We first examined transcript levels for genes related to CDC42EP3 and CDC42EP4 signaling and dendritic spine morphology. The mean relative expression of CDC42EP3 mRNA was significantly (F1,53
=10.3, p=0.002) increased by 19.7% in the subjects with schizophrenia (). The mean level of CDC42EP4 mRNA was also increased by 10.4% in the schizophrenia subjects, although this difference did not achieve statistical significance (; F1,53
=2.4, p=0.13). Consistent with our previous findings by in situ
), mean CDC42 mRNA expression was decreased by 7.5% in the subjects with schizophrenia, although this finding showed only a trend level of significance (; F1,53
=2.7, p=0.10). In contrast, SEPT7 mRNA was significantly (F1,53
=10.3, p=0.002) decreased by 7.2% in the subjects with schizophrenia. The other transcripts in the first set of RT-qPCR analyses (anillin, PSD-95 and spinophilin) did not significantly (all F1,53
<1.5, all p>0.22) differ between subject groups, consistent with previous findings for spinophilin (46
) and PSD-95 (47
) mRNAs in the DLPFC of subjects with schizophrenia. Similarly, in the second set of qPCR analyses, mRNA levels for synaptopodin, another marker of spines, also did not differ (F1,53
=0.1, p=0.74) between subject groups.
Figure 1 Relative expression levels of (A) CDC42EP3, (B) CDC42EP4, and (C) CDC42 mRNAs in the DLPFC of schizophrenia and matched comparison subjects. Values for subjects with schizophrenia and their matched comparison subjects are indicated as filled circles, (more ...)
In order to examine the validity and specificity of the group difference in SEPT7 mRNA expression level, we conducted the second RT-qPCR study. In this study, we used the original primer set for SEPT7 mRNA, and another primer set (SEPT7-R) that amplifies a different portion of the molecule. Consistent with the first RT-qPCR study, the original primer set revealed a significant (F1,53
=13.9, p=0.0005) 6.9% mean decrease of SEPT7 mRNA in the schizophrenia subjects (), and the SEPT7-R primer set confirmed a significant (F1,53
=8.5, p=0.005) 10.2% mean decrease. The relative expression level of SEPT7 mRNA was significantly correlated (r=0.71, p=0.048, n=8) with the density of dendritic spines on layer 3 pyramidal neurons in the same subjects (11
), and the within-pair percent difference of SEPT7 mRNA was significantly correlated with that of CDC42 mRNA (r=0.68, p<0.0001, n=31).
Figure 2 Relative expression levels of (A) SEPT7 and (B) SEPT11 mRNAs in the DLPFC of schizophrenia and matched comparison subjects. Values for subjects with schizophrenia and their matched comparison subjects are indicated as filled circles, and those for subjects (more ...)
The correlations of SEPT7 mRNA levels with spine density and with CDC42 mRNA levels in the DLPFC of subjects with schizophrenia suggest that the three might be causally related. If the SEPT7 mRNA reduction is related to the spine deficits, SEPT5 and/or SEPT11 proteins, which are also localized to spines (35
), might also altered in schizophrenia, because down-regulation of one septin protein can lead to either a parallel decrease (35
) or a compensatory increase (48
) of other septins. On the other hand, since SEPT7 protein is also present in other cellular components such as cell bodies and axon terminals (35
), the SEPT7 decrease might reflect disturbances in those components. To clarify whether the SEPT7 reduction is related to dendriticspines or other cellular components, we examined septins present in spines (SEPT5, SEPT11), in other neuronal subcellular locations (SEPT3, SEPT6, SEPT8), or in glial cells (SEPT2) (33
) in the second RT-qPCR study. Of the two other septin proteins localized to spines, SEPT11 mRNA was significantly (F1,53
=6.0, p=0.017) increased by 20.1% in schizophrenia (), whereas SEPT5 mRNA levels were unchanged (F1,53
=2.6, p=0.11). This increase of SEPT11 mRNA could be a compensation for decreased SEPT7 mRNA, although the within-pair percent differences of SEPT11 and SEPT7 mRNA levels were not significantly correlated (r=0.26, p=0.16, n=31). In addition, the expression levels of all other septins examined (SEPT2, SEPT3, SEPT6, SEPT8), which are not localized to spines, did not significantly differ (all F1,53
<1.9, all p>0.17) between subject groups, suggesting that the alteration in SEPT7 mRNA expression might be specific to alterations in dendritic spines, and not to disturbances in other neuronal subcellular locations or in glial cells.
In order to control for possible false positive findings due to multiple comparisons, we employed a Bonferroni correction for all of the transcript comparisons reported above across both RT-qPCR analyses. Only the alterations in CDC42EP3 and SEPT7 mRNAs remained significant.
In order to determine whether the alterations of CDC42EP3 or SEPT7 mRNAs in the subjects with schizophrenia might reflect the effects of chronic treatment with antipsychotic medications, we studied monkeys chronically exposed to haloperidol, olanzapine or sham. Neither the expression of CDC42EP3 (F2,10=1.3, p=0.31) nor SEPT7 (F2,10=0.08, p=0.93) mRNA significantly differed across these three groups of animals (). We also tested the effects of potential confounding factors on the relative mRNA levels of CDC42EP3 and SEPT7 in the schizophrenia subjects (). None of these factors (sex; diagnosis of schizoaffective disorder; treatment with antipsychotics, benzodiazepine or sodium valproate, or antidepressants at time of death; history of substance dependence/abuse; tobacco use at time of death; or death by suicide) showed a significant effect on the relative expression level of either transcript in the schizophrenia subjects (all F<2.5, all p>0.13).
Mean (SD) relative expression levels of (A) CDC42EP3 and (B) SEPT7 mRNAs in the DLPFC of matched triads of sham-, olanzapine- and haloperidol-exposed monkeys.
Figure 4 Relative expression levels for individual subjects (circles) and mean values for the indicated group (bars) for (A) CDC42EP3 mRNA and (B) SEPT7 mRNA for the subjects with schizophrenia grouped by potential confounding factors. Numbers in bars indicate (more ...)