Deficiency in the transcription factor NRF2 worsens inflammatory parameters in a mouse model with combined tauopathy and amyloidopathy

Abstract

Chronic neuroinflammation is a hallmark of the onset and progression of brain proteinopathies such as Alzheimer disease (AD) and it is suspected to participate in the neurodegenerative process. Transcription factor NRF2, a master regulator of redox homeostasis, controls acute inflammation but its relevance in low-grade chronic inflammation of AD is inconclusive due to lack of good mouse models. We have addressed this question in a transgenic mouse that combines amyloidopathy and tauopathy with either wild type (AT-NRF2-WT) or NRF2-deficiency (AT-NRF2-KO). AT-NRF2-WT mice died prematurely, at around 14 months of age, due to motor deficits and a terminal spinal deformity but AT-NRF2-KO mice died roughly 2 months earlier. NRF2-deficiency correlated with exacerbated astrogliosis and microgliosis, as determined by an increase in GFAP, IBA1 and CD11b levels. The immunomodulatory molecule dimethyl fumarate (DMF), a drug already used for the treatment of multiple sclerosis whose main target is accepted to be NRF2, was tested in this preclinical model. Daily oral gavage of DMF during six weeks reduced glial and inflammatory markers and improved cognition and motor complications in the AT-NRF2-WT mice compared with the vehicle-treated animals. This study demonstrates the relevance of the inflammatory response in experimental AD, tightly regulated by NRF2 activity, and provides a new strategy to fight AD.

Graphical abstract

Fig. 1

Death of the AT-mice occurs faster in the absence of NRF2. A, Kaplan-Meier curves of AT-NRF2-WT (n = 269) and AT-NRF2-KO (n = 209) over 3 years of observation. Statistical analysis was performed and a value of ***p < 0.001 was obtained comparing AT-NRF2-WT and AT-NRF2-KO curves. B, pictures from the indicated genotypes illustrating terminal postures. C, X-ray images of mice from the indicated genotypes showing the spinal deformity termed kyphosis. D, representative images of the clasping behavior of wild type and AT-mice when hanging from the tail. E, average score of the motor test performed in which we analyzed ledge, clasping, gait and kyphosis. We tested AT-NRF2-WT and AT-NRF2-KO animals at the indicated ages. Statistical analysis was performed with Student's t-test. *p < 0.5 comparing AT-NRF2-KO vs. AT-NRF2-WT groups.

Fig. 2

Neurodegeneration in the brainstem was exacerbated in the absence of NRF2. A and B, silver staining of sagittal brain sections from 11-months old mice of the indicated genotypes. Pictures show dystrophic neurites (A) and neurons with arginophilic inclusions (B) in the pontine reticular formation. C, quantification of the number of positive neurons with arginophilic inclusions from B. Data are mean ± SEM (n = 3). Statistical analysis was performed with Student's t-test. *p < 0.05, comparing AT-NRF2-KO vs. AT-NRF2-WT groups.

Fig. 3

Gliosis is exacerbated by NRF2 deficiency in hippocampi of AT-NRF2-KO mice. A and B, brain sections from 11-months old AT-NRF2-WT and AT-NRF2-KO mice were stained with GFAP or CD11b, respectively. Pictures show CA1 and subiculum regions of the hippocampus. C and E, mRNA levels determined by qRT-PCR of the indicated genes normalized to the expression of ActB. Data are mean ± SEM (n = 4). Statistical analysis was performed with Student's t-test. *p < 0.05, comparing AT-NRF2-KO vs. AT-NRF2-WT mice. D, number of CD11b-positive microglial cells in CA1 and subiculum of the indicated genotypes. Data are mean ± SEM (n = 3) represented as % of the total number of cells. Statistical analysis was performed with Student's t-test. *p < 0.05, comparing AT-NRF2-KO vs. AT-NRF2-WT mice.

Fig. 4

Increased glial activation in the brainstem of AT-NRF2-KO mice. A, immunostaining with anti-GFAP and anti-IBA1 in sagittal sections of the pontine reticular formation of the brainstem in the indicated genotypes. B, immunoblot analysis of GFAP and IBA1 levels in brainstem and spinal cord homogenates from 11-month old AT-NRF2-WT and AT-NRF2-KO mice. Protein levels of GAPDH were analyzed to ensure similar load per lane. C, densitometric quantification of representative blots from B. Data are mean ± SEM (n = 3). Statistical analysis was performed with Student's t-test. *p < 0.05, comparing AT-NRF2-KO vs. AT-NRF2-WT mice.

Fig. 5

Glial activation in the spinal cord of AT-NRF2-WT and AT-NRF2-KO mice. A, immunostaining with anti-GFAP or anti-IBA1 in sagittal sections of the spinal cord in the indicated genotypes. Pictures show white (a) and grey (b) matter of the anterior horn. Spinal cord sections were counterstained with hematoxylin. B, immunoblot analysis of GFAP and IBA1 levels in spinal cord homogenates from 11-month old AT-NRF2-WT and AT-NRF2-KO mice. Protein levels of GAPDH were analyzed to ensure similar load per lane. C, densitometric quantification of representative blots from B. Data are mean ± SEM (n = 3). Statistical analysis was performed with t-Student test.

Fig. 6

DMF improves memory and motor deficits in AT-NRF2-WT mice. 9-month old AT-NRF2-WT mice received intragastric doses of vehicle (n = 10) or DMF (100 mg/kg, n = 10) once every two days during six weeks. A, qRT-PCR determination of brain mRNA levels of the NRF2-regulated genes Nrf2, Nqo1, Osgin1, and Gstm1 normalized by the average of Actb, Tbp and Gapdh. Data are mean ± SEM (n = 4). Statistical analysis was performed with Student's t-test. **p < 0.01 and *p < 0.05, comparing DMF vs. vehicle treated mice. B and C, immunostaining with anti-GFAP (B) and anti-IBA1 (C) in sagittal brain sections of the indicated groups. Pictures show CA1 and subiculum of the hippocampus as well as the pontine reticular formation of the brainstem. D, immunoblot analysis of the indicated protein levels in brain homogenates from vehicle- or DMF-treated mice. Protein levels of ACTB were analyzed to ensure similar load per lane. E, densitometric quantification of representative blots from D. Data are mean ± SEM (n = 3). Statistical analysis was performed with Student's t-test. **< 0.01 and *p < 0.05, comparing DMF vs. vehicle treated mice. F, average score of the motor test performed in which we analyzed ledge, clasping, gait and kyphosis at the indicated times. Statistical analysis was performed with two-way ANOVA followed by Bonferroni post-hoc test. *p < 0.05, comparing DMF vs. vehicle treated mice. G, discrimination index obtained in the novel object recognition task (total time spent with new object/total time of object exploration) by the two experimental groups. Statistical analysis was performed with two-way ANOVA followed by Bonferroni post-hoc test. *p < 0.05, comparing DMF vs. vehicle treated mice.