Reversal of depressed behaviors in mice by p11 gene therapy in the nucleus accumbens

Abstract

The etiology of major depression remains unknown, but dysfunction of serotonergic signaling has long been implicated in the pathophysiology of this disorder. p11 is an S100 family member recently identified as a serotonin 1B [5-hydroxytryptamine 1B (5-HT(1B))] and serotonin 4 (5-HT(4)) receptor-binding protein. Mutant mice in which p11 is deleted show depression-like behaviors, suggesting that p11 may be a mediator of affective disorder pathophysiology. Using somatic gene transfer, we have now identified the nucleus accumbens as a key site of p11 action. Reduction of p11 with adeno-associated virus (AAV)-mediated RNA interference in the nucleus accumbens, but not in the anterior cingulate, of normal adult mice resulted in depression-like behaviors nearly identical to those seen in p11 knockout mice. Restoration of p11 expression specifically in the nucleus accumbens of p11 knockout mice normalized depression-like behaviors. Human nucleus accumbens tissue shows a significant reduction of p11 protein in depressed patients when compared to matched healthy controls. These results suggest that p11 loss in rodent and human nucleus accumbens may contribute to the pathophysiology of depression. Normalization of p11 expression within this brain region with AAV-mediated gene therapy may be of therapeutic value.

Fig 1. Focal inhibition of p11 expression in adult mouse NAcc induces depression-like behaviors

(A)Western blot analysis confirming effective knockdown of a p11-GFP fusion protein in HEK 293 cells co-transfected with shRNA to p11 but not with luciferase shRNA, analyzed using an antibody to GFP. Band sizes are in kDa. (B) Quantitative PCR analysis of endogenous p11 mRNA levels in primary NAcc neurons following AAV-mediated gene transfer of p11 shRNA compared with luciferase control shRNA (C) Coronal brain section of 3 month old male mouse with injection of p11 shRNA virus co-expressing YFP confirms local transduction within the NAcc following YFP immunostaining. AC=Anterior commissure. (D) Confocal micrographs of YFP (green) and p11 (red) immunofluorescense in the NAcc after injection of control virus (AAV.siLuc.YFP, top 2 panels) and knockdown virus (AAV.sip11.YFP, bottom 2 panels) in vivo. Arrows, YFP-positive cells; arrowhead, YFP-negative cell still expressing p11 at normal levels (scale bars, 20 μm). (E-G) Induction of depression-like behaviors in adult mice following AAV shRNA-mediated focal knockdown of p11 in NAcc. (E) Increased immobility of p11 knockdown compared with controls on tail suspension test. (F) Increased immobility of p11 knockdown compared with controls on forced swim test * p<0.05, ** p<0.01, two-tailed t-test. (G) Effect of focal p11 knockdown in the NAcc on behavioral response to anti-depressant treatment in the tail suspension test. The loss of NAcc p11 expression does not abolish the anti-depressant effect of imipramine (open bars) compared with saline controls (closed bars) ††† p<0.001 post-imipramine compared with matched saline control, two-tailed t-test. However, there remains a significant increase in immobility following drug treatment in p11 siRNA mice compared with siluc controls *p<0.05, **p<0.01, two-tailed t-test.

Fig 2. Overexpression of p11 in NAcc neurons potentiates 5-HT_1B agonist binding and signaling

(A) Immunohistochemistry (left, low power magnification; right, high-power magnification) demonstrates focal restoration of p11 expression in the NAcc of p11 KO mice following AAV mediated gene therapy with a p11 cDNA but not with control YFP, which demonstrates only light background staining. AC=Anterior commissure (scale bars= 80 μm). (B,C) 5-HT_1B receptor binding levels in p11 KO mice after intraccumbal injection of p11 overexpression (OE) vector (AAV.siLuc.p11) or the control vector (AAV.siLuc.YFP) using 10pM [125I]-cyanopindolol. (B) Autoradiography revealed increased binding in the pallidum terminal regions of accumbens neurons in the hemisphere overexpressing p11 following AAV-mediated gene transfer (p11 OE) compared with the control hemisphere expressing the YFP marker gene (ctrl). (C) Quantification confirmed the significant increase in 5-HT_1B binding in the p11 overexpressing hemisphere (** p<0.01, two-tailed t-test). (D) Treatment of primary striatal neurons with the 5HT1B-agonist anpirtoline (10μM) resulted in a repeated increase in fluorescence as measured by the area under the curve using the calcium-sensitive dye Fluor 4 (black bar=exposure to anpirtoline; green bar=exposure to glutamate as a positive control for calcium fluorescence). (E) Effect of anpirtoline (25μM; black bar) was completely blocked by exposure to the 5-HT_1B antagonist GR127935 (10μM, red bar); green bar=exposure to glutamate as a positive control for calcium fluorescence. (F) Calcium fluorescence was significantly increased by AAV-mediated overexpression of p11 (black bars) in ventral striatal neurons compared with control RFP (gray bars) expression after exposure to 10μM, 25μM and 50μM anpirtoline (****p<0.0001).

Fig 3. Focal restoration of NAcc p11 via AAV gene therapy reverses depression-like behaviors in transgenic p11 KO mice

(A) Immobility times on tail suspension test following AAV gene therapy to restore NAcc p11 expression (hatched bars). Depression-like behaviors were reversed in these animals compared with both untreated mice (filled bars) and control AAV-treated mice (open bars). (B) Immobility times on the forced swim test in the same animals as in fig. 3A. (C) Increased sucrose preference in p11 KO mice following restoration of NAcc p11 expression. Volume of sucrose intake over 24 hrs. per gram of body weight was significantly increased in p11 KO mice compared with controls while there was no significant difference between groups in water intake. The percent of sucrose as a measure of overall fluid intake was also significantly increased (not shown). **p<0.01, ***p<0.005, two-tailed t-test.

Fig 4. p11 expression in post-mortem human NAcc brain tissue

(A) Representative western blot of protein lysates from post-mortem NAcc brain tissue from normal controls (NC) and depressed patients (Dep) (N=17 per group). (B) Quantitative densitometry of western blots demonstrates reduced expression of p11 in post-mortem NAcc tissue from depressed patients (DEP) compared with normal controls (NC). *p<0.05, two-tailed t-test.