Mast cell tetrahydrobiopterin contributes to itch in mice.

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.

Tetrahydrobiopterin Attenuates DSS-evoked Colitis in Mice by Rebalancing Redox and Lipid Signalling.

BACKGROUND AND AIMS: Guanosine triphosphate cyclohydrolase [GCH1] governs the production of the enzyme cofactor tetrahydrobiopterin [BH4] which is essential for biogenic amine synthesis, lipid metabolism via alkylglycerol monooxygenase [AGMO], and redox coupling of nitric oxide synthases [NOSs]. Inflammation-evoked unequal regulation of GCH1 and NOS or AGMO may cause redox stress and lipid imbalances. METHODS: The present study assessed potential therapeutic effects of rebalancing these systems with BH4 in experimental colitis in mice. RESULTS: Oral treatment with BH4 as a suspension of crushed tablets attenuated colitis, whereas inhibition of its production had opposite effects: aggravated weight loss, epithelial haemorrhages and ulcers, neutrophil infiltrates, production of reactive oxygen species, and unfavourable profile changes of endocannabinoids, ceramides, and lysophosphatidic acids. Conversely, oral BH4 normalised biopterin, reduced in vivo activity of oxidases and peroxidases in the inflamed gut, favoured nitric oxide over hydrogen peroxide, and maintained normal levels of lipid signalling molecules. BH4 favoured thereby resident CD3+CD8+ and regulatory CD3+CD25+ intraepithelial T cells that are important for epithelial integrity. CONCLUSIONS: BH4 protected against colitis in mice via two major pathways: [i] by reduction of oxidative stress; and [ii] by re-orchestration of alkyl- and acylglycerolipid signalling via AGMO. Oral treatment with BH4 is a safe approved supplementary therapy for genetic BH4 deficiency and did not excessively increase systemic BH4 levels. Therefore, one may consider repurposing of oral BH4 as an adjunctive treatment for colitis.

Activation of Mitochondrial Complex II-Dependent Respiration Is Beneficial for α-Synucleinopathies.

Parkinson's disease and dementia with Lewy bodies are major challenges in research and clinical medicine world-wide and contribute to the most common neurodegenerative disorders. Previously, specific mitochondrial polymorphisms have been found to enhance clearance of amyloid-ß from the brain of APP-transgenic mice leading to beneficial clinical outcome. It has been discussed whether specific mitochondrial alterations contribute to disease progression or even prevent toxic peptide deposition, as seen in many neurodegenerative diseases. Here, we investigated α-synuclein-transgenic C57BL/6J mice with the A30P mutation, and a novel A30P C57BL/6J mouse model with three mitochondrial DNA polymorphisms in the ND3, COX3 and mtRNA(Arg) genes, as found in the inbred NOD/LtJ mouse strain. We were able to detect that the new model has increased mitochondrial complex II-respiration which occurs in parallel to neuronal loss and improved motor performance, although it exhibits higher amounts of high molecular weight species of α-synuclein. High molecular weight aggregates of different peptides are controversially discussed in the light of neurodegeneration. A favourable hypothesis states that high molecular weight species are protective and of minor importance for the pathogenesis of neurodegenerative disorders as compared to the extreme neurotoxic monomers and oligomers. Summarising, our results point to a potentially protective and beneficial effect of specific mitochondrial polymorphisms which cause improved mitochondrial complex II-respiration in α-synucleinopathies, an effect that could be exploited further for pharmaceutical interventions.

Regulation of circulating endocannabinoids associated with cancer and metastases in mice and humans.

BACKGROUND AND AIMS: Endocannabinoids may modify cancer development, progression and associated pain. We determined whether cancer-evoked dysregulations in this system become manifest in altered tissue and plasma endocannabinoids. METHODS: Endocannabinoid changes due to cancer were explored in a local and metastatic syngeneic mouse melanoma model. Endocannabinoid stratification in human cancer was cross-sectionally assessed in the plasma of 304 patients (147 men, 157 women, aged 32 - 87 years) suffering from several types of cancer at Roman Numeral Staging between I and IVc, mostly IV (n = 220), and compared with endocannabinoids of healthy controls. RESULTS: In mice with local tumor growth, ethanolamide endocannabinoids, i.e., anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were downregulated, whereas 2-arachidonoylglycerol (2-AG) was increased. Upon spreading of the cancer cells particularly 2-AG steadily increased in parallel to disease progression while OEA modulated cell migration. Results translated into humans, in whom cancer was associated with a decreased AEA, increased 2-AG and increased OEA correlating with the number of metastases. CONCLUSIONS: The endocannabinoid system was subject to cancer-associated regulations to an extent that led to measurable changes in circulating endocannabinoid levels, emphasizing the importance of the endocannabinoid system in the pathophysiology of cancer.

Hypoxia-inducible factor 1 regulates heat and cold pain sensitivity and persistence.

AIMS: The present study assessed the functions of the transcription factor hypoxia-inducible factor (HIF) in sensory neurons in models of acute, inflammatory, ischemic, and neuropathic pain. The alpha subunit, HIF1α, was specifically deleted in neurons of the dorsal root ganglia by mating HIF1α(fl/fl) mice with SNScre mice. RESULTS: SNS-HIF1α(-/-) mice were more sensitive to noxious heat and cold pain stimulation than were HIF1α(fl/fl) control mice. They also showed heightened first-phase nociceptive responses in the formalin and capsaicin tests with increased numbers of cFos-positive neurons in the dorsal horn, and intensified hyperalgesia in early phases after paw inflammation and hind limb ischemia/reperfusion. The behavioral cold and heat pain hypersensitivity was explained by increased calcium fluxes after transient receptor potential channel activation in primary sensory neurons of SNS-HIF1α(-/-) mice and lowered electrical activation thresholds of sensory fibers. SNS-HIF1α(-/-) mice however, developed less neuropathic pain after sciatic nerve injury, which was associated with an abrogation of HIF1-mediated gene up-regulation. INNOVATION: The results suggest that HIF1α is protective in terms of acute heat and cold pain but in case of ongoing activation in injured neurons, it may promote the development of neuropathic pain. CONCLUSION: The duality of HIF1 in pain regulation may have an impact on the side effects of drugs targeting HIF1, which are being developed, for example, as anticancer agents. Specifically, in patients with cancer neuropathy, however, temporary HIF1 inhibition might provide a welcome combination of growth and pain reduction.

Decreased expression of nardilysin in SH-SY5Y cells under ethanol stress and reduced density of nardilysin-expressing neurons in brains of alcoholics.

There is evidence for a genetic link between the metalloendopeptidase nardilysin and alcohol dependence, but the functional implication of the enzyme in alcoholism is unknown. Interestingly, some of the enzyme's substrates and interaction partners are altered in neural and non-neural tissues under the influence of ethanol consumption. To learn more about putative roles of nardilysin in alcohol dependence we studied the expression of the enzyme protein in human neuroblastoma cells under chronic ethanol exposure as well as in four brain regions of alcoholics and matched controls. Cultured SH-SY5Y cells were exposed for 96 h to two different concentrations of ethanol (50 and 200 mM). Nardilysin expression was determined using Western blotting with densitometric analysis. Furthermore, we morphometrically studied the cellular expression of nardilysin in postmortem brains of eight chronic alcoholics and nine controls by counting the number of nardilysin-immunopositive neurons in left frontal limbic area, Nuc. basalis of Meynert, paraventricular and supraoptic hypothalamic nuclei and calculating numerical cell densities. Nardilysin expression was significantly reduced after 96 h of SH-SY5Y cells exposure to 200 mM ethanol. In human brains nardilysin protein was localized to multiple neurons. In heavy drinkers there was a significantly reduced density of nardilysin immunoreactive neurons in Nuc. basalis of Meynert, paraventricular, and supraoptic nuclei. The alcohol-dependent reduction of nardilysin in cell culture and nervous tissue points to an implication of the enzyme in the pathophysiology of alcoholism.