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. 2023 Mar 14;15(1):53.
doi: 10.1186/s13195-023-01202-z.

Attenuation of Alzheimer's brain pathology in 5XFAD mice by PTH1-34, a peptide of parathyroid hormone

Li Chen #  1   2 , Lei Xiong #  1   3 , Lingling Yao  1 , Jinxiu Pan  1   3 , Emily Arzola  1 , Xiaojuan Zhu  2 , Lin Mei  1   3 , Wen-Cheng Xiong  4   5
Affiliations

Attenuation of Alzheimer's brain pathology in 5XFAD mice by PTH1-34, a peptide of parathyroid hormone

Li Chen et al. Alzheimers Res Ther. .

Abstract

Background: Alzheimer's disease (AD) and osteoporosis are two distinct diseases but often occur in the same patient. Their relationship remains poorly understood. Studies using Tg2576 AD animal model demonstrate bone deficits, which precede the brain phenotypes by several months, arguing for the independence of bone deficits on brain degeneration and raising a question if the bone deficits contribute to the AD development. To address this question, we investigated the effects of PTH1-34, a peptide of parathyroid hormone analog and a well-recognized effective anabolic therapy drug for patients with osteoporosis, on 5XFAD animal model.

Methods: 5XFAD mice, an early onset β-amyloid (Aβ)-based AD mouse model, were treated with PTH1-34 intermittently [once daily injection of hPTH1-34 (50 μg/Kg), 5 days/week, starting at 2-month old (MO) for 2-3 month]. Wild type mice (C57BL/6) were used as control. The bone phenotypes were examined by microCT and evaluated by measuring serum bone formation and resorption markers. The AD relevant brain pathology (e.g., Aβ and glial activation) and behaviors were assessed by a combination of immunohistochemical staining analysis, western blots, and behavior tests. Additionally, systemic and brain inflammation were evaluated by serum cytokine array, real-time PCR (qPCR), and RNAscope.

Results: A reduced trabecular, but not cortical, bone mass, accompanied with a decrease in bone formation and an increase in bone resorption, was detected in 5XFAD mice at age of 5/6-month old (MO). Upon PTH1-34 treatments, not only these bone deficits but also Aβ-associated brain pathologies, including Aβ and Aβ deposition levels, dystrophic neurites, glial cell activation, and brain inflammatory cytokines, were all diminished; and the cognitive function was improved. Further studies suggest that PTH1-34 acts on not only osteoblasts in the bone but also astrocytes in the brain, suppressing astrocyte senescence and expression of inflammatory cytokines in 5XFAD mice.

Conclusions: These results suggest that PTH1-34 may act as a senolytic-like drug, reducing systemic and brain inflammation and improving cognitive function, and implicate PTH1-34's therapeutic potential for patients with not only osteoporosis but also AD.

Keywords: Alzheimer’s disease; Astrocytes; Aβ; Neuroinflammation; PTH.

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Conflict of interest statement

A patent of this study, entitled “PTH1-34 as a therapeutic drug for Alzheimer’s disease,” has been filed.

Figures

Fig. 1
Fig. 1
Trabecular bone loss in 5XFAD mice was diminished by PTH1-34 treatments. A Schematic of hPTH1-34 or vehicle (0.9% sodium chloride) intermittent treatment and tissue samples collection in WT/5XFAD female mice. Female WT/5XFAD mice were administered a once-daily injection of hPTH1–34, or veh (0.9% NaCl) via subcutaneous injection, starting at 2 ~ MO old, 5 days per week. Mice were sacrificed at 6 ~ MO old to detect bone phenotypes. B–F μCT analysis of femurs from 6 ~ MO WT and 5XFAD female mice with PTH1-34 or Veh treatment. Representative images are shown in B and quantification analyses of trabecular bone volume over total volume (Tb. BV/TV), trabecular bone number (Tb. N), trabecular bone thickness (Tb. Th), and cortical bone volume over total volume (Cb. BV/TV) by the direct model of μCT analysis are presented in B–F. G Serum osteocalcin levels analyses, measured by ELISA assays, in 6 ~ MO female mice. H Serum PYD levels analyses, measured by ELISA assays, in 6 ~ MO female mice. Three different female mice from each group were examined. The data were presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with two-stage step-up method of Benjamini, Krieger, and Yekutieli multiple comparisons test was used
Fig. 2
Fig. 2
PTH1-34 attenuation of cognition decline and memory loss in 5XFAD female mice. A Illustration of PTH1-34 or vehicle intermittent treatment in WT/5XFAD female mice and behavioral testing schedule. All mice were tested for behavior from the age of 5 ~ MO old and treated with PTH1-34/Veh continuously during the testing procedure. The results shown in this figure were for female mice. B, C Novel object recognition (NOR): Representative tracing images (B), and the quantification of discrimination index ([novel object explore time − old object explore time]/[total explore time]) of NOR (C) were shown. D Y maze: Quantifications of the total arm entries and spontaneous alternation. E–H Morris water maze (MWM): The latencies to reach the hidden platform during the training period (E), the representative tracing images (F), quantification of time spent in the target quadrant (G), and target zone crossovers (H) on the testing day were showed. All quantification data were presented as mean ± SD (n = 6–8 female mice per group). *P < 0.05, **P < 0.01, two-way ANOVA with two-stage step-up method of Benjamini, Krieger, and Yekutieli multiple comparisons test was used
Fig. 3
Fig. 3
PTH1-34 reduction of soluble Aβ level and in-soluble Aβ deposition in 5XFAD brain. A Illustration of PTH1-34 intermittent treatment in 5XFAD mice. Mice were sacrificed at 5 ~ MO old to detect Aβ level and accumulation. B, C ELISA analyses of human Aβ40(B) and Aβ42(C) levels in the soluble fraction of brain homogenates including cortex and hippocampus (200 μg total protein) from female 5XFAD mice with PTH1-34 or Veh treatment (n = 4 mice per group). D, E Representative images of ThioS staining for Aβ plaque depositions analysis in the cortex and hippocampus of 5XFAD-Veh (control) and 5XFAD-PTH1-34 mice. Representative images of female mice were shown in D and representative images of male mice were shown in E. F, G Quantification of plaque density (the amount of plaque deposition in each sub-region) in the cortex of 5XFAD female and male mice. H Quantification of the total plaque density in the cortex of female and male 5XFAD mice. I, J Quantification of plaque density in subregions of hippocampus in 5XFAD female and male mice. K Quantification of the total plaque density in the hippocampus of female and male 5XFAD mice. L Quantification of average plaque size in 5XFAD female and male mice. n = 8 per group for female mice and n = 6 per group for male mice in F–L. Scale bars as indicated in each panel. All data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Sidak’s multiple comparisons test was used
Fig. 4
Fig. 4
PTH1-34 diminishment of GFAP + reactive astrocytes. A Representative overall images of co-immunostaining with ThioS (green) and GFAP (red) of cortex and hippocampus from 6 ~ MO 5XFAD-Veh and 5XFAD-PTH1-34 female mice. B Quantification of relative GFAP fluorescence intensity in each layer of cortex and subregional of hippocampus. C Representative images and high-magnification images in Aβ deposition regions of co-immunostaining with ThioS (green) and GFAP (red) of cortex and hippocampus from 6 ~ MO 5XFAD-Veh and 5XFAD-PTH1-34 female mice. D Quantification of Abeta-associated GFAP fluorescence intensity. The Abeta-associated GFAP fluorescence intensity was defined by the intensity of GFAP-positive astrocytes in a plaque-centered circle within 50 μm in diameter (marked by dashed white circles). n = 8 mice per group. E Representative Western blots using antibodies against IBA1 and GFAP in homogenates of cortex and hippocampus from 6 ~ MO WT and 5XFAD female mice with PTH1-34 or Veh treatment. GAPDH was used as a loading control. F Quantification of relative protein level in E (n = 5 mice per group). All quantification data were presented as mean ± SD. Scale bars were indicated in each panel. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Sidak’s multiple comparisons test was used
Fig. 5
Fig. 5
PTH1-34 diminishment of brain inflammation in 5XFAD mice. A Real-time PCR (RT-PCR) analysis of indicated gene expressions in the cortex of 6 ~ MO WT and 5XFAD female mice with PTH1-34 or Veh treatment. The level of GAPDH was normalized to 1, n = 3 per group. B Total quantification of relative gene expression levels in A. The level of WT group was normalized to 1, n = 10 gene per group. C The summary of altered genes in the 5XFAD-PTH1-34 treatment group compared with the 5XFAD-Veh group in cortex. D RT-PCR analysis of indicated gene expressions in the hippocampus of 6 ~ MO female mice, n = 3 per group. E Total quantification of relative gene expression levels in D. F The summary of altered genes in the 5XFAD-PTH1-34 treatment group compared with the 5XFAD-Veh group in hippocampus. All quantification data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey’s multiple-comparison test was used
Fig. 6
Fig. 6
PTH1-34 reduction of serum inflammatory cytokines. A Representative images of serum L-Series label-multiplex antibody arrays of 6 ~ MO WT and 5XFAD female mice with PTH1-34 or Veh treatment. Proteins with changes are marked by dashed blue circles. B Total quantification analyses of the data in A. WT-Veh group were normalized to 1, n = 36 proteins per group. C Quantification of relative serum protein levels in A. The data showed those proteins that were significantly changed and arranged into different groups according to their characteristics. The data were presented as mean ± SD (n = 4 mice per group), *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey’s multiple-comparison test. D Heat map of serum protein. n = 4, significant difference was set at P < 0.05. E Comparison of the PTH1-34 downregulated cytokines in serum of 5XFAD mice (downregulated cytokines in serum of PTH1-34 treated 5XFAD mice over 5XFAD control mice) to those detected in the cortex or hippocampus of 5XFAD-PTH1-34 mice. F Comparison between the 5XFAD with Tg2576 antibody array of secreted proteins in serum
Fig. 7
Fig. 7
More PTH1-34 association with astrocytes in 5XFAD brain than that of WT control. A Schematic diagram of experimental design. 2.5 ~ MO WT and 5XFAD female mice were administered PTH1-34-Biotin (100 μg/100 μl) or vehicle (phosphate buffer, 100 μl) by tail-intravenous injection to detect PTH1-34 diffusion in vivo. Mice were sacrificed after 30 min for brain isolation and tissue sectioning, and immunofluorescence staining was used to analyze the distribution of PTH1-34-Biotin. B Representative images and high-magnification images of Biotin (green) co-immunostaining with GFAP, IBA1, SLC16A1, and MAP2 (red) respectively from 5XFAD + Veh, 5XFAD + PTH1-34-Biotin and WT + PTH1-34-Biotin female mice. All images were obtained from the cortex regions. Scale bars were indicated in the panel. C Quantification of relative Biotin fluorescence intensity in these three groups (mean ± SD; n = 8 mice per group). **P < 0.01, ***P < 0.001, one-way ANOVA with Kruskal–Wallis multiple-comparison test. D Quantification of PTH1-34-Biotin distribution in 5XFAD brain cells from B. The distribution of PTH1-34 in various cells was shown as a percentage, with a total proportion of 100%, derived from the mean value of data collected from all mice in the 5XFAD + PTH1-34-Biotin group
Fig. 8
Fig. 8
PTH1-34 decrement of senescence-like astrocytes from 5XFAD mice. A RT-PCR analysis of senescence-related gene expression level in the cultured astrocytes with PTH1-34 or Veh treatment from WT and 5XFAD P3 female pups. GAPDH expression level was normalized to 1, n = 3 independent experiments. B SA-β-gal staining of cultured WT and 5XFAD astrocytes with PTH1-34 or Veh treatment. Scale bars as indicated in the panel. C Quantification of relative SA-β-gal + cell intensity in B (mean ± SD; n = 3 independent experiments). D RT-PCR analysis of senescence-related gene expression level in the hippocampus from 6 ~ MO WT or 5XFAD female mice with PTH1-34 or Veh treatment. GAPDH expression level was normalized to 1 (n = 3). E Western blot analysis of indicated protein expression in homogenates of hippocampus of 6 ~ MO WT and 5XFAD female mice with PTH1-34 or Veh treatment. F Quantification of relative protein level in E, n = 4 mice per group. All quantification data were presented as mean ± SD. *P < 0.05, **P < 0.01, one-way ANOVA with Tukey’s multiple-comparison test was used. G Summary of AD brain pathology and cognitive function differences in 5XFAD mice with Veh or PTH1-34 intermittent treatment. H Illustration of the working model
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