Neuronal DAMPs exacerbate neurodegeneration via astrocytic RIPK3 signaling

Abstract

Astrocyte activation is a common feature of neurodegenerative diseases. However, the ways in which dying neurons influence the activity of astrocytes is poorly understood. RIPK3 signaling has recently been described as a key regulator of neuroinflammation, but whether this kinase mediates astrocytic responsiveness to neuronal death has not yet been studied. Here, we used the MPTP model of Parkinson's disease to show that activation of astrocytic RIPK3 drives dopaminergic cell death and axon damage. Transcriptomic profiling revealed that astrocytic RIPK3 promoted gene expression associated with neuroinflammation and movement disorders, and this coincided with significant engagement of DAMP signaling. Using human cell culture systems, we show that factors released from dying neurons signal through RAGE to induce RIPK3-dependent astrocyte activation. These findings highlight a mechanism of neuron-glia crosstalk in which neuronal death perpetuates further neurodegeneration by engaging inflammatory astrocyte activation via RIPK3.

Figure 1.. Astrocytic RIPK3 signaling promotes pathogenesis in the MPTP model of Parkinson’s disease.

(A-B) IHC analysis of tyrosine hydroxylase (TH) staining in the substantia nigra pars compacta (SNpc) in indicated genotypes 7 days following either saline or MPTP treatment (scale bar = 200 μm). (C) IHC analysis of TH⁺ axons with colabeling with the damaged axon marker SMI-32 in the striatum in indicated genotypes 7 days following either saline or MPTP treatment (scale bar = 20 μm). (F) Schematic diagram for the vertical grid test. (G) Behavioral performance in the vertical grid test 6 days after injection with MPTP or saline. *p<0.05, **p < 0.01, ***p < 0.001. See also Figure S1.

Figure 2.. RIPK3 drives inflammatory transcriptional activation but not proliferation in midbrain astrocytes.

(A-B) IHC analysis of GFAP staining in the substantia nigra pars compacta (SNpc) in indicated genotypes 3 days post-MPTP treatment (scale bar = 200 μm). (C-D) Flow cytometric analysis of GLAST+ astrocytes in midbrain homogenates derived from indicated genotypes 3 days post-MPTP treatment. (E-F) qRT-PCR analysis of indicated genes in midbrain homogenates derived from astrocyte-specific Ripk3 knockouts (E) or astrocyte-specific Ripk3 overexpressing (F) mice 3 days post-MPTP treatment. (G-H) Schematic of inducible RIPK3 activation system (G) and stereotactic delivery of dimerization drug into the ventral midbrain (H). (I) qRT-PCR analysis of indicated genes in midbrain homogenates derived from Ripk3-2xFV^fl/fl Aldh1l1-Cre+ mice 24 hours following administration of B/B homodimerizer or vehicle control. *p<0.05, **p < 0.01, ***p < 0.001. See also Figure S2.

Figure 3.. Astrocytic RIPK3 signaling has minimal impact on microgliosis in the MPTP model.

(A-B) IHC analysis of IBA1 staining in the substantia nigra pars compacta (SNpc) in indicated genotypes 3 days post-MPTP treatment (scale bar = 200 μm). (C) Representative flow cytometric plot depicting leukocyte populations in midbrain homogenates derived from indicated genotypes 3 days pos-MPTP treatment. (D) Quantification of absolute numbers of microglia derived from flow cytometric analysis. (E-F) Representative histogram (E) and quantification of geometric mean fluorescence intensity (GMFI) (F) derived from analysis of CD80 expression on microglial populations in (D). (G) Quantification of absolute numbers of CD45^hi leukocytes derived from flow cytometric analysis. **p < 0.01

Figure 4.. Astrocytic RIPK3 activation drives a transcriptomic state associated with inflammation and neurodegeneration in the midbrain.

(A-I) Midbrains were harvested from mice of indicated genotypes 3 days post-treatment with MPTP or saline and subjected to bulk RNA-seq. (A) Principal component analysis demonstrating separation of treatment groups and genotypes in the RNA-seq dataset. (B-D) Volcano plots showing differentially expressed genes derived from indicated comparisons. Data points in red are genes exhibiting upregulated expression, while those in blue exhibit downregulated expression. Genes with an FDR <0.05 were considered significant. (E-F) Bubble plots showing selected significantly enriched disease and function terms (E) or canonical pathways (F) derived from Ingenuity Pathway Analysis comparing Cre- vs. Cre+ MPTP-treated groups. (G-I) Heatmaps showing significantly differentially expressed genes for selected pathways.

Figure 5.. Secreted factors from dying neurons drive RIPK3-dependent astrocyte activation.

(A) Schematic of experimental design for DAMP transfer experiments. Differentiated SH-SY5Y cells were treated with MPP⁺ or saline for 24h and media (NCM) was then transferred to cultures of primary human midbrain astrocytes. Astrocytes were treated with NCM in the presence of GSK 872 or control for 24h prior to qRT-PCR profiling. (B) Heatmap showing expression of astrocyte activation-associated genes in astrocyte cultures treated as in (A). (C-D) qRT-PCR profiling of indicated genes in astrocytes treated for 24h with clarified NCM supernatants (C) or pelleted SH-SY5Y debris (D). (E) Schematic of experimental design for neurotoxicity assay. Astrocytes were treated with NCM as in (A) for 24h. Astrocytes were then washed and media replaced for another 24h. This new astrocyte conditioned medium (ACM) was then transferred to fresh SH-SY5Y cells for cell viability measurement. (F) Cell Titer Glo analysis of SH-SY5Y viability 24h following treatment with ACM derived from indicated conditions. *p<0.05, **p < 0.01, ***p < 0.001. See also Figures S3 and S4.

Figure 6.. DAMP signaling via RAGE drives inflammatory activation in midbrain astrocytes.

(A) Schematic of experimental design for DAMP transfer experiments. Differentiated SH-SY5Y cells were treated with MPP⁺ or saline for 24h and media (NCM) was then transferred to cultures of primary human midbrain astrocytes. Astrocytes were treated with NCM in the presence of FPS-ZM1 or control for 24h prior to qRT-PCR profiling. (B) qRT-PCR profiling of indicated genes in astrocytes treated for 24h with NCM derived from indicated conditions. (C-D) ELISA analysis of HMGB1 protein levels in supernatants of SH-SY5Y cells treated with MPP⁺ (C) or midbrain homogenates from WT mice 3 days post-MPTP treatment (D) n=4–8 replicates per time point in (C). (E-G) qRT-PCR analysis of indicated genes in WT murine midbrain astrocytes (E) or midbrain astrocytes derived from indicated genotypes (F-G) 24h following treatment with recombinant HMGB1 (E-F) or S100b (G). *p<0.05, **p < 0.01, ***p < 0.001.

Figure 7.. Activation of RIPK3 by DAMP signaling drives pathogenic functional changes in midbrain astrocytes.

(A) Schematic of experimental design for neurotoxicity experiments. Differentiated SH-SY5Y cells were treated with MPP⁺ or saline for 24h and media (NCM) was then transferred to cultures of primary human midbrain astrocytes. Astrocytes were treated with NCM in the presence of FPS-ZM1 or control for 24h. Astrocytes were then washed and media replaced for another 24h. This new astrocyte conditioned medium (ACM) was then transferred to fresh SH-SY5Y cells for cell viability measurement. (B) Cell Titer Glo analysis of SH-SY5Y viability 24h following treatment with ACM derived from indicated conditions. (C) Schematic showing treatment of primary human midbrain astrocytes with recombinant DAMPs for 24h prior to transfer of ACM to SH-SY5Y cultures and measurement of cell viability. (D) Cell Titer Glo analysis of SH-SY5Y viability 24h following treatment with ACM derived from indicated conditions. (F) Schematic showing generation and transfer of CSFE-labeled neuronal debris to midbrain astrocytes treated with recombinant DAMPs with or without GSK 872. Astrocytes were cultured in the presence of labelled debris for 24h and then CSFE internalization was measured via flow cytometry. (G-H) Representative histograms (G) and quantification of GMFI (H) of CSFE signal in astrocytes treated as in (F). (I) GMFI of CSFE internalization in astrocytes treated as in (F) but with NCM rather than recombinant DAMPs and FPS-ZM1 rather than GSK 872. **p < 0.01, ***p < 0.001. See also Figure S5.