Thirty peripheral blood samples were collected from 15 patients with AD prior to and following four weeks (25.9±2.7 days) on divalproex sodium therapy. Subjects had mean Mini Mental Status Examination scores of 15.5±1.3 and Clinical Dementia Rating scores of 1.3±0.2 at the time of study enrollment. The mean 4-week sodium divalproex dose was 832±50 mg/day (12.3±0.9 mg/kg), resulting in a mean plasma valproate level of 64.9±9.3 mcg/mL. All data are expressed as mean ± SEM. The majority of patients were concurrently taking cholinesterase inhibitors (11 of 15) and vitamin C (11) as well as other concomitant medications, as is common in these populations [see (Profenno et al., 2005
Peripheral leukocyte protein lysates were prepared subjected to 2DE and computer-aided densitometric analysis of the silver stained gels (). From this leukocyte proteome 978 unique peptide spots were identified on at least one of the 2D gels, while 457 spots were matched on at least two gels. A total of 253 spots were matched between all 30 gels with an additional 78 spots matched on 28 gels and 41 more spots matched on 26 gels. Thus, approximately 81% (372 of 457 spots) of these spots that were matched on at least two gels were also matched in at least 13 of the 15 subjects’ samples. Aggregate comparisons of the 2D gel spot pattern were made between each subject’s baseline sample and 4-week sample and identified 16 spots that were differentially expressed in subjects following VPA treatment. Using separate preparative gels these spots were localized, excised, and subjected to MALDI-TOF MS for protein identification. Nine of these spots were successfully identified based upon an average ~43% amino acid coverage (range: 21.4%–54.5%) and an average of nine peptides matched (range: 5–16) for each protein (; Supplemental Table 1
). A tenth spot (#450, mean fold difference between 4-week and baseline, −1.368±0.297, mean ± SEM, p<0.0035) had a good mass spectrum, but was not identified in any of the databases. The remaining spots were identified as either keratin or hemoglobin contaminants (three of six spots), an actin fragment (one of six spots), or were unidentifiable due to poor spectra (three of six spots). The nine identified proteins belong to a number of functional classes and two of these proteins were significantly up-regulated following VPA treatment: 14-3-3 protein ε (YWHAE) and peroxiredoxin 2 (PRDX2). The remaining seven proteins were significantly down-regulated after four weeks of VPA therapy and included: WD-repeat protein 1/actin-interacting protein 1 (AIP1, WDR1), mitogen activated protein kinase 1/extracellular signal-regulated kinase 2 (MAPK1, ERK2), beta actin (ACTB), annexin A1 (ANXA1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), transforming protein RhoA (RHOA), and acidic leucine-rich nuclear phosphoprotein 32 family member B/acidic protein rich in leucines (ANP32B, APRIL).
Representative silver-stained 2D gel of human peripheral leukocytes
Differentially Expressed 2DE Gel Spots Following 4-Week VPA Treatment
We next performed a limited validation of the VPA-responsive targets both in patient samples and in a series of in vitro experiments. In a demonstration of the sensitivity and specificity of the 2DE gel studies we compared relative expression levels of identified ANXA1 and PRDX2 silver-stained spots with Western immunoblotting data using specific antibodies to these two proteins (). While the relative fold changes are different, there was good correspondence of expression levels in specific subjects using both techniques. Furthermore, in an examination of 11 of the 15 patient samples we found good correspondence and correlation between the 2DE data and Western blot analyses for PRDX2 (7 of 11 show same change; r=0.59, p=0.055) and GAPDH (8 of 11 show same change; r=0.70, p=0.016), but a non-significant correlation in the ANXA1 data (6 of 11 subjects show same change in direction; r=0.31, p=0.364). We did not undertake further proteomic or transcriptomic studies from this patient population because of a limited supply of samples.
Correspondence of relative expression levels of two proteins between 2DE analysis and Western immunoblot analysis following VPA treatment
As an additional set of validation studies we examined alterations in transcript and protein expression in cultured human peripheral blood mononuclear cells (PBMCs; lymphocytes and monocytes) treated with VPA. We hypothesized that VPA-induced alterations identified in the in vivo
studies would be generalizable to these culture experiments. PBMCs were isolated from healthy lab volunteers and cultured for two days in vitro
(2 DIV) in the presence of varying concentrations of VPA (0–5 mM). This range of concentrations comports with other studies in a variety of cell types (Bradbury et al., 2005
; Fraser et al., 1999
; Fuller et al., 2002
; Gurvich et al., 2004
; Marchion et al., 2005
; Shen et al., 2005
) and has been shown to substantially inhibit GSK3β [0.5 mM, see (Chen et al., 1999
; Kim et al., 2005
)] and HDAC activities [0.1–20 mM, see (Gottlicher et al., 2001
; Gurvich et al., 2004
; Phiel et al., 2001
)]. Cell viability as assayed by Trypan blue exclusion was significantly reduced at the highest doses in cultures following 2 DIV VPA treatment (data not shown). Next, we examined the effects of in vitro
treatment on two putative VPA targets, the HDACs and GSK3β. In our culture system we found increased acetylation by Western blot analysis of the histones H3 and H4, which is an indirect measure of HDAC inhibition (data not shown). Likewise, we also observed an increase in phosphorylated GSK3β in cultures treated with VPA compared to controls with no observed increase in total GSK3β levels (data not shown). While we did not detect a dose-response relationship in these measures, these data are consistent with VPA inhibiting both HDACs and GSK3β in cultured human PBMCs.
Finally, we investigated a number of potential VPA targets identified from the 2DE studies both on the transcript and the protein levels in our PBMC cultures following VPA treatment. On the transcript level we found good correlation between five of the eight targets examined: ACTB, ANXA1, MAPK1, PRDX2, and RHOA (). The GAPDH data were more complex and trended toward decreased expression levels between the two middle VPA concentrations and the highest concentration (overall p=0.0709). We found no significant change in APRIL transcript levels in these cultures, while YWHAE transcript levels showed changes in the opposite direction compared to the proteomic data from our initial patient cohort. In separate sets of cultures, there was a significant dose-dependent decrease in ANXA1 and APRIL at the protein level and a trend toward increased PRDX2 protein expression in PBMC cultures treated with VPA, mimicking the data from our in vivo patient study (). Interestingly, ACTB protein expression was relatively stable across the range of VPA concentration, while GAPDH expression levels were in the opposite direction from those observed in the patient study (overall p=0.0624). Thus, three of the five targets surveyed in the in vitro study demonstrate consistent changes on the protein level with those observed in the initial patient study. While we acknowledge differences in these cultures, both in terms of complexity and the origins of these samples (e.g. normal volunteers versus patients with AD), these results suggest commonalities in VPA-dependent biochemical alterations between this culture system and the patient population we studied.
VPA-responsive targets identified in patients on valproate therapy are also differentially regulated at the transcript level in cultured human PBMCs following VPA treatment
VPA-responsive targets identified in patients on valproate therapy are also differentially regulated at the protein level in cultured human PBMCs following VPA treatment