Deficiency of Aph1B/C-gamma-secretase disturbs Nrg1 cleavage and sensorimotor gating that can be reversed with antipsychotic treatment

Abstract

Regulated intramembrane proteolysis by gamma-secretase cleaves proteins in their transmembrane domain and is involved in important signaling pathways. At least four different gamma-secretase complexes have been identified, but little is known about their biological role and specificity. Previous work has demonstrated the involvement of the Aph1A-gamma-secretase complex in Notch signaling, but no specific function could be assigned to Aph1B/C-gamma-secretase. We demonstrate here that the Aph1B/C-gamma-secretase complex is expressed in brain areas relevant to schizophrenia pathogenesis and that Aph1B/C deficiency causes pharmacological and behavioral abnormalities that can be reversed by antipsychotic drugs. At the molecular level we find accumulation of Nrg1 fragments in the brain of Aph1BC(-/-) mice. Our observations gain clinical relevance by the demonstration that a Val-to-Leu mutation in the Nrg1 transmembrane domain, associated with increased risk for schizophrenia, affects gamma-secretase cleavage of Nrg1. This finding suggests that dysregulation of intramembrane proteolysis of Nrg1 could increase risk for schizophrenia and related disorders.

Fig. 1.

Expression of Aph1B/C in neurons of the adult mouse brain. (a and b) Color-coded sagittal images of Aph1B/C mRNA in situ hybridizations on adult mouse brain using radioactively labeled antisense (a) and sense (b) probes. High expression is seen in hippocampus (HC), olfactory bulb (OB), PFC (PFC-CTX), and cerebellar cortex (CER). In contrast, expression levels are relatively low in striatum (STR). (c and e) Dark-field microscopy images of Aph1B/C mRNA in situ hybridizations of PFC using radioactively labeled antisense (c) and sense (e) probes. (d) Nonradioactive VGLUT1 labeling shows the density of glutamatergic neurons. Aph1B/C expression is enriched in cortical layers 5 and layers 2–3, is low in layers 4 and 6, and is absent in layer 1. (f and h) Dark-field microscopy images of Aph1B/C mRNA in situ hybridizations of hippocampus using radioactively labeled antisense (f) and sense (h) probes. (g) Nonradioactive VGLUT1 labeling shows the density of glutamatergic neurons. Aph1B/C expression is high in hippocampal area CA1 and almost absent in CA3 and dentate gyrus (DG).

Fig. 2.

Aph1BC^−/− mice have a sensorimotor gating deficit that is alleviated by antipsychotics. (a) PPI was measured in Aph1BC^−/− mice (n = 25, empty bars) and WT littermates (n = 24, filled bars), using different combinations of prepulse and startle pulse intensities (indicated on the x axis). PPI in Aph1BC^−/− mice was significantly lower than in WTs (genotype effect, P < 0.001) when a pulse of 110 db was used as startle stimulus [post hoc Tukey test, P = 0.005 for pp74, P = 0.008 for pp78 (**)]. (b and c) Treatment with clozapine (clz) or haloperidol (hal) (both 1 mg/kg, i.p) alleviated the PPI deficit in the knockout [compared with saline injected (ctr)], as shown for prepulse/pulse combinations pp74-p110 (b) and pp78-p110 (c) (treatment effect, P = 0.003 for pp74, P = 0.005 for pp78; genotype × treatment effect, P < 0.001 for both b and c). Post hoc Tukey tests, P = 0.009 and P = 0.007 (**) for the control group in b and c, respectively. P > 0.05 [not significant (n.s.)] for the clozapine and the haloperidol group in both b and c. n = 20 for both genotypes in b and c.

Fig. 3.

Abnormalities in the dopaminergic mesolimbic sytem of Aph1BC^−/− mice. (a) Mice were habituated to a novel cage and after 1 h they were injected i.p. with amphetamine or saline (arrow) and monitored for 2 more hours. Aph1BC WT, saline (diamonds, n = 20); Aph1BC^−/−, saline (squares, n = 19); Aph1BC WT, 3 mg/kg amphetamine i.p. (triangles, n = 20); Aph1BC^−/−, 3 mg/kg i.p. (rectangles, n = 19). Amphetamine elicits hyperactivity in WTs and knockouts (drug effect, P < 0.001), but the response to the drug is significantly stronger in Aph1BC^−/− mice (genotype effect, P = 0.008). (b) Activity of Aph1BC WT and knockout mice was summated (area under the curve) over 2 h after injection and normalized to total activity after saline injection. Amphetamine in three different doses (0.3, 1, and 3 mg/kg) causes a dose-dependent hyperactivity phenotype in WT and Aph1BC^−/− mice, but the phenotype is more severe in knockouts (genotype effect, P = 0.004). Post hoc Tukey tests show a significant difference both at 1 mg/kg (*, P = 0.038) and 3 mg/kg (**, P = 0.005). n = 20 for WT, n = 19 for Aph1BC^−/− mice. (c–e) Dopamine metabolites were measured in Aph1BC^−/− mice (empty bars, n = 9) and WT littermates (filled bars, n = 8). In the ventral striatum the dopamine catabolites, expressed as ng/mg wet tissue weight, DOPAC (c) (Student's t test; *, P = 0.018) and HVA (d) [Student's t test; P = 0.1 (borderline)] are increased. In the dorsal striatum, no significant differences are measured. (e) Dopamine turnover, expressed as the sum of the levels of HVA and DOPAC relative to total dopamine, is slightly enhanced in Aph1BC^−/− mice (Student's t test; *, P = 0.026).

Fig. 4.

Impaired working memory and hypersensitivity to a noncompetitive NMDA receptor antagonist in Aph1BC^−/− mice. (a) Aph1BC^−/− mice (squares, n = 20) and WT littermates (diamonds, n = 20) were trained in the standard version of the Morris water maze task. Latency to find the hidden platform decreased gradually with number of training days, indicating acquisition of reference memory for the platform location. Learning curves were similar for both genotypes (no genotype effect). (b) After 2 weeks of training, Aph1BC^−/− mice (empty bars, n = 20) and WT littermates (filled bars, n = 20) were subjected to a probe trial. Both mice preferred the target quadrant (TQ), where the platform was located during the acquisition phase, above the opposite (OQ) and adjacent (AQ1 and AQ2) quadrants (no genotype effect). Preference was quantified as the total amount of time spent in a specific quadrant during a 100-s probe trial. (c) Subsequently, Aph1BC^−/− mice (squares, n = 20) and WT littermates (diamonds, n = 20) were assessed for working memory capacity. In daily trial blocks of five swims (cue trial and test trials 1–4), mice had to find a hidden platform that changed location daily. Aph1BC^−/− mice needed significantly more time to find the hidden platform expressed as mean latency of test trials 1–4 (genotype effect; P = 0.041). (d) Savings in escape latency between the cue trial and test trial 1 are similar (t test, P > 0.05) for Aph1BC^−/− mice (squares) and WT littermates (diamonds), but savings between test trial 1 and test trial 3 are smaller for Aph1BC^−/− mice (t test, P = 0.027). Data are given as means of latencies on the specified trials, averaged over 6 trial days, and are expressed for both genotypes as the percentage of the escape latency compared with the cue trial. (e and f) PPI was measured after i.p. injection with saline or different concentrations of MK-801 in Aph1BC^−/− mice (squares, n = 15) and WT littermates (diamonds, n = 16). Results are shown for two different prepulse/pulse combinations: pp74-p110 (e) and pp78-p110 (f). PPI under each drug condition is plotted as the percentage of PPI under placebo. For both trial types, Aph1BC^−/− mice are hypersensitive to MK-801 (genotype effect; P = 0.002 for pp74, P = 0.004). Post hoc Tukey tests reveal significant differences at the 0.5 mg/kg dose (**, P = 0.003 for pp74/p110 trials and *, P = 0.036 for pp78/p110 trials).

Fig. 5.

Effect of V321L mutation and Aph1BC deficiency on Nrg1 cleavage by γ-secretase. (a and b) Western blot analysis of membrane fractions of mouse brain homogenates. (a) Antibody recognizing a C-terminal epitope in Nrg1 with “a”-type tail conformation, identifies a ≈50- to 55-kDa Nrg1-CTF that accumulates in specific brain areas of the knockout mice. (b) No differences in the levels of the 80-kDa ErbB4-CTF are seen. (c–e) Western blot analysis of COS1 cells transiently overexpressing Nrg1βa. (c) The anti-Nrg1 a-type tail antibody reveals ≈50- to 55-kDa Nrg1-CTF accumulation upon treatment with γ-secretase inhibitors DAPT and L-685,458. Full-length (FL) Nrg1 and secreted Nrg1 (SUP) are unaffected (CE, cell extract; SUP, supernatant). (d and e) The schizophrenia-associated V321L mutation in type III Nrg1βa (empty bar) leads to significantly elevated levels of the ≈50- to 55-kDa Nrg1-CTF compared with WT Nrg1βa (filled bar). **, P = 0.009; n = 4 for both genotypes.