Proteomic analysis of peripheral leukocytes in Alzheimer's disease patients treated with divalproex sodium

Abstract

The molecular profiling of peripheral tissues, including circulating leukocytes, may hold promise in the discovery of biomarkers for diagnosing and treating neurodegenerative diseases, including Alzheimer's disease (AD). As a proof-of-concept, we performed a proteomics study on peripheral leukocytes from patients with AD both before and during treatment with divalproex sodium. Using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, we identified 10 differentially expressed proteins: two up-regulated proteins, 14-3-3 protein epsilon and peroxiredoxin 2; and eight down-regulated proteins, actin-interacting protein, mitogen activated protein kinase 1, beta actin, annexin A1, glyceraldehyde 3-phosphate dehydrogenase, transforming protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B, and a currently unidentified protein. A subset was validated on both the transcript and protein levels in normal human peripheral blood mononuclear cell cultures treated with valproic acid. These proteins comprise a number of functional classes that may be important to the biology of AD and to the therapeutic action of valproate. These data also suggest the potential of using peripheral leukocytes to monitor pharmaceutical action for neurodegenerative diseases.

Figure 1. Representative silver-stained 2D gel of human peripheral leukocytes

Protein lysates from human peripheral leukocytes were prepared and analyzed via 2DE. Individual spot intensities were normalized to total spot intensity for each gel and differences were expressed as a ratio of 4 week VPA treatment to baseline. Nine spots were significantly differentially expressed following VPA treatment (p<0.05) and were identified via MALDI-TOF MS (see Table 1; Supplementary Table 1). A tenth spot (#450) that was significantly differentially expressed was not identifiable using MS. The differentially expressed proteins are indicated by spot number and arrows. This figure was taken from an original scanned gel that was contrast enhanced, despeckled, and sharpened using Adobe ImageReady CS2 for illustration purposes only. Gels were analyzed using the Progenesis software system as detailed in Materials and Methods with detailed manual checking of the spot finding and matching functions.

Figure 2. Correspondence of relative expression levels of two proteins between 2DE analysis and Western immunoblot analysis following VPA treatment

Examination of annexin a1 (Spot #278) and peroxiredoxin 2 (Spot #460) expression in two study patients using 2DE differential spot analysis and immunoblot analysis demonstrate similar relative levels of expression between the two methodologies. Expression levels are indicated as fold-level changes between the 4-week treatment and baseline samples. 2D, 2D gel analysis; WB, Western blot analysis; #1, subject #1; #2, subject #2.

Figure 3. VPA-responsive targets identified in patients on valproate therapy are also differentially regulated at the transcript level in cultured human PBMCs following VPA treatment

PBMCs were isolated from healthy lab volunteers and cultured in the presence of varying concentrations of VPA for two days in vitro. Eight of the nine targets identified in the 2DE experiments were analyzed using qRT-PCR and all data were normalized to 18S rRNA (n=5 separate experiments for MAPK1; n=6 separate experiments all other targets). All data are expressed as a percent of the 0 mM (control) treatment condition (mean ± SEM). *, significantly different between “0 mM VPA”, p<0.05; inverted brackets, significantly different between the two bracketed treatments, p<0.05.

Figure 4. VPA-responsive targets identified in patients on valproate therapy are also differentially regulated at the protein level in cultured human PBMCs following VPA treatment

PBMCs were isolated from healthy lab volunteers and cultured in the presence of varying concentrations of VPA for two days in vitro. Five of the nine targets identified in the 2DE experiments were analyzed using Western blots (n=3 separate experiments). All data are expressed as a percent of the 0 mM (control) treatment condition (mean ± SEM). *, significantly different between “0 mM VPA”, p<0.05; inverted brackets, significantly different between the two bracketed treatments, p<0.05.