Potential Alzheimer’s early biomarkers in a transgenic rat model and benefits of diazoxide/dibenzoylmethane co-treatment on spatial memory and AD-pathology

Alzheimer’s disease (AD) is the major form of dementia prevalent in older adults and with a high incidence in females. Identification of early biomarkers is essential for preventive intervention to delay its progression. Furthermore, due to its multifactorial nature, a multi-target approach could be therapeutically beneficial. Our studies included 4- (pre-pathology) and 11-month (mild-pathology) TgF344-AD rats, a transgenic Alzheimer’s model that exhibits age-dependent AD progression. We identified two potential early biomarker genes for AD, early growth response 2 (EGR2) and histone 1H2AA (HIST1H2AA), in the hippocampus of 4-month females. Out of 17,168 genes analyzed by RNA sequencing, expression of these two genes was significantly altered in 4-month TgF344-AD rats compared to wild-type littermates. We also evaluated co-treatment with diazoxide (DZ), a potassium channel activator, and dibenzoylmethane (DIB), which inhibits eIF2α-P activity, on TgF344-AD and wild-type rats. DZ/DIB-treatment mitigated spatial memory deficits and buildup of hippocampal Aβ plaques and tau PHF in 11-month TgF344-AD rats but had no effect on wild-type littermates. To our knowledge, this preclinical study is the first to report EGR2 and HIST1H2AA as potential AD biomarkers in females, and the benefits of DZ/DIB-treatment in AD. Evaluations across multiple AD-related models is warranted to corroborate our findings.

Supplemental Tables 2A, 2B and 2C -RNA sequence analysis for differentially expressed genes in (A) 4-month female transgenic AD non-treated (TGNT) compared to age-matched female wild-type non-treated (WTNT) rats.(B) 4-month female transgenic AD DZ-DIB treated (TGTR) rats compared to age-matched female TGNT rats.(C) 11-month female transgenic AD non-treated (TGNT) compared to age-matched female wild-type non-treated (WTNT) rats.Genes are listed with significant fold change difference in expression.Upregulation in gene expression is represented with an up-arrow and downregulation in gene expression represented with a down-arrow.Fold change, p values, false discovery rates (FDR), means, and standard errors of the mean (SEM) for RPMs (reads per million).All genes with an FDR under 0.05 were included in A and B, but not in C.

Gene symbol
11 month-old wild-type (A and B), 4-month Wild-type (C and D), and 4-month transgenic (E and F) rats stained by IHC for Amyloid Beta across hippocampal regions CA1, CA3, DG, and SB.Scale bar = 1000µm (panels A, C, and E).Scale bar = 200µm (panels B, D and F).Full hippocampus are visible in panels A, C and E, with magnifications of the DG (panels B, D, and F) below the respective hippocampal image.CA = cornu ammonis, DG = dentate gyrus, SB = subiculum.-old wild-type (A, B, and C), 11-month Wild-type (D, E, and F), and 4-month transgenic (G, H, and I) rats stained by IHC for Tau paired helical filaments (PHF1), an early precursor of neurofibrillary tangles across hippocampal regions CA1, CA3, DG, and SB.Scale bar = 1000µm (panels A, D and G).Scale bar = 100µm (panels B, C, E, F, H, and I).Full hippocampus are visible in panels A, D and G, with magnifications of CA3 (panels B, E, and H) and the DG (panels C, F, and I) below the respective hippocampal image.CA = cornu ammonis, DG = dentate gyrus, SB = subiculum.

Table 6 : DZ/DIB effects on NeuN signal across the hippocampus and subregions Overall 4-way ANOVA Results Source Type III Sum of
Pairwise Comparisons (Genotype x Age x Region x Treatment) -Sidak's Post-hoc tests

Table 7 :
A five-way ANOVA assessing DZ/DIB drug treatment, genotype, age, morphology, and brain region effects (top table).Sidak's multiple comparisons tests were used for relevant post-hoc analyses (bottom table).

Table 8 : Tg-AD rats show higher levels of full-length APP at both 4-months and 11-months; DZ/DIB treatment attenuates increases in APP levels.
Values represent the percentage of the pixel ratio for full-length APP over actin loading control.

Table 9 : Tg-AD rats show higher levels of eIF2-alpha at both 4-months and 11-months; DZ/DIB treatment attenuates increases in eIF2-alpha.
Values represent the percentage of the pixel ratio for eIF2-alpha over beta-tubulin loading control.