Lithium rescues dendritic abnormalities in Ank3 deficiency models through the synergic effects of GSK3β and cyclic AMP signaling pathways

Abstract

Bipolar disorder (BD) is a highly heritable mood disorder with intermittent episodes of mania and depression. Lithium is the first-in-line medication to treat BD, but it is only effective in a subset of individuals. Large-scale human genomic studies have repeatedly linked the ANK3 gene (encoding ankyrin-G, AnkG) to BD. Ank3 knockout mouse models mimic BD behavioral features and respond positively to lithium treatment. We investigated cellular phenotypes associated with BD, including dendritic arborization of pyramidal neurons and spine morphology in two models: (1) a conditional knockout mouse model which disrupts Ank3 expression in adult forebrain pyramidal neurons, and (2) an AnkG knockdown model in cortical neuron cultures. We observed a decrease in dendrite complexity and a reduction of dendritic spine number in both models, reminiscent of reports in BD. We showed that lithium treatment corrected dendrite and spine deficits in vitro and in vivo. We targeted two signaling pathways known to be affected by lithium using a highly selective GSK3β inhibitor (CHIR99021) and an adenylate cyclase activator (forskolin). In our cortical neuron culture model, CHIR99021 rescues the spine morphology defects caused by AnkG knockdown, whereas forskolin rescued the dendrite complexity deficit. Interestingly, a synergistic action of both drugs was required to rescue dendrite and spine density defects in AnkG knockdown neurons. Altogether, our results suggest that dendritic abnormalities observed in loss of function ANK3 variants and BD patients may be rescued by lithium treatment. Additionally, drugs selectively targeting GSK3β and cAMP pathways could be beneficial in BD.

Fig. 1. AnkG maintains dendritic spine density and dendrite complexity in pyramidal neurons in vivo and in vitro.

A–F Bright-field images of Golgi-Cox staining in somatosensory cortical slices from (A) wild-type (WT) and (D) Ank3 cKO mice, scale = 100 µm. Dendrite pictures (B and E, scale = 5 µm) and representative traces of basal dendrites (C and F, scale = 20 µm) from pyramidal neurons from cortical layer 2/3 (G) Scatter plot of spine density from WT and AnkG cKO mice (46 cells, 3 brains per group, t-test, ± SEM, ***p ≤ 0.001). H Graph showing Sholl analysis for basal dendrites (20 neurons, 3 brains per group, 2-way ANOVA with Sidak’s post-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ±SEM. (I) Representative traces of cultured rat neurons expressing control or AnkG RNAi with GFP (no AnkG), GFP-AnkG-190, GFP-AnkG-270. Scale = 100 µm. Graphs showing Sholl analysis for total (J), basal (K), and apical (L) dendrites (15–22 neurons, 2-way ANOVA with Dunnett’s post-test and row repeated measures, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ±SEM).

Fig. 2. Chronic lithium treatment rescues dendrite and spine deficits in AnkG–deficient neurons in Ank3 cKO mice.

A Bright-field dendrite pictures of Golgi-Cox staining from somatosensory cortical pyramidal neurons from cortical layer 2/3 of wild-type and Ank3 cKO mice treated with or without lithium carbonate (scale = 20 µm). B Scatter plot of spine density from WT and AnkG cKO mice (37–45 cells, 3 brains per group, 2-way ANOVA with Dunnett’s post-test, ±SEM, ^#p ≤ 0.0001). C Representative traces of basal dendrites (scale = 20 µm) from somatosensory cortical pyramidal neurons from cortical layer 2/3. D Graph showing Sholl analysis for basal dendrites (23–29 neurons, 3 brains per group, 2-way ANOVA with Dunnett’s post-test and row repeated measures, ***p ≤ 0.001, ±SEM).

Fig. 3. Lithium treatment rescues the spine and dendrite deficits in AnkG knockdown neurons.

A Confocal images of mCherry from cultured rat neurons transfected for three days with control or AnkG RNAi and treated with lithium chloride, scale = 10 µm. B–D Scatter plots showing spine density, spine length, and spine head width (15–44 neurons from 3 independent experiments, 2-way ANOVA with Tukey’s post-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ±SEM). E Representative traces of cultured rat neurons expressing control or AnkG RNAi, ±lithium chloride, scale = 100 µm. F–H Sholl analysis graph for total, basal, and apical dendrites (28–74 neurons from 3 independent experiments, 2-way ANOVA with Dunnett’s post-test row repeated measures, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ±SEM).

Fig. 4. CHHIR99021 treatment rescues only spine deficits in AnkG knockdown neurons.

A Confocal images of mCherry from cultured rat neurons were transfected for three days with control or AnkG RNAi and treated with CHHIR99021 (scale = 10 µm). B–D Scatter plots showing spine density, spine length, and spine head width (21 neurons from 3 independent experiments, 2-way ANOVA with Dunnett’s post-test, *p ≤ 0.05, ***p ≤ 0.001, ±SEM). E Representative traces of cultured rat neurons expressing control or AnkG RNAi, ±CHHIR99021, scale = 100 µm. F–H Sholl analysis graph for total, basal, and apical dendrites (27–30 neurons from 3 independent experiments, 2-way ANOVA with Dunnett’s post-test and row repeated measures, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ±SEM).

Fig. 5. Forskolin treatment rescues only dendrite deficit and synergizes with CHIR99021 to rescue spine deficits in AnkG knockdown neurons.

A Confocal images of mCherry from cultured rat neurons transfected for three days with control or AnkG RNAi and treated with or without forskolin or forskolin + CHIR99021, scale = 10 µm. B–D Scatter plots showing spine density, spine length, and spine head width (23–30 neurons from 3 independent experiments, 2-way ANOVA with Dunnett’s post-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ±SEM). E Representative traces of cultured rat neurons expressing control or AnkG RNAi, ±forskolin or forskolin + CHIR99021, scale = 100 µm. F–H Sholl analysis graph for total, basal, and apical dendrites (41–46 neurons from 3 independent experiments, 2-way ANOVA with Dunnett’s post-test row repeated measures, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ±SEM).

Fig. 6. Inhibition of PKA and expression of GSK3βS9A prevent the lithium rescue in AnkG knockdown neurons.

A Confocal images of GFP from cultured rat neurons transfected for three days with control or AnkG RNAi with or without HA-GSK3βS9A and treated with or without lithium chloride and Rp-8-Br-cAMPS, scale = 10 µm. B–D Scatter plots showing spine density, spine length, and spine head width (20 neurons from 2 independent experiments, 1-way ANOVA with Dunnett’s post-test, **p < 0.01, #p < 0.0001, ±SEM). E Representative traces of cultured rat neurons expressing control or AnkG RNAi, +/- HA-GSK3βS9A and treated with or without lithium chloride and Rp-8-Br-cAMPS, scale = 100 µm. F–H Sholl analysis graph for total, basal, and apical dendrites (41–46 neurons from 3 independent experiments, 2-way ANOVA with Dunnett’s post-test row repeated measures, *p < 0.05, #p < 0.0001, ±SEM).