C5aR1 antagonism suppresses inflammatory glial gene expression and alters cellular signaling in an aggressive Alzheimer's model

Abstract

Alzheimer's disease (AD) is the leading cause of dementia in older adults, and the need for effective, sustainable therapeutic targets is imperative. Pharmacologic inhibition of C5aR1 reduces plaque load, gliosis and memory deficits in animal models. However, the cellular basis underlying this neuroprotection and which processes were the consequence of amyloid reduction vs alteration of the response to amyloid were unclear. In the Arctic model, the C5aR1 antagonist PMX205 did not reduce plaque load, but deficits in short-term memory in female mice were prevented. Hippocampal single cell and single nucleus RNA-seq clusters revealed C5aR1 dependent and independent gene expression and cell-cell communication. Microglial clusters containing neurotoxic disease-associated microglial genes were robustly upregulated in Arctic mice and drastically reduced with PMX205 treatment, while genes in microglia clusters that were overrepresented in the Arctic-PMX205 vs Arctic group were associated with synapse organization and transmission and learning. PMX205 treatment also reduced some A-1 astrocyte genes. In spite of changes in transcript levels, overall protein levels of some reactive glial markers were relatively unchanged by C5aR1 antagonism, as were clusters associated with protective responses to injury. C5aR1 inhibition promoted signaling pathways associated with cell growth and repair, such as TGFβ and FGF, in Arctic mice, while suppressing inflammatory pathways including PROS, Pecam1, and EPHA. In conclusion, pharmacologic C5aR1 inhibition prevents cognitive loss, limits microglial polarization to a detrimental inflammatory state and permits neuroprotective responses, as well as leaving protective functions of complement intact, making C5aR1 antagonism an attractive therapeutic strategy for individuals with AD.

Fig.1:. Clustering of transcriptome from single nucleus and single cells in wild type and Arctic mice.

(A) Experimental timeline for PMX205 treatments (B) Seurat cluster identification of combined cells/nuclei transcriptome. (C) Seurat cluster identification of cells derived from single cells (SC) or single nucleus (SN) sequencing. (D) Proportion of cells in each cluster originating from SC or SN transcriptome. (E) Proportion of cells in each cluster by treatment/genotype. (F) Expression of complement pathway components, receptors or regulators by cell type.

Fig.2:. Single cell and single nucleus RNA-Seq clustered by cell type reveals cell-specific complement gene expression.

Microglia or nuclei were isolated from hippocampi, fixed, and sequenced. (A) U-Map of all cell types with all treatment groups included (counts per cell type were: Neurons 37512, Astrocytes 4612, Oligodendrocytes 3478, Microglia 4891, OPCs 1259, Endothelial 898, Mixed 1017, Pericytes 490). (B) Proportion of isolated cell types in the single nucleus RNA-seq by genotype and treatment group. (C) Differential cell type-specific expression of complement pathway components and regulators of the complement system derived from single nucleus RNA-seq.

Fig.3. DAM1 gene expression is suppressed in Arctic-PMX205 hippocampal microglia.

(A) Cells/nuclei identified as microglia were re-clustered separately. (B) Proportion of microglia (SC+SN) clusters in WT-Veh, WT-PMX, Arc-Veh, and Arc-PMX samples. (C) Proportion of cells in each cluster originating from SC or SN transcriptome. (D) Proportion of cells/nuclei in each cluster by treatment/genotype. (E) Relative expression of genes representative of homeostatic microglia, DAM1, DAM2, or other genes of interest within the different microglial clusters. (F) Pie chart demonstrating proportion of SC microglia samples derived from different treatment groups. (G) Relative expression of homeostatic, DAM1, or DAM2 genes in different treatment groups, with SC data alone.

Fig.4:. Reactive Astrocyte gene expression is largely suppressed in Arctic-PMX205 hippocampus.

(A) Cells identified as astrocytes were re-clustered separately. (B) Proportion of astrocyte populations in WT-Veh, WT-PMX, Arc-Veh, and Arc-PMX samples. (C) Proportion of cells in each cluster by treatment/genotype. (D) Relative expression of genes representative of homeostatic astrocytes, pan-reactive, A-1 neurotoxic, or A-2 neuroprotective astrocytes within the different astrocyte clusters. (E) Relative expression of homeostatic, pan-reactive, A-1, or A-2 genes in the different treatment groups.

Fig.5:. Relative information flow of pathways significantly enhanced or suppressed in Arctic mice and altered by PMX205 treatment.

(A) Relative information flow of pathways altered between WT-veh (blue) and Arc-veh (salmon). (B) Relative information flow of pathways altered between Arc-veh (salmon) and Arc-PMX (teal). (C) Cellular senders of pathways altered in WT-veh (left), Arc-veh (middle), and Arc-PMX (right).

Fig.6. PMX205 induces dynamic changes in hippocampal microglia and astrocyte protein markers:

(A) Representative images of hippocampal region CA1 stained for Iba1 (A₁, A₄), CD11b (A₂, A₅) and CD11c (A₃, A₆) in Arc-veh (top panel) and Arc-PMX (bottom panel) 20X magnification, scale bar 100 μm. (B-D) Quantification of staining volume normalized to Arc-veh levels for Iba1 (B), CD11b (C), and CD11c (D). (E) Representative images of dorsal hippocampus stained for S100a6 (E₁, E₄), LCN2 (E₂, E₅), and GFAP (E₃, E₆) in Arc-veh (top panel) and Arc-PMX (bottom panel) 10X magnification, scale bar 200 μm. (F-H) Quantification of percent field area of S100a6 (F), LCN2 (G), and GFAP (H) was done separately in sections of the dorsal, middle, and ventral hippocampus. Data shown as mean ± SEM. * p < 0.05, t-test (B-D) and One-way ANOVA with Tukey’s post hoc (F-H). N = 4–7 mice/genotype/treatment.

Fig.7. PMX205 protects against spatial memory deficits in Arctic females

(A) Overview of Y maze experiment. (B-C) Time spent in the novel arm during the test trial in females (B) and males (C). Data shown as mean ± SEM. * p < 0.05; *** p < 0.001, Two-way ANOVA with Tukey’s post hoc. N = 6–13 mice/sex/genotype/treatment.

Fig.8:. Early treatment with PMX205 suppresses neurofilament light levels in plasma of Arc mice.

(A) Plasma NfL levels in 4.5 mo WT and Arc prior to treatment and in 7 mo WT and Arctic mice after PMX205 treatment for 2, 6, or 12 weeks compared to WT and Arctic age-matched controls. (B) Plasma NfL levels of WT and Arctic mice before and after PMX205 or Vehicle (Veh) treatment from 7.5 to 10 months. Data shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001, Repeated measures Two-way ANOVA with Dunnet’s post hoc. N = 3–6 mice/treatment (A) and Two-way ANOVA with Sidak’s post hoc, N = 10–16 mice/genotype/treatment (B). Males and females were included in both studies with no apparent sex differences evident in any genotype/treatment group.