Schizophrenia is associated with dysregulation of a Cdk5 activator that regulates synaptic protein expression and cognition

Abstract

Cyclin-dependent kinase 5 is activated by small subunits, of which p35 is the most abundant. The functions of cyclin-dependent kinase 5 signalling in cognition and cognitive disorders remains unclear. Here, we show that in schizophrenia, a disorder associated with impaired cognition, p35 expression is reduced in relevant brain regions. Additionally, the expression of septin 7 and OPA1, proteins downstream of truncated p35, is decreased in schizophrenia. Mimicking a reduction of p35 in heterozygous knockout mice is associated with cognitive endophenotypes. Furthermore, a reduction of p35 in mice results in protein changes similar to schizophrenia post-mortem brain. Hence, heterozygous p35 knockout mice model both cognitive endophenotypes and molecular changes reminiscent of schizophrenia. These changes correlate with reduced acetylation of the histone deacetylase 1 target site H3K18 in mice. This site has previously been shown to be affected by truncated p35. By restoring H3K18 acetylation with the clinically used specific histone deacetylase 1 inhibitor MS-275 both cognitive and molecular endophenotypes of schizophrenia can be rescued in p35 heterozygous knockout mice. In summary, we suggest that reduced p35 expression in schizophrenia has an impact on synaptic protein expression and cognition and that these deficits can be rescued, at least in part, by the inhibition of histone deacetylase 1.

Figure 1

p35 and p25-regulated proteins are downregulated in schizophrenia post-mortem brain. Total lysates of post-mortem samples from the hippocampus (A and B) and the prefrontal cortex (C and D) were western blotted against p35/p25, Cdk5, homer, PSD95 and the p25-regulated proteins septin 7 and OPA1. Protein levels were normalized against β-actin. Hippocampal total lysate from a p25 transgenic mouse (P25) was loaded as a control for specificity of staining on the western blots against p35/p25. (C) p35 and homer were decreased in hippocampus. However, in this tissue OPA1, but not septin 7, was reduced. Cdk5 levels remained unchanged. (D) There was a significant downregulation of p35, homer and septin 7 in prefrontal cortex of patients with schizophrenia. No changes were detected for OPA1, PSD95 or Cdk5 levels. (E and F) The downregulation of p35 was confirmed by immunohistochemistry on the prefrontal cortex (layer 3). (E) In control subjects, p35 antibody revealed evenly distributed labelling of moderate intensity in the neuronal cytoplasm and cell processes (arrows) with no significant nuclear staining. (F) In patients with schizophrenia, weak or absent cytoplasmic and cell process labelling was noted with moderate nuclear labelling. (G–H) Quantification of immunostaining against p35+25. (G) Somatic p35+p25 was strongly reduced in all prefrontal cortical layers in patients with schizophrenia. (H) Dendritic p35+p25 was reduced in layers 3, 5 and 6 of prefrontal cortex from patients with schizophrenia. (A and B): n = 13–15 per group; (C and D): n = 8–9 per group; (G and H): n = 95–105 cells per group. *P < 0.05; **P < 0.01; ***P < 0.001; average ± SEM is shown; (E and F): scale bar = 50 micrometer; 25 µm (insets). A.U = arbitrary units.

Figure 2

Cdk5 activators and downstream proteins are not affected by the antipsychotic drug clozapine. (A) Purified synaptic fractions from prefrontal cortex of rats that had been treated with clozapine or vehicle were western blotted against p35, the p25-regulated proteins septin 7 and OPA1 as well as against homer. (B) None of the analysed proteins were affected by clozapine treatment. (n = 4–7; average ± SEM is shown).

Figure 3

p35^+/− mice display sex-specific cognitive deficits. (A) Prepulse inhibition (PPI) was measured as the mean startle response to a set of prepulses of sound intensities of 69, 73 and 77 dB within a background noise of 65 dB. They were followed after 100 ms by a sound of 120 dB. (B) Another set of prepulses of 77 dB were given at interval times of 50, 100, 200 and 300 ms before a 120 dB sound. Female p35^+/− mutants showed reduced prepulse inhibition for all tested parameters. (C and D) Female p35^+/− mutants also showed a reduced maximum startle amplitude (ΔV_max) for various interval times at 77 dB. (E and F) The baseline startle response of female p35^+/− mice was not altered. (G and H) p35^+/− mice and wild-type (WT) littermates of both sexes were trained in the hidden platform version of the Morris water maze. (G) After reversal learning, female p35^+/− mutants spent significantly less time in the platform quadrant and made fewer crosses through the platform area (H). (A–F): n = 12–18 per group, asterisks represent sex by genotype interaction and post hoc data; (G and H): n = 7–9 per group, asterisks represent post hoc data. *P < 0.05; average ± SEM is shown. F = female; M = male. AdJL = quadrants left of the platform quadrant; R: quadrants right of the platform quadrant; **P < 0.01, ***P < 0.001.

Figure 4

Female p35^+/− mice have reduced OPA1 expression and acetylation of H3K18 in hippocampus, while male p35^+/− mice show decreased H3K18 acetylation and septin 7 expression in prefrontal cortex. (A) Hippocampal total lysates from p35^+/− mice (P35) and wild-type (WT) littermates were probed by western blot for the acetylated forms of H3K18, H3K14 and H4K12 as well as septin 7 and OPA1. (B) In the hippocampus, acetylation of H3K18 and OPA1 expression were specifically reduced in female p35^+/− mice. (C) Total lysates from prefrontal cortex of p35^+/− mice and wild-type littermates were western blotted against acetyl-H3K18, septin 7 and OPA1. (D) Septin 7 expression and H3K18 acetylation were specifically reduced in the prefrontal cortex of male p35^+/− mice. The downregulation in hippocampal OPA1 levels in response to reduced p35 is in contrast to an upregulation of septin 7 in response to p25 over-expression in our two mouse models. This might be due to differences in expression of transgene in neurons under the control of the alphaCaMKII promoter versus knockout in all p35-containing neurons (n = 10 per group; *P < 0.05; **P < 0.01; asterisks represent sex by genotype interaction and post hoc data; average ± SEM is shown). F = female; M = male.

Figure 5

Expression of septin 7 and OPA1 are affected by administration of HDAC inhibitors. (A) Scheme of drug administration: cohorts were administered 10 days of vehicle (0d), 5 days of vehicle followed by 5 days of MS-275 (5d) or 10 days of MS-275 or SAHA (10d). (B and C) Total hippocampal lysates from mice that had obtained MS-275 injections or vehicle were western blotted against acetylated forms of H3K18, H3K14 and H4K12, against p35/p25, septin 7 and OPA1, and normalized against β-actin. (C) Acetylation of H3K18 was increased after MS-275 administration, indicating successful drug treatment. Acetylation of H3K14 and H4K12 were not changed, confirming the specificity of the drug. The expression of septin 7 and OPA1 was significantly increased after 10 days of MS-275 administration, while p35/p25 levels were unaltered. (D and E) After 10 days of SAHA treatment, p25 and septin 7 levels were significantly upregulated. SAHA increased acetylation of H3K18 as well as H4K12 in agreement with its unspecific inhibition of class I HDACs. Expression of p35, H3K14 and OPA1 were not affected by SAHA treatment (n = 6–8 per group; *P < 0.05; **P < 0.01; average ± SEM shown).

Figure 6

Administration of MS-275 rescues molecular endophenotypes and prepulse inhibition in female p35^+/− mice. (A–G) p35^+/− mice and wild-type (WT) littermates were injected intraperitoneally for 10 days with either the HDAC1-inhibitor MS-275 or vehicle. (A) Scheme of the experiment. (B and C) Total hippocampal lysates were western blotted against acetyl-H3K18, septin 7 and OPA1. (C) There was a significant reduction in expression of septin 7 and OPA1 and H3K18 acetylation in p35 mutants, which was rescued by administration of MS-275. (D and E) Prepulse inhibition (PPI) was tested with a protocol comprising four different interval times. Weight was analysed as covariate as weight correlated with detected startle amplitudes and therefore affected the measurements. There was a significant treatment by genotype interaction for the prepulse inhibition as measured by change in the mean (ΔPPI) and the maximum (ΔV_max) startle amplitudes. Post hoc analysis revealed that this change arises from reduced prepulse inhibition in female p35^+/− mice in the vehicle group as well as from an effect of drug treatment within p35 mutants and within wild-type mice. (F and G) The baseline startle response was not affected by administration of MS-275 [− = vehicle; + = MS-275; n = 4–8 per group; (D and E) asterisks represent sex by genotype interaction and post hoc data; *P < 0.05; **P < 0.01; ***P < 0.001; average ± SEM shown].