A mouse model of Rubinstein-Taybi syndrome: defective long-term memory is ameliorated by inhibitors of phosphodiesterase 4

Abstract

Mice carrying a truncated form of cAMP-responsive element binding protein (CREB)-binding protein (CBP) show several developmental abnormalities similar to patients with Rubinstein-Taybi syndrome (RTS). RTS patients suffer from mental retardation, whereas long-term memory formation is defective in mutant CBP mice. A critical role for cAMP signaling during CREB-dependent long-term memory formation appears to be evolutionarily conserved. From this observation, we reasoned that drugs that modulate CREB function by enhancing cAMP signaling might yield an effective treatment for the memory defect(s) of CBP+/- mice. To this end, we designed a cell-based drug screen and discovered inhibitors of phosphodiesterase 4 (PDE4) to be particularly effective enhancers of CREB function. We extend previous behavioral observations by showing that CBP+/- mutants have impaired long-term memory but normal learning and short-term memory in an object recognition task. We demonstrate that the prototypical PDE4 inhibitor, rolipram, and a novel one (HT0712) abolish the long-term memory defect of CBP+/- mice. Importantly, the genetic lesion in CBP acts specifically to shift the dose sensitivity for HT0712 to enhance memory formation, which conveys molecular specificity on the drug's mechanism of action. Our results suggest that PDE4 inhibitors may be used to treat the cognitive dysfunction of RTS patients.

Fig. 2.

Object recognition memory in CBP^+/− mutant mice and their normal siblings. (A) One-day memory retention in object recognition depends on duration of training. Wild-type mice were subjected to a 3.5-min (n = 24), 5-min (n = 24), 8-min (n = 16), 15-min (n = 46), or 20-min (n = 12) training duration and then tested 24 h later. Memory retention was quantified as a DI (see Methods). Maximal memory retention was reached with a training duration of 15 min. In contrast, 1-day memory was not detected after a 3.5-min training duration. (B) CBP^+/− mutant mice have impaired long-term memory of an object recognition task. Wild-type mice and CBP^+/− mutant mice were trained for 15 min and tested 3h or 24h later. Three-hour memory levels were similar for normal mice and CBP^+/− mutants (P = 0.76; n = 6 for each genotype), but 24-h memory was significantly lower than normal in mutant mice (P < 0.01; n = 10 for each genotype). (C) PDE4 inhibitors ameliorate the long-term memory defect of CBP^+/− mutant mice. Wild-type mice and CBP^+/− mutant mice received i.p. injections of 0.1 mg/kg HT 0712 or Rolipram 20 min before training. Animals were trained with a 15-min training session, and memory retention was tested 24 h later (see Methods). In vehicle-injected CBP^+/− mutants, memory was significantly lower than in vehicle-injected wild-type mice (P < 0.01; n = 12 and n = 6, respectively). In HT0712-injected CBP^+/− mutants, memory was significantly higher than in vehicle-injected mutants (P < 0.05, n = 10 and n = 12, respectively). Similarly, memory retention in Rolipram-injected animals was significantly higher than that in vehicle-alone-treated animals (P < 0.05; n = 8 and n = 12, respectively). There was no significant difference between HT0712-treated CBP^+/− mutants and HT0712-treated wild-type mice (P = 0.78) or between Rolipram-treated CBP^+/− mutants and Rolipram-treated wild-type mice (P = 0.19). N values reflect number of observations (repetitions) per treatment.

Fig. 3.

HT0712 dose sensitivity is decreased in mutant CBP^+/− mice. (A) Dose-response curve for HT 0712 in wild-type mice. Mice received a single i.p. injection of drug or vehicle alone 20 min before training. Doses of 0.001, 0.005, 0.01, 0.05, 0.10, 0.15, 0.20, and 0.5 mg/kg were used. Animals experienced a 3.5-min training duration and were tested 24 h later (see Methods). Memory retention in drug-injected animals was significantly higher than that in vehicle-alone treated animals (n = 35) at doses of 0.05 mg/kg (n = 20; P < 0.05), 0.10 mg/kg (n = 22; P < 0.0001), 0.15 mg/kg (n = 18; P < 0.001), and 0.20 mg/kg (n = 17; P < 0.05). There were no significant effects at doses 0.001 mg/kg (P = 0.91; n = 6 for HT0712), 0.005 mg/kg (P = 0.72; n = 22 for HT0712), or 0.01 mg/kg (P = 0.34; n = 10 for HT0712). Because each session included vehicle-injected mice, the data for vehicle-injected mice were pooled. N values indicate a number of observations (repetitions) per treatment. (B) CBP^+/− mutant mice are less sensitive than wild-type mice to the enhancing effects of HT 0712. CBP^+/− mutants and wild-type mice received a single i.p. injection of vehicle or drug 20 min before training. They experienced a 3.5-min training duration and were tested 24-h later (see Methods). In wild-type mice, memory retention in drug-treated groups again was higher than in the vehicle-alone group (n = 26) at doses of 0.05 mg/kg (n = 12; P < 0.005), 0.10 mg/kg (n = 8; P < 0.0001), 0.15 mg/kg (n = 18; P < 0.005), and 0.2 mg/kg (n = 14; P < 0.005). In CBP^+/− mutants, memory retention in drug-treated groups was higher than in the vehicle-alone group (n = 26) at doses of 0.10 mg/kg (n = 8; P < 0.0001), 0.15 mg/kg (n = 10; P < 0.0001), and 0.20 mg/kg (n = 14; P < 0.0001). In contrast to wild-type mice, a 0.05 mg/kg dose of HT0712 failed to enhance memory (n = 26; P = 0.79) in CBP^+/− mutants. N values reflect number of observations (repetitions) per dose per genotype.

Fig. 1.

PDE4 inhibitors enhance forskolin-induced gene expression in human neuroblastoma cells. (A) Cells were stably transfected with a CRE-luciferase reporter gene and exposed to vehicle alone or drug (HT0712 or rolipram) for 2 h before stimulation by a suboptimal dose of forskolin. Relative light units (RLU) emitted from luciferase were quantified. Both drugs increased forskolin-induced CRE-luciferase expression 1.9-fold above forskolin alone (P < 0.001 in each case). (B) Real-time PCR was used to quantify expression of somatostatin, an endogenous cAMP-responsive gene. Expression levels induced by forskolin or by forskolin plus drug are quantified as differences in threshold cycle number (ΔC_t) above vehicle alone control groups (not shown). HT0712 and rolipram produced 4.6-fold (P < 0.001) and 2.3-fold (P < 0.001) increases, respectively, in forskolin-induced expression of somatostatin.