The novel estrogen receptor modulator STX attenuates Amyloid-β neurotoxicity in the 5XFAD mouse model of Alzheimer's disease

Abstract

Based on previous evidence that the non-steroidal estrogen receptor modulator STX mitigates the effects of neurotoxic Amyloid-β (Aβ) in vitro, we have evaluated its neuroprotective benefits in a mouse model of Alzheimer's disease. Cohorts of 5XFAD mice, which begin to accumulate cerebral Aβ at two months of age, were treated with orally-administered STX starting at 6 months of age for two months. After behavioral testing to evaluate cognitive function, biochemical and immunohistochemical assays were used to analyze key markers of mitochondrial function and synaptic integrity. Oral STX treatment attenuated Aβ-associated mitochondrial toxicity and synaptic toxicity in the brain, as previously documented in cultured neurons. STX also moderately improved spatial memory in 5XFAD mice. In addition, STX reduced markers for reactive astrocytosis and microgliosis surrounding amyloid plaques, and also unexpectedly reduced overall levels of cerebral Aβ in the brain. The neuroprotective effects of STX were more robust in females than in males. These results suggest that STX may have therapeutic potential in Alzheimer's Disease.

Keywords: Alzheimer's; Amyloid; Behavior; Estrogen; Microgliosis; Mitochondria; Reactive astrocytosis; Synaptic loss.

Fig. 1.. Summary of experimental design used in this analysis.

1A). Number of mice included for each genotype, sex, and treatment group. 1B). Treatment regimen used for this study. 5XFAD mice and their wildtype (wt) littermates were weaned at 21 days, genotyped at 2 months of age, and then maintained under normal rearing conditions until 6 months old. Separate groups of females and males of both genotypes were then treated every other day (BID) with either vehicle (Veh) or STX for 2 months, administered orally by gavage. Behavioral testing commenced at 7 months, and the animals were euthanized at 8 months of age to harvest brain tissue for subsequent analysis.

Fig. 2.. Oral STX treatment mitigated the reduction in Electron Transport Chain (ETC) protein expression in the hippocampus of 5XFAD mice.

2A): Montage of representative western blots of protein lysates prepared from the hippocampi of each treatment group at the conclusion of behavioral testing (at 8 months of age). Samples were run in replicate and normalized by comparison with identical samples of control cortical lysates included on each gel (not shown). Antibodies against the following ETC proteins were included in the total OXPHOS Rodent Western Blot Antibody Cocktail (Abcam): ATP5A (Complex V ATP synthase F1 subunit alpha; 55 kDa); UQCR2 (Complex III ubiquinol-cytochrome C reductase Core Protein 2; 48 kDa); SDHB (Complex II Succinate dehydrogenase [ubiquinone] iron‑sulfur subunit, 30 kDa); and NDUFB8 (Complex I subunit NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8; 20 kDa). Westerns were subsequently stripped and re-probed with anti-GAPDH (37 kDa) as a loading control (boxed row). 2B). Comparison of OXPHOS protein expression levels in 5XFAD females and wildtype (wt) female littermates that had been treated with vehicle or STX for two months. Values for each treatment group (indicated in relative units) were first calculated as a ratio with GAPDH per lane and then normalized against replicate samples of control mouse cortex (to control for differences in loading and exposure between gels). STX treatment significantly improved the expression of all four OXPHOS proteins in 5XFAD females, maintaining levels that were similar to vehicle-treated wt females, and moderately increased OXPHOS proteins in wt females. 2C). STX also attenuated the reduction in OXPHOS proteins in 5XFAD males. Results from the Kruskal-Wallis tests are reported in each fig. N ≥ 10. Dunn’s pairwise comparisons are denoted with the following: ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

Fig. 3.. Oral STX treatment attenuated the loss of ETC gene expression in the hippocampus of 5XFAD females.

Hippocampal lysates from animals that had been treated for 2 months with vehicle or STX were used to evaluate the relative expression of the following mitochondrial electron transport genes by qRT-PCR: Mt-ATP6 (mitochondrially encoded ATP synthase), Mt-CYB (mitochondrially encoded cytochrome B), Mt-ND1 (mitochondrially encoded NADH dehydrogenase 1), and Mt-CO1 (mitochondrially encoded cytochrome c oxidase 1).

Fig. 4.. Oral STX treatment mitigated the reduction in synaptic protein expression in the hippocampus 5XFAD mice.

4A). Montage of representative western blots of protein lysates prepared from the hippocampi of each treatment group at the conclusion of behavioral testing (at 8 months of age). As in Fig. 3, samples were normalized by comparison with identical control cortical lysates included on each gel (not shown). Western blots were labeled with rabbit antibodies against the postsynaptic protein PSD95 (95 kDa) and the presynaptic protein synaptophysin (38 kDa) that were applied and visualized simultaneously; the blots were then stripped re-probed with anti-GAPDH (boxed row). 4B). Comparison of synaptic protein expression levels in 5XFAD females versus wildtype (wt) female littermates treated with vehicle or STX for two months. STX treatment significantly improved the expression of both synaptophysin and PSD95 in 5XFAD females, and moderately increased their expression in wt littermate females. 4C). STX treatment also mitigated the reduction in synaptophysin expression seen in 5XFAD males and moderately improved PSD95 levels. Results from the Kruskal-Wallis tests are reported in each fig. N ≥ 10. Dunn’s pairwise comparisons are denoted with the following: *p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

Fig. 5.. Effects of oral STX treatment on synaptic gene expression in the hippocampus of 5XFAD females.

Hippocampal lysates from animals treated with vehicle or STX were used to evaluate the relative expression of mRNA encoding the pre-synaptic marker synaptophysin and the post-synaptic marker PSD95. STX treatment mitigated the loss of normal expression levels in 5XFAD females. Results from Kruskal-Wallis tests are reported in each fig. N ≥ 10.

Fig. 6.. Oral STX treatment protected against the decline in hippocampal-dependent memory in 5XFAD female mice.

Animals that had been orally treated with vehicle or STX for 2 months (from 6 to 8 months of age) were analyzed for cognitive responses using the Morris Water Maze (MWM) test and novel object location memory test. 6A). In the MWM test, STX treatment did not significantly alter the performance of wild type or 5XFAD females in either the visible or hidden platform assays. 6B). Vehicle-treated 5XFAD females exhibited moderately reduced performance in the probe test, which was mitigated by STX. 6C). In the object location memory (OLM) test, vehicle-treated 5XFAD females spent significantly less time exploring the object in the novel location (compared with vehicle-treated wild type females), whereas STX treatment prevented this loss in performance. 6D). STX moderately improved spatial memory performance in both wild type and 5XFAD males (not significant. Results from the Kruskal-Wallis tests are reported in each fig. N ≥ 13 for OLM tests; N ≥ 8 for MWM tests. In C, *p < 0.05 (Dunn’s pairwise comparison).

Fig. 7.. Oral STX treatment reduced Aβ plaque burden in 5XFAD female mice.

Brain slices from 5XFAD animals that had been orally treated with vehicle or STX for 2 months (from 6 to 8 months of age) were immunostained with anti-Aβ antibodies. Aβ pathology was expressed as a percentage of the hippocampus and cortex occupied by detectable immunoreactive staining. Scale bar = 1 mm. 7A). Brain section from an 8 month old vehicle-treated 5XFAD female contained abundant amyloid plaques throughout the hippocampus (h) and cortex (c). 7B). Section from an 8 month old STX-treated 5XFAD female contained substantially less Aβ pathology in all brain regions. 7C). lower plaque densities within hippocampal regions of 5XFAD females, with a more moderate reduction in males (consistent with our other assays). 7D) A similar trend was apparent in the cerebral cortex, although this effect did not reach statistical significance. Plaque density (calculated as percentage of brain area occupied by detectable immunoreactive staining). N ≥ 17. Results from Kruskal-Wallis tests with p values are shown in each panel.

Fig. 8.. Oral STX treatment reduced markers of reactive gliosis in both male and female 5XFAD mice.

8A). Brain slices from 5XFAD animals treated with vehicle or STX from 6 to 8 months of age were immunostained with antibodies against GFAP (a marker for reactive astrocytes). 8B). Brain sections from 8 month old mice labeled with the GSL (a marker for activated microglia. Scale bar = 1 mm. 8C). Quantification of reactive astrocytosis (based on anti-GFAP staining) in hippocampal regions of females and males. 8D). Quantification of reactive astrocytosis (based on anti-GFAP staining) in cortical regions of females and males. 8E-F) Quantification of activated microglia (based on biotinylated GSL labeling) in different brain regions of females and males. N ≥ 6. Results from the Kruskal-Wallis tests are reported in each figure. Dunn’s pairwise comparisons are denoted with the following: *p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.