A heterozygous mutation in tubulin, beta 2B ( Tubb2b ) causes cognitive deficits and hippocampal disorganization

Abstract

Development of the mammalian forebrain requires a significant contribution from tubulin proteins to physically facilitate both the large number of mitoses in the neurogenic brain (in the form of mitotic spindles) as well as support cellular scaffolds to guide radial migration (radial glial neuroblasts). Recent studies have identified a number of mutations in human tubulin genes affecting the forebrain, including TUBB2B . We previously identified a mouse mutation in Tubb2b and we show here that mice heterozygous for this missense mutation in Tubb2b have significant cognitive defects in spatial learning and memory. We further showed reduced hippocampal long-term potentiation consistent with these defects. In addition to the behavioural and physiological deficits, we show here abnormal hippocampal morphology. Taken together, these phenotypes suggest that heterozygous mutations in tubulin genes result in cognitive deficits not previously appreciated. This has implications for design and interpretation of genetic testing for humans with intellectual disability disorders.

Keywords: CA3; CNS; congenital malformation; development; hippocampus; learning; mouse; neural development; tubb2b; tubulin.

Figure 1. Tubb2b^brdp/+ heterozygous mice exhibit increased locomotion

Horizontal activity (beam breaks) over the 60 min interval are shown in the top panel with the main effect in the inset. Central horizontal activity (beam breaks) over the 60 min interval are shown in the bottom panel with the main effect in the inset. Data are LS Mean ± SEM. * p < 0.05, ^†p < 0.1 vs. WT. WT: n = 19 mice; Tubb2b^brdp/+: n = 19 mice

Figure 2. Tubb2b^brdp/+ heterozygous mice exhibit learning deficits

Morris water maze (MWM) acquisition (A,C,E,G) and MWM reversal (B,D,F,H) for path efficiency, path length, latency, and swim speed, respectively. Data are LS Mean ± SEM. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for main effect vs. WT. WT: n = 19 mice; Tubb2b^brdp/+: n = 19 mice

Figure 3. Tubb2b^brdp/+ heterozygous mice exhibit memory deficits

MWM probe trials for average distance to the former platform location given 24 h after the last platform trial of each phase. A, acquisition probe; B, reversal probe. Data are LS Mean ± SEM plus individual data points. *p < 0.05 vs. WT. WT: n = 19 mice; Tubb2b^brdp/+: n = 19 mice

Figure 4. Tubb2b^brdp/+ heterozygous mice exhibit cued trials

MWM cued random trials as a test of proximal cue learning (latency, s). There was a significant genotype effect but no interaction. The effect is shown on the same scale as latency in Figure 1 for comparison. As can be seen the latency effect on cued trials was small and did not change across days. **p < 0.01 vs. WT. WT: n = 18 mice; Tubb2b^brdp/+: n = 18 mice (1 litter was inadvertently missed)

Figure 5. Tubb2b^brdp/+ heterozygous mice have reduced long-term potentiation

Electrophysiological analyses of long-term potentiation (LTP). The amplitude (A) and slope (B) of resulting EPSPs from the parasagittal sections (350 μm) of hippocampal CA1 region were recorded. Both the baseline amplitude and slopes of the EPSP (LTP) in 35 days Tubb2b^brdp/+ (black circle) was significantly (p= 0.0016 for amplitude and p=0.0419 for slope) decreased compared with WT (open circle) mice after recording for 60 min following stimulation. WT,n = 4 mice; Tubb2b^brdp/+, n = 4 mice. **p<0.01, *p<0.05.

Figure 6. Tubb2b^brdp/+ heterozygous mice have abnormal hippocampal structure

(A-D) Histological analysis revealed hippocampal phenotypes in the CA3 region of heterozygous mice. C,D are higher magnification of approximate areas indicated in A,B. Note the disorganized and dispersed appearance of cells in the CA3 region of the heterozygous animal relative to control (asterisks). (E,F) Immunohistochemistry for Calbindin shows disorganization of fibers in CA3 region with a web-like appearance in the heterozygous animals (arrows). (G,H) NeuN immunohistochemistry further demonstrates disorganization of the CA3 region in the heterozygous animals with a number of ectopic cells (arrow heads). (I) Quantification of NeuN staining shows that there is a reduced staining profile density in the heterozygous animals relative to wild-type (*p<0.05, n=3).

Figure 7. Thy1-GFP highlights structural deficits in the adult dentate gyrus

Staining shows mossy fiber projections in 4 month old control Thy1-GFP/+;Tubb2b^+/+ (A) and Thy1-GFP/+;Tubb2b^brdp/+ (B) animals in the CA3 region. Note that in the control animals there is both a suprapyramidal bundle (SPB, white brackets) and an infrapyramidal bundle (IPB, yellow arrow heads). In contrast, mossy fibers appear less dense and there is a lack of separation between the SPB and IPB in the Tubb2b^brdp/+ animals (asterisks).