βCaMKII in lateral habenula mediates core symptoms of depression

Abstract

The lateral habenula (LHb) has recently emerged as a key brain region in the pathophysiology of depression. However, the molecular mechanism by which LHb becomes hyperactive in depression remains unknown. Through a quantitative proteomic screen, we found that expression of the β form of calcium/calmodulin-dependent protein kinase type II (βCaMΚΙΙ) was significantly up-regulated in the LHb of animal models of depression and down-regulated by antidepressants. Increasing β-, but not α-, CaMKII in the LHb strongly enhanced the synaptic efficacy and spike output of LHb neurons and was sufficient to produce profound depressive symptoms, including anhedonia and behavioral despair. Down-regulation of βCaMKII levels, blocking its activity or its target molecule the glutamate receptor GluR1 reversed the depressive symptoms. These results identify βCaMKII as a powerful regulator of LHb neuron function and a key molecular determinant of depression.

Fig. 1. βCaMKII is upregulated in the LHb of animal models of depression

(A) Depression phenotypes of cLH rats. Numbers in the bars indicate number of animals used. Note in LH test, maximal number of bar presses is 15. (B) Experimental outline of the high-throughput quantitative proteomics based on stable isotope labeling. Briefly, habenula of unlabeled (¹⁴N) WT or cLH rats were dissected, homogenized, and mixed in a 1:1 ratio with total brain homogenate from a ¹⁵N-labeled rat. Membrane fraction was enriched, and 100 µg protein sample was used for standard mass spectra analysis. ¹⁴N /¹⁵N ratio for each identified peptide was calculated. Peptide ratios for each protein were then compared between cLH and control sample. Details see methods. (C) Proteomic analysis of βCaMKII, based either on total peptides, or unique peptides (peptides not shared by other CaMKII family members) identified in 3 independent proteomic runs. (D, E) Western blot analysis showing change of βCaMKII in membrane fraction of habenula (D) or hippocampus (E) of cLH rats. Tissue amounts of tubulin were used as loading control. Protein expression was normalized by control amount. (F) qPCR analysis of βCaMKII mRNA in habenula. (G) βCaMKII level increase in acute learned helpless and chronic mild stress (CMS) depression models. aLH and aNLH were rat groups subjecting to LH stress but did (aLH), or did not (aNLH) display LH symptom. (H) Western blot analysis showing level of βCaMKII in membrane fraction of habenula of cLH rats treated with saline or antidepressant imipramine. Data are mean ± SEM. * p < 0.05 ** p < 0.01, *** p < 0.001 compared to control group, n.s., not significant, two-tailed Student’s t-tests for two-group comparison, one-way ANOVA with Bonferroni post hoc analysis for multiple-group comparison.

Fig. 2. Overexpression of βCaMKII but not αCaMKII in LHb caused depressive-like behaviors in both mice and rats

A) Schematics of AAV vectors engineered to overexpress a control construct, βCaMKII, αCaMKII, or a kinase-dead mutant of βCaMKII. ITR, inverted terminal repeats; CMV, cytomegalovirus promoter; Ubi: ubiquitin promoter; 2A: viral 2A linker peptide allowing translation of multiple unfused proteins. (B) Experimental paradigm for behavioral testing of WT mice or rats. (C) Illustration of bilateral viral injection of AAV-βCaMKII in mouse LHb (counter-stained with anti-GFP and nuclear marker Hoechst). Scale bars, 50 µm. (D-G) Behavioral effects of expressing various viral constructs in LHb in animal models of depression in mice (D-E) or rats (H, I). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with non-injected WT, # p < 0.05, ## p < 0.01 compared with AAV-control, one-way ANOVA with Bonferroni post hoc analysis.

Fig. 3. Overexpression of βCaMKII increased synaptic activity and spike output of LHb neurons

(A) Schematics of paired recording configuration in LHb (left). Right: paired patching of a βCaMKII-infected LHb neuron (pointed by green arrow) and a neighboring uninfected neuron (pointed by red arrow) under transmitted and fluorescent light microscopy. Scale bars, 10 µm. (B) Example mEPSC traces, measured in a whole-cell configuration, from LHb neurons of control neurons, or neurons infected by AAV-βCaMKII or AAV-αCaMKII. (C, D) Cumulative distribution of mEPSC inter-events interval and average frequency (left), or mEPSC amplitude (right) of neurons infected by AAV-βCaMKII (C) or AAV-αCaMKII (D). Each line represents values from a pair of control and neighboring viral-infected neurons. (E) Example traces of spontaneous spiking, measured in a cell-attached configuration, from LHb neurons of control neurons, or neurons infected by AAV-βCaMKII or AAV-αCaMKII. (F - H) Average spontaneous spiking frequency of neurons infected by AAV-βCaMKII (F), AAV-αCaMKII (G), or AAV-GFP (H). ** p < 0.01, *** p < 0.001, Wilcoxon signed-rank test.

Fig. 4. Knocking-down of βCaMKII in LHb rescued depression-like phenotypes of cLH rats and reduced synaptic activity of LHb neurons

(A) Schematics of the AAV vector engineered to overexpress an RNAi form of βCaMKII. H1: human H1 promoter. (B) Specific knocking down of βCaMKII but not αCaMKII by the βCaMKII RNAi construct. pSuper-βCaMKII-RNAi construct was co-transfected with AAV-βCaMKII or AAV-αCaMKII plasmid in 293TN cells, and expressed for 48 hrs before Western analysis. Left: representative western blot. Right: quantification of knock down efficiency. (C) Experimental paradigm for behavioral testing of cLH rats infected by virus. (D-E) Behavioral effects of expressing AAV-βRNAi and AAV-βK43R in the LHb of cLH rats in forced swim (D) or learned helpless test (EF). * p < 0.05, *** p < 0.001, one-way ANOVA with Bonferroni post hoc test. (F) Percentage of animals in each category. LH: learned helpless rats with ≤ 5 lever presses. NLH: non-learned helpless rats with ≥10 lever presses. (G) Sub-regional characterization of viral infection. Top: illustration of the division of lateral LHb (LHb-L) and medial LHb (LHb-M) in an AAV-βRNAi virus infected cLH rat brain slice. Scale bar, 100 µm. Bottom: Behavioral response in the learned helpless test plotted against infection rates in the LHb-L and LHb-M of cLH rats. (H) mEPSCs of AAV-βRNAi infected LHb neurons from cLH rats were altered. Top: example mEPSC traces. Cumulative distribution of mEPSC inter-events interval and average frequency (bottom left) or mEPSC amplitude (bottom right) of cLH LHb neurons infected by AAV-βRNAi. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with cLH/AAV-control, Wilcoxon signed-rank test.