Neuroprotective Properties of Dimethyl Fumarate Measured by Optical Coherence Tomography in Non-inflammatory Animal Models.

While great advances have been made in the immunomodulatory treatment of multiple sclerosis (MS), there is still an unmet need for drugs with neuroprotective potential. Dimethyl fumarate (DMF) has been suggested to exert both immunomodulatory and neuroprotective effects in MS. To investigate if DMF has neuroprotective effects independent of immunomodulation we evaluated its effects in the non-inflammatory animal models of light-induced photoreceptor loss and optic nerve crush. This might also reveal applications for DMF besides MS, such as age related macular degeneration. Retinal neurodegeneration was longitudinally assessed by in vivo retinal imaging using optical coherence tomography (OCT), and glutathione (GSH) measurements as well as histological investigations were performed to clarify the mode of action. For light-induced photoreceptor loss, one eye of C57BL/6J mice was irradiated with a LED cold light lamp while for optic nerve crush the optic nerve was clamped behind the eye bulb. The other eye served as control. GSH was measured in the optic nerve, choroid and retina and immunohistological staining of retinal microglia (Iba1) was performed. Mice were treated with 15 or 30 mg DMF/kg bodyweight or vehicle. While no protective effects were observed in optic nerve crush, in the light-induced retinal degeneration model DMF treatment significantly reduced retinal degeneration. In these mice, GSH levels in the retina and surrounding choroid were increased and histological investigations revealed less microglial activation in the outer retinal layers, suggesting both antioxidant and anti-inflammatory effects.

Early alpha-lipoic acid therapy protects from degeneration of the inner retinal layers and vision loss in an experimental autoimmune encephalomyelitis-optic neuritis model.

BACKGROUND: In multiple sclerosis (MS), neurodegeneration is the main reason for chronic disability. Alpha-lipoic acid (LA) is a naturally occurring antioxidant which has recently been demonstrated to reduce the rate of brain atrophy in progressive MS. However, it remains uncertain if it is also beneficial in the early, more inflammatory-driven phases. As clinical studies are costly and time consuming, optic neuritis (ON) is often used for investigating neuroprotective or regenerative therapeutics. We aimed to investigate the prospect for success of a clinical ON trial using an experimental autoimmune encephalomyelitis-optic neuritis (EAE-ON) model with visual system readouts adaptable to a clinical ON trial. METHODS: Using an in vitro cell culture model for endogenous oxidative stress, we compared the neuroprotective capacity of racemic LA with the R/S-enantiomers and its reduced form. In vivo, we analyzed retinal neurodegeneration using optical coherence tomography (OCT) and the visual function by optokinetic response (OKR) in MOG35-55-induced EAE-ON in C57BL/6J mice. Ganglion cell counts, inflammation, and demyelination were assessed by immunohistological staining of retinae and optic nerves. RESULTS: All forms of LA provided equal neuroprotective capacities in vitro. In EAE-ON, prophylactic LA therapy attenuated the clinical EAE score and prevented the thinning of the inner retinal layer while therapeutic treatment was not protective on visual outcomes. CONCLUSIONS: A prophylactic LA treatment is necessary to protect from visual loss and retinal thinning in EAE-ON, suggesting that a clinical ON trial starting therapy after the onset of symptoms may not be successful.

Aryl hydrocarbon receptor repressor (AhRR) function revisited: repression of CYP1 activity in human skin fibroblasts is not related to AhRR expression.

The skin reacts to environmental noxae by inducing cytochrome P450 (CYP)-catalyzed reactions via activation of the aryl hydrocarbon receptor (AhR). A drawback of this response is the generation of oxidative stress, which is especially dangerous for postreplicative cells such as dermal fibroblasts, in which damage may accumulate over time. Accordingly, in dermal fibroblasts, CYP1 expression is repressed and it has been proposed that this is due to the AhR repressor (AhRR), which is supposedly overexpressed in fibroblasts as compared with other skin cells. Here, we revisited this "AhRR hypothesis", which has been mainly based on ectopic overexpression studies and correlation analyses of high AhRR gene expression with CYP1A1 repression in certain cell types. In primary human skin fibroblasts (NHDFs) of 25 individuals, we found that (i) the AhRR was expressed only at moderate RNA copy numbers and that, against the common view, (ii) in some fibroblast strains, CYP1A1 mRNA expression could be induced by AhR activators. However, even the highest induction did not translate into measurable CYP1 enzyme activity, and neither basal expression nor mRNA inducibility correlated with AhRR expression. In addition, enhancement of CYP1A1 mRNA expression by trichostatin A, which inhibits AhRR-recruited histone deacetylases at the CYP1A1 promoter, failed to induce measurable CYP1 activity. Finally, AhRR-deficient ((-/-)) mouse embryonic fibroblasts were not induced to biologically relevant CYP1 enzyme activity despite impressive mRNA induction. These data clearly indicate that repressed CYP1 activity in NHDFs is not causally related to AhRR expression, which may serve a different, yet unknown, biological function.

Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression.

Urea is an endogenous metabolite, known to enhance stratum corneum hydration. Yet, topical urea anecdotally also improves permeability barrier function, and it appears to exhibit antimicrobial activity. Hence, we hypothesized that urea is not merely a passive metabolite, but a small-molecule regulator of epidermal structure and function. In 21 human volunteers, topical urea improved barrier function in parallel with enhanced antimicrobial peptide (AMP; LL-37 and ß-defensin-2) expression. Urea stimulates the expression of, and is transported into, keratinocytes by two urea transporters (UTs), UT-A1 and UT-A2, and by aquaporins 3, 7, and 9. Inhibitors of these UTs block the downstream biological effects of urea, which include increased mRNA and protein levels of (i) transglutaminase-1, involucrin, loricrin, and filaggrin, (ii) epidermal lipid synthetic enzymes, and (iii) cathelicidin/LL-37 and ß-defensin-2. Finally, we explored the potential clinical utility of urea, showing that topical urea applications normalized both barrier function and AMP expression in a murine model of atopic dermatitis. Together, these results show that urea is a small-molecule regulator of epidermal permeability barrier function and AMP expression after transporter uptake, followed by gene regulatory activity in normal epidermis, with potential therapeutic applications in diseased skin.

Effects of the genotoxic compounds, benzo[a]pyrene and cyclophosphamide on phase 1 and 2 activities in EpiDerm™ models.

The micronucleus assay in the 3D human reconstructed EpiDerm™ skin model (RSMN) is a promising new assay for evaluating genotoxicity of dermally applied chemicals. To complement the testing of metabolically activated chemicals, such as cyclophosphamide (CPA) and benzo[a]pyrene (B[a]P), we measured phase 1 (ethoxyresorufin O-deethylation (EROD) and testosterone metabolism) and 2 activities (UGTs and GSTs) in non-treated and genotoxin treated EpiDerm™ models in a study design which mimics the RSMN assay. The assay involved a three-dose dosing regimen over 72 h to take into account effects e.g. enzyme induction, which requires longer than the standard 2 dose 48-h assay. These studies demonstrated the presence of basal phase 1 and 2 activities of EpiDerm™ models. With the exception of GST, all of the activities measured did not reproducibly change over time. It was possible to measure enzyme induction using this assay design. EROD activity was significantly induced by B[a]P but not by CPA. CPA and B[a]P had little or no reproducible effects on GST and UGT activities. In conclusion, a number of metabolic enzyme activities were present in the EpiDerm™ skin model and at least the CYP1 family was inducible.

Photoprotection against UVAR: effective triterpenoids require a lipid raft stabilizing chemical structure.

UVA(Ultraviolet A)-induced gene expression is supposed to be a hallmark for inflammation, for immunosuppression and in long-term cancer formation. In previous studies, we have shown for keratinocytes that physiological doses of UVA radiation result in the upregulation of gene expression mediated by ceramide formation from sphingolipids/cholesterol-rich microdomains (rafts), which can be blocked by preloading keratinocytes with cholesterol or plant sterols. Here, we show that besides stigmasterol and ß-sitosterol, also sterols like 14-dehydroergosterol, ergosterol-peroxide and 29-norcycloartenol inhibit the UVA response. Moreover, we present evidence that natural material-derived triterpenoids such as oleanolic acid can abrogate UVA-induced gene expression by raft stabilization. This effect depends on the structure of the molecule, because its isomer ursolic acid also integrates within the rafts without inhibiting ceramide formation and upregulation of gene expression.