Apoptosis-related proteins and proliferation markers in the orbitofrontal cortex in major depressive disorder

Abstract

Background: In major depressive disorder (MDD), lowered neural activity and significant reductions of markers of cell resiliency to degeneration occur in the prefrontal cortex (PFC). It is still unclear whether changes in other relevant markers of cell vulnerability to degeneration and markers of cell proliferation are associated with MDD.

Methods: Levels of caspase 8 (C8), X-linked inhibitor of apoptosis protein (XIAP), direct IAP binding protein with low pI (DIABLO), proliferating cell nuclear antigen (PCNA) and density of cells immunoreactive (-IR) for proliferation marker Ki-67 were measured in postmortem samples of the left orbitofrontal cortex (OFC) of subjects with MDD, and psychiatrically-normal comparison subjects.

Results: There was significant increase in C8, a higher ratio of DIABLO to XIAP, lower packing density of Ki-67-IR cells, and an unexpected age-dependent increase in PCNA in subjects with MDD vs. controls. PCNA levels were significantly higher in MDD subjects unresponsive to antidepressants or untreated with antidepressants. The DIABLO/XIAP ratio was higher in MDD subjects without antidepressants than in comparison subjects.

Limitations: Qualitative nature of responsiveness assessments; definition of resistance to antidepressant treatment is still controversial; and unclear role of PCNA.

Conclusions: Markers of cell vulnerability to degeneration are increased and density of Ki67-positive cells is low MDD, but accompanied by normal XIAP levels. The results suggest increased vulnerability to cell pathology in depression that is insufficient to cause morphologically conspicuous cell death. Persistent but low-grade vulnerability to cell degeneration coexisting with reduced proliferation readiness may explain age-dependent reductions in neuronal densities in the OFC of depressed subjects.

Keywords: Comorbidity; Depression; Postmortem; Prefrontal cortex; Vulnerability.

Fig. 1

Western blot detection and quantification of caspase 8 (C8). A) Lanes from two different representative Western blots of OFC proteins (40 μg per lane) from 8 different subjects with major depressive disorder (MDD, 4 in each blot) and 4 different comparison subjects (COMP, 2 in each blot). The blots were probed with an anti-caspase 8 antibody. Bands at the approximate molecular weights of 50, 36 and 14 kDa are singled out. Bands at about 14 and 50 kDa were quantified against β-actin bands. B) Bar graph representing the normalized levels of C8-14 in COMP (n=23) and MDD (n=23) subjects. Quantification of the 50 kDa band is presented in supplemental figure 1. Whiskers represent the standard error of the mean. *p<0.001.

Fig. 2

Western blot immunodetection and quantification of the proteins DIABLO and XIAP in the OFC from COMP and MDD subjects (abbreviations as in figure 1). A) Reactive bands in three different Western blot membranes, each including subjects of the two groups. Subjects in each blot were different from the subjects in the other blots. B) Bar graph of the ratio of the normalized levels of DIABLO to the normalized levels of XIAP in COMP (n=15) and MDD (n=14) subjects. C) and D) Bar graphs representing the mean normalized levels of DIABLO and XIAP, respectively, in the diagnostic groups. Whiskers represent the standard error of the mean. *p=0.023 relative to COMP.

Fig 3

Western blot immunodetection and quantification of PCNA in the OFC the MDD and COMP subjects (abbreviations as in figure 1). A) Two representative Western blots with subjects from the two diagnostic groups; subjects in the left blot were different from the subjects in the right blot. B) Bar graph representing the normalized levels of PCNA in COMP (n=23) and MDD (n=23) subjects.. Whiskers represent the standard error of the mean. *p=0.046.

Fig. 4

Immunohistochemistry and cell counting of Ki-67-immunoreactive cell nuclei in the gray matter of the OFC. A) and B) Low power micrographs of immunoreactive nuclei in the upper cortical layers in COMP and MDD subjects, respectively. C) and D) Higher power micrographs of Ki-67-immunoreactive cell nuclei in the gray matter of the OFC of a COMP subject. E) Bar graph of the average packing density of Ki-67-immunoreactive nuclei in MDD (n=23) and COMP (n=23) subjects. LI=cortical layer I; LII=cortical layer II. Other abbreviations as in figure 1. *p=0.033.

Fig. 5

Scatter graphs illustrating the correlation between levels of the 14 kDa fragment of caspase 8 and age at the time of death in COMP (A) and MDD (B) subjects. There was a significant positive correlation in COMP, but not in MDD subjects. (Abbreviations as in figure 1)

Fig. 6

Scatter graphs illustrating the correlation between levels of PCNA and age at the time of death in COMP subjects (A), MDD subjects (B). There was a significant positive correlation in MDD, but only a non-significant trend for a correlation in the COMP group. (Abbreviations as in figure 1).