Rescue of Learning and Memory Deficits in the Human Nonsyndromic Intellectual Disability Cereblon Knock-Out Mouse Model by Targeting the AMP-Activated Protein Kinase-mTORC1 Translational Pathway

Abstract

A homozygous nonsense mutation in the cereblon (CRBN) gene results in autosomal recessive, nonsyndromic intellectual disability that is devoid of other phenotypic features, suggesting a critical role of CRBN in mediating learning and memory. In this study, we demonstrate that adult male Crbn knock-out (Crbn^KO) mice exhibit deficits in hippocampal-dependent learning and memory tasks that are recapitulated by focal knock-out of Crbn in the adult dorsal hippocampus, with no changes in social or repetitive behavior. Cellular studies identify deficits in long-term potentiation at Schaffer collateral CA1 synapses. We further show that Crbn is robustly expressed in the mouse hippocampus and Crbn^KO mice exhibit hyperphosphorylated levels of AMPKα (Thr172). Examination of processes downstream of AMP-activated protein kinase (AMPK) finds that Crbn^KO mice have a selective impairment in mediators of the mTORC1 translation initiation pathway in parallel with lower protein levels of postsynaptic density glutamatergic proteins and higher levels of excitatory presynaptic markers in the hippocampus with no change in markers of the unfolded protein response or autophagy pathways. Acute pharmacological inhibition of AMPK activity in adult Crbn^KO mice rescues learning and memory deficits and normalizes hippocampal mTORC1 activity and postsynaptic glutamatergic proteins without altering excitatory presynaptic markers. Thus, this study identifies that loss of Crbn results in learning, memory, and synaptic defects as a consequence of exaggerated AMPK activity, inhibition of mTORC1 signaling, and decreased glutamatergic synaptic proteins. Thus, Crbn^KO mice serve as an ideal model of intellectual disability to further explore molecular mechanisms of learning and memory.SIGNIFICANCE STATEMENT Intellectual disability (ID) is one of the most common neurodevelopmental disorders. The cereblon (CRBN) gene has been linked to autosomal recessive, nonsyndromic ID, characterized by an intelligence quotient between 50 and 70 but devoid of other phenotypic features, making cereblon an ideal protein for the study of the fundamental aspects of learning and memory. Here, using the cereblon knock-out mouse model, we show that cereblon deficiency disrupts learning, memory, and synaptic function via AMP-activated protein kinase hyperactivity, downregulation of mTORC1, and dysregulation of excitatory synapses, with no changes in social or repetitive behaviors, consistent with findings in the human population. This establishes the cereblon knock-out mouse as a model of pure ID without the confounding behavioral phenotypes associated with other current models of ID.

Keywords: AMPK; cereblon; excitatory; glutamatergic; intellectual disability; mTOR.

Figure 1.

Validation of cereblon antibody by Western blot analysis and immunohistochemistry. A, Western blot with total hippocampal protein lysates from Crbn^WT and Crbn^KO mice. A band at the expected 51 KDa size present in Crbn^WT mice but absent in Crbn^KO mice confirms the specificity of the antibody. B, Representative images of hippocampal sections stained for NeuN (left) and cereblon (middle). Cereblon staining is visible in all subregions of the Crbn^WT but not the Crbn^KO hippocampus.

Figure 2.

Crbn^KO mice demonstrate learning and memory deficits in the Morris water maze and water-based Y-maze tests, with no deficits in a working memory test. A, In the MWM training, Crbn^KO mice displayed increased latency to find a hidden platform compared to Crbn^WT mice (n = 10/genotype). B, In the MWM probe test, Crbn^WT mice spent significantly more time in the goal zone compared to either zone 1 or zone 2, whereas Crbn^KO mice did not. ***p < 0.001 (n = 10/genotype). C, In the Y-maze training, Crbn^KO mice demonstrated increased latency to find the hidden platform compared to Crbn^WT mice (n = 7/genotype). D, During the Y-maze probe test, Crbn^KO mice took longer to locate the submerged platform compared to Crbn^WT mice. *p < 0.05 (n = 7/genotype). E, Crbn^KO mice did not show impaired working memory in the spontaneous alternation task compared to Crbn^WT mice (Crbn^WT, n = 11; Crbn^KO, n = 10). F, In the social interaction task, both Crbn^WT and Crbn^KO mice spent significantly more time with the stranger mouse compared than the empty cup. G, In the social preference test, both Crbn^WT and Crbn^KO mice spent significantly more time with the novel stranger mouse than the familiar mouse. H, Crbn^WT and Crbn^KO mice showed no differences in repetitive grooming behavior (Crbn^WT, n = 7; Crbn^KO, n = 8). Data are displayed as mean ± SEM.

Figure 3.

Focal knockdown of CRBN in the dorsal hippocampus induced deficits in learning and memory. A, Representative image of GFP expressed by microinjection of AAV-Cre into the dorsal hippocampus of CRBN^fl/fl mice. B, Injection of AAV-Cre into the dorsal hippocampus of CRBN^fl/fl mice resulted in a significant decrease in Crbn mRNA expression in the dorsal hippocampus compared to injection of AAV-GFP (AAV-GFP, n = 6; AAV-Cre, n = 8). C, In the MWM training, knockdown of CRBN in the dorsal hippocampus increased latency to find a hidden platform during 5 d of training (AAV-GFP, n = 6; AAV-Cre, n = 8). D, In the MWM probe test, mice injected with AAV-GFP spent significantly more time in the goal zone compared to zone 1, zone 2, or zone 3 (AAV-GFP, n = 6; AAV-Cre, n = 8), while mice injected with AAV-Cre did not. E, During the Y-maze training, mice injected with AAV-Cre displayed increased latency to find a hidden platform compared to mice injected with AAV-GFP (AAV-GFP, n = 6; AAV-Cre, n = 8). F, During the y-maze probe test, mice injected with AAV-Cre demonstrated increased latency to find the hidden platform compared to mice injected with AAV-GFP (*p < 0.05; AAV-GFP, n = 6; AAV-Cre, n = 8). G, In a test of contextual fear conditioning, mice injected with AAV-Cre spent a significantly lower percentage of time freezing compared to mice injected with AAV-GFP (AAV-GFP, n = 6; AAV-Cre, n = 8). *p < 0.05; **p < 0.01 (Bonferroni post hoc). Data are displayed as mean ± SEM.

Figure 4.

Crbn^KO mice have impairments in long-term potentiation and synaptic plasticity. A, Input/output curves expressed as EPSP slopes (in millivolts per millisecond) plotted against stimulus intensity (in millivolts). No differences between genotypes were found (n = 5/genotype). B, LTP represented as the normalized fEPSP slope over the course of 60 min. Crbn^KO mice showed a significant deficit in LTP during the first 40 min compared to Crbn^WT mice (n = 5/genotype). C, Crbn^KO mice displayed a deficit in chemical LTP-induced increase in GluA1 protein levels in the PSD, which was rescued by overexpression of human CRBN (n = 5/genotype). ^#p < 0.05 (Bonferroni post hoc test, WT basal vs KO basal); *p < 0.05; **p < 0.01, ****p < 0.0001 (Bonferroni post hoc test, basal vs chemical LTP). Data displayed as mean ± SEM.

Figure 5.

Crbn^KO mice show altered mTORC1 signaling in the hippocampus. A, Schematic of proposed molecular mechanism. B, Compared to Crbn^WT mice, Crbn^KO mice displayed significantly higher synaptosomal levels of phosphorylated AMPKα (Thr172) in the hippocampus (Crbn^WT, n = 10; Crbn^KO, n = 14), with no difference found in levels of total AMPKα (Crbn^WT, n = 6; Crbn^KO, n = 7). C, Crbn^WT and Crbn^KO mice displayed similar levels of synaptosomal eIF2α and phosphorylated eIF2α well as cytoplasmic ATG5 and ATG12 in the hippocampus (n = 5/genotype). D, Crbn^KO mice displayed decreased levels of synaptosomal phosphorylated mTOR (n = 5/genotype), phosphorylated S6 (Crbn^WT, n = 9; Crbn^KO, n = 12), and phosphorylated 4E-BP1 (Crbn^WT, n = 4; Crbn^KO, n = 7) compared to Crbn^WT mice, with no differences found in total mTOR (n = 5/genotype), S6 (Crbn^WT, n = 11; Crbn^KO, n = 12), or 4E-BP1 (Crbn^WT, n = 5; Crbn^KO, n = 6) between Crbn^WT and Crbn^KO mice. Crbn^WT and Crbn^KO mice displayed similar levels of phosphorylated eEF2 Thr56 and total eEF2 (n = 5/genotype). *p < 0.05; **p < 0.01; ***p < 0.001. Data are displayed as mean ± SEM. Western blot images are representative samples taken from the same blot.

Figure 6.

Crbn^KO mice show decreased levels of synaptic proteins in the hippocampus. A, PSD enriched hippocampal fractions from Crbn^KO mice showed lower protein levels of CaMKIIα (Crbn^WT, n = 12; Crbn^KO, n = 11), CaMKIIβ (Crbn^WT, n = 7; Crbn^KO, n = 6), GluA1 (Crbn^WT, n = 12; Crbn^KO, n = 11), GluA2 (Crbn^WT, n = 7; Crbn^KO, n = 6), GluN1 (Crbn^WT, n = 12; Crbn^KO, n = 11), GluN2A (n = 4/genotype), and GluN2B (n = 11/genotype) compared to Crbn^WT mice. B, No differences were found in mRNA expression of Camk2a Gria1, Gria2, Grin1, Grin2a, or Grin2b in the hippocampi of Crbn^WT and Crbn^KO mice (n = 7/genotype). C, Compared to Crbn^WT mice, Crbn^KO mice displayed significantly increased synaptosomal protein levels of PSD-95 (Crbn^WT, n = 7; Crbn^KO, n = 6), synaptophysin (Crbn^WT, n = 6; Crbn^KO, n = 7), VGLUT1 (n = 7/genotype), and VGLUT2 (n = 7/genotype), with no change in VGAT expression observed between genotypes (n = 7/genotype). Crbn^KO mice displayed a significantly higher VGLUT1/VGAT ratio and VGLUT2/VGAT ratio compared to Crbn^WT mice (n = 7/genotype). *p < 0.05; **p < 0.01; ***p < 0.001. Data displayed as mean ± SEM. Western blot images are representative samples taken from the same blot.

Figure 7.

Pharmacological inhibition of AMPK rescues learning and memory deficits in Crbn^KO mice. A, During the Y-maze training, Compound C–treated Crbn^KO mice displayed decreased latency to find the hidden platform compared to vehicle-treated Crbn^KO mice, similar to the level observed in Crbn^WT mice. Compound C had no effect on latency in Crbn^WT mice (Crbn^WT + vehicle, n = 8; Crbn^WT + Compound C, n = 4; Crbn^KO + vehicle, n = 7; Crbn^KO + Compound C, n = 5). B, During the Y-maze probe test, treatment with Compound C significantly reduced the latency of Crbn^KO mice to find a hidden platform, to a level similar to that of Crbn^WT mice (Crbn^WT + vehicle, n = 8; Crbn^WT + Compound C, n = 4; Crbn^KO + vehicle, n = 7; Crbn^KO + Compound C, n = 5). *p < 0.05 (Bonferroni post hoc test, WT + vehicle vs KO vehicle); ^#p < 0.05 (Bonferroni post hoc test, KO + vehicle vs KO + Compound C). C, In a test of contextual fear conditioning, treatment with Compound C significantly increased percentage of time freezing in Crbn^KO mice to the level of Crbn^WT mice (Crbn^WT + vehicle, n = 8; Crbn^WT + Compound C, n = 4; Crbn^KO + vehicle, n = 7; Crbn^KO + compound C, n = 5). *p < 0.05 (Bonferroni post hoc test, WT + vehicle vs WT + Compound C); ^#p < 0.05 (Bonferroni post hoc test, Crbn^KO + vehicle vs Crbn^KO + Compound C). D, Treatment with Compound C decreased synaptosomal expression of phosphorylated AMPK in Crbn^KO mice but not in Crbn^WT mice (Crbn^WT + vehicle, n = 5; Crbn^WT + Compound C, n = 6; Crbn^KO + vehicle, n = 5 Crbn^KO + compound C, n = 6). Treatment with Compound C increased phosphorylated mTORC1 (WT + vehicle, n = 5; WT + Compound C, n = 5; KO + vehicle, n = 4; KO + Compound C, n = 6), phosphorylated S6 (WT + vehicle, n = 5; WT + Compound C, n = 6; KO + vehicle, n = 5; KO + Compound C, n = 6), and phosphorylated 4E-BP1 (WT + vehicle, n = 5; WT + Compound C, n = 5; KO + vehicle, n = 5; KO + Compound C, n = 4) in Crbn^WT and Crbn^KO mice. *p < 0.05 (Bonferroni post hoc test, Crbn^KO + vehicle vs Crbn^KO + Compound C). E, Treatment with Compound C increased protein levels of GluA1, GluA2, and GluN2B, but not GluN2A (WT + vehicle, n = 5; WT + Compound C, n = 6; KO + vehicle, n = 5; KO + Compound C, n = 6) in the hippocampal PSD of Crbn^WT and Crbn^KO mice, and increased expression of GluN1 in Crbn^KO mice. **p < 0.01 (Bonferroni post hoc test, KO + vehicle vs KO + Compound C). F, Treatment with Compound C did not alter synaptosomal protein levels of VGLUT1 or VGLUT2 in Crbn^WT or Crbn^KO mice (WT + vehicle, n = 5; WT + Compound C, n = 6; KO + vehicle, n = 5; KO + Compound C, n = 6), nor did it alter the ratio of VGLUT1/VGAT or VGLUT2/VGAT (WT + vehicle, n = 5; WT + Compound C, n = 6; KO + vehicle, n = 5; KO + Compound C, n = 5). Data are displayed as mean ± SEM. Western blot images are representative samples taken from the same blot.