Figure 1

Gain-of-function model for conditional IKK/NF-κB signalling in excitatory forebrain neurons. (A) Generation of the conditional transgenic IKK2^nCA mouse model using the tetracycline-regulated gene expression system (tet-off). The Camk2a promoter drives the expression of the tetracycline transactivator protein (tTA). The tTA-activated promoter (7xtetO) directs the transcription of luciferase and the constitutively active IKK2-CA allele (IKK2^nCA). Doxycycline (DOX) administration blocks transgene expression. (B) In vivo luciferase measurement of IKK2^nCA mice indicates forebrain-restricted transgene expression (left panel). Luciferase expression (p/s/cm2/sr) showed rapid transgene expression already within one week after DOX removal that sustains with age (right panel) (n = 14). (C) Robust transgene expression over time. 3M, 6M, 9M = 3, 6 and 9 month (n = 13). (D) Luciferase activity was measured in different organs and brain regions of 3M old mice. RLU/μg = relative light units per μg protein (n = 4). (E) IKK2-CA transgene expression (hIKK2) was monitored in different brain regions of 3M old mice. Erk2 is used as loading control (n = 3). (F) Immunohistochemical analysis of IKK2-CA expression (hIKK2, red; NeuN; green; 9M). Arrows indicate IKK2-CA expression in CA1 co-expressed with NeuN (yellow). DAPI (blue). Scale bar: 50 μm. (G) IKK2^nCA mice (9M) show increased nuclear RelA staining in the DG. Inserts depict magnification of marked areas. (H) Increased IKK activity in striatal lysates of IKK2^nCA mice (3M) was monitored by in vitro kinase assay using GST-IκBα as IKK substrate. Immunoprecipitated NEMO protein levels serve as loading control. (A-H) Co = wild type and single transgenic littermates, IKK2^nCA = mice with neuron-specific IKK2-CA expression. Hi, hippocampus; cx, cortex, str, striatum; Ol.B, Olfactory bulbi; H.thal, hypothalamus; Thal, thalamus, M.B, midbrain; Cb, cerebellum; Med, medulla; Sp.co., spinal cord. All data are shown as mean ± SEM. All p-values are derived from two-tailed-unpaired student’s t test.