Rescue of Alzheimer's disease phenotype in a mouse model by transplantation of wild-type hematopoietic stem and progenitor cells

Abstract

Alzheimer's disease (AD) is the most prevalent cause of dementia; microglia have been implicated in AD pathogenesis, but their role is still matter of debate. Our study showed that single systemic wild-type (WT) hematopoietic stem and progenitor cell (HSPC) transplantation rescued the AD phenotype in 5xFAD mice and that transplantation may prevent microglia activation. Indeed, complete prevention of memory loss and neurocognitive impairment and decrease of β-amyloid plaques in the hippocampus and cortex were observed in the WT HSPC-transplanted 5xFAD mice compared with untreated 5xFAD mice and with mice transplanted with 5xFAD HSPCs. Neuroinflammation was also significantly reduced. Transcriptomic analysis revealed a significant decrease in gene expression related to "disease-associated microglia" in the cortex and "neurodegeneration-associated endothelial cells" in the hippocampus of the WT HSPC-transplanted 5xFAD mice compared with diseased controls. This work shows that HSPC transplant has the potential to prevent AD-associated complications and represents a promising therapeutic avenue for this disease.

Keywords: Alzheimer's disease; CP: Neuroscience; CP: Stem cell research; hematopoietic stem and and progenitor cells; microgliosis; neuroinflammation.

Figure 1.. Transplantation of WT HSPCs prevents neurocognition impairments in 5xFAD mice

(A) Schematic representation of the experimental design and timeline. (B) Memory recognition test evaluated by discrimination index and preference index in mice at 6 months of age. A representative tracking plot of the 2 day test is shown for 5xFAD/5xFAD HSPC and 5xFAD/WT HSPC mice in the right panels. (C) Elevated plus maze test in the different mouse groups with time expressed in seconds (s) spent in the closed arms and in the open arms and the ratio of time spent in the open arms versus the total time of the test. A representative tracking plot of the test is shown for 5xFAD/5xFAD HSPC and 5xFAD/WT HSPC mice in the right panels. (D) Open field test with the total distance and periphery distance expressed in meters (m) traveled by the different mouse groups, as well as the average speed over a duration of 5 min. A representative tracking plot of the test is shown for 5xFAD/5xFAD HSPC and 5xFAD/WT HSPC mice in the right panels. All data are indicated as mean ± SEM. **p < 0.005 and ***p < 0.0005 determined by one-way ANOVA followed by Tukey’s multiple comparisons, and each dot represent individual samples.

Figure 2.. Transplantation of WT HSPCs led to the decrease of Aβ plaque deposition in the cortex and hippocampus in 5xFAD mice

(A) Representative sagittal sections of the cortex and hippocampus stained against the Aβ plaque marker 6E10 of 5xFAD/5xFAD HSPC and 5xFAD/WT HSPC mice. Scale bars, 500 μm. (B) Quantification of the plaque number, area occupied by the plaques, and percentage of total area occupied in the cortex and hippocampus in 5xFAD, 5xFAD/5xFAD HSPC, and 5xFAD/WT HSPC mice. (C) Triton-X-soluble and guanidine insoluble of Aβ_1–42 (ELISA) in the brain of WT, 5xFAD, 5xFAD/5xFAD HSPC, and 5xFAD/WT HSPC mice. All data are indicated as mean ± SEM. **p < 0.005, ***p < 0.0005, and ****p < 0.0001 determined by one-way ANOVA followed by Bonferroni’s multiple comparisons, and each dot represent individual samples.

Figure 3.. Infiltration of transplanted GFP⁺ WT HSPCs into the brain of 5xFAD mice and differentiation into microglia-like cells

(A) Representative immunohistochemistry image of a sagittal section of the brain from 5xFAD/WT HSPC mice at 4 months post-transplantation showing GFP⁺ cells; insets show GFP⁺ cells in hippocampus and cortex. Scale bars, 500 μm (whole brain) and 100 μm (insets). (B) Representative immunofluorescent image of the brain showing engrafted GFP⁺ HSPC-derived cells (green) and anti-Iba1 (red) and anti-Cd11b (magenta) antibodies. Scale bars, 100 μm. Inset showing HSCP-derived microglia-like cells. Scale bar, 20 μm (inset). (C) Representative 3D reconstitution of immunofluorescence image of brain sections from 5xFAD/5xFAD HSPC (top panel) and 5xFAD/WT HSPC mice (bottom panel) stained with anti-GFP (green), anti-Iba1 (red), and anti-6E10 (magenta) antibodies. Scale bars, 10 μm. The top inset shows inflamed active Iba1⁺ in close proximity to 6E10⁺ plaques of 5xFAD/5xFAD HSPC mice. The bottom inset shows ramified GFP⁺ Iba1⁺ microglia around 6E10⁺ plaques of 5xFAD/WT HSPC mice. Scale bar, 2 μm. (D) Flow cytometry plots of live and GFP⁺ cells in the whole brain of WT, 5xFAD, 5xFAD/5xFAD HSPC, and 5xFAD/WT HSPC mice. Brain cells were labeled with Zombie Aqua for live-/dead-cell populations. (E) Flow cytometry of microglia (CD45⁺ CD11b^high) and of GFP⁺ microglia in 5xFAD/WT HSPC mice. (F) Percentage of Iba1⁺ Cd11b⁺ microglia derived from the GFP⁺ HSPCs quantified in the whole brain, hippocampus, and cortex. Each dot represents individual samples.

Figure 4.. Transplantation of WT HSPCs led to microglia activation and neuroinflammation reduction

(A) Representative images of hippocampus sections immunostained for the microglial marker Iba1; inset shows dentate gyrus region of the hippocampus. Scale bars, 100μm. (B) Quantification of the area occupied by Iba1⁺ cells, as well as Iba1⁺ cell density, in the hippocampus. (C) Representative chromogenic image showing Iba1⁺ microglia (green) and 6E10⁺ plaques (purple). Scale bars, 50 μm. (D) Representative images of Imaris rendering of Iba1⁺ microglia. Sholl analysis quantification of number of Sholl intersections in the cortex and hippocampus brain sections in the different groups of mice. (E) Quantitative PCR quantification of murine cytokine (Ifnγ, Tnfα, and Il6) mRNA expression in whole brains from WT, 5xFAD, 5xFAD/5xFAD HSPC, and 5xFAD/WT HSPC mice. Data are represented as fold change relative to WT and were normalized with GAPDH. (F) Level of IFN-γ, TNF-α, and IL-6 proteins measured by ELISA. Data are means ± SEM. **p < 0.005, ***p < 0.0005, and ****p < 0.0001 determined by one-way ANOVA, and each dot represent individual samples.

Figure 5.. Transplantation of WT HSPCs led to preservation of neurovascular integrity in the hippocampus of 5xFAD mice

(A) Representative immunofluorescence image of brain sections from WT, 5xFAD, 5XFAD/5XFAD HSPC, and 5XFAD/WT HSPC mice stained with anti-Cdh5 (red) antibodies and anti-PECAM1 (magenta). Scale bars, 50 μm. (B) Quantification of total number of Cdh5⁺ vessels, vessel density, area, and vessel diameter in the hippocampus in the different groups of mice. Quantification of vascular PECAM1 coverage (percentage of total PECAM1 staining area co-localizing with Cdh5). Data are means ± SEM. *p < 0.05 and **p < 0.005 determined by one-way ANOVA followed by Tukey’s multiple comparisons. (C) Representative immunofluorescence image of an 5xFAD/WT HSPC hippocampus showing an HSPC-derived microglia-like cell (arrow) in close proximity to a cerebral vessel from mice. Anti-GFP is shown in green, anti-PECAM1 in red, and anti-Cdh5 in magenta. Scale bars, 20 μm. Each dot represent individual samples.