Benutzerspezifische Werkzeuge


In this project, we will pursue the following three objectives:

(I) Expression and proteomic analysis after overexpression and downregulation of AAAS in the human adrenal cell line NCI-H295R: impact for adrenocortical cell function and steroidogenesis.
With overexpression and a stable knockdown of the AAAS gene in the human adrenal cell line NCI-H295R we will study the qualitative and quantitative effects of ALADIN overexpression and knockdown on the adrenal gene expression and proteomic levels. From the pattern of expression and protein changes we aim at the identification of further interaction partners of ALADIN in the adrenal gland which are important for steroidogenesis and cell survival. As ALADIN has been shown to impair nucleocytoplasmic transport processes in patient fibroblasts, we use the NCI-H295R cell model to assess nucleocytoplasmic transport processes important for normal adrenal microenvironment.

(II) Investigation of the role of ALADIN for the interplay between adrenocortical and medullary function.
The obvious impairment of adrenal medullary function seen in triple A patients leads to the hypotheses that ALADIN is either primarily important for adrenomedullary function or that the absence of ALADIN in adrenocortical cells influences secondarily the function of medullary cells. These hypotheses will be studied in primary co-cultures of bovine adrenocortical and chromaffin cells in which the AAAS gene will be downregulated by siRNA-knockdown experiments. The interaction between adrenocortex and medulla will be studied under normal and enhanced oxidative stress conditions.

(III) Investigation of the role of ALADIN for adrenocortical function under oxidative stress.
The pathogenetic role of ALADIN for adrenocortical function under oxidative stress conditions will be studied in double knockout mice crossing Aaas-/- and Sod2+/- mice strains. Triple A syndrome is associated with an increased oxidative stress level in ALADIN-deficient cells. The mitochondrial antioxidant manganese superoxide dismutase-2 (Sod2) might protect against the stress-induced changes. By reducing the protecting Sod2 activity in Aaas-/- mice to 50 % the phenotype of ALADIN-deficient adrenocortical cells under oxidative stress will be studied on genomic, proteomic, histologic and biochemical levels.