FTO (fat-mass and obesity-associated protein) deficiency aggravates age-dependent depression-like behaviors and cognitive impairment

Abstract

Background: The demethylase fat mass and obesity-related associated protein (FTO) is strongly associated with depression. Aging is a risk factor for synaptic plasticity damage in the brain and leads to neurocognitive dysfunctions. FTO-dependent m6A modification plays an important role in neurodevelopment and cognitive function. However, whether FTO is associated with susceptibility to depression in different age groups remains unknown.

Methods: We subjected 3-and 12-month-old C57BL/6J male mice to chronic unpredictable mild stress (CUMS) for 6 weeks, of which 3 weeks were used for hippocampal injection of FTO knockdown adeno-associated virus 9 shRNA (FTO-KD AAV9). Finally, 36 male mice in each 3-month-old and 12-month-old groups were divided into three groups (n = 12): Sham, CUMS, and FTO-KD. After 6 weeks, we assessed behavioral deficits (depressive and anxiety-like behaviors and cognitive impairment) by behavioral tests and hippocampal neuronal damage (dendritic spine density, neuronal atrophy, and expression of proteins associated with synaptic plasticity) by molecular biochemical experiments.

Results: The results showed that 12-month-old C57BL/6J mice were more likely to develop depression-like behavior and spatial learning and memory impairment induced by CUMS than 3-month-old mice. Chronic stress-induced depression-like behavior and cognitive impairment worsened after the FTO-KD intervention. In the hippocampus of 3- and 12-month-old mice, CUMS induced the downregulation of FTO, nerve growth factor (NGF), reelin, and synaptic plasticity-related proteins. It also caused abnormal brain-derived neurotrophic factor (BDNF)- the tropomyosin-related kinase B (TrkB) signaling, reduced density of dendritic spines, and an increased number of neuronal pyknotic nuclei, leading to neuronal disarray, which was more significant in 12-month-old animals. FTO deficiency accelerated neuronal damage in the hippocampus of 12-month-old CUMS mice.

Conclusions: This study provides rodent evidence that FTO deficiency may increase the susceptibility to depression in older adults by impairing hippocampal neuronal function and neuronal synaptic plasticity in an age-dependent manner. This suggests that the development of FTO activators may be an effective treatment for depression in older adults.

Keywords: BDNF-TrkB signaling pathway; Chronic unpredictable mild stress (CUMS); Cognitive impairment; FTO; Synaptic plasticity.

Fig. 1

Experimental design and protocol. The CUMS protocol includes 10 stressors. CUMS Chronic Unpredictable Mild Stress, AAV9 adeno-associated virus 9, SPT Sucrose preference test, OFT Open field test, EPM the elevated plus maze, TST tail suspension test, FST forced swimming test, MWM Morris Water Maze

Fig. 2

FTO deficiency aggravates CUMS-induced depression-like and anxiety-like behavior in an age-dependent manner. A Weight changes in 3-month-old and 12-month-old mice. B Sucrose preference percentage in SPT. C Movement distance (cm) in OFT. D Representative picture of the movement trajectory in OFT. The red box represents the central area. E Time spent in the central area is in OFT (s). F The traveled distance in open arms (cm). G The time of residence in open arms (s). H Representative picture of the movement trajectory in EPM. The cross patterns represent open and closed arms, respectively. I Total traveled distance in open arms (s). J Immobility time (s) in TST. K Immobility time (s) in FST. N = 12 per group. CUMS Chronic Unpredictable Mild Stress, FTO-KD FTO knockdown, SPT Sucrose preference test, OFT open field test, EPM the elevated plus maze, TST tail suspension test, FST forced swimming test. Date was expressed as mean ± SD and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. ^*P < 0.05, Sham vs. CUMS; ^#P < 0.05, CUMS vs FTO-KD; ^&P < 0.05, FTO-KD (3-month-old) vs. FTO-KD (12-month-old)

Fig. 3

FTO deficiency aggravates CUMS-induced cognitive impairment in Morris Water Maze. A Time spent in target quadrant (s); B number of entries in platform. C number of entries in target quadrant. D Swimming speed (cm/s). E Representative picture of the movement trajectory in MWM. N = 12 per group. CUMS Chronic Unpredictable Mild Stress, FTO-KD FTO knockdown, MWM Morris Water Maze. Date was expressed as mean ± SD and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. ^*P < 0.05, Sham vs. CUMS; ^#P < 0.05, CUMS vs FTO-KD; ^&P < 0.05, FTO-KD (3-month-old) vs. FTO-KD (12-month-old)

Fig. 4

Brain stereotaxic surgery and expression of FTO in the hippocampus. A FTO knockdown AAV9 shRNA virus injection. B Representative Western blotting bands of FTO in the hippocampus. Quantification of OFT in hippocampus. C Immunohistochemical staining of anti-FTO in the hippocampus. The anti-FTO-positive neurons were shown as dark brown dots. Scale bar = 20 µm. N = 3 per group. CUMS Chronic Unpredictable Mild Stress, FTO-KD FTO knockdown. Date was expressed as mean ± SD and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. ^*P < 0.05, Sham vs. CUMS; ^#P < 0.05, CUMS vs FTO-KD; ^&P < 0.05, FTO-KD (3-month-old) vs. FTO-KD (12-month-old)

Fig. 5

NGF expression in the hippocampus. Representative picture of NGF immunohistochemical staining in hippocampus. The anti-NGF-positive neurons were shown as dark brown dots. NGF-positive cells in CA1, CA2 and DG regions of the hippocampus were quantified. Scale bar = 20 µm. N = 3 per group. CUMS Chronic Unpredictable Mild Stress, FTO-KD FTO knockdown. Date was expressed as mean ± SD and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. ^*P < 0.05, Sham vs. CUMS; ^#P < 0.05, CUMS vs FTO-KD; ^&P < 0.05, FTO-KD (3-month-old) vs. FTO-KD (12-month-old); ns, no significance

Fig. 6

Reelin expression in the hippocampus. Representative picture of Reelin immunohistochemical staining in hippocampus. The anti-Reelin-positive neurons were shown as brown area. Reelin-positive cells in CA1, CA2 and DG regions of the hippocampus were quantified. Scale bar = 20 µm. N = 3 per group. CUMS Chronic Unpredictable Mild Stress, FTO-KD FTO knockdown. Date was expressed as mean ± SD and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. ^*P < 0.05, Sham vs. CUMS; ^#P < 0.05, CUMS vs FTO-KD; ^&P < 0.05, FTO-KD (3-month-old) vs. FTO-KD (12-month-old); ns, no significance

Fig. 7

FTO deficiency aggravates CUMS-induced hippocampal neuronal damage. Representative image of H&E staining of the hippocampus. Quantification of neuronal pyknotic nuclei in CA1, CA2, and DG regions. Pyknotic nuclei are shrunken, hyperchromatic nuclei with condensed chromatin, characteristic of apoptotic or degenerating cells. Scale bar = 20 µm. N = 3 per group. CUMS Chronic Unpredictable Mild Stress, FTO-KD FTO knockdown. Date was expressed as mean ± SD and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. ^*P < 0.05, Sham vs. CUMS; ^#P < 0.05, CUMS vs FTO-KD; ^&P < 0.05, FTO-KD (3-month-old) vs. FTO-KD (12-month-old); ns, no significance

Fig. 8

FTO deficiency aggravated CUMS-induced hippocampal dendritic spine abnormalities. Representative image of hippocampal dendritic spines. Quantification of hippocampal dendritic spine density. Scale bar = 10 µm. N = 3 per group. CUMS Chronic Unpredictable Mild Stress, FTO-KD FTO knockdown. Date was expressed as mean ± SD and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. ^*P < 0.05, Sham vs. CUMS; ^#P < 0.05, CUMS vs FTO-KD; ^&P < 0.05, FTO-KD (3-month-old) vs. FTO-KD (12-month-old)

Fig. 9

FTO deficiency aggravates synaptic plasticity damage in hippocampus. A Representative image of Western blotting bands. B–F. Quantification of hippocampal BDNF, TrkB, PSD-95, SYN, and SAP97 expression. N = 3 per group. CUMS Chronic Unpredictable Mild Stress, FTO-KD FTO knockdown, BDNF brain-derived neurotrophic factor, TrkB tyrosine Kinase receptor B, PSD-95 postsynaptic density protein 95, SYN synaptophysin, SAP97 synapse associated protein of 97. Date was expressed as mean ± SD and were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons post hoc test. ^*P < 0.05, Sham vs. CUMS; ^#P < 0.05, CUMS vs FTO-KD; ^&P < 0.05, FTO-KD (3-month-old) vs. FTO-KD (12-month-old)