Franziska D. Weber1, Isabelle Weinhofer1, Sonja Forss-Petter1, Harald Maier2, Johannes Berger1, Willi H.A. Weber1
(1) Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria (2) Department of Dermatology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
Objectives: X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disease with impaired very long-chain fatty acid (VLCFA) metabolism. Mutations in the ABCD1 gene, encoding a peroxisomal membrane protein (ABCD1), cause this clinically heterogeneous disease. X-ALD can manifest as a rapidly progressive, inflammatory cerebral demyelination (CALD). The only curative therapies are transplantations of allogeneic or genetically corrected autologous hematopoietic stem cells. Thus, alternative therapies are urgently needed. Recently, we demonstrated that monocytes but not lymphocytes are affected in X-ALD, implying that metabolic correction of monocytes/macrophages (MO/MP) should halt the inflammation in CALD (1). As overexpression of ABCD2, the closest homologue of ABCD1, restores VLCFA metabolism in vitro (2) and in vivo (3), we investigated candidate drugs (e.g. retinoids) to induce ABCD2 in MO/MP.
Methods: The monocytic cell line THP-1, human primary MO and in vitro differentiated MP were treated with retinoids for 24h, followed by qRT-PCR analysis of ABCD1/2/3 and HPRT (reference gene) mRNA levels. MO, B cells and enriched T cells were isolated from peripheral blood of controls and acne patients before and during retinoid therapy for analysis of ABCD1/2/3 mRNA levels. A linear mixed effect (lme) model was used with the log-transformed mRNA copies as response and two primary covariates, a gene factor (HPRT, ABCD1/2/3) and a population (Acne patients, controls) or treatment (drug exposure) factor. Using the lme function of the nlme R-package (4, 5) with the treatment contrast, we obtained means of ABCD1/2/3 mRNA normalized to HPRT of controls and differences to the population/treatment factor.
Results: In THP-1 cells, 13-cis-retinoic acid (13-CRA) revealed the highest, 5-fold increase (Emax) of ABCD2 mRNA with a potency (EC50) of 0.03 µM. ABCD2 mRNA levels in MO of untreated and 13-CRA-treated (0.75 mg/kg over 6 months) acne patients remained in the range of control MO. In differentiated MP treated in vitro, we observed a 4-fold induction of the ABCD2 mRNA with 7 µM 13-CRA.
Conclusions: Apparently, the accessibility of the ABCD2 promoter changes during differentiation of MO into MP. However, the relative induction of ABCD2 expression by 13-CRA alone would not suffice to compensate for ABCD1 deficiency in these cells. Lme with the treatment contrast was an effective tool to analyse these data, as the results directly reflect the mean log ratio of ABCD1/2/3 to HPRT.
References:
[1] Weber, F.D., Wiesinger, C., Forss-Petter, S., Regelsberger, G., Einwich, A., Weber, W.H.A., Köhler, W., Stockinger, H. and Berger, J. (2014) X-linked adrenoleukodystrophy: Very long-chain fatty acid metabolism is severely impaired in monocytes but not in lymphocytes. Hum. Mol. Genet., 10.1093/hmg/ddt645.
[2] Netik, A., Forss-Petter, S., Holzinger, A., Molzer, B., Unterrainer, G. and Berger, J. (1999) Adrenoleukodystrophy-related protein can compensate functionally for adrenoleukodystrophy protein deficiency (X-ALD): implications for therapy. Hum. Mol. Genet., 8, 907–913.
[3] Pujol, A., Ferrer, I., Camps, C., Metzger, E., Hindelang, C., Callizot, N., Ruiz, M., Pàmpols, T., Giròs, M. and Mandel, J.L. (2004) Functional overlap between ABCD1 (ALD) and ABCD2 (ALDR) transporters: a therapeutic target for X-adrenoleukodystrophy. Hum. Mol. Genet., 13, 2997–3006.
[4] Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. and R Development Core Team (2013) nlme: Linear and Nonlinear Mixed Effects Models. R package version 3.1-111.
[5] R Core Team (2013) R: A language and environment for statistical computing R Foundation for Statistical Computing, Vienna, Austria.
Reference: PAGE 23 (2014) Abstr 3273 [www.page-meeting.org/?abstract=3273]
Poster: Drug/Disease modeling - Other topics