Memory deficits due to familial British dementia BRI2 mutation are caused by loss of BRI2 function rather than amyloidosis

Abstract

Familial dementias, which include Alzheimer disease (AD), familial British dementia (FBD), and familial Danish dementia (FDD), are caused by dominantly inherited autosomal mutations and are characterized by the production of amyloidogenic peptides, neurofibrillary tangles (NFTs) and neurodegeneration (St George-Hyslop and Petit, 2005; Garringer et al., 2009). The prevailing pathogenic theory, the "amyloid cascade hypothesis" (Hardy and Selkoe, 2002), posits that the accumulation of amyloidogenic peptides triggers tauopathy, neurodegeneration, and cognitive and behavioral changes. However, this hypothesis is yet to be validated, and causes of dementia may be multifaceted and involve other mechanisms, such as loss of function due to pathogenic mutations. Mouse models of human dementia invariably use transgenic expression systems (LaFerla and Oddo, 2005; McGowan et al., 2006; Vidal et al., 2009; Coomaraswamy et al., 2010) that do not reflect the genotypes of human disease and cannot replicate loss of function. Therefore, we generated a knock-in (KI) mouse model of FBD (FBD(KI)) genetically congruous with the human disease. FBD is caused by a missense mutation at the stop codon of the BRI2 gene (Vidal et al., 1999) and, like FBD patients, FBD(KI) mice carry this mutation in one of the two murine Bri2 alleles. We report that the British mutation drastically reduces expression of mature BRI2 in both KI mice and human FBD brains. This deficit is associated with severe hippocampal memory deficits in FBD(KI) mice. Remarkably, these animals showed no cerebral amyloidosis and tauopathy. Bri2(+/-) mice present memory deficits similar to those in FBD(KI) animals. Collectively, these results indicate that the British BRI2 mutation underlies abnormal memory due to loss of BRI2 function and independently of histopathological alterations typically evident in advanced neurodegenerative disease.

Figure 1.

FBD_KI mouse generation. A, Schematic representation of the construct that was injected in 129 ES cells, showing the ABri mutation on Exon 6, primer sites, site of Southern blot probe, LoxP, pgk-dt and pgk-Neo sites. The bottom graphics depict the construct with and without the pgk-Neo cassette that has been removed by means of Cre recombinase. B, Examples of 2 ES clones positive for the homologous recombination of the mutated allele: left arm (a–c: 1.67 kb) and right arm (b–d, 3.4 kb) PCRs are shown. C, Southern blot showing a shift from the 11.9 kb of the wild-type genome to the 8.9 kb band of the FBD_KI mice, due to the insertion of a new BamHI site. FBD_KI mice show both bands, indicating heterozygosity.

Figure 2.

FBD mice have neither amyloid lesions nor inflammatory response. A–F, Sections from a FBD_KI/KI mouse (A, C, E), a WT mouse (F), and a patient with FBD (B, D), were stained with ThS (A, B), Ab 1705, specific for the ABri amyloid peptide (C, D) and anti-GFAP (E, F). No significant differences were observed between WT and knock-in mice. G, H, Iba1 staining is also similar in hippocampi of FBD (G) and control (H) mice, as well in the other areas (cerebellum, cortices, brainstem and olfactory bulb: not shown). Scale bars, 50 μm. I–L, NeuN staining shows similar cellularity in hippocampus (I, FBD_KI/KI; J, WT) and adjacent cortex (K, FBD_KI/KI; L, WT) as well as in other areas (cerebellum, cortices, brainstem, olfactory bulb: data not shown). Scale bars, 260 μm.

Figure 3.

FBD mice do not present tauopathy. A–D, Hippocampal sections of FBD_KI/KI mice (A) show intracellular DA9 (total Tau) staining similar to control mice (B). FBD_KI/KI (C) and control mice (D) also show similar Tau immunoreactivity in gray and white matter of the temporal lobe. E, F, Hippocampi of FBD_KI/KI mice (E) and control mice (F) show CP13 cytoplasmic staining, with some neurons showing initial neuritic involvement, without significant difference between the knock-in and the WT animals. G–L, Similarly, the entorhinal cortex (G, FBD_KI/KI; H, WT) shows CP13 immunostaining, with no difference between the genotypes. PHF staining was not different in FBD_KI/KI and control mice in the hippocampus (I, FBD_KI/KI; J, WT) with rare neuritc staining (insets), in the temporal cortical and subcortical region (K, FBD_KI/KI; L, WT). Scale bars: A–L, 50 μm. M, Quantification of total Tau (DA9) and of 3 Tau phosphorylation sites (CP13: pSer202; CP17: pThr231; PHF1: pSer396/Ser404) by sandwich ELISA shows no significant difference in whole forebrains from FBD_KI and control mice at 24 months of age. N, Western blot analysis of 4 FBD_KI mice and 4 control littermates, at 6 months, fails to show differences in total Tau and 3 phosphorylation sites (CP13: pSer202; CP9: pThr231; PHF1: pSer396/Ser404) in these mice.

Figure 4.

Mature BRI2 levels are reduced in FBD_KI mice and in human FBD. A, Lysates from a control and an FBD case were blotted with antibodies against Bri2 and actin. Mature BRI2 is reduced the FBD brain. B, In FBD_KI mice, Bri2 is reduced in total brain lysates (S1). C, Bri2 is enriched in synaptic membranes (LP1) in WT animals. D, However synaptic levels of Bri2 (LP1) are reduced in FBD_KI mice. * indicates nonspecific bands also present in Bri2KO (Bri2 ^−/−) mice (Matsuda et al., 2008).

Figure 5.

Memory is impaired in FBD_KI mice in an ethologically relevant, nonaversive behavioral context. A, Ten-month-old FBD_KI and WT animals spent the same amount of time exploring the two identical novel objects. B, Twenty-four hours later, FBD_KI mice do not recognize the new object, while the WT mice do.

Figure 6.

Short-term memory is impaired in FBD_KI mice. FBD_KI mice present spatial working memory deficits at 9 months of age. FBD_KI mice do not learn the position of the hidden platform compared with WT littermates [FBD_KI: n = 10 males; WT: n = 10 males; no difference was found at A1 (p = 0.65) and A2 (0.49); however, a significant difference was seen at A3 (p = 0.027), at A4 (p = 0.0089), and the retention trail (R) (p < 0.0001). *p < 0.05, **p < 0.01, ***p < 0.0001.

Figure 7.

Contextual fear-conditioning deficits in 9-month-old FBD_KI mice. A, FBD_KI and WT littermates show no difference in freezing during training (baseline; p = 0.19), whereas contextual fear conditioning performed 24 h after training shows a reduction of freezing responses in FBD_KI mice compared with WT littermates [the freezing time of FBD_KI mice was ∼50% of WT mice; n = 10 males, WT littermates, n = 10 males (p = 0.0068). B, Nine-month-old FBD_KI mice and WT littermates show similar freezing during cued conditioning, both in the pre-CS group (no sound, p = 0.67), and in the CS group (Sound, p = 0.95).

Figure 8.

Bri2^+/− mice present cognition deficits similar to those of FBD_KI mice. A, Seven-month-old Bri2 ^+/− mice present spatial working memory deficits [no difference was found at A1 (p = 0.58) and A2 (0.076), however, a significant difference was seen at A3 (p = 0.0019), at A4 (p = 0.0008), and the retention trail (R) (p < 0.0001). B, Seven-month-old Bri2 ^+/− animals have a defect in object recognition memory. C, Bri2 ^+/− and WT littermates show similar freezing during cued conditioning (both in the pre-CS group, p = 0.96, and in the CS group, p = 0.94). D, Bri2 ^+/− and WT littermates show no difference in freezing during training (baseline; p = 0.8), whereas contextual fear conditioning performed 24 h after training shows a reduction of freezing responses in Bri2 ^+/− mice compared with WT littermates [the freezing time of Bri2 ^+/− mice was ∼50% of WT mice; n = 8 males, WT littermates, n = 8 males (p = 0.0021). *p < 0.002; **p < 0.001; ***p < 0.0001. E, Bri2 levels are reduced in synaptic fractions (LP1) of Bri2^+/− mice compared with littermates. The absence of the signal in Bri2 ^−/− mice attests the specificity of the band. Levels of APP are comparable in all genotypes.