Multi-target neuroprotection by dl-PHPB in APP/PS1 mice: a proteomic analysis

Abstract

Introduction: Dl-PHPB [potassium 2-(1-hydroxypentyl) benzoate] demonstrates robust neuroprotective effects in preclinical models of Alzheimer's disease (AD), significantly ameliorating cognitive deficits and pathological hallmarks. However, the underlying mechanism remains largely unclear. The current study primarily focused on elucidating dl-PHPB's neuroprotective mechanisms and identifying potential targets in preclinical AD models.

Methods: Comparative proteomic analyses were performed on APP/PS1 mice orally administered either dl-PHPB (30 mg/kg) or vehicle daily for 3 months, alongside vehicle-treated wild-type (WT) non-transgenic littermates as controls. Total proteins were separated using two dimensional difference gel electrophoresis, and differentially expressed protein spots were identified via LC-MS/MS.

Results and discussion: Our results revealed 11 altered proteins in the cortex and 10 in the hippocampus between the WT and APP/PS1 groups treated with vehicle. Following dl-PHPB treatment, 12 differentially expressed proteins were identified in the cortex and 9 in the hippocampus of APP/PS1 mice. These proteins are primarily involved in energy metabolism, neuronal structure, protein trafficking, inflammatory and oxidative responses, and amyloid β (Aβ) and Tau processes, among which several proteins were validated as potential therapeutic targets. Notably, the expression levels of cofilin-2 and VDAC1 in APP/PS1 mice were restored to near-normal levels by the treatment with dl-PHPB, memantine, or donepezil, and further clinical validation is required to establish their utility as AD biomarkers for therapeutic efficacy.

Keywords: Alzheimer’s disease; LC-MS/MS; biomarker; dl-PHPB; proteomics.

FIGURE 1

Chemical structure of dl-PHPB.

FIGURE 2

Long-term dl-PHPB treatment ameliorated spatial learning and memory deficits in APP/PS1 mice. The latency (A) and error times (B) were evaluated using the step-down passive avoidance test. Compared with vehicle treatment, dl-PHPB, memantine, and donepezil treatments significantly attenuated learning and memory deficits in APP/PS1 mice. Statistical analysis: Between-group differences were analyzed by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Data were presented as mean ± SEM (n = 12–16). ^## p < 0.01 vs. vehicle-treated WT group; **p < 0.01 vs. vehicle-treated APP/PS1 group.

FIGURE 3

Representative 2D gel images of differentially expressed proteins in the cortex (A,B) and hippocampus (C,D) across groups. A and C, APP/PS1+H₂O vs. WT + H₂O. B and D, APP/PS1+dl-PHPB vs. APP/PS1+H₂O. Approximately 120 µg of protein was loaded per gel to detect protein expression. n = 10. Differentially expressed protein spots (ratio>2, q < 0.05) were labeled in the images. Detailed identification information was shown in Tables 1–4, respectively.

FIGURE 4

Western blot analysis of several proteins with altered abundance in the cortex or hippocampus of WT + H₂O, APP/PS1+H₂O, APP/PS1+dl-PHPB, APP/PS1+memantine, and APP/PS1+donepezil mice. The changes in PDHE1α (A), VDAC1 (B), DRP-2 (C), cofilin-2 (D), peroxiredoxin-6 (E), Pin 1 (F), and cathepsin B (G) detected by Western blotting were consistent with the proteomic data. The Western blot images correspond to: lane 1, WT mice treated with normal drinking water; lane 2, APP/PS1 mice treated with normal drinking water; lane 3, APP/PS1 mice treated with dl-PHPB; lane 4, APP/PS1 mice treated with memantine; and lane 5, APP/PS1 mice treated with donepezil. Quantified results were normalized to β-actin expression. Values were expressed as percentages relative to vehicle-treated WT mice (WT + H₂O) (set to 100%) and presented as the group mean ± SEM. Statistical analysis: Between-group differences were analyzed by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. n = 8–10 mice per group. ^# p < 0.05, ^## p < 0.01 versus the vehicle-treated WT group. *p < 0.05, **p < 0.01 versus the vehicle-treated APP/PS1 group.