Sustained Molecular Pathology Across Episodes and Remission in Major Depressive Disorder

Abstract

Background: Major depressive disorder (MDD) is a debilitating mental illness and a major cause of lost productivity worldwide. MDD patients often suffer from lifelong recurring episodes of increasing severity, reduced therapeutic response, and shorter remission periods, suggesting the presence of a persistent and potentially progressive pathology.

Methods: Subgenual anterior cingulate cortex postmortem samples from four MDD cohorts (single episode, n = 20; single episode in remission, n = 15; recurrent episode, n = 20; and recurrent episode in remission, n = 15), and one control cohort (n = 20) were analyzed by mass spectrometry-based proteomics (n = 3630 proteins) combined with statistical analyses. The data was investigated for trait and state progressive neuropathologies in MDD using both unbiased approaches and tests of a priori hypotheses.

Results: The data provided weak evidence for proteomic differences as a function of state (depressed/remitted) or number of previous episodes. Instead it suggested the presence of persistent MDD effects, regardless of episodes or remitted state, namely on proteomic measures related to presynaptic neurotransmission, synaptic function, cytoskeletal rearrangements, energy metabolism, phospholipid biosynthesis/metabolism, and calcium ion homeostasis. Selected proteins (dihydropyrimidinase-related protein 1, synaptosomal-associated protein 29, glutamate decarboxylase 1, metabotropic glutamate receptor 1, and excitatory amino acid transporter 3) were validated by Western blot analysis. The findings were independent of technical, demographic (sex or age), or other clinical parameters (death by suicide and drug treatment).

Conclusions: Collectively, the results provide evidence for persistent MDD effects across current episodes or remission, in the absence of detectable progressive neuropathology.

Keywords: Bioinformatics; Major depressive disorder; Mass spectrometry; Progressive neuropathologies; Remission; Subgenual cingulate cortex.

Figure 1. Overview of MDD cohorts and control subjects used in the study

Hypothesized progressive model of major depressive disorder (MDD) showing recurring episodes of increasing severity, reduced therapeutic response and shorter remission periods. MDD episodes and treatment phases are indicated by valleys and crests, respectively. A gray arrow depicts the predicted trajectory of MDD pathology across various diseases stages. Abbreviations used: MDD-SE, MDD single episode; MDD-SE R, MDD single episode remission; MDD-Rec, MDD-recurrent and MDD-Rec R, MDD-recurrent remission. Groups are indicated by I–V (adapted from Sibille 2013 (53)).

Figure 2. Summary of identified proteins across MDD episodes and remission phases

A. Venn diagram of the differentially expressed proteins associated with all MDD patients (MDD-ALL, gray shading) and those in current episodes (MDD-E, red shading) or remission phases (MDD-R, blue shading) at p≤0.05 and RIM coefficient effect (log 2 fold change) ≥±0.26 thresholds. Differentially expressed proteins associated only with patients in current episodes (n=13) or remission (n=25) are indicated with black line shading. B. Volcano plot indicating distribution of identified proteins based on their RIM effect Coefficient (log 2 fold change) and −log 10 (p-value). Upregulated and downregulated proteins are highlighted in black (see right and left panels, respectively). Upregulated (DRP-1 and SNAP-29) and downregulated (GAD-67, mGluR1 and EAAT3) proteins selected for Western Blot confirmation (Fig. 3) are indicated in the plot. C. Scatter (gg) plots of selected differentially expressed proteins across in subgroups of all MDD patients. #, * and ** denote trend, statistical significance of p≤0.05 and 0.01, respectively.

Figure 3. Western blot analysis of selected differentially expressed proteins in MDD cohorts

(A) Immunoblotting with DRP-1, SNAP-29, GAD-67, mGluR1 and EAAT3 antibodies showed increased protein expression of DRP-1 (62 kDa) and SNAP-29 (29 kDa), but reduced protein expression of GAD-67, mGluR1, EAAT3 (67, 62 and 57 kDa, respectively) for MDD patients in current episodes compared to control subjects. Anti-beta actin (ACTB, 42 kDa) and anti-beta tubulin (TUB1, 56 kDa) were used to assess equal loading of protein lysates. The two protein standards (labelled as M) used were: All Blue Prestained Protein Standards (#1610373, BioRad Canada) and BLUeye Prestained Protein Ladder (ThermoScientific). (B) Quantitation of the bands using Image Lab software (Biorad) indicated statistical significance, as denoted by * and ** for p≤0.05 and 0.01, respectively.