular membrane trafficking, and mitochondria functions. Detection of alterations in glycosphingolipid sulfate species suggested modifications in ceramide generation, and turnover is frequently described in cancer cell survival and resistance to chemotherapy. Blockade of ceramide generation may explain autophagic default, resistance to apoptosis, and the onset of senescence.

Relationship of Sulfatides Physiological Function and Peroxisome Proliferator-Activated Receptor α.

Sulfatides are unique sphingolipids present in the serum and the plasma membrane. Sulfatides exert important functions in a number of systems in the human body, including the nervous, immune, cardiovascular, and coagulation systems.Furthermore, it is closely related to tumor occurrence, development, and metastasis. Peroxisome proliferators-activated receptor α (PPARα) is a class of the nuclear receptor superfamily of transcription factors, which is a potential regulator of sulfatides. This review not only summarizes the current knowledge on the physiological functions of sulfatides in various systems, but also discusses the possible PPARα regulatory mechanisms in sulfatide metabolism and functions. The results of the present analysis provide deep insights and further novel ideas for expanding the research on the physiological function and clinical application of sulfatides.

Disrupted myelin lipid metabolism differentiates frontotemporal dementia caused by GRN and C9orf72 gene mutations.

Heterozygous mutations in the GRN gene and hexanucleotide repeat expansions in C9orf72 are the two most common genetic causes of Frontotemporal Dementia (FTD) with TDP-43 protein inclusions. The triggers for neurodegeneration in FTD with GRN (FTD-GRN) or C9orf72 (FTD-C9orf72) gene abnormalities are unknown, although evidence from mouse and cell culture models suggests that GRN mutations disrupt lysosomal lipid catabolism. To determine how brain lipid metabolism is affected in familial FTD with TDP-43 inclusions, and how this is related to myelin and lysosomal markers, we undertook comprehensive lipidomic analysis, enzyme activity assays, and western blotting on grey and white matter samples from the heavily-affected frontal lobe and less-affected parietal lobe of FTD-GRN cases, FTD-C9orf72 cases, and age-matched neurologically-normal controls. Substantial loss of myelin-enriched sphingolipids (sulfatide, galactosylceramide, sphingomyelin) and myelin proteins was observed in frontal white matter of FTD-GRN cases. A less-pronounced, yet statistically significant, loss of sphingolipids was also observed in FTD-C9orf72. FTD-GRN was distinguished from FTD-C9orf72 and control cases by increased acylcarnitines in frontal grey matter and marked accumulation of cholesterol esters in both frontal and parietal white matter, indicative of myelin break-down. Both FTD-GRN and FTD-C9orf72 cases showed significantly increased lysosomal and phagocytic protein markers, however galactocerebrosidase activity, required for lysosomal catabolism of galactosylceramide and sulfatide, was selectively increased in FTD-GRN. We conclude that both C9orf72 and GRN mutations are associated with disrupted lysosomal homeostasis and white matter lipid loss, but GRN mutations cause a more pronounced disruption to myelin lipid metabolism. Our findings support the hypothesis that hyperactive myelin lipid catabolism is a driver of gliosis and neurodegeneration in FTD-GRN. Since FTD-GRN is associated with white matter hyperintensities by MRI, our data provides important biochemical evidence supporting the use of MRI measures of white matter integrity in the diagnosis and management of FTD.

Identification of the Lipid Antigens Recognized by rHIgM22, a Remyelination-Promoting Antibody.

Failure of the immune system to discriminate myelin components from foreign antigens plays a critical role in the pathophysiology of multiple sclerosis. In fact, the appearance of anti-myelin autoantibodies, targeting both proteins and glycolipids, is often responsible for functional alterations in myelin-producing cells in this disease. Nevertheless, some of these antibodies were reported to be beneficial for remyelination. Recombinant human IgM22 (rHIgM22) binds to myelin and to the surface of O4-positive oligodendrocytes, and promotes remyelination in mouse models of chronic demyelination. Interestingly, the identity of the antigen recognized by this antibody remains to be elucidated. The preferential binding of rHIgM22 to sulfatide-positive cells or tissues suggests that sulfatide might be part of the antigen pattern recognized by the antibody, however, cell populations lacking sulfatide expression are also responsive to rHIgM22. Thus, we assessed the binding of rHIgM22 in vitro to purified lipids and lipid extracts from various sources to identify the antigen(s) recognized by this antibody. Our results show that rHIgM22 is indeed able to bind both sulfatide and its deacylated form, whereas no significant binding for other myelin sphingolipids has been detected. Remarkably, binding of rHIgM22 to sulfatide in lipid monolayers can be positively or negatively regulated by the presence of other lipids. Moreover, rHIgM22 also binds to phosphatidylinositol, phosphatidylserine and phosphatidic acid, suggesting that not only sulfatide, but also other membrane lipids might play a role in the binding of rHIgM22 to oligodendrocytes and to other cell types not expressing sulfatide.

Metabolic Regulation and Lipidomic Remodeling in Relation to Spermidine-induced Stress Tolerance to High Temperature in Plants.

Beneficial effects of spermidine (Spd) on alleviating abiotic stress damage have been explored in plants for hundreds of years, but limited information is available about its roles in regulating lipids signaling and metabolism during heat stress. White clover (Trifolium repens) plants were pretreated with 70 µM Spd and then subjected to high temperature (38/33 °C) stress for 20 days. To further investigate the effect of Spd on heat tolerance, transgenic Arabidopsisthaliana overexpressing a TrSAMS encoding a key enzyme involved in Spd biosynthesis was exposed to high temperature (38/33 °C) stress for 10 days. A significant increase in endogenous Spd content in white clover by exogenous application of Spd or the TrSAMS overexpression in Arabidopsisthaliana could effectively mitigate heat-induced growth retardation, oxidative damage to lipids, and declines in photochemical efficiency and cell membrane stability. Based on the analysis of metabolomics, the amino acids and vitamins metabolism, biosynthesis of secondary metabolites, and lipids metabolism were main metabolic pathways regulated by the Spd in cool-season white clover under heat stress. Further analysis of lipidomics found the TrSAMS-transgenic plants maintained relatively higher accumulations of total lipids, eight phospholipids (PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; CL, cardiolipin; LPA, lysophosphatidic acid; LPC, lyso phosphatidylcholine; LPG, lyso phosphatidylglycerol; and LPI, lyso phosphatidylinositol), one glycoglycerolipid (DGDG, digalactosyl diacylglycerol), and four sphingolipids (Cer, ceramide; CerG2GNAc1, dihexosyl N-acetylhexosyl ceramide; Hex1Cer, hexosyl ceramide; and ST, sulfatide), higher ratio of DGDG: monogalactosyl diacylglycerol (MGDG), and lower unsaturation level than wild-type Arabidopsisthaliana in response to heat stress. Spd-induced lipids accumulation and remodeling could contribute to better maintenance of membrane stability, integrity, and functionality when plants underwent a long period of heat stress. In addition, the Spd significantly up-regulated PIP2 and PA signaling pathways, which was beneficial to signal perception and transduction for stress defense. Current findings provide a novel insight into the function of Spd against heat stress through regulating lipids signaling and reprograming in plants.

A model of metformin mitochondrial metabolism in metachromatic leukodystrophy: first description of human Schwann cells transfected with CRISPR-Cas9.

Metachromatic leukodystrophy is a neurological lysosomal deposit disease that affects public health despite its low incidence in the population. Currently, few reports are available on pathophysiological events related to enzyme deficiencies and subsequent sulfatide accumulation. This research aims to examine the use of metformin as an alternative treatment to counteract these effects. This was evaluated in human Schwann cells (HSCs) transfected or non-transfected with CRISPR-Cas9, and later treated with sulfatides and metformin. This resulted in transfected HSCs showing a significant increase in cell reactive oxygen species (ROS) production when exposed to 100 µM sulfatides (p = 0.0007), compared to non-transfected HSCs. Sulfatides at concentrations of 10 to 100 µM affected mitochondrial bioenergetics in transfected HSCs. Moreover, these analyses showed that transfected cells showed a decrease in basal and maximal respiration rates after exposure to 100 µM sulfatide. However, maximal and normal mitochondrial respiratory capacity decreased in cells treated with both sulfatide and metformin. This study has provided valuable insights into bioenergetic and mitochondrial effects of sulfatides in HSCs for the first time. Treatment with metformin (500 µM) restored the metabolic activity of these cells and decreased ROS production.

LncRNA lncAY is upregulated by sulfatide via Myb/MEF2C acetylation to promote the tumorigenicity of hepatocellular carcinoma cells.

LncRNA (long noncoding RNA) is often dysregulated in tumors especially hepatocellular carcinoma (HCC). However, the dysregulation mechanism of lncRNAs is largely unknown. Here, we showed that lncRNA lncAY expression was stimulated in HCC by either endogenous or exogenous sulfatide. Elevated lncAY promoted HCC cell migration or angiogenesis, whereas lncAY silence suppressed HCC cell migration and proliferation. Interestingly, the activity of lncAY gene promoter was enhanced by sulfatide. Then Myb and MEF2C were identified as the transcription factors responsible for the stimulation of lncAY promoter activity and transcription by sulfatide. Both Myb and MEF2C enrichment on lncAY promoter was further confirmed, and their occupancy on lncAY promoter was strengthened by sulfatide for Myb or MEF2C was acetylated. Mutant Myb-K456A exhibited reduced acetylation and weak stimulation for lncAY transcription. However, Myb mutation K456/503A prevented Myb from acetylation induced by sulfatide. The mutant Myb K456/503A further was unable to occupy lncAY promoter and enhance lncAY transcription. In conclusion, this study demonstrated lncAY transcription was abnormally upregulated by sulfatide in HCC through Myb/MEF2C to promote HCC progression.

The PGRMC1 Antagonist AG-205 Inhibits Synthesis of Galactosylceramide and Sulfatide.

Sulfatide synthesis in the human renal cancer cell line SMKT-R3 was strongly inhibited in the presence of low µM concentrations of AG-205, a progesterone receptor membrane component 1 (PGRMC1) antagonist. This was also the case in Chinese hamster ovary (CHO) cells stably transfected with UDP-galactose: ceramide galactosyltransferase and cerebroside sulfotransferase, the two enzymes required for sulfatide synthesis. In CHO cells synthesizing galactosylceramide but not sulfatide, galactosylceramide was also strongly reduced, suggesting an effect at the level of galactolipid synthesis. Notably, AG-205 inhibited galactosylceramide synthesis to a similar extent in wild type CHO cells and cells that lack PGRMC1 and/or PGRMC2. In vitro enzyme activity assays showed that AG-205 is an inhibitor of UDP-galactose: ceramide galactosyltransferase, but not cerebroside sulfotransferase. This study shows that PGRMC1 is only one of several targets of AG-205 and should be used with caution, especially in studies using cells synthesizing galactosylceramide and sulfatide.

A Lipidomics Atlas of Selected Sphingolipids in Multiple Mouse Nervous System Regions.

Many lipids, including sphingolipids, are essential components of the nervous system. Sphingolipids play critical roles in maintaining the membrane structure and integrity and in cell signaling. We used a multi-dimensional mass spectrometry-based shotgun lipidomics platform to selectively analyze the lipid species profiles of ceramide, sphingomyelin, cerebroside, and sulfatide; these four classes of sphingolipids are found in the central nervous system (CNS) (the cerebrum, brain stem, and spinal cord) and peripheral nervous system (PNS) (the sciatic nerve) tissues of young adult wild-type mice. Our results revealed that the lipid species profiles of the four sphingolipid classes in the different nervous tissues were highly distinct. In addition, the mRNA expression of sphingolipid metabolism genes-including the ceramidase synthases that specifically acylate the N-acyl chain of ceramide species and sphingomyelinases that cleave sphingomyelins generating ceramides-were analyzed in the mouse cerebrum and spinal cord tissue in order to better understand the sphingolipid profile differences observed between these nervous tissues. We found that the distinct profiles of the determined sphingolipids were consistent with the high selectivity of ceramide synthases and provided a potential mechanism to explain region-specific CNS ceramide and sphingomyelin levels. In conclusion, we portray for the first time a lipidomics atlas of select sphingolipids in multiple nervous system regions and believe that this type of knowledge could be very useful for better understanding the role of this lipid category in the nervous system.

GM3 Ganglioside Linked to Neurofibrillary Pathology in a Transgenic Rat Model for Tauopathy.

Glycosphingolipids (GSLs) are amphipathic lipids composed of a sphingoid base and a fatty acyl attached to a saccharide moiety. GSLs play an important role in signal transduction, directing proteins within the membrane, cell recognition, and modulation of cell adhesion. Gangliosides and sulfatides belong to a group of acidic GSLs, and numerous studies report their involvement in neurodevelopment, aging, and neurodegeneration. In this study, we used an approach based on hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (HRMS/MS) to characterize the glycosphingolipid profile in rat brain tissue. Then, we screened characterized lipids aiming to identify changes in glycosphingolipid profiles in the normal aging process and tau pathology. Thorough screening of acidic glycosphingolipids in rat brain tissue revealed 117 ganglioside and 36 sulfatide species. Moreover, we found two ganglioside subclasses that were not previously characterized-GT1b-Ac2 and GQ1b-Ac2. The semi-targeted screening revealed significant changes in the levels of sulfatides and GM1a gangliosides during the aging process. In the transgenic SHR24 rat model for tauopathies, we found elevated levels of GM3 gangliosides which may indicate a higher rate of apoptotic processes.

Treatment of adult metachromatic leukodystrophy model mice using intrathecal administration of type 9 AAV vector encoding arylsulfatase A.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by an arylsulfatase A (ARSA) deficiency and characterized by severe neurological symptoms resulting from demyelination within the central and peripheral nervous systems. We investigated the feasibility and efficacy of intrathecal administration of a type 9 adeno-associated viral vector encoding ARSA (AAV9/ARSA) for the treatment of 6-week-old MLD model mice, which are presymptomatic, and 1-year-old mice, which exhibit neurological abnormalities. Immunohistochemical analysis following AAV9/ARSA administration showed ARSA expression within the brain, with highest activities in the cerebellum and olfactory bulbs. In mice treated at 1 year, alcian blue staining and quantitative analysis revealed significant decreases in stored sulfatide. Behaviorally, mice treated at 1 year showed no improvement in their ability to traverse narrow balance beams as compared to untreated mice. By contrast, MLD mice treated at 6 weeks showed significant decreases in stored sulfatide throughout the entire brain and improved ability to traverse narrow balance beams. These findings suggest intrathecal administration of an AAV9/ARSA vector is a promising approach to treating genetic diseases of the central nervous system, including MLD, though it may be essential to begin therapy before the onset of neurological symptoms.

Adult-onset CNS myelin sulfatide deficiency is sufficient to cause Alzheimer's disease-like neuroinflammation and cognitive impairment.

BACKGROUND: Human genetic association studies point to immune response and lipid metabolism, in addition to amyloid-beta (Aß) and tau, as major pathways in Alzheimer's disease (AD) etiology. Accumulating evidence suggests that chronic neuroinflammation, mainly mediated by microglia and astrocytes, plays a causative role in neurodegeneration in AD. Our group and others have reported early and dramatic losses of brain sulfatide in AD cases and animal models that are mediated by ApoE in an isoform-dependent manner and accelerated by Aß accumulation. To date, it remains unclear if changes in specific brain lipids are sufficient to drive AD-related pathology. METHODS: To study the consequences of CNS sulfatide deficiency and gain insights into the underlying mechanisms, we developed a novel mouse model of adult-onset myelin sulfatide deficiency, i.e., tamoxifen-inducible myelinating glia-specific cerebroside sulfotransferase (CST) conditional knockout mice (CSTfl/fl/Plp1-CreERT), took advantage of constitutive CST knockout mice (CST-/-), and generated CST/ApoE double knockout mice (CST-/-/ApoE-/-), and assessed these mice using a broad range of methodologies including lipidomics, RNA profiling, behavioral testing, PLX3397-mediated microglia depletion, mass spectrometry (MS) imaging, immunofluorescence, electron microscopy, and Western blot. RESULTS: We found that mild central nervous system (CNS) sulfatide losses within myelinating cells are sufficient to activate disease-associated microglia and astrocytes, and to increase the expression of AD risk genes (e.g., Apoe, Trem2, Cd33, and Mmp12), as well as previously established causal regulators of the immune/microglia network in late-onset AD (e.g., Tyrobp, Dock, and Fcerg1), leading to chronic AD-like neuroinflammation and mild cognitive impairment. Notably, neuroinflammation and mild cognitive impairment showed gender differences, being more pronounced in females than males. Subsequent mechanistic studies demonstrated that although CNS sulfatide losses led to ApoE upregulation, genetically-induced myelin sulfatide deficiency led to neuroinflammation independently of ApoE. These results, together with our previous studies (sulfatide deficiency in the context of AD is mediated by ApoE and accelerated by Aß accumulation) placed both Aß and ApoE upstream of sulfatide deficiency-induced neuroinflammation, and suggested a positive feedback loop where sulfatide losses may be amplified by increased ApoE expression. We also demonstrated that CNS sulfatide deficiency-induced astrogliosis and ApoE upregulation are not secondary to microgliosis, and that astrogliosis and microgliosis seem to be driven by activation of STAT3 and PU.1/Spi1 transcription factors, respectively. CONCLUSION: Our results strongly suggest that sulfatide deficiency is an important contributor and driver of neuroinflammation and mild cognitive impairment in AD pathology.

Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice.

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.