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. 2021 Dec 1;109(23):3793-3809.e8.
doi: 10.1016/j.neuron.2021.09.008. Epub 2021 Oct 5.

Mitophagy in the basolateral amygdala mediates increased anxiety induced by aversive social experience

Kaizheng Duan  1 , Qinhua Gu  1 , Ronald S Petralia  2 , Ya-Xian Wang  2 , Debabrata Panja  1 , Xing Liu  1 , Michael L Lehmann  3 , Huiwen Zhu  1 , Jun Zhu  4 , Zheng Li  5
Affiliations

Mitophagy in the basolateral amygdala mediates increased anxiety induced by aversive social experience

Kaizheng Duan et al. Neuron. .

Abstract

Psychosocial stress is a common risk factor for anxiety disorders. The cellular mechanism for the anxiogenic effect of psychosocial stress is largely unclear. Here, we show that chronic social defeat (CSD) stress in mice causes mitochondrial impairment, which triggers the PINK1-Parkin mitophagy pathway selectively in the amygdala. This mitophagy elevation causes excessive mitochondrial elimination and consequent mitochondrial deficiency. Mitochondrial deficiency in the basolateral amygdalae (BLA) causes weakening of synaptic transmission in the BLA-BNST (bed nucleus of the stria terminalis) anxiolytic pathway and increased anxiety. The CSD-induced increase in anxiety-like behaviors is abolished in Pink1-/- and Park2-/- mice and alleviated by optogenetic activation of the BLA-BNST synapse. This study identifies an unsuspected role of mitophagy in psychogenetic-stress-induced anxiety elevation and reveals that mitochondrial deficiency is sufficient to increase anxiety and underlies the psychosocial-stress-induced anxiety increase. Mitochondria and mitophagy, therefore, can be potentially targeted to ameliorate anxiety.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. The size and mass of mitochondria decrease in the amygdala of defeated mice.
Mito-YFP mice were subjected to 30 days of CSD or untreated. (A, B, E, F) Representative images of mito-YFP in brain sections; scale bar: 150 μm in A; 500 μm in D; in B and E, 15 μm for images in the left two columns and 10 μm for images in the right column. (C, G) Quantification of average mitochondrial area in designated brain regions. (D, H) Quantification of integrated mito-YFP intensity in designated brain regions. The numbers below the bars indicate the number of brain sections from 3 control and 6 defeated mice. Data are presented as mean ± SEM. ** p < 0.01; *** p < 0.001. See also Figures S1 and S2, Table S1.
Figure 2.
Figure 2.. MMP, COX activity, and the p-AMPK level are altered in the amygdala of defeated mice.
C57BL/6 mice were subjected to 30 days of CSD or untreated. (A) Representative images of acute brain slices stained with TMRE; 10x (for the amygdala) or 4x (for the hippocampus) objective was used for image acquisition; scale bar, 25 μm (amygdala) and 100 μm (hippocampus). (B) Quantification of the integrated TMRE intensity in designated brain areas; the number in the bar indicates the number of brain slices taken from 3 control and 6 defeated mice. (C) COX activity in total lysates; Amyg: amygdala; Hippo: hippocampus. (D) COX activity in the mitochondrial fraction. (E) Representative immunoblots. (F) Quantification for the ratio of pAMPK to AMPK. (G) Quantification for the ratio of pAMPK to GAPDH. The number in the bar indicates the number of animals in C, D, F, G. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01. See also Figures S3 and S4, Table S1.
Figure 3.
Figure 3.. The effect of CSD on mtDNA.
C57BL/6 mice were subjected to 30 days of CSD or untreated. (A) Representative images of double labeling for EdU and Tom20 at the BLA in brain sections from mice injected with EdU; the yellow lines indicate the location of the orthogonal views along the XZ and YZ axis; scale bar, 5 μm for the left three images and 2 μm for the “XY” image. (B) Quantification for the proportion of Tom20+ structures containing EdU in the BLA. (C) Representative images of double labeling for EdU and Tom20 at the dentate gyrus in brain sections from mice injected with EdU; scale bar, 5 μm for the left three images and 2 μm for the “XY” image. (D) Quantification for the proportion of Tom20+ structures containing EdU in the dentate gyrus. (E) The ratio of mtDNA to nuclear DNA. (F, G) The average mtDNA mutation frequency per mtDNA site. (H) mRNA levels of PGC1α and TFAM in control and defeated mice. (I) Representative immunoblots of PGC1α and TFAM using lysates from control and defeated mice. (J) Quantification for PGC1α protein expression. (K) Quantification for TFAM protein expression. Amyg: amygdala; Hippo: hippocampus. The number in and below the bar indicates animal numbers in E-G, H, J, K, and the number of brain sections from 3 mice for each group in B and D. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01. See also Figure S5, Table S1.
Figure 4.
Figure 4.. Mitophagy is increased in the amygdala of defeated mice.
Brain sections or brain lysates prepared from the amygdala of C57BL/6 mice subjected to 30 days of social defeat or untreated were used for immunostaining (A–D), EM (G–M), or immunoblotting (N–Q). In E and F, mice were injected with mtKeima AAV, subjected to 30 days of social defeat or untreated, then used for the preparation of acute brain slices and imaging. (A, C) Representative images of staining for Tom20 and LC3; the yellow lines indicate the location of the orthogonal views along the XZ and YZ axis; scale bar: 5 μm for the left 3 images, 2 μm for the “XY” images. (B, D) Quantification for the proportion of Tom20 positive structures containing LC3 or optineurin. (E) Representative images of mtKeima excited at 543 nm (red) and 458 nm (blue); scale bar, 10 μm. (F) Quantification for the ratio of mtKeima fluorescence excited at 543 nm to that excited at 458 nm. (G) Representative electron micrographs of mitophagosome-like structures; red arrows and red dash lines indicate mitophagosome-like structures; m: mitochondria; scale bar, 200 nm. (H) Quantification for the number of mitophagosome-like structures. (I) Representative electron micrographs of abnormal mitochondria indicated by red arrows; scale bar, 500 nm. (J) Quantification for mitochondrial number. (K) Quantification for the number of abnormal mitochondria. (L) Representative electron micrographs of autophagosome-like structures; arrows indicate autophagosome-like structures; scale bar, 500 nm. (M) Quantification for the number of autophagosome-like structures. (N) Representative immunoblots of LC3-II and SQSTM1/p62. (O) Quantification for the ratio of LC3-II to LC3-I. (P) Quantification for the ratio of LC3-II to tubulin. (Q) Quantification for the ratio of SQSTM1/p62 to GAPDH. The numbers in and below the bar and above the x-axis indicate the number of brain sections from 3-4 mice in B, D and F; the number of micrographs from 3 control and 6 defeated mice in H, J, K, M; animal numbers in O–Q. Data are presented as mean ± SEM in B, D, F, O–Q or violin plots (H, J, K, M) illustrating kernel probability density, i.e. the width of the outlined area represents the proportion of the data located there. * p < 0.05, ** p < 0.01, *** p < 0.001. See also Figure S6, Table S1.
Figure 5.
Figure 5.. PINK1 and Parkin are required for CSD-induced mitophagy increase and increase in anxiety-like behaviors.
Fixed brain slices were prepared from WT, PINK1−/−, and Parkin−/− mice subjected to 30 days of CSD or untreated. (A) Representative images of staining for Tom20, LC3, and optineurin; the yellow lines indicate the location of the orthogonal views along the XZ and YZ axis; scale bar, 5 μm for images in the left 3 columns, and 2 μm for the “XY” images. (B) Quantification for the proportion of Tom20 positive structures containing LC3. (C) Quantification for the proportion of Tom20 positive structures containing optineurin. (D) Quantification for integrated Tom20 intensity. The number in and below the bar indicates the number of brain sections from 3 animals in B–D. (E) Schematic diagram of the experimental procedure. Defeated and control mice were divided into two groups for different testing schedules. (F–J) Quantification of the open field, light/dark box, social interaction, and forced swim tests after CSD in the same group of mice. (K and L) Quantification of the elevated plus maze and social interaction tests in the same group of mice. The number below the bar indicates the number of animals combined from multiple cohorts (For mice tested for open field, light/dark box and forced swim tests: WT, 4; PINK1 KO, 5; Parkin KO; 5. For mice tested in elevated plus maze: WT, 2; PINK1 KO, 3; Parkin KO, 3). Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01; *** p < 0.001. See also Figures S7-S11, Table S1.
Figure 6.
Figure 6.. Illumination of mtKillerRed increases anxiety-like behaviors and mitophagy.
C57BL/6 mice were injected with mtKillerRed AAV or mtKeima AAV in the BLA, then tested for anxiety-like behavior. Animals were used for EM after behavioral testing. (A) Schematic diagram of the experimental procedure. (B) Representative maps of the time that an animal spent in the light box; color indicates total time spent at each location. (C) Quantification for the total time that animals spent in the light compartment of the light/dark box; the same animals are connected by lines. (D) Quantification for the total time that animals spent in the open arm of the elevated plus maze; the same animals are connected by lines. (E) Representative electron micrographs of mitochondria in photo-stimulated and sham-stimulated mice (m, mitochondria; arrows indicate abnormal mitochondria); scale bar, 500 nm. (F) Quantification for the number of abnormal mitochondria. (G) Representative electron micrographs of mitophagosome-like structures (indicated by the red arrow); scale bar, 500 nm for the left image and 100 nm for the enlarged image. (H) Quantification of the number of mitophagosome-like structures. (I) Quantification of the number of mitochondria. (J) Representative electron micrographs of autophagosome-like structures (indicated by the red arrow); scale bar, 500 nm for the left image and 100 nm for the enlarged image. (K) Quantification for the number of autophagosome-like structures. The numbers in and below the bar and above the x-axis indicate the animal number in C and D and the number of micrographs from 3 mice in F, H, I, K. Data are presented as mean ± SEM in C and D, or violin plots (F, H, I, K) illustrating kernel probability density, i.e. the width of the outlined area represents the proportion of the data located there. * p < 0.05, ** p < 0.01. See also Figure S12, Table S1.
Figure 7.
Figure 7.. BLA-adBNST synaptic transmission in defeated mice was reduced in a PINK1-dependent manner and optogenetic activation of the BLA-BNST projection abolishes the effect of CSD on anxiety-like behaviors.
The BLA was injected with ChR2 AAV (A–F) alone or along with lentivirus expressing siRNAs or scramble nucleotides (G–J) and subjected to 30 days of CSD or untreated. BNST slices were prepared after CSD, and EPSCs evoked by 473-nm light pulses were recorded in adBNST neurons using whole-cell patch-clamp. C57BL/6 mice (A, B, G–J), WT littermates of PINK1−/− mice (C, D), and PINK1−/− mice (E, F) were used. (A, C, E, G, I) EPSCs evoked by light pulses at various intensities. (B, D, F, H, J) Paired-pulse ratio analyzed by stimulating the BLA to BNST projections with pairs of light pulses (50 ms interpulse interval) at various intensities. N represents the number of neurons from 5 mice in A–J. (K–P) The BLA of C57BL/6 mice was injected with ChR2 AAV, tested for baseline anxiety-like behaviors, then subjected to 30 days of CSD or untreated. After CSD, mice were assessed for anxiety-like behaviors again in the absence or presence of 473-nm light pulses. (K) Schematic diagram of the experimental procedure. (L) Representative image of ChR2 expression at the BNST; the white bar indicates the implanted optic fiber. (M) The average time spent in the central arena during the 5-min block in the baseline test, and the time spent in the central arena during the 5-min block without or with light stimulation in the post-CSD test. (N) The average time spent in the open arms during the 5-min block in the baseline test, and the time spent in the open arms during the 5-min block without or with light stimulation in the post-CSD test. (O) Total distance traveled during the 5-min block in the baseline test, and total distance traveled during the 5-min block without or with light stimulation in the post-CSD test. (P) Total distance traveled in the elevated plus maze during the 5-min block in the baseline test, and total distance traveled in the elevated plus maze during the 5-min block without or with light stimulation in the post-CSD test. The number below the bar indicates the number of animals in K–P. The same animal in different test sessions is connected with lines in M–P. Data are presented as mean ± SEM; ** p < 0.01, *** p < 0.001.See also Figures S13-S15, Table S1.
Figure 8.
Figure 8.. Chemogenetic inhibition of the amygdala blocks CSD-induced increases in mitophagy and anxiety-like behaviors.
C57BL/6 mice were injected with Gi-mCherry AAV in the BLA and subjected to 30 days of CSD or untreated, then used for immunostaining or behavioral tests. (A) Schematic diagram of the experimental procedure. (B) Representative image of Gi-mCherry in the BLA; scale bar: 100 μm. (C) Representative images of double staining for LC3 and Tom20 in the BLA; the yellow lines indicate the location of the orthogonal views along the XZ and YZ axis; scale bar, 5 μm for images in the left 3 columns, and 2 μm for the “XY” images. (D) Quantification for the proportion of Tom20 positive structures that contain LC3. (E) Time spent in the central arena of the open field box. (F) Total distance traveled in the open field box. (G) Time spent in the light compartment of the light/dark box. (H) Quantification for the social interaction test. (I) Time spent in the open arms of the elevated plus maze. The number in and below the bar indicates the number of brain sections from 4 mice for each group in D and animal number in E–I. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01. See also Figure S16, Table S1.
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Comment in

  • Excessive mitophagy for anxiety.
    Wang H, Xiong WC, Mei L. Wang H, et al. Neuron. 2021 Dec 1;109(23):3715-3716. doi: 10.1016/j.neuron.2021.11.007. Neuron. 2021. PMID: 34856130

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