Cerebrospinal fluid biomarkers for major depression confirm relevance of associated pathophysiology

Abstract

Individual characteristics of pathophysiology and course of depressive episodes are at present not considered in diagnostics. There are no biological markers available that can assist in categorizing subtypes of depression and detecting molecular variances related to disease-causing mechanisms between depressed patients. Identification of such differences is important to create patient subgroups, which will benefit from medications that specifically target the pathophysiology underlying their clinical condition. To detect characteristic biological markers for major depression, we analyzed the cerebrospinal fluid (CSF) proteome of depressed vs control persons, using two-dimensional polyacrylamide gel electrophoresis and time-of-flight (TOF) mass spectrometry peptide profiling. Proteins of interest were identified by matrix-assisted laser desorption ionization TOF mass spectrometry (MALDI-TOF-MS). Validation of protein markers was performed by immunoblotting. We found 11 proteins and 144 peptide features that differed significantly between CSF from depressed patients and controls. In addition, we detected differences in the phosphorylation pattern of several CSF proteins. A subset of the differentially expressed proteins implicated in brain metabolism or central nervous system disease was validated by immunoblotting. The identified proteins are involved in neuroprotection and neuronal development, sleep regulation, and amyloid plaque deposition in the aging brain. This is one of the first hypothesis-free studies that identify characteristic protein expression differences in CSF of depressed patients. Proteomic approaches represent a powerful tool for the identification of disease markers for subgroups of patients with major depression.

Figure 1

(a) Representative Coomassie-stained two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gel image of cerebrospinal fluid (CSF) from a depressed patient. Differentially regulated proteins and isoforms between disease and control groups are indicated, and were identified as 1. transthyretin precursor; 2. cystatin C; 3. prostaglandin D2 synthase; 4. apolipoprotein E; 5 and 6. pigment epithelium derived factor; 7. vitamin D-binding protein; 8. β-2-glycoprotein; 9. hemopexin; and 10. alpha-1B-glycoprotein. (b) 2D-PAGE comparison of proteins that were further validated by immunoblots.

Figure 2

ProQ Diamond-stained two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gel image of cerebrospinal fluid (CSF) from a control person. Differentially phosphorylated proteins and isoforms between disease and control groups are circled and identified proteins are numbered as 1. apolipoprotein E; 2. heterogeneous nuclear ribonucleoprotein H; 3. heat-shock cognate 71 kDa protein; 4. cAMP-dependent protein kinase catalytic subunit alpha; 5. fructose-bisphosphate aldolase C.

Figure 3

Hierarchical clustering tree for molecular features extracted from the liquid chromatography mass spectrometry (LCMS) analysis of depressed and control cerebrospinal fluid (CSF) samples. The clustering analysis can be used to monitor the reproducibility of the analysis (technical replicates), as well as the similarity of the biological replicates. The clustering analysis was performed for the feature list from t-test of features found in 100% of at least one group.

Figure 4

Log 2 plot of molecular feature abundance from cerebrospinal fluid (CSF) samples of depressed and control individuals. Features that are significantly upregulated or downregulated are shown.

Figure 5

Cystatin C SDS gel western blot (a) and isoelectric focusing (IEF) western blot (b). Total cystatin C protein, as well as the most acidic isoform of the protein show higher expression levels in cerebrospinal fluid (CSF) from control persons ((a)+(b): ^*p<0.01).

Figure 6

(a) Pigment epithelium-derived factor western blot. Two bands are detectable due to different molecular weights of the isoforms. The upper band isoform showed a significant increase in cerebrospinal fluid (CSF) of depressed individuals compared with controls (b), ^*p<0.05.

Figure 7

Prostaglandin D2 synthase SDS gel western blot (a) and isoelectric focusing (IEF) western blot. (b) Statistical analysis of western blot band intensities did not reveal a significant difference between depressed patients and controls ((a), p=0.134), whereas analysis of individual isoforms showed a significant decrease of isoform 2 in depressed patients ((b), ^*p<0.05).

Figure 8

3D principal component analysis (PCA) for feature list from t-test of features found in 100% of at least one group allows patient categorization.