Erythropoietin-derived peptide treatment reduced neurological deficit and neuropathological changes in a mouse model of tauopathy

Abstract

Background: Prominent activation of microglial immune/inflammatory processes is a characteristic feature of brains of patients with tauopathies including Alzheimer's disease (AD), suggesting that neuroinflammation may be a critical factor in their pathogenesis. Strategies aimed at developing new therapeutics for tauopathies based on anti-inflammation or immunomodulation are likely to be promising avenues of research. We previously developed JM4-a 19'mer cyclic peptide derived from the first loop of human erythropoietin. This peptide possesses beneficial immune modulatory and tissue protective effects while lacking the undesirable side effects of full-length erythropoietin. In this preclinical study, we investigated the effect of chronic JM4 treatment on the PS19 mouse that carries the P301S mutant human tau gene, linked to a form of frontotemporal dementia. This transgenic mouse has been widely used as a model of tauopathies including AD and related dementias.

Methods: Daily subcutaneous treatment of female PS19 mice with JM4 was initiated before disease onset and continued on for the animals' lifespan. The progression of neurological deficit and the lifespan of these mice were assessed. To evaluate the effect of JM4 treatment on cognition of these animals, the PS19 mice underwent Barnes maze test and elevated plus maze test. In addition, neuronal loss, phosphorylated tau aggregation, and microglial activation were assessed using immunohistochemistry of PS19 mouse brain sections.

Results: JM4 treatment of PS19 mice initiated before disease onset reduced neurological deficit, prolonged lifespan, and rescued memory impairment. The beneficial effects of JM4 were accompanied by reductions in neuronal loss, phosphorylated tau aggregation, and microglial activation in the PS19 mouse brain.

Limitations: Use of a single dose of JM4 and female mice only.

Conclusion: JM4 is a potential novel therapeutic agent for the treatment of tauopathies including AD and related dementias.

Keywords: Alzheimer’s disease; Erythropoietin; Microglia; Mouse model; Neuroinflammation; Tauopathy.

Fig. 1

Chronic JM4 treatment reduced neurological deficit and prolonged lifespan in PS19 mice. a In a cohort of mice in which JM4 treatment was initiated at 2 months of age, the PBS sham-treated PS19 mice (open circles) showed an earlier onset of disease than the JM4-treated PS19 mice (filled circles). Disease onset was defined as the age at which mice displayed clasping of at least one hind limb for 30 s when suspended by their tail (clinical score 2) (p = 0.03, log rank (Mantel-Cox) test). b Kaplan-Meier curves show that JM4 treatment (black line) prolongs lifespan of PS19 mice compared to PBS sham-treatment (red line) (p = 0.02, log rank (Mantel-Cox) test). c In another cohort of mice in which JM4 treatment was initiated at one month of age, JM4-treated PS19 mice (filled squares) display less severe neurological deficit compared to PBS sham-treated PS19 mice (open circles) from 5.5 months and on (*p < 0.05, **p < 0.01, two-tailed unpaired t test for each time point)

Fig. 2

Chronic JM4 treatment rescued behavioral abnormalities in PS19 mice. a JM4-treated PS19 mice at 7 months of age took less time to find the target hole than PBS sham-treated group in the Barnes maze test (*p < 0.05, two-tailed unpaired t test). b PBS sham-treated PS19 mice traveled a longer distance to find the target hole in the Barnes maze test (*p < 0.05, two-tailed unpaired t test). c PS19 mice spent more time on the open arm than the wild-type mice suggesting PS19 mice have less anxiety-like behavior when compared to wild-type mice (***p < 0.001, PBS sham-treated PS 19 mice vs. wild-type mice, one-way ANOVA followed by Tukey’s multiple comparison test). Chronic JM4 treatment significantly reduced this behavioral abnormality in PS19 mice (*p < 0.05, PBS sham-treated PS19 mice vs. JM4-treated PS19 mice). d There was no significant difference in total distance traveled among PBS sham-treated PS19 mice, JM4-treated PS19 mice, and wild-type control (ns, not significant; one-way ANOVA followed by Tukey’s multiple comparison test)

Fig. 3

Chronic JM4 treatment blocked neuron loss in PS19 mouse brains. a Images of coronal sections of mouse brains containing the hippocampus at 10 months of age showing NeuN immunoreactivity. × 2 objective. Rectangles mark CA1 region and dashed rectangles mark frontal cortex region selected for quantification. Scale bar 500 μm. b Magnified images of the hippocampus CA1 region showing NeuN immunoreactivity. × 10 objective. Scale bar 100 μm. There is marked decrease in the number of neuronal cell bodies stained by NeuN antibody in PBS sham-treated PS19 mice brain compared to JM4-treated PS19 mice brain. c Quantification of NeuN immunoreactivity in the hippocampus CA1 region. The values were normalized to the area in wild-type control mice brain sections (***p < 0.001, PBS sham-treated PS19 mice vs. wild-type mice, and PBS sham-treated PS19 mice vs. JM4-treated PS19 mice, one-way ANOVA followed by Tukey’s multiple comparison test). d Quantification of the number of NeuN positive neurons per a field of view in the hippocampus CA1 region (***p < 0.001, PBS sham-treated PS19 mice vs. wild-type mice, and *p < 0.05 PBS sham-treated PS19 mice vs. JM4-treated PS19 mice, one-way ANOVA followed by Tukey’s multiple comparison test). e Magnified images of the frontal cortex region showing NeuN immunoreactivity. × 10 objective. Scale bar 100 μm. f Quantification of NeuN immunoreactivity in the frontal cortex. The values were normalized to the area in wild-type control mice brain sections. No statistically significant differences were noted. g Quantification of the number of NeuN positive neurons per a field of view in the frontal cortex. No statistically significant differences were noted

Fig. 4

Chronic JM4 treatment reduced hyperphosphorylated tau aggregates in PS19 mouse brains. a Top row: Images of coronal sections of mouse brains containing the hippocampus at 10 months of age showing AT8 immunoreactivity. Rectangles mark dentate gyrus region and dashed rectangles mark frontal cortex region selected for quantification. × 2 objective. Scale bar 500 μm. Middle row: Magnified images of the hippocampus dentate gyrus region showing AT8 immunoreactivity. × 10 objective. Scale bar 100 μm. Bottom row: Magnified images of the frontal cortex region showing AT8 immunoreactivity. × 10 objective. Scale bar 100 μm. The left panels show abundant strongly positive hyperphosphorylated tau aggregates recognized by the AT8 antibody in PBS sham-treated PS19 mouse brain. The middle panels show dramatically reduced levels of AT8 immunoreactivity in JM4-treated PS19 mouse brain. The right panels show essentially no AT8 immunoreactivity in wild-type mouse brain. b Quantification of AT8 immunoreactivity in the hippocampus dentate gyrus region (*p < 0.05, one-way ANOVA followed by Bonferroni test to compare JM4-treated group and PBS sham-treated group). c Western blots of mouse brain homogenates of PBS sham-treated PS19 mice, JM4-treated PS19 mice, and wild-type (WT) mice showing the immunoreactivity of RAB fraction, RIPA fraction, and urea fraction detected by AT8 antibody. GAPDH served as a loading control. d Quantification of TAU-5 immunoreactivity in the brain slices containing the hippocampus. No statistically significant difference was noted on two-tailed unpaired t test. e Quantitative RT-PCR analysis of human MAPT mRNA levels relative to mouse GAPDH mRNA level in 10 months of age mouse brain lysates. The data was normalized to the mean value of the PBS sham-treated group. No statistically significant difference was noted on two-tailed unpaired t test

Fig. 5

Chronic JM4 treatment reduced microglial activation in PS19 mouse brains. a Top row: Images of coronal sections of mouse brains containing the hippocampus at 10 months of age showing IBA-1 immunoreactivity. Rectangles mark dentate gyrus region. × 2 objective. Scale bar 500 μm. Bottom row: Magnified images of the hippocampus dentate gyrus region showing IBA-1 immunoreactivity. × 10 objective. Scale bar: 50 μm. b Magnified images of mouse brain sections containing the hippocampus dentate gyrus region at 10 months of age showing strong MHC II immunoreactivity displaying numerous reactive microglia with thickened appendages in PBS sham-treated P19 mouse brain (left). In contrast, there is diminished MHC II signal in the JM4-treated PS19 mouse brain (right). Scale bar 50 μm. c Magnified mages of mouse brains sections containing the hippocampus dentate gyrus region at 10 months of age showing strong C3 immunoreactivity in PBS sham-treated P19 mouse brain (left). In contrast, there is diminished C3 immunoreactivity in the JM4-treated PS19 mouse brain (right). Scale bar 100 μm. d Quantification of IBA-1 immunoreactivity in the hippocampus dentate gyrus region (*p < 0.05, one-way ANOVA followed by Bonferroni test to compare JM4-treated group and PBS sham-treated group and to compare wild-type and PBS sham-treated group). e Quantification of MHC II immunoreactivity (*p < 0.05, two-tailed unpaired t test). f Quantification of C3 immunoreactivity (*p < 0.05, two-tailed unpaired t test). g Quantitative RT-PCR analysis of mouse IBA-1, CD68, IL1β, IL6, and TNFα mRNA levels relative to mouse GAPDH mRNA level in 10 months of age mouse brain lysates (*p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant; one-way ANOVA followed by Bonferroni test to compare JM4-treated group and PBS sham-treated group or to compare wild-type and PBS sham-treated group). The data was normalized to the mean value of wild-type