Laszlo Bodai

A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila

The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntingtons disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds.

ERK Activation by the Polyphenols Fisetin and Resveratrol Provides Neuroprotection in Multiple Models of Huntington's Disease

Huntingtons disease (HD) is an inherited, progressive and ultimately fatal neurodegenerative disorder that is characterized by psychiatric, cognitive and motor symptoms. Among the pathways implicated in HD are those involving mitogen-activated protein kinase signaling and particularly the Ras-extracellular signal-regulated kinase (ERK) cascade. Studies in both cells and animal models suggest that ERK activation might provide a novel therapeutic target for the treatment of HD but compounds that specifically activate ERK are few. To test the hypothesis that pharmaceutical activation of ERK might be protective for HD, a polyphenol, fisetin, which was previously shown to activate the Ras-ERK cascade, was tested in three different models of HD: PC12 cells expressing mutant Httex1 under the control of an inducible promoter, Drosophila expressing mutant Httex1 and the R6/2 mouse model of HD. The results indicate that fisetin can reduce the impact of mutant huntingtin in each of these disease models. Prompted by this observation, we determined that the related polyphenol, resveratrol, also activates ERK and is protective in HD models. Notably, although more than a dozen small molecule inhibitors of ERK activation are in clinical trials, very few small molecule activators of ERK signaling are reported. Thus, fisetin, resveratrol and related compounds might be useful for the treatment of HD by virtue of their unique ability to activate ERK.

Targeting H3K4 trimethylation in Huntington disease

Significance Transcriptional dysregulation is an early and reproducible feature of Huntington disease (HD); however, mechanisms underlying this dysregulation are unclear. This article describes a unique pattern of the chromatin mark H3K4me3 at transcriptionally repressed promoters in HD mouse and human brain identified by genome-wide analysis. Reducing the levels of the demethylase SMCX/Jarid1c in primary neurons reversed down-regulation of key neuronal genes caused by mutant Huntingtin expression and was neuroprotective in a Drosophila HD model. These results suggest that targeting epigenetic signatures may be an effective strategy to ameliorate the consequences of HD and other neurodegenerative diseases. Transcriptional dysregulation is an early feature of Huntington disease (HD). We observed gene-specific changes in histone H3 lysine 4 trimethylation (H3K4me3) at transcriptionally repressed promoters in R6/2 mouse and human HD brain. Genome-wide analysis showed a chromatin signature for this mark. Reducing the levels of the H3K4 demethylase SMCX/Jarid1c in primary neurons reversed down-regulation of key neuronal genes caused by mutant Huntingtin expression. Finally, reduction of SMCX/Jarid1c in primary neurons from BACHD mice or the single Jarid1 in a Drosophila HD model was protective. Therefore, targeting this epigenetic signature may be an effective strategy to ameliorate the consequences of HD.

The homologous Drosophila transcriptional adaptors ADA2a and ADA2b are both required for normal development but have different functions

ABSTRACT In Drosophila and several other metazoan organisms, there are two genes that encode related but distinct homologs of ADA2-type transcriptional adaptors. Here we describe mutations of the two Ada2 genes of Drosophila melanogaster. By using mutant Drosophila lines, which allow the functional study of individual ADA2s, we demonstrate that both Drosophila Ada2 genes are essential. Ada2a and Ada2b null homozygotes are late-larva and late-pupa lethal, respectively. Double mutants have a phenotype identical to that of the Ada2a mutant. The overproduction of ADA2a protein from transgenes cannot rescue the defects resulting from the loss of Ada2b, nor does complementation work vice versa, indicating that the two Ada2 genes of Drosophila have different functions. An analysis of germ line mosaics generated by pole-cell transplantation revealed that the Ada2a function (similar to that reported for Ada2b) is required in the female germ line. A loss of the function of either of the Ada2 genes interferes with cell proliferation. Interestingly, the Ada2b null mutation reduces histone H3 K14 and H3 K9 acetylation and changes TAF10 localization, while the Ada2a null mutation does not. Moreover, the two ADA2s are differently required for the expression of the rosy gene, involved in eye pigment production, and for Dmp53-mediated apoptosis. The data presented here demonstrate that the two genes encoding homologous transcriptional adaptor ADA2 proteins in Drosophila are both essential but are functionally distinct.

Differentiation-regulated expression of Toll-like receptors 2 and 4 in HaCaT keratinocytes.

Toll-like receptors (TLRs) play an important role in the recognition of pathogens in keratinocytes. In this study, we investigated whether the differentiation state of HaCaT keratinocytes correlates with the expression of TLR2 and TLR4 genes. The expression levels of TLR2 and TLR4 in a HaCaT differentiation model system were determined using quantitative real-time RT-PCR (Q-RT-PCR) and flow cytometry. The progression of keratinocyte differentiation was monitored by determining the level of involucrin gene expression using Q-RT-PCR. The expression levels of TLR2 and TLR4 increased with the stage of differentiation and there were strong correlations between the expression level of the involucrin gene and those of the TLR2 gene (r=0.809, P<0.0001) and the TLR4 gene (r=0.568, P<0.02). Increased cell surface expression of TLR2 and TLR4 was also found in differentiated HaCaT keratinocytes by flow cytometric analysis. Our findings suggest that upregulation of TLR expression during differentiation in keratinocytes could be a part of the differentiation process of keratinocytes and could have biological significance in protecting skin against microbes.

Treatment of atopic dermatitis with the xenon chloride excimer laser

Background  Narrow‐band ultraviolet B phototherapy is an effictive and safe treatment for atopic dermatitis. We have previously found that the 308 nm xenon chloride excimer laser was more effective than the narrow‐band ultraviolet B light for the treatment of psoriasis, suggesting that ultraviolet B laser might offer advantages over narrow‐band ultraviolet B.

The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

In Drosophila, the dADA2b-containing dSAGA complex is involved in histone H3 lysine 9 and 14 acetylation. Curiously, although the lysine 9- and 14-acetylated histone H3 levels are drastically reduced in dAda2b mutants, these animals survive until a late developmental stage. To study the molecular consequences of the loss of histone H3 lysine 9 and 14 acetylation, we compared the total messenger ribonucleic acid (mRNA) profiles of wild type and dAda2b mutant animals at two developmental stages. Global gene expression profiling indicates that the loss of dSAGA-specific H3 lysine 9 and 14 acetylation results in the expression change (up- or down-regulation) of a rather small subset of genes and does not cause a general transcription de-regulation. Among the genes up-regulated in dAda2b mutants, particularly high numbers are those which play roles in antimicrobial defense mechanisms. Results of chromatin immunoprecipitation experiments indicate that in dAda2b mutants, the lysine 9-acetylated histone H3 levels are decreased both at dSAGA up- and down-regulated genes. In contrast to that, in the promoters of dSAGA-independent ribosomal protein genes a high level of histone H3K9ac is maintained in dAda2b mutants. Our data suggest that by acetylating H3 at lysine 9, dSAGA modifies Pol II accessibility to specific promoters differently.

Genes of the ecdysone biosynthesis pathway are regulated by the dATAC histone acetyltransferase complex in Drosophila.

ABSTRACT Uncovering mechanisms that regulate ecdysone production is an important step toward understanding the regulation of insect metamorphosis and processes in steroid-related pathologies. We report here the transcriptome analysis of DrosophilamelanogasterdAda2a and dAda3 mutants, in which subunits of the ATAC acetyltransferase complex are affected. In agreement with the fact that these mutations lead to lethality at the start of metamorphosis, both the ecdysone levels and the ecdysone receptor binding to polytene chromosomes are reduced in these flies. The cytochrome genes (spookier, phantom, disembodied, and shadow) involved in steroid conversion in the ring gland are downregulated, while the gene shade, which is involved in converting ecdysone into its active form in the periphery, is upregulated in these dATAC subunit mutants. Moreover, driven expression of dAda3 at the site of ecdysone synthesis partially rescues dAda3 mutants. Mutants of dAda2b, a subunit of the dSAGA histone acetyltransferase complex, do not share phenotype characteristics and RNA profile alterations with dAda2a mutants, indicating that the ecdysone biosynthesis genes are regulated by dATAC, but not by dSAGA. Thus, we provide one of the first examples of the coordinated regulation of a functionally linked set of genes by the metazoan-specific ATAC complex.

Endogenous Phospholipid Metabolite Containing Topical Product Inhibits Ultraviolet Light-Induced Inflammation and DNA Damage in Human Skin

Background: N-palmitoylethanolamine (PEA) and organic osmolytes are endogenous components of the human epidermis and are generated from phospholipids in the stratum granulosum. PEA has been shown to exert potent antioxidant and anti-inflammatory activities. The endogenous organic osmolytes such as betaine and sarcosine control skin humidity, but have also been shown to inhibit ultraviolet (UV) light-induced oxidative stress in keratinocytes. Objectives: To investigate the effect of a PEA- and organic osmolyte-containing topical product (Physiogel AI®) on the development of UV light-induced erythema, thymine dimer formation and p53 tumor suppressor gene activation, as well as intercellular adhesion molecule 1 (ICAM-1) and Ki67 expression in normal human skin. Methods: The UV-induced erythema was measured by a spectrofluorometric method. Thymine dimers, p53, ICAM-1 and Ki67 were detected in skin biopsies using immunohistochemistry. Results: Physiogel AI cream significantly inhibited the development of UV light-induced erythema and thymine dimer formation in normal human skin, but did not alter the number of Ki67+ proliferating keratinocytes and the expression of p53 and ICAM-1. Conclusions: Our results suggest that PEA and organic osmolytes might represent a new generation of compounds which suppress UV-induced photodamage.

The Arg160Trp allele of melanocortin-1 receptor gene might protect against vitiligo.

Melanocortin‐1 receptor (MC1R) and agouti signaling protein (ASIP) play pivotal roles in the regulation of human pigmentation. We aimed to study whether single nucleotide polymorphisms (SNPs) of the MC1R and ASIP genes contribute to the pathogenesis of the polygenic pigment skin disorder, vitiligo. The PCR‐amplified, full‐length MC1R gene was studied with sequence analysis, and the 3′ untranslated region (3′ UTR) SNP of ASIP was detected using restriction fragment length polymorphism. The allele frequency of the ASIP SNP did not show any difference between the skin type, hair color and eye color‐matched 97 vitiligo patients and the 59 healthy control individuals. As one of the MC1R polymorphisms showed significantly higher incidence among fair‐skinned individuals (Fitzpatrick I + II, n = 140) than among dark‐skinned individuals (Fitzpatrick III + IV, n = 90), both vitiligo patients and controls were divided into two groups and the frequency of the MC1R alleles was studied separately in fair‐skinned and dark‐skinned subgroups of diseased and healthy groups. C478T, one of the MC1R SNPs studied in 108 fair‐skinned vitiligo patients and in 70 fair‐skinned healthy control individuals, showed a significant difference (P = 0.0262, odds ratio [95% confidence interval] = 3.6 [0.0046–0.1003]) in allele frequency between the two groups: the allele frequency was higher in the control group, suggesting protection against vitiligo. Computer prediction of antigenicity has revealed that the Arg160Trp amino acid change caused by this SNP results in a decrease in antigenicity of the affected peptide epitope.