The known functions of Par-4 include well-established pro-apoptotic effects (Guo et al., 1998
) as well as more recently discovered involvement in D2DR signaling (Park et al., 2005
). In this study, we sought to determine whether Par-4 protein was altered in temporal cortex of subjects with schizophrenia, major depression and bipolar disorder. Although Par-4 levels appeared somewhat lower in subjects with schizophrenia and bipolar disorder compared to normal controls, these differences did not reach statistical significance. However, mean Par-4 levels were decreased by 67% in major depression compared to controls. Interestingly, this supports a Par-4 mutant mouse model of depression in which Par-4 binding to D2DR is disrupted, effectively reducing Par-4 availability for normal D2DR signaling (Park et al., 2005
). Par-4 mutant mouse behavioral phenotype included increased immobility during the forced swim test, increased latency to contact novel food, and decreased open-field exploration. The substantial reduction of Par-4 abundance in postmortem cortex in major depression may functionally replicate altered Par-4/D2DR signaling seen in the Par-4 mouse model and suggests pathophysiological relevance of low cortical Par-4 levels.
Cortical levels of the D2DR 98 kDa dimer were approximately 50% lower in schizophrenia, major depression and bipolar disorder compared to normal controls, although only the schizophrenia group reached statistical significance. A role for dopamine dysregulation has long been demonstrated in schizophrenia (reviewed by Guillin et al., 2007
), including several lines of evidence for altered D2DR signaling in temporal cortex, such as PET studies of D2/3 receptors showing decreased binding in the temporal cortex of drug-naïve subjects (Tuppurainen et al., 2003
; Buchsbaum et al., 2006
) altered distribution of D2DR in postmortem temporal lobe (Goldsmith et al., 1997
) and reduced Akt1 levels, an anti-apoptotic protein downstream from D2DR that helps regulate dopamine neurotransmission in frontal cortex and hippocampus (Chang et al., 2003
; Beaulieu et al., 2005
; Emamian et al., 2004
; Zhao et al., 2006
). Additionally, Par-4 and D2DR levels were positively correlated across all samples, further evidence supporting a functional interaction between these two factors. Calmodulin, which competes with Par-4 for D2DR binding, was unchanged across the diagnostic groups. Taken together, the data suggest that the Par-4/D2DR signaling pathway is altered in major depression and, to a lesser extent, in schizophrenia, potentially involving mechanisms of both apoptosis and dopamine signaling.
cAMP is a well established second messenger of D2DR signaling. Normally, D2DR activity is associated with inhibition of cAMP signaling (Bonci and Hopf, 2005
). However, when Par-4 expression was knocked down by RNA interference, D2DR activation increased cAMP activity suggesting that Par-4 is necessary for optimizing D2DR efficacy. The Par-4 mouse model of depression lacking the leucine zipper domain where Par-4 interacts with the D2DR had increased cAMP levels (Park et al., 2005
). It should be noted that the leucine zipper domain of Par-4 could potentially interact with proteins other than D2DR (Sells et al., 1997
; Guo et al., 1998
) although in a yeast two-hybrid assay the Par-4/D2DR interaction appeared relatively specific (Park et al., 2005
). These data demonstrate how the integrity of Par-4/D2DR interactions can directly affect cAMP signaling, providing additional insight into the mechanism by which Par-4 deficits may contribute to major depression.
Several other groups have also studied Par-4 in psychiatric illnesses. Par-4 protein levels in lymphocytes and in postmortem frontal cortex of subjects with schizophrenia and bipolar disorder were unchanged compared to normal matched controls (Amar et al., 2008
). No evidence for variation in the Par-4 gene was found in Japanese patients with schizophrenia, bipolar disorder or major depression (Kishi et al., 2008
). However, Chinese female patients with schizophrenia were found to have two missense single nucleotide polymorphisms of the Par-4 gene (Wang et al., 2008
Transgenic mice overexpressing Par-4 have demonstrated that Par-4 inhibits the DNA-binding activity of NF-κB (Camandola and Mattson, 2000
; El-Guendy and Rangnekar, 2003
), an anti-apoptotic transcription factor (Won et al., 2002
). Interestingly, increased levels of NF-κB have been found in the frontal cortex of patients with schizophrenia, bipolar disorder, and major depression compared to normal control subjects (Sun et al., 2001
). Our findings of decreased Par-4 in subjects with major depression could potentially contribute to evidence for a reciprocal increase in NF-κB expression. In addition, levels of anti-apoptotic Bcl-2 protein were decreased in the temporal cortex of the same subjects with schizophrenia that were examined in the current study (Jarskog et al., 2000
). On the other hand, since Par-4 upregulation is known to suppress Bcl-2 expression (Camandola and Mattson, 2000
), the current data indicate that Par-4 likely did not account for low cortical Bcl-2 in schizophrenia. In fact, the evidence for slightly lower Par-4 in schizophrenia is consistent with data that apoptosis may be downregulated in schizophrenia, at least in later stages of illness, including postmortem studies showing reduced DNA fragmentation in cingulate cortex (Benes et al., 2003
), downregulation of multiple pro-apoptotic genes in hippocampus (Benes et al., 2006
), and reduced ARTS/Sept 4 protein in frontal cortex (Gottfried et al., 2007
Several potentially confounding variables were considered. Because Par-4 is preferentially localized to the postsynaptic density (Guo et al., 2001
), decreased Par-4 could potentially reflect the effects of reduced synaptic density. A number of studies have demonstrated decreased cortical levels of synaptic marker proteins such as synaptophysin (Perrone-Bizzozero et al., 1996
; Glantz and Lewis, 1997
) and SNAP-25 (Thompson et al., 1998
; Karson et al., 1999
) in schizophrenia compared to controls. However, the current study found no difference in synaptophysin levels in any of the patient groups compared to normal controls, even when stratifying for antidepressant treatment at the time of death. The effects of psychotropic treatment were also considered. Rats that received 2 weeks of daily antidepressant treatment showed no effect on cortical Par-4 levels. Further, previous studies indicate that D2DR binding is upregulated (not downregulated) in monkey prefrontal and temporal cortices after antipsychotic treatment (Lidow and Goldman-Rakic, 1994
), suggesting that antipsychotic treatment did not confound the evidence for lower D2DR levels in schizophrenia. Postmortem stability of Par-4 showed a reduction of 54% in Par-4 levels over 24 hours PMI. While this is a substantial reduction, the diagnostic groups were matched for PMI and it is unlikely that PMI exerted a differentially greater effect on Par-4 in the major depression group compared to the other groups. D2DR levels showed no change over 24 hours PMI. Finally, while samples were also group-matched on age, gender, ethnicity, brain hemisphere, and brain pH, the duration of illness was significantly shorter in the depression group compared to the schizophrenia and bipolar groups. A linear regression analysis found no association between duration of illness and Par-4 levels (r=0.019, p=0.898). Considered together, these potentially confounding variables do not appear to have exerted a significant effect on the results.
In summary, this study found changes in the Par-4/D2DR receptor pathway in postmortem temporal cortex in patients with major depression, with more subtle changes also found in patients with schizophrenia. The pathophysiological significance of low Par-4 levels remains uncertain; however, mechanisms involving both the regulation of apoptosis and dopamine signaling must be considered. Given that the current findings appear to support the Par-4 mouse model of depression, further study of Par-4 in model systems and in human postmortem brain tissue is warranted.