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Depletion of the enzyme cofactor, tetrahydrobiopterin (BH4), in T-cells was shown to prevent their proliferation upon receptor stimulation in models of allergic inflammation in mice, suggesting that BH4 drives autoimmunity. Hence, the clinically available BH4 drug (sapropterin) might increase the risk of autoimmune diseases. The present study assessed the implications for multiple sclerosis (MS) as an exemplary CNS autoimmune disease. Plasma levels of biopterin were persistently low in MS patients and tended to be lower with high Expanded Disability Status Scale (EDSS). Instead, the bypass product, neopterin, was increased. The deregulation suggested that BH4 replenishment might further drive the immune response or beneficially restore the BH4 balances. To answer this question, mice were treated with sapropterin in immunization-evoked autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Sapropterin-treated mice had higher EAE disease scores associated with higher numbers of T-cells infiltrating the spinal cord, but normal T-cell subpopulations in spleen and blood. Mechanistically, sapropterin treatment was associated with increased plasma levels of long-chain ceramides and low levels of the poly-unsaturated fatty acid, linolenic acid (FA18:3). These lipid changes are known to contribute to disruptions of the blood–brain barrier in EAE mice. Indeed, RNA data analyses revealed upregulations of genes involved in ceramide synthesis in brain endothelial cells of EAE mice (LASS6/CERS6, LASS3/CERS3, UGCG, ELOVL6, and ELOVL4). The results support the view that BH4 fortifies autoimmune CNS disease, mechanistically involving lipid deregulations that are known to contribute to the EAE pathology.
Trehalose, a sugar from fungi, mimics starvation due to a block of glucose transport and induces Transcription Factor EB- mediated autophagy, likely supported by the upregulation of progranulin. The pro-autophagy effects help to remove pathological proteins and thereby prevent neurodegenerative diseases such as Alzheimer’s disease. Enhancing autophagy also contributes to the resolution of neuropathic pain in mice. Therefore, we here assessed the effects of continuous trehalose administration via drinking water using the mouse Spared Nerve Injury model of neuropathic pain. Trehalose had no effect on drinking, feeding, voluntary wheel running, motor coordination, locomotion, and open field, elevated plus maze, and Barnes Maze behavior, showing that it was well tolerated. However, trehalose reduced nerve injury-evoked nociceptive mechanical and thermal hypersensitivity as compared to vehicle. Trehalose had no effect on calcium currents in primary somatosensory neurons, pointing to central mechanisms of the antinociceptive effects. In IntelliCages, trehalose-treated mice showed reduced activity, in particular, a low frequency of nosepokes, which was associated with a reduced proportion of correct trials and flat learning curves in place preference learning tasks. Mice failed to switch corner preferences and stuck to spontaneously preferred corners. The behavior in IntelliCages is suggestive of sedative effects as a 'side effect' of a continuous protracted trehalose treatment, leading to impairment of learning flexibility. Hence, trehalose diet supplements might reduce chronic pain but likely at the expense of alertness.
High glucosylceramides and low anandamide contribute to sensory loss and pain in Parkinson's disease
(2020)
Background: Parkinson's disease (PD) causes chronic pain in two‐thirds of patients, in part originating from sensory neuropathies. The aim of the present study was to describe the phenotype of PD‐associated sensory neuropathy and to evaluate its associations with lipid allostasis, the latter motivated by recent genetic studies associating mutations of glucocerebrosidase with PD onset and severity. Glucocerebrosidase catalyzes the metabolism of glucosylceramides.
Methods: We used quantitative sensory tests, pain ratings, and questionnaires and analyzed plasma levels of multiple bioactive lipid species using targeted lipidomic analyses. The study comprised 2 sets of patients and healthy controls: the first 128 Israeli PD patients and 224 young German healthy controls for exploration, the second 50/50 German PD patients and matched healthy controls for deeper analyses.
Results: The data showed a 70% prevalence of PD pain and sensory neuropathies with a predominant phenotype of thermal sensory loss plus mechanical hypersensitivity. Multivariate analyses of lipids revealed major differences between PD patients and healthy controls, mainly originating from glucosylceramides and endocannabinoids. Glucosylceramides were increased, whereas anandamide and lysophosphatidic acid 20:4 were reduced, stronger in patients with ongoing pain and with a linear relationship with pain intensity and sensory losses, particularly for glucosylceramide 18:1 and glucosylceramide 24:1.
Conclusions: Our data suggest that PD‐associated sensory neuropathies and PD pain are in part caused by accumulations of glucosylceramides, raising the intriguing possibility of reducing PD pain and sensory loss by glucocerebrosidase substituting or refolding approaches. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
GTP cyclohydrolase (GCH1) governs de novo synthesis of the enzyme cofactor, tetrahydrobiopterin (BH4), which is essential for biogenic amine production, bioactive lipid metabolism and redox coupling of nitric oxide synthases. Overproduction of BH4 via upregulation of GCH1 in sensory neurons is associated with nociceptive hypersensitivity in rodents, and neuron‐specific GCH1 deletion normalizes nociception. The translational relevance is revealed by protective polymorphisms of GCH1 in humans, which are associated with a reduced chronic pain. Because myeloid cells constitute a major non‐neuronal source of BH4 that may contribute to BH4‐dependent phenotypes, we studied here the contribution of myeloid‐derived BH4 to pain and itch in lysozyme M Cre‐mediated GCH1 knockout (LysM‐GCH1−/−) and overexpressing mice (LysM‐GCH1‐HA). Unexpectedly, knockout or overexpression in myeloid cells had no effect on nociceptive behaviour, but LysM‐driven GCH1 knockout reduced, and its overexpression increased the scratching response in Compound 48/80 and hydroxychloroquine‐evoked itch models, which involve histamine and non‐histamine dependent signalling pathways. Mechanistically, GCH1 overexpression increased BH4, nitric oxide and hydrogen peroxide, and these changes were associated with increased release of histamine and serotonin and degranulation of mast cells. LysM‐driven GCH1 knockout had opposite effects, and pharmacologic inhibition of GCH1 provided even stronger itch suppression. Inversely, intradermal BH4 provoked scratching behaviour in vivo and BH4 evoked an influx of calcium in sensory neurons. Together, these loss‐ and gain‐of‐function experiments suggest that itch in mice is contributed by BH4 release plus BH4‐driven mediator release from myeloid immune cells, which leads to activation of itch‐responsive sensory neurons.
Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis
(2017)
Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the ‘symptom-free’ intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach.
Aims: Parkinson's disease (PD) is frequently associated with a prodromal sensory neuropathy manifesting with sensory loss and chronic pain. We have recently shown that PD-associated sensory neuropathy in patients is associated with high levels of glucosylceramides. Here, we assessed the underlying pathology and mechanisms in Pink1−/−SNCAA53T double mutant mice. Methods: We studied nociceptive and olfactory behaviour and the neuropathology of dorsal root ganglia (DRGs), including ultrastructure, mitochondrial respiration, transcriptomes, outgrowth and calcium currents of primary neurons, and tissue ceramides and sphingolipids before the onset of a PD-like disease that spontaneously develops in Pink1−/−SNCAA53T double mutant mice beyond 15 months of age. Results: Similar to PD patients, Pink1−/−SNCAA53T mice developed a progressive prodromal sensory neuropathy with a loss of thermal sensitivity starting as early as 4 months of age. In analogy to human plasma, lipid analyses revealed an accumulation of glucosylceramides (GlcCer) in the DRGs and sciatic nerves, which was associated with pathological mitochondria, impairment of mitochondrial respiration, and deregulation of transient receptor potential channels (TRPV and TRPA) at mRNA, protein and functional levels in DRGs. Direct exposure of DRG neurons to GlcCer caused transient hyperexcitability, followed by a premature decline of the viability of sensory neurons cultures upon repeated GlcCer application. Conclusions: The results suggest that pathological GlcCer contribute to prodromal sensory disease in PD mice via mitochondrial damage and calcium channel hyperexcitability. GlcCer-associated sensory neuron pathology might be amenable to GlcCer lowering therapeutic strategies.
Monoclonal antibodies (mAb) are promising therapeutics in multiple sclerosis and multiple new candidates have been developed, hence increasing the need for some agreement for preclinical mAb studies. We systematically analyzed publications of experimental autoimmune encephalomyelitis (EAE) studies showing effects of monoclonal antibodies. A PubMed search retrieved 570 records, out of which 122 studies with 253 experiments were eligible based on experimental design, number of animals and presentation of time courses of EAE scores. Analysis of EAE models, treatment schedules, single and total doses, routes of administration, and onset of treatment from pre-immunization up to 35 days after immunization revealed high heterogeneity. Total doses ranged from 0.1 to 360 mg/kg for observation times of up to 35 days after immunization. About half of experiments (142/253) used total doses of 10–70 mg/kg. Employing this range, we tested anti-Itga4 as a reference mAb at varying schedules and got no, mild or substantial EAE-score reductions, depending on the mouse strain and onset of the treatment. The result agrees with the range of outcomes achieved in 10 reported anti-Itga4 experiments. Studies comparing low and high doses of various mAbs or early vs. late onset of treatment did not reveal dose-effect or timing-effect associations, with a tendency towards better outcomes with preventive treatments starting within the first week after immunization. The systematic comparison allows for extraction of some “common” design characteristics, which may be helpful to further assess the efficacy of mAbs and role of specific targets in preclinical models of multiple sclerosis.
Based on increasing evidence suggesting that MS pathology involves alterations in bioactive lipid metabolism, the present analysis was aimed at generating a complex serum lipid-biomarker. Using unsupervised machine-learning, implemented as emergent self-organizing maps of neuronal networks, swarm intelligence and Minimum Curvilinear Embedding, a cluster structure was found in the input data space comprising serum concentrations of d = 43 different lipid-markers of various classes. The structure coincided largely with the clinical diagnosis, indicating that the data provide a basis for the creation of a biomarker (classifier). This was subsequently assessed using supervised machine-learning, implemented as random forests and computed ABC analysis-based feature selection. Bayesian statistics-based biomarker creation was used to map the diagnostic classes of either MS patients (n = 102) or healthy subjects (n = 301). Eight lipid-markers passed the feature selection and comprised GluCerC16, LPA20:4, HETE15S, LacCerC24:1, C16Sphinganine, biopterin and the endocannabinoids PEA and OEA. A complex classifier or biomarker was developed that predicted MS at a sensitivity, specificity and accuracy of approximately 95% in training and test data sets, respectively. The present successful application of serum lipid marker concentrations to MS data is encouraging for further efforts to establish an MS biomarker based on serum lipidomics.
Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin (BH4)-dependent enzyme with major expression in the liver and white adipose tissue that cleaves alkyl ether glycerolipids. The present study describes the disclosure and biological characterization of a candidate compound (Cp6), which inhibits AGMO with an IC50 of 30–100 µM and 5–20-fold preference of AGMO relative to other BH4-dependent enzymes, i.e., phenylalanine-hydroxylase and nitric oxide synthase. The viability and metabolic activity of mouse 3T3-L1 fibroblasts, HepG2 human hepatocytes and mouse RAW264.7 macrophages were not affected up to 10-fold of the IC50. However, Cp6 reversibly inhibited the differentiation of 3T3-L1 cells towards adipocytes, in which AGMO expression was upregulated upon differentiation. Cp6 reduced the accumulation of lipid droplets in adipocytes upon differentiation and in HepG2 cells exposed to free fatty acids. Cp6 also inhibited IL-4-driven differentiation of RAW264.7 macrophages towards M2-like macrophages, which serve as adipocyte progenitors in adipose tissue. Collectively, the data suggest that pharmacologic AGMO inhibition may affect lipid storage.
Progranulin deficiency in mice is associated with deregulations of the scavenger receptor signaling of CD36/SCARB3 in immune disease models, and CD36 is a dominant receptor in taste bud cells in the tongue and contributes to the sensation of dietary fats. Progranulin-deficient mice (Grn−/−) are moderately overweight during middle age. We therefore asked if there was a connection between progranulin/CD36 in the tongue and fat taste preferences. By using unbiased behavioral analyses in IntelliCages and Phenomaster cages we showed that progranulin-deficient mice (Grn−/−) developed a strong preference of fat taste in the form of 2% milk over 0.3% milk, and for diluted MCTs versus tap water. The fat preference in the 7d-IntelliCage observation period caused an increase of 10% in the body weight of Grn−/− mice, which did not occur in the wildtype controls. CD36 expression in taste buds was reduced in Grn−/− mice at RNA and histology levels. There were no differences in the plasma or tongue lipids of various classes including sphingolipids, ceramides and endocannabinoids. The data suggest that progranulin deficiency leads to a lower expression of CD36 in the tongue resulting in a stronger urge for fatty taste and fatty nutrition.