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Published: 27 May 2024

NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

Nature Neuroscience (2024)Cite this article

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Abstract

Oligodendrocyte-lineage cells, including NG2 glia, undergo prominent changes in various neurodegenerative disorders. Here, we identify a neuroprotective role for NG2 glia against prion toxicity. NG2 glia were activated after prion infection in cerebellar organotypic cultured slices (COCS) and in brains of prion-inoculated mice. In both model systems, depletion of NG2 glia exacerbated prion-induced neurodegeneration and accelerated prion pathology. Loss of NG2 glia enhanced the biosynthesis of prostaglandin E2 (PGE2) by microglia, which augmented prion neurotoxicity through binding to the EP4 receptor. Pharmacological or genetic inhibition of PGE2 biosynthesis attenuated prion-induced neurodegeneration in COCS and mice, reduced the enhanced neurodegeneration in NG2-glia-depleted COCS after prion infection, and dampened the acceleration of prion disease in NG2-glia-depleted mice. These data unveil a non-cell-autonomous interaction between NG2 glia and microglia in prion disease and suggest that PGE2 signaling may represent an actionable target against prion diseases.

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Main

Neurodegenerative diseases such as Alzheimer disease (AD), Parkinson’s disease (PD) and prion diseases involve several cell types and various genetic and environmental factors¹. Accumulating evidence suggest that both cell-autonomous and non-cell-autonomous mechanisms contribute to the neurodegenerative process^2,3. Among the common neurodegenerative disorders, prion diseases can be best mimicked with laboratory animal models⁴. Prion-inoculated mice develop a fatal neurodegenerative condition that is almost indistinguishable from its human counterpart at the neuropathological level, providing an invaluable model system for investigating the cellular and molecular mechanisms of chronic neurodegeneration relevant to humans.

Different neurodegenerative diseases have distinct pathogenic triggers and clinical manifestations, yet certain molecular and cellular alterations, such as abnormal protein aggregation and activation of glial cells, are common to all these disorders¹. Large amounts of data support vital roles of microglia and astrocytes in the initiation and progression of neurodegeneration. However, the function of other cell types, including oligodendrocyte-lineage cells, in the pathogenesis of neurodegenerative diseases has been elusive, and investigations on this crucial aspect have largely been ignored by the neurodegenerative disease community. Recent advances in high resolution gene expression analyses have enabled a deeper understanding of this complex cellular landscape. In addition to confirming the alterations of microglia and astrocytes, single-cell and spatial transcriptomics analyses of brain tissues from patients and animal models have unraveled prominent changes of oligodendrocyte-lineage cells in several neurodegenerative disorders^5,6,7, including prion diseases^8,9.

Oligodendrocyte-lineage cells are traditionally considered as supportive cells in the central nervous system (CNS), producing myelin sheath, which insulates nerve fibers and helps speed up transmission of electrical signals along neuronal axons. It is unclear how myelinating cells interact with other cell types of the brain in the context of neurodegenerative process. Here, we investigated the role of oligodendrocyte precursor cells (NG2 glia) in chronic neurodegeneration induced by prion infections. We found that NG2 glia were neuroprotective, and played a crucial role in influencing the microglial pathway that is responsible for the biosynthesis of PGE2, which promotes prion-induced neurodegeneration through binding to the EP4 receptor. These data suggest that NG2 glia have an impact on an intricate cell–cell interaction network in prion diseases, and highlight NG2 glia and PGE2 signaling as potential targets for disease-modifying therapies against neurodegenerative disorders.

Results

NG2 glia activation in prion disease models

Our previous gene expression analyses of prion-inoculated mice¹⁰ and prion-infected COCS¹¹, as well as recent single-cell transcriptomics from other groups^8,9, point to possible transcriptional changes in NG2 glia during prion diseases. However, it is unclear how these cells respond to prion infections at the cellular level. We therefore examined the markers of NG2 glia, including NG2 and platelet-derived growth factor receptor alpha (Pdgfrα), in prion-infected COCS and mouse brains by western blotting and immunofluorescence. We found an increase in NG2 protein levels in both paradigms (Fig. 1a–d). The protein level of Pdgfrα was also upregulated in mouse brains after prion infection, although not as much as that of NG2 (Fig. 1c,d). Similarly, we observed enhanced NG2 immunoreactivity in prion-infected C57BL/6J and Tga20 COCS as well as in prion-inoculated mouse brains (Fig. 1e–h and Extended Data Fig. 1a,b). After prion infection, NG2 glia exhibited enlargement of cell bodies and arborization of cellular processes (Fig. 1g), reminiscent of the NG2 glia phenotypes in mouse models of brain injuries^12,13, suggesting that neuronal damage may play a role in prion-induced NG2 glia activation.

Loss of NG2 glia accelerates prion disease

To facilitate functional investigations of NG2 glia, we previously developed efficient and selective NG2 glia depletion strategies ex vivo and in vivo¹⁴. We used the PDGFR inhibitor CP673451 to deplete NG2 glia from COCS, and combined cell-type-selective diphtheria toxin (DT) receptor (DTR) expression with systemic DT injections for in vivo NG2 glia depletion¹⁴.

To determine the functional relevance of NG2 glia in prion-induced neurodegeneration, we depleted NG2 glia from COCS by continuous (Extended Data Fig. 1c,d) or transient (Extended Data Fig. 2a) CP673451 treatment in the presence or absence of prions, and compared them with dimethylsulfoxide (DMSO)-treated controls. Again, we saw enhanced NG2 immunoreactivity in prion-infected C57BL/6J and Tga20 COCS and—as expected—NG2 glia loss in both uninfected and prion-infected COCS after continuous CP673451 treatment (Fig. 2a,b and Extended Data Fig. 1e,f). Although NG2 glia depletion did not influence neuronal survival in the absence of prions, it enhanced neurodegeneration in prion-exposed C57BL/6J and Tga20 COCS (Fig. 2a,b and Extended Data Fig. 1e,f), suggesting a neuroprotective role of NG2 glia under pathological conditions. A similar enhancement of neurodegeneration was also observed in prion-infected COCS after transient CP673451 treatment (Extended Data Fig. 2b,c). NG2 glia number was largely restored 3 weeks after transient CP673451 treatment in prion-infected COCS (Extended Data Fig. 2b,c), suggesting that short-term destruction of NG2 glia suffices to influence prion neurotoxicity.

**Fig. 2: NG2 glia depletion enhances prion neurotoxicity and accelerates prion disease.**

To investigate the effects of NG2 glia depletion on prion disease in vivo, we crossed Pdgfrα-CreER mice with iDTR mice and generated Pdgfrα-CreER/iDTR (Pdgfrα^iDTR) mice. Pdgfrα-CreER and iDTR littermates were used for controls. We inoculated mice with prions 1 month after tamoxifen treatment, and administered DT daily for 5 days at 12 or 16 weeks postinoculation (wpi) (Extended Data Fig. 3a). According to immunofluorescent examinations, there was decrease of NG2 glia number of ~50% in the brains of Pdgfrα^iDTR mice compared with controls after DT injection¹⁴. Prion disease was accelerated in NG2-glia-depleted Pdgfrα^iDTR mice of both sexes when NG2 glia depletion was induced at 16 wpi (Fig. 2c), and in male mice when NG2 glia depletion was induced at 12 wpi (Extended Data Fig. 3b), possibly attributable to the sex effects on the incubation time of prion disease in animal models^15,16.

Early neurodegeneration in prion diseases is characterized by severe dendritic pathology¹⁷, which can be best visualized in the hippocampus. To investigate whether loss of NG2 glia affects prion-induced neurodegeneration in vivo, we inoculated a second cohort of Pdgfrα^iDTR and control mice after tamoxifen treatment, administered DT at 16 wpi and collected brains 4 weeks thereafter at ~21 wpi. Immunostaining of NG2 confirmed reduction of NG2 glia number in Pdgfrα^iDTR mice compared with controls (Fig. 2d,e). We then examined hippocampal dendritic pathology by Map2 immunofluorescence. As expected, we observed dendritic damages characterized by dendritic varicosities and fragmentation in both NG2-glia-intact and NG2-glia-depleted mice after prion infection (Fig. 2d). However, the density of surviving dendrites was less in Pdgfrα^iDTR mice than in control mice (Fig. 2d,e), suggesting that NG2 glia deficiency enhances prion-induced neurodegeneration in vivo. HE staining indicated similar spongiform changes in the brains of prion-inoculated Pdgfrα^iDTR and control mice (Extended Data Fig. 4).

Unaltered prion replication and overall neuroinflammation

PrP^C is the substrate of prion replication¹⁸, and is essential for prion disease pathogenesis¹⁹. Tissue abundance of PrP^C correlates with the incubation time of prion disease in animal models^20,21. We therefore examined the levels of PrP^C and PrP^Sc (the proteinase K-resistant pathological PrP) in NG2-glia-depleted COCS and mice by western blotting. We found that neither the PrP^C (Extended Data Fig. 5a,b) nor the PrP^Sc levels (Extended Data Fig. 5c,d) noticeably changed in COCS treated with CP673451 compared with controls. In addition, similar levels of PrP^C (Extended Data Fig. 5e,f) and PrP^Sc (Extended Data Fig. 5g,h) were observed in the brains of NG2-glia-depleted Pdgfrα^iDTR mice and control mice. Hence, enhanced neurodegeneration and accelerated prion disease after NG2 glia depletion were not caused by alterations of PrP^C expression and prion replication.

We found previously that NG2 glia were required for maintaining the homeostatic microglia state¹⁴. However, depletion of NG2 glia in cultured brain slices and adult mice did not influence microglia numbers or induce notable changes of tissue-level neuroinflammatory responses under normal conditions¹⁴. To determine whether loss of NG2 glia affects prion-induced microglia activation and neuroinflammation, we compared the density of Cd68⁺ reactive microglia between NG2-glia-depleted and -intact COCS in the presence or absence of prions. We found that reactive microglia were rare in control COCS, but their number increased after prion infection (Extended Data Fig. 5i,j). Nevertheless, the levels of microglia activation were comparable between NG2-glia-depleted and -intact COCS, with or without prion infection (Extended Data Fig. 5i,j). Similarly, we did not observe any changes in Iba1 and Cd68 immunoreactivity in NG2-glia-depleted brains after prion inoculation (Extended Data Fig. 5k,l). Furthermore, the mRNA levels of the proinflammatory factors, tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β) and IL12β, were largely unaltered by NG2 glia depletion in COCS and mouse brains after prion infection (Extended Data Fig. 5m,n). Collectively, these data suggest that loss of NG2 glia does not change prion-induced microglia activation and neuroinflammation at tissue level.

Loss of NG2 glia enhances microglial PGE2 biosynthesis

When evaluating gene expression changes in NG2-glia-depleted COCS with RNA sequencing¹⁴, we noticed possible dysregulation of genes related to the biosynthesis of PGE2, which was reported to be increased in the cerebral spinal fluid (CSF) of prion disease patients^22,23.

To confirm the potential influence of NG2 glia on PGE2, we depleted NG2 glia in COCS with CP673451, and examined the expression levels of prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox2) and prostaglandin E synthase (Ptges), two genes encoding the main enzymes responsible for PGE2 biosynthesis under pathological conditions. Both immunofluorescence and quantitative real-time-PCR (qRT-PCR) results confirmed efficient NG2 glia depletion after CP673451 treatment (Fig. 3a–c). We found that the expression levels of both Cox2 and Ptges were upregulated in NG2-glia-depleted COCS compared with NG2-glia-intact COCS (Fig. 3c). Furthermore, we treated COCS with PDGFAA, the ligand for Pdgfrα, which promotes NG2 glia proliferation²⁴. Immunofluorescent and qRT-PCR results indicated increase of NG2 glia density in PDGFAA-treated COCS compared with controls (Fig. 3d–f). We found that increase of NG2 glia number decreased the expression levels of both Cox2 and Ptges (Fig. 3f). These data indicate that NG2 glia regulate the PGE2 biosynthesis pathway. In line with the above findings, upregulation of both Cox2 and Ptges were also observed in NG2-glia-depleted COCS after prion infection (Fig. 3g). As a comparison, we found that neither the expression level of prostaglandin-endoperoxide synthase 1 (Ptgs1, also known as Cox1) nor the expression level of prostaglandin D2 synthase (Ptgds) changed upon NG2 glia depletion (Extended Data Fig. 6).

**Fig. 3: Loss of NG2 glia upregulates Cox2–Ptges expression.**

To investigate whether loss of NG2 glia induces similar expression changes of Cox2 and Ptges in vivo, we examined the protein levels of Cox2 and Ptges as well as their cellular localization in the brain by immunofluorescence. We found that the immunoreactivities of both Cox2 and Ptges were relatively low under normal condition (Fig. 3h,i); however, prion infection increased their levels (Fig. 3h,i), especially in microglia (Fig. 3h). Furthermore, NG2 glia depletion increased prion-induced upregulation of Cox2 and Ptges (Fig. 3j,k), suggesting that NG2 glia also regulate this pathway in vivo.

NG2 glia regulate microglial PGE2 via several mechanisms

To molecularly characterize PGE2-producing microglia in prion-infected brains, we analyzed a previously generated single-cell RNA sequencing dataset⁸ based on a prion infection model similar to that used in the current study. In normal brains, microglia were largely (~99%) Cox2⁻ or Ptges⁻; however, the fractions of Cox2⁺ and Ptges⁺ microglia were increased after prion infection in both the cerebral cortex (Fig. 4a,b) and hippocampus (Extended Data Fig. 7a,b). Expression analysis identified 943 differentially expressed genes (DEGs) between Cox2⁺ and Cox2⁻ microglia (Supplementary Table 1), and 827 DEGs between Ptges⁺ and Ptges⁻ microglia (Supplementary Table 2), respectively, in the cerebral cortex of prion-infected mice (Fig. 4c). Most (~80–90%) of DEGs identified in Cox2⁺ microglia were also dysregulated in Ptges⁺ microglia (Fig. 4c), suggesting that Cox2⁺ and Ptges⁺ microglia largely overlap. Similar results were also observed in the hippocampus of prion-infected mice (Extended Data Fig. 7c and Supplementary Tables 3 and 4).

**Fig. 4: NG2 glia regulate microglial Cox2–Ptges through several mechanisms.**

The signature genes of homeostatic microglia, including Tmem119, P2ry12 and Cx3cr1, were downregulated in Cox2⁺/Ptges⁺ microglia (Fig. 4d and Extended Data Fig. 7d), whereas disease-associated microglia (DAM)²⁵ signature genes such as Itgax, Cst7 and Apoe as well as MHC-II microglia²⁶ signature genes such as H2⁻D1, H2⁻Aa and Cd74 were upregulated in Cox2⁺/Ptges⁺ microglia (Fig. 4d and Extended Data Fig. 7d). No interferon response microglia²⁶ signature genes were significantly altered in Cox2⁺/Ptges⁺ microglia. In addition, we found that Cox2⁺/Ptges⁺ microglia expressed higher levels of inflammatory genes (Fig. 4d and Extended Data Fig. 7d). These data suggest that Cox2⁺/Ptges⁺ microglia represent either a novel microglia population or a mixture of different microglia populations that are involved in neurodegenerative diseases.

To investigate the mechanisms by which NG2 glia influence the generation of Cox2⁺/Ptges⁺ microglia in prion diseases, we analyzed cell–cell communication between NG2 glia and Cox2⁺ or Cox2⁻ microglia at the single-cell level with CellChat²⁷. We found that the number of interactions between NG2 glia and Cox2⁺ or Cox2⁻ microglia was unchanged (Fig. 4e and Extended Data Fig. 7e). However, the strength of interactions between NG2 glia and Cox2⁺ microglia was weakened compared with those of NG2 glia and Cox2⁻ microglia (Fig. 4e and Extended Data Fig. 7e). The weakened NG2 glia inputs on Cox2⁺ microglia were associated with several mechanistic categories, including secreted signaling such as transforming growth factor beta 2 (Tgfb2), pleiotrophin (Ptn) and midkine (Mdk), ECM-receptor interaction such as tenascin R (Tnr), laminin subunit gamma 1 (Lamc1) and collagen type IX alpha 3 chain (Col9a3), and cell–cell contact such as vascular cell adhesion protein 1 (Vcam1), nectin cell adhesion molecule 3 (Nectin3) and junctional adhesion molecule 3 (Jam3) (Fig. 4f and Extended Data Fig. 7f).

We previously reported that Tgfb signaling disruption in NG2-glia-depleted COCS might be responsible for the loss of homeostatic microglia state¹⁴. These observations not only validate the Cellchat analysis results (Fig. 4f and Extended Data Fig. 7f), but also suggest that some of the weakened NG2 glia to Cox2⁺ microglia signaling might be causally linked to the enhanced cellular state transition from Cox2⁻ microglia into Cox2⁺ microglia in the NG2-glia-depleted mouse brains after prion infection. To test this hypothesis, we established high-purity serum-free microglia cultures from mouse brains through Cd11b immunopanning (Extended Data Fig. 8a,b). After 3 days in culture, we treated primary microglia with several of the NG2-glia-derived factors identified by the cell–cell communication analysis (Fig. 4f and Extended Data Fig. 7f), including Tgfb2, Ptn, Mdk, bone morphogenetic protein 7 (Bmp7) and semaphorin3d (Sema3d) in the presence or absence of prions, and examined Ptges levels with immunofluorescence. We found that Tgfb2, Ptn, Mdk but not Bmp7 and Sema3d suppressed microglial Ptges expression under both experimental conditions (Fig. 4g–i). These data suggest that NG2 glia can directly influence the microglial Cox2–Ptges pathway through several mechanisms.

Inhibition of PGE2 biosynthesis decelerates prion disease

Although previous studies indicated that the level of PGE2 in the CSF was increased in prion disease patients^22,23, it is unclear whether PGE2 plays a causal role in prion-induced neurodegeneration. To investigate this, we treated normal and prion-infected COCS with PGE2, PGD2 or DMSO as control, and examined neurodegeneration by NeuN immunofluorescence. PGE2 treatment had no effect on neuronal survival under normal conditions but enhanced neuronal death in prion-infected COCS (Fig. 5a,b). In contrast, prion-induced neurodegeneration in PGD2-treated COCS was largely unaltered compared with DMSO (Fig. 5a,b). To investigate whether inhibition of PGE2 biosynthesis protects against prion-induced neurodegeneration, we treated normal and prion-infected COCS with C118 or C934 (ref. ²⁸)—two high-selective Ptges inhibitors. Both Ptges inhibitors reduced neurodegeneration in prion-exposed COCS (Fig. 5c,d). These results suggest that PGE2 is a potent enhancer of prion neurotoxicity, and that inhibition of PGE2 biosynthesis is neuroprotective.

**Fig. 5: Cox2–Ptges inhibition diminishes prion neurotoxicity and decelerates prion disease.**

To investigate the effects of PGE2 biosynthesis inhibition on prion disease in vivo, we inoculated Cox2Luc mice, in which the Cox2 gene was replaced with the coding sequence of luciferase, and control mice with prions. We found that Cox2Luc mice survived longer than their littermate controls after prion infection (Fig. 5e), suggesting that elevation of Cox2 and PGE2 production during prion disease are detrimental in vivo. Consistent with this, immunofluorescent staining of Map2 demonstrated ameliorated prion-induced dendritic pathology in the hippocampus of the Cox2Luc mice compared with controls (Fig. 5f,g).

PGE2 inhibition rescues accelerated prion disease

Next, we investigated whether the upregulation of Cox2 and Ptges and the subsequent increase of PGE2 biosynthesis were responsible for the enhanced prion neurotoxicity and accelerated prion disease after NG2 glia depletion. We examined neuronal survival upon inhibition of Ptges activity by C118 and C934 in normal and prion-infected COCS with or without NG2 glia depletion. Consistent with the aforementioned findings (Fig. 2a,b), loss of NG2 glia enhanced prion-induced neurodegeneration (Fig. 6a,b). However, this enhancement was largely blocked by C118 and C934 treatment (Fig. 6a,b). These data suggest that the enhanced neurodegeneration in NG2-glia-depleted COCS is mediated by PGE2.

**Fig. 6: Cox2–Ptges inhibition rescues enhanced neurodegeneration and accelerated prion disease after NG2 glia depletion.**

We then crossed Pdgfrα-CreER mice and iDTR mice with Cox2Luc mice to generate Pdgfrα^iDTR/Cox2Luc mice. Pdgfrα-CreER/Cox2Luc and iDTR/Cox2Luc mice served as controls (Ctrl/Cox2Luc). We infected Pdgfrα^iDTR/Cox2Luc and Ctrl/Cox2Luc mice with prions and compared them with prion-inoculated Pdgfrα^iDTR and genetically matched wild-type (WT) control mice (designated as Pdgfrα^iDTR/WT and Ctrl/WT mice, respectively). All mice were treated with tamoxifen 1 month before prion inoculation and injected with DT at 16 wpi. As expected, loss of NG2 glia accelerated prion disease in Pdgfrα^iDTR/WT mice compared with Ctrl/WT mice (Fig. 6c). However, the accelerated prion disease in NG2-glia-depleted mice was almost completely rescued in Pdgfrα^iDTR/Cox2Luc mice compared with Ctrl/Cox2Luc mice (Fig. 6c), and both Pdgfrα^iDTR/Cox2Luc mice and Ctrl/Cox2Luc mice survived longer than Pdgfrα^iDTR/WT and Ctrl/WT mice (Fig. 6c). Loss of Cox2 also rescued the enhanced prion-induced dendritic pathology in Pdgfrα^iDTR/Cox2Luc mice compared with Pdgfrα^iDTR/WT mice (Fig. 6d,e). These findings confirm that increased PGE2 biosynthesis underlies the enhanced prion neurotoxicity and accelerated prion disease in NG2 glia deficient mice.

PGE2 enhances prion neurotoxicity via the EP4 receptor

To investigate how PGE2 promotes prion-induced neurodegeneration, we first examined the cellular localization of its receptors, including prostaglandin E receptor 1 (Ptger1, also known as EP1), Ptger2 (also known as EP2), Ptger3 (also known as EP3) and Ptger4 (also known as EP4), in the adult mouse brain by immunofluorescence. We found that all four PGE2 receptors were expressed by neurons (Extended Data Fig. 9a). To determine which receptor might be responsible for the enhanced prion neurotoxicity, we expressed them one by one together with GFP in HovS cells chronically infected with prions²⁹ through lentiviral transduction, and examined death of HovS cells in the presence or absence of PGE2. We found that the number of GFP⁺ HovS cells were comparable between PGE2- and DMSO-treated groups when Ptger2, Ptger3 or a control construct was expressed (Fig. 7a,b). However, expression of Ptger1 and Ptger4 reduced the survival of HovS cells in PGE2-treated groups compared with DMSO-treated controls (Fig. 7a,b). Furthermore, we observed an abnormal morphology of surviving HovS cells, characterized by retraction of cellular processes and shrinkage of cell bodies, after PGE2 treatment only when Ptger1 and Ptger4 were expressed (Fig. 7a). Therefore, PGE2 may enhance prion-induced neurodegeneration through activating Ptger1- and Ptger4-mediated signaling.

**Fig. 7: PGE2 enhances prion neurotoxicity mainly through the EP4 receptor (Ptger4).**

To further confirm the possible involvement of Ptger1 and Ptger4 in PGE2-mediated enhancement of neurodegeneration after prion infection, we established primary neuronal cultures using cerebellum from C57BL/6J mice. Immunofluorescence indicated expression of both Ptger1 and Ptger4 in primary neurons (Extended Data Fig. 9b). We infected primary neurons with prion-containing brain homogenates at day 5, and treated them with different concentrations of Ptger1 agonist 17‑phenyl‑trinor‑PGE2 (17-pt-PGE2), Ptger4 agonist L902688 or control solvents 10 days later for 48 h. We found that L902688 enhanced death of prion-infected neurons and damage to neuronal processes in a concentration-dependent manner (Fig. 7c,d). High concentration of L902688 also induced neuronal death in the absence of prions (Extended Data Fig. 10a,b), suggesting activation of Ptger4 signaling is highly toxic to neurons. In contrast, we found that activation of Ptger1 with several concentrations of 17-pt-PGE2 had no effects on neuronal survival in the presence of prions (Extended Data Fig. 10c,d). Similarly, we found that activation of Ptger4 but not Ptger1 enhanced prion-induced neurodegeneration in COCS in a concentration-dependent manner (Fig. 7e,f and Extended Data Fig. 10e,f). These results confirm that PGE2 enhances prion-induced neurodegeneration primarily by activating the Ptger4-mediated signaling.

Discussion

Oligodendrocyte-lineage cells have long been considered as supportive components of the CNS and bystanders in neurogenerative diseases³⁰. However, accumulating evidence suggest that this cell lineage may have additional functions other than producing myelin sheath^31,32,33, and may be actively involved in neurodegenerative processes^7,34. Here, we found that loss of oligodendrocyte precursor cells unleashed a microglial pathway that is responsible for PGE2 biosynthesis, which enhances prion-induced neurotoxicity and accelerates prion disease in animal models (Fig. 8). These data uncover a crucial role of oligodendrocyte precursor cells in a non-cell-autonomous interaction network during prion infection, and support a protective function of these cells during chronic neurodegeneration. Developing approaches that promote the beneficial activities of oligodendrocyte precursor cells may hold potential for new disease-modifying therapies.

**Fig. 8: Diagram summarizing the main findings.**

The study of functions of oligodendrocyte-lineage cells was limited by the lack of efficient and specific ways to manipulate them ex vivo and in vivo. To overcome this difficulty, we previously established experimental approaches to deplete oligodendrocyte precursor cells in cultured brain slices and in the adult mouse brain¹⁴, which formed the basis for the discoveries described here. Adaption of these tools to other models of neurodegeneration, such as AD and PD, would facilitate the investigation of disease-specific roles of oligodendrocyte precursor cells.

We found that depletion of oligodendrocyte precursor cells augmented the PGE2 biosynthesis pathway in cultured brain slices and adult mouse brains after prion infection, revealing a previously unknown regulatory mechanism of PGE2 signaling in the CNS. The role of PGE2 signaling in prion disease and prion-induced neurodegeneration was undefined, but PGE2 levels were found to be increased in the CSF of prion disease patients^22,23. Our findings indicate that PGE2 signaling is a strong enhancer of prion neurotoxicity and a potent driver of prion disease development, which underlies the enhanced neurodegeneration and accelerated prion disease in mice deficient of oligodendrocyte precursor cells. Furthermore, we found that PGE2 enhanced prion-induced neurodegeneration through binding directly to its receptor EP4 on neuronal cells. These data suggest that Ptges inhibitors blocking PGE2 biosynthesis, such as C118 and C934 (ref. ²⁸), which were tested in the current study, as well as EP4 signaling antagonists, may represent good drug candidates for the treatment of prion diseases.

Microglia play important roles in prion disease pathogenesis^35,36,37. Besides neurons, glial cells, especially activated microglia, also express PGE2 receptors³⁸. Manipulation of PGE2 signaling in microglia influences their reaction in models of neurodegeneration^39,40. Therefore, in addition to directly acting on neurons, PGE2 may modulate microglial phenotypes through autocrine signaling, and contribute to neurodegeneration indirectly.

We reported previously that oligodendrocyte precursor cells were required for maintaining the homeostatic state of microglia in the adult mouse brain; however, their loss did not result in microglia activation and neuroinflammation under normal conditions¹⁴, in contrast to NG2 glia depletion in rats⁴¹. Although we now confirm this even in the context of prion infections, we found that deficiency of oligodendrocyte precursor cells dysregulates the PGE2 biosynthesis pathway in microglia. Single-cell RNA sequencing data indicate that PGE2-producing microglia exhibit the molecular features of previously reported DAM²⁵ and of MHC-II microglia²⁶ but not interferon responsive microglia²⁶, and express higher levels of inflammatory genes. PGE2-producing microglia may represent a novel microglia population of diseased brains, or a mixture of several different microglia populations identified previously. Profiling larger numbers of microglial cells in the prion-inoculated brain with single-cell RNA sequencing in future studies may help clarify this crucial question.

Using CellChat²⁷ to analyze NG2 glia-microglia communications in the prion-infected brain at the single-cell level, we identified several weakened NG2-glia-to-microglia signals that might be linked mechanistically to the enhanced generation of PGE2-producing microglia in NG2-glia-depleted brains after prion infection. Of these, we validated Tgfb2, Ptn and Mdk experimentally in primary microglia cultures. Targeting these signals or other CellChat-identified NG2-glia-derived factors may represent promising ways for modulating microglia phenotypes in neurodegenerative diseases.

Methods

Mouse experiments

C57BL/6J mice were obtained from Charles River. Tga20 mice²⁰ were obtained from Laboratory Animal Services Center at University of Zurich. Pdgfrα-CreER mice (stock no. 018280), iDTR mice (stock no. 007900) and Cox2Luc mice (stock no. 030853) were obtained from the Jackson Laboratory. Double transgenic Pdgfrα-CreER/iDTR (Pdgfrα^iDTR) mice were generated by crossing Pdgfrα-CreER mice with iDTR mice. To induce Cre activity and DTR expression in NG2 glia, Pdgfrα^iDTR mice were fed with tamoxifen-containing diet (ENVIGO, cat. no. TD. 55125.I) for 4 weeks as previously reported¹⁴. To generate Pdgfrα^iDTR mice on a Cox2Luc background, Pdgfrα-CreER mice and iDTR mice were first crossed with the Cox2Luc mice, then the resulting Pdgfrα-CreER mice and iDTR mice on the Cox2Luc background were crossed with each other. Littermates of the experimental groups were used as controls. Female mice were used in Cox2Luc related experiments. Both sexes were used in all other experiments. Postnatal day 12 pups were used for preparing brain slice cultures. Postnatal day 5 pups were used for primary microglia and neuronal cultures. For other experiments, the mice were 2 to 6 months old at the beginning of the experiments. Mice were housed under a temperature- and humidity-controlled environment (temperature between 21 °C and 24 °C, and humidity between 45% and 50%) with 12 h light/12 h dark cycle and ad libitum access to water and chow. All animal experiments in the current study were performed according to Swiss federal guidelines, and had been approved by the Animal Experimentation Committee of the Canton of Zurich under permits 040/2015, 139/2016, 243/2018 and 236/2019.

Prion inoculation

Intracerebral prion inoculation was performed as previously described¹¹. Briefly, adult mice were anesthetized with isoflurane and injected in the right hemisphere of the brain with 30 μl 0.01% w/v brain homogenates derived from adult C57BL/6J mice suffering from terminal prion disease. The prion strain used in the current study was the Rocky Mountain Laboratory strain of scrapie, passage 6. Mice inoculated with 30 μl 0.01% w/v noninfectious brain homogenates (NBH) were used as controls. After prion inoculation, the health status of mice was monitored closely, and bodyweights of mice were recorded once per week. For experiments evaluating survival time, mice were euthanized when showing terminal disease symptoms or reached more than 20% loss of bodyweight. For biochemical and immunohistochemical analysis, mice were euthanized at specific timepoints after prion inoculation for tissue collection. To avoid potential influences of tamoxifen on prion disease development, mouse strains (for example, Pdgfrα-CreER, iDTR and Pdgfrα^iDTR mice on WT or Cox2Luc background) undergoing tamoxifen food treatment were inoculated at least 4 weeks after switching back to the normal diet.

Cerebellar organotypic cultured slices

COCS were prepared according to a previously published protocol⁴². Briefly, cerebella from 12-day-old C57BL/6J or Tga20 pups were dissected, embedded in low melting point agarose (Invitrogen, cat. no. 15517-022) and cut into 350-μm thick slices with a vibratome (Leica, cat. no. VT1000S) in cold Gey’s balanced salt solution (GBSS) supplemented with the glutamate receptor antagonist kynurenic acid (1 mM, Sigma, cat. no. K3375) and glucose (33.33 mM, Sigma, cat. no. G8769). Slices with intact morphology were collected and washed briefly three times in GBSS supplemented with kynurenic acid and glucose. Afterwards, brain slices were exposed to either 0.01% w/v prion-containing brain homogenates or 0.01% w/v brain homogenates derived from healthy mice for 1 h at 4 °C and washed five times in GBSS supplemented with kynurenic acid and glucose. Six to eight slices were put on a Millicell-CM Biopore PTFE membrane insert (Millipore, cat. no. PICM 03050) and kept on slice culture medium containing 50% minimum essential medium (MEM), 25% basal medium eagle, 25% inactivated horse serum, 0.65% w/v glucose, 1% GlutaMax (ThermoFisher Scientific, cat. no. 35050061) and 1% penicillin/streptomycin (ThermoFisher Scientific, cat. no. 10378016) at 37 °C in a tissue culture incubator. Culture medium was changed three times per week.

Treatment of brain slice cultures

Prostaglandin E2 (PGE2, sc-201225), prostaglandin D2 (PGD2, sc-201221) and EP1 receptor agonist 17-phenyl-trinor-prostaglandin E2 (17-pt-PGE2, sc-201255) were purchased from Santa Cruz Biotechnology. EP4 receptor agonist L902688 (cat. no. HY-119163) was purchased from MedChemExpress. Prostaglandin E synthase (Ptges) inhibitors C118 and C934 (ref. ²⁸) were obtained from P.-J.J. at Karolinska Institutet. All the above compounds except L902688, which was dissolved in methanol, were dissolved in DMSO (Sigma, cat. no. 472301) and stored at −80 °C in aliquots. Treatments of COCS with PGE2 (1 μM), PGD2 (1 μM), C118 (2 μM) and C934 (2 μM), 17-pt-PGE2 (1 μM and 5 μM) and L902688 (1 μM and 5 μM) were started from 14 days after the cultures were established and lasted to the end of experiments. COCS treated with same amounts of DMSO (or methanol for L902688) were used as controls. Recombinant human PDGFAA (110-13A) was purchased from Peprotech, dissolved in Opti-MEM, and stored at −20 °C in aliquots. Treatment of COCS with PDGFAA (40 ng ml⁻¹) was performed following the same protocol as with the compounds, except that COCS treated with Opti-MEM were used as controls.

NG2 glia depletion

NG2 glia depletion in COCS and in vivo was performed as previously described¹⁴. Briefly, to achieve ex vivo NG2 glia depletion, CP673451 (MedChemExpress, cat. no. HY-12050) was supplemented in slice culture medium with the concentration of 1 μM. Treatments were started from 14 days after the cultures were established and lasted to the end of experiments (for long-term continuous depletion) or 10 days (for short-term transient depletion). Fresh CP673451 was added every time the culture medium was changed. COCS treated with same amounts of DMSO were used as controls. To achieve in vivo NG2 glia depletion, tamoxifen-treated Pdgfrα^iDTR mice were injected intraperitoneally with DT (Sigma, cat. no. D0564) diluted in saline for 5 consecutive days (two injections per day with an 8-h interval, 200 ng per injection). DT-injected tamoxifen-treated Pdgfrα-CreER mice and iDTR mice were pooled together and used as controls.

Lentiviral production

Lenti-vectors used for expressing human PGE2 receptors EP1 (pLenti-PTGER1-mGFP-P2A-Puro, cat. no. RC208597L4), EP2 (pLenti-PTGER2-mGFP-P2A-Puro, cat. no. RC210883L4), EP3 (pLenti-PTGER3-mGFP-P2A-Puro, cat. no. RC220173L4) and EP4 (pLenti-PTGER4-mGFP-P2A-Puro, cat. no. RC210932L4) were purchased from OriGene Technologies. A control vector (pLenti-HygR-mGFP-P2A-Puro) was produced in house by replacing the EP2 sequence in the pLenti-PTGER2-mGFP-P2A-Puro plasmid with the sequence of the Hygromycin resistant gene (HygR). All plasmids were verified by Sanger sequencing before lentiviral production in HEK293T cells (ATCC, cat. no. CRL-3216) maintained in Opti-MEM supplemented with 10% fetal bovine serum (FBS). Briefly, HEK293T cells were seeded in 10-cm cell culture dishes and transfected at ~80% confluency with a packaging plasmid mixture (transgene plasmid; VSVG plasmid; PAX2 plasmid) using FuGENE HD transfection reagent (Promega, cat. no. E2311). At 24 h after transfection, the culture medium was changed to remove the transfection reagent. After another 48 h, the culture medium containing lentivirus was collected, centrifuged at 1,500g for 10 min and filtered through 0.45-micron Whatman filter units (GE Healthcare, cat. no. 10462100). High-titer lentivirus was produced by concentrating the filtered supernatant with Lenti-X concentrator (Takara, cat. no. 631231) and stored at −80 °C in aliquots.

Primary microglia culture

Serum-free mouse microglia cultures were established using the immunopanning method according to previously published protocols with small modifications. Briefly, brain tissues (olfactory bulb, cerebellum and subcortical regions removed) from 5-day-old C57BL/6J mice were dissected in cold Hanks’ Balanced Salt solution (HBSS, ThermoFisher Scientific, cat. no. 14175095) under a stereomicroscope. After removing meninges, brain tissues were minced and washed three times with HBSS. Afterwards, the minced brain tissues were digested in papain solution (Worthington Biochemical Corporation, cat. no. LK003178) with DNase1 (Worthington Biochemical Corporation, cat. no. LK003172) for 20 min at 37 °C and pipetted into single-cell suspension. The digestion solution was removed by centrifugation (1,000g for 5 min), and cell pellets were resuspended in Opti-MEM and filtered through a 70-μm cell strainer for immunopanning at room temperature. Each Petri dish used for immunopanning was coated with 30 μl goat anti-rat IgG (Jackson ImmunoResearch, cat. no. 112-005-167) diluted in 10 ml of sterile 50 mM Tris-HCl (pH 9.5) overnight at 4 °C. After washing five times in PBS, the dishes were further coated with 30 μl rat anti-mouse CD11b antibody (ThermoFisher Scientific, cat. no. 14-0112-82) diluted in 10 ml of the same buffer overnight at 4 °C and washed five times in PBS before use. After immunopanning for 45 min (gently swirl the dishes every 15 min), the floating cells in the suspension were removed from the dishes, and cells attached to the dishes were washed five times with PBS and digested with trypsin-EDTA (ThermoFisher Scientific, cat. no. 25200056) for 10 min at 37 °C. Afterwards, the cells were collected by centrifugation, resuspended and seeded in ibiTreat eight-well slide chambers (Ibidi, cat. no. 80806) in Opti-MEM-based medium containing 1% Sato Mix, 1% GlutaMax, 1% penicillin/streptomycin, 2 ng ml⁻¹ human Tgfb2 (ThermoFisher Scientific, cat. no. 100-35B), 10 ng ml⁻¹ murine Csf1 (Biolegend, cat. no. 576404) and 1.5 mg ml⁻¹ cholesterol (Merck, cat. no. C3045). After 3 days in culture, primary microglia were treated with recombinant human Tgfb2 (100 ng ml⁻¹), human Pleiotrophin (100 ng ml⁻¹, ThermoFisher Scientific, 450-15), human Midkine (100 ng ml⁻¹, ThermoFisher Scientific, 450-16), human Bmp7 (100 ng ml⁻¹, ThermoFisher Scientific, cat. no. 120-03 P) or human Semaphorin3d (400 ng ml⁻¹, Novus Biologicals, cat. no. H00223117-P01) for 72 h in the presence or absence of 0.01% w/v prion-containing brain homogenates, followed by paraformaldehyde (PFA) fixation and immunofluorescence.

Primary neuronal culture

Primary neuronal cultures were established using cerebellar tissues from 5-day-old C57BL/6J mice according to previously published protocols. Briefly, after removing meninges, cerebellar tissues were minced, washed three times with HBSS, digested in papain solution with DNase1 for 15 min at 37 °C and pipetted into single-cell suspension. After centrifugation, cell pellets were resuspended in Opti-MEM and incubated in uncoated tissue culture dishes for 10 min to remove astrocytes and microglia. Afterwards, the cells were recollected, centrifuged, resuspended in neuronal culture medium containing Neurobasal medium (ThermoFisher Scientific, cat. no. 21103049), 1% N2 (ThermoFisher Scientific, cat. no. 17502048), 1% B27 (ThermoFisher Scientific, cat. no. 17504044), 1% MEM nonessential amino acids (MEM-NEAA, ThermoFisher Scientific, cat. no. 11140050), 1% GlutaMax and 1% penicillin/streptomycin, and seeded in 48-well plates coated with poly-d-lysine (ThermoFisher Scientific, cat. no. A3890401). After 1 day, the cultures were treated with 2 μM AraC (British Pharmacopoeia, cat. no. 383) for 24 h to further remove glial cells. For prion infection, 0.01% w/v prion-containing brain homogenates were added into the cultures at day 5. After 10 days of prion infection, the cultures were treated with different concentrations of 17-pt-PGE2 and L902688 for 48 h or controls (DMSO for 17-pt-PGE2 and methanol for L902688), followed by fixation and immunofluorescence.

HovS cell culture

HovS cells²⁹—a subclone of the human SH-SY5Y cell line—where the human PRNP gene was replaced with the ovine PRNP VRQ allele, were maintained in Opti-MEM medium supplemented with 10% FBS, 1% MEM-NEAA, 1% GlutaMax, 1% penicillin/streptomycin and 400 μg ml⁻¹ Geneticin (ThermoFisher Scientific, cat. no. 10131027). To establish a cellular model of chronic prion infection, HovS cells were exposed to brain homogenates derived from PG127-prion-infected tg338 mice and passaged for at least ten times before being used for experiments. To investigate the effects of PGE2 and the involved PGE2 receptors in prion-induced cell toxicity, PG127-HovS cells were seeded into 96-well plates. At 24 h after seeding, cells were transduced with either control lentivirus or lentivirus harboring one of the four EP receptor transgenes. After 48 h, the lentivirus-containing culture medium was removed, and fresh culture medium supplemented with DMSO or PGE2 (10 μM) was added in the wells and incubated for another 48 h. Finally, GFP⁺ cells (cells infected by lentivirus) were imaged with a Nikon Eclipse Ti2-E fluorescent microscope and quantified with ImageJ.

Western blotting

Western blotting was performed as previously described⁴³. Briefly, cultured brain slices or collected brain tissues were lysed with a bead-mill homogenizer in RIPA buffer supplemented with proteinase inhibitor cocktail cOmplete (Merck, cat. no. 11697498001) and phosphatase inhibitor cocktail PhosSTOP (MERCK, cat. no. 4906845001). After centrifuging, protein concentrations in the supernatant were quantified with the bicinchoninic acid method. Protein samples were then mixed with western blotting loading buffer and heated for 5 min at 95 °C before being loaded onto gels. For western blotting aimed to detect prions, samples were first digested with 10 μg ml⁻¹ (for COCS) or 20 μg ml⁻¹ (for brain tissues) proteinase K for 30 min at 37 °C, then mixed with western blotting loading buffer and boiled for 5 min at 95 °C. The following primary antibodies were used: mouse monoclonal antibody against actin (1:10,000, Merck Millipore, cat. no. MAB1501R); mouse monoclonal antibody against PrP (POM1, 1:5,000, homemade); rabbit polyclonal antibody against NG2 (1:500, MERCK, cat. no. AB5320); rabbit polyclonal antibody against PDGFRα (1:500, Santa Cruz, cat. no. sc-338); rabbit monoclonal antibody against NeuN (1:2,000, Abcam, ab177487). Depending on the primary antibodies used, suitable HRP-conjugated goat anti-mouse IgG antibody (1:10,000, Jackson ImmunoResearch, cat. no. 115-035-003) or HRP-conjugated goat anti-rabbit IgG antibody (1:10,000, Jackson ImmunoResearch, cat. no. 111-035-003) were chosen. Membranes were developed with Crescendo Western HRP Substrate (Merck, cat. no. WBLUR0500), visualized and digitized with ImageQuant (cat. no. LAS-4000; Fujifilm). Optical densities of bands were analyzed using ImageJ.

Immunofluorescence

Immunofluorescent staining of cultured brain slices, primary cells and cryosections was performed according to procedures published previously¹⁴. For cultured brain slices, after removing culture medium, brain slices were washed with PBS and fixed in 4% PFA for 30 min at room temperature. After several washes to remove residual PFA, brain slices were then permeabilized with PBST (PBS + 0.1% Triton X-100) for 2 h at room temperature and blocked with 5% goat serum (GS) overnight at 4 °C before adding primary antibodies. For primary cells, after removing culture medium, cells were washed with PBS and fixed in 4% PFA for 30 min at room temperature. After several washes to remove residual PFA, cells were blocked with 5% GS for 2 h at room temperature before adding primary antibodies. For cryosections, mice were perfused transcardially with 20 ml PBS and 20 ml 4% PFA. The dissected brains were then postfixed with 4% PFA for 4 h or overnight in the fridge. After removing PFA and brief washing with PBS, brains were transferred into 30% sucrose solution for dehydration, and kept at 4 °C. After dehydration was complete, brains were cut into 25-μm thick cryosections. To perform immunostaining, cryosections were permeabilized in PBST for 2 h and blocked with 5% GS for 2 h at room temperature before incubation in primary antibodies. The following primary antibodies were used: rabbit polyclonal antibody against NG2 (1:500, a gift from W. Stallcup), rabbit monoclonal antibody against NeuN (1:1,000, Abcam, cat. no. ab177487), rabbit polyclonal antibody against Iba1 (1:500, Wako, cat. no. 019-19741), rat monoclonal antibody against Cd68 (1:200, BioRad, cat. no. MCA1957), rabbit polyclonal antibody against Map2 (1:200, Biolegend, cat. no. 840601), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, cat. no. MN1010), chicken polyclonal antibody against NeuN (1:1,000, Merck, cat. no. ABN91), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, cat. no. sc-166475), mouse monoclonal antibody against Ptges (1:200, Santa Cruz, cat. no. sc-365844), rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, cat. no. BS-6316R), rabbit monoclonal antibody against EP2 (1:200, Abcam, cat. no. ab167171), rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, cat. no. 101760) and mouse monoclonal antibody against EP4 (1:200, ProteinTech, cat. no. 66921-1-Ig). Cultured brain slices were incubated in primary antibody at 4 °C for 3 days. Cryosections and primary cells were incubated in primary antibody at 4 °C overnight. After several washes in PBST, cultured brain slices were incubated in suitable secondary antibodies Alexa488-conjugated goat anti-mouse IgG antibody (1:3,000, ThermoFisher Scientific, cat. no. A32723), Alexa594-conjugated goat anti-rabbit IgG antibody (1:3,000, ThermoFisher Scientific, cat. no. A32740) or Alexa647-conjugated goat anti-chicken IgG antibody (1:3,000, ThermoFisher Scientific, cat. no. A32933) overnight at 4 °C, and cryosections and primary cells were incubated in suitable secondary antibodies Alexa488-conjugated goat anti-mouse IgG antibody (1:3,000, ThermoFisher Scientific, cat. no. A32723), Alexa594-conjugated goat anti-rabbit IgG antibody (1:3,000, ThermoFisher Scientific, cat. no. A32740) or Alexa647-conjugated goat anti-chicken IgG antibody (1:3,000, ThermoFisher Scientific, cat. no. A32933) for 2 h at room temperature. Immunofluorescent images were captured using a FLUOVIEW FV10i confocal microscope (Olympus Life Science) or Nikon Eclipse Ti2-E fluorescent microscope, and were quantified using ImageJ.

Quantitative real-time PCR

To perform qRT-PCR analysis, total RNA from cultured brain slices or collected brain tissues was extracted using TRIzol reagent (ThermoFisher Scientific, cat. no. 15596026). QuantiTect Reverse Transcription kit (Qiagen, cat. no. 205311) was used to synthesize cDNA from the extracted RNA samples. qRT-PCR was performed on a ViiA7 Real-Time PCR system (Applied Biosystems) using the SYBR Green PCR Master Mix (ThermalFisher Scientific, cat. no. 4309155). We used the following primers synthesized by Microsynth AG for qRT-PCR analysis: mouse actin: sense, 5′-AGATCAAGATCATTGCTCCTCCT-3′, antisense, 5′-ACGCAGCTCAGTAACAGTCC-3′. Mouse NG2: sense, 5′-ACCCAGGCTGAGGTAAATGC-3′, antisense, 5′-ACAGGCAGCATCGAAAGACA-3′. Mouse Pdgfrα: sense, 5′-ATTAAGCCGGTCCCAACCTG-3′, antisense, 5′-AATGGGACCTGACTTGGTGC-3′. Mouse TNFα: sense, 5′-ACGTCGTAGCAAACCACCAA-3′, antisense, 5′-ATAGCAAATCGGCTGACGGT-3′. Mouse IL1β: sense, 5′-TGCAGCTGGAGAGTGTGGATCCC-3′, antisense, 5′-TGTGCTCTGCTTGTGAGGTGCTG-3′. Mouse IL12β: sense, 5′-TGGTTTGCCATCGTTTTGCTG-3′, antisense, 5′-ACAGGTGAGGTTCACTGTTTCT-3′. Mouse Cox1: sense, 5′-TCCATCCACTCCCAGAGTCAT-3′, antisense, 5′-ACAACAGGGATTGACTGGTGA-3′. Mouse Cox2: sense, 5′-GGGCCATGGAGTGGACTTAAA-3′, antisense, 5′-ACTCTGTTGTGCTCCCGAAG-3′. Mouse Ptges: sense, 5′-TCTCACTCTCAGTCCCGGTG-3′, antisense, 5′-GGGGTTGGCAAAAGCCTTC-3′. Mouse Ptgds: sense, 5′-GCTCCTTCTGCCCAGTTTTCC-3′, antisense, 5′-CCCCAGGAACTTGTCTTGTTGA-3′.

Single-cell RNA sequencing analysis

Cerebral cortex and hippocampal single-cell RNA sequencing dataset from prion-infected or control mice⁸ were obtained from the single-cell portal at the Broad Institute through the following link: https://singlecell.broadinstitute.org/single_cell/study/SCP1962. After loading the raw single-cell RNA sequencing data into R, doublets and ambient RNA were removed using scrublet⁴⁴ and decontX⁴⁵, respectively. Cells with nFeature_RNA less than 1,000 or more than 7,000 or mitochondrial RNA percentage more than 5% were filtered out using Seurat⁴⁶. After normalizing the data and regressing out the mitochondrial genes and cell cycle effects with Seurat, data from different animals were integrated using Harmony⁴⁷. After data integration, cells were clustered using uniform manifold approximation and projection (UMAP), and the resulting clusters were annotated based on known cell-type markers. To molecularly characterize Cox2⁺ and Ptges⁺ microglia populations in the prion-infected mouse brain, microglia clusters were selected for further analysis, and differentially expressed genes between the Cox2⁺ and Cox2⁻ or Ptges⁺ and Ptges⁻ microglia were identified using the FindMarkers function with Wilcoxon Rank Sum test in Seurat based on the cutoff false discovery rate (FDR) < 0.05 and logfc.threshold > 0.25. Changes in cell–cell interactions between NG2 glia and Cox2⁺ or Cox2⁻ microglia at the single-cell level in the prion-infected mouse brain were analyzed using the CellChat package²⁷.

Statistical analyses

Unpaired two-tailed t-test was used for comparing data from two groups. For comparisons between more than two groups, Benjamini–Hochberg FDR was used for adjusting P values. Data distribution was assumed to be normal, but this was not formally tested. To compare the incubation time of prion-inoculated mice, survival curves were estimated using the Kaplan–Meier method and compared statistically with the log-rank test. Sample sizes used in the current study were based on relevant literature, and were not predetermined by statistical methods. In all experiments, mice, brain slices, cell cultures or other samples were assigned randomly to experimental conditions and timepoints. The investigators were blinded for animal grouping, treatments and tissue collection for in vivo experiments, but were not blinded for some of the ex vivo experiments due to the fact that blinding is practically infeasible or temporary shortages of manpower. Statistical analysis and data visualization were done using GraphPad Prism v.9 or R. No datapoints were excluded from analysis unless otherwise mentioned. P < 0.05 was considered statistically significant. In all figures and Extended Data figures: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Single-cell RNA sequencing data were obtained from the single-cell portal at the Broad Institute through the link: https://singlecell.broadinstitute.org/single_cell/study/SCP1962. Other data associated with the findings of the current study are provided as figures, Extended data figures or Supplementary information. Source data are provided with this paper.

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Acknowledgements

We thank W. Stallcup for providing the polyclonal antibodies against NG2, P. Schwarz, J. Domange, M. Delic, M. Svercelova and M. Mihailova for excellent assistance. This work was supported by a University of Zurich Forschungskredit postdoctoral grant (FK Liu 19-042 to Y.L.), an Advanced Grant of the European Research Council (670958 to A.A.), the Swiss National Science Foundation (179040 to A.A.) and the Nomis Foundation (A.A.).

Funding

Open access funding provided by University of Zurich.

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Authors and Affiliations

Institute of Neuropathology, University of Zurich, Zurich, Switzerland
Yingjun Liu, Jingjing Guo, Maja Matoga & Adriano Aguzzi
Karolinska Institutet, Department of Medicine Solna, Division of Rheumatology, Stockholm, Sweden
Marina Korotkova & Per-Johan Jakobsson
Karolinska University Hospital at Solna, Stockholm, Sweden
Marina Korotkova & Per-Johan Jakobsson

Authors

Yingjun Liu
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Jingjing Guo
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Maja Matoga
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Marina Korotkova
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Per-Johan Jakobsson
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Adriano Aguzzi
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Contributions

Y.L. and A.A. conceptualized the study. Y.L. designed and performed the experiments with help from J.G., M.M. and M.K. P.-J.J. provided the Ptges inhibitors and helpful guidance on relevant experiments. Y.L. and A.A. wrote the manuscript.

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Correspondence to Yingjun Liu or Adriano Aguzzi.

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Extended data

Extended Data Fig. 1 Enhanced prion neurotoxicity in continuously NG2-glia-depleted C57BL/6 J COCS.

a, NG2 immunofluorescence showing NG2 glia activation in prion-infected C57BL/6 J COCS vs C57BL/6 J COCS exposed to NBH. Nuclei were stained with DAPI (blue). b, Quantification of NG2 immunointensity shown in a. n = 8 slices for NBH; n = 11 slices for Prion. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.0007. c-d, Experimental design of continuous NG2 glia depletion in Tga20 COCS (c) and C57BL/6 J COCS (d). e, NeuN and NG2 immunofluorescence showing enhanced neurodegeneration in continuously NG2-glia-depleted (CP673451) C57BL/6 J COCS compared to NG2 glia intact (DMSO) C57BL/6 J COCS after prion infection. Nuclei were stained with DAPI (blue). f, Quantification of NG2 immunointensity and NeuN positive area shown in e. n = 14 slices/condition for NeuN; NG2: 8 slices for NBH + DMSO; 10 slices for Prion+DMSO; 15 slices for NBH + CP673451 and Prion+CP673451. Data are presented as mean ± SEM. One-way ANOVA with Benjamini-Hochberg FDR adjustment for multiple comparisons. NeuN: P = 0.2466 (NBH + DMSO vs. NBH + CP673451); P = 0.0035 (NBH + DMSO vs. Prion+DMSO); P = 0.0288 (Prion+DMSO vs. Prion+CP673451). NG2: P < 0.0001 (NBH + DMSO vs. NBH + CP673451); P < 0.0001 (NBH + DMSO vs. Prion+DMSO); P < 0.0001 (Prion+DMSO vs. Prion+CP673451).

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Extended Data Fig. 2 Enhanced prion neurotoxicity in transiently NG2-glia-depleted Tga20 COCS.

a, Experimental design of transient NG2 glia depletion in Tga20 COCS. b, NeuN and NG2 immunofluorescence showing enhanced neurodegeneration in transiently NG2-glia-depleted (CP673451) Tga20 COCS compared to NG2 glia intact (DMSO) Tga20 COCS after prion infection. NG2 glia density is largely recovered in prion-infected, transiently NG2-glia-depleted COCS at the end of the experiment. Nuclei were stained with DAPI (blue). c, Quantification of NG2 immunointensity and NeuN positive area shown in b. NeuN: n = 20 slices for NBH + DMSO and NBH + CP673451; n = 28 slices for Prion+DMSO; n = 31 slices for Prion+CP673451. NG2: n = 12 slices for NBH + DMSO; n = 16 slices for Prion+DMSO, NBH + CP673451 and Prion+CP673451. Data are presented as mean ± SEM. One-way ANOVA with Benjamini-Hochberg FDR adjustment for multiple comparisons. NeuN: P = 0.1057 (NBH + DMSO vs. NBH + CP673451); P < 0.0001 (NBH + DMSO vs. Prion+DMSO); P < 0.0001 (Prion+DMSO vs. Prion+CP673451). NG2: P = 0.0690 (NBH + DMSO vs. NBH + CP673451); P < 0.0001 (NBH + DMSO vs. Prion+DMSO); P = 0.6641 (Prion+DMSO vs. Prion+CP673451).

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Extended Data Fig. 3 Early NG2 glia depletion accelerates prion disease in male but not female mice.

a, Experimental design of NG2 glia depletion in prion inoculated Pdgfrα^iDTR mice. Littermate Pdgfrα-CreER and iDTR mice were pooled together as control (Ctrl) and treated with tamoxifen and DT the same way as the Pdgfrα^iDTR mice. b, Survival curves showing accelerated prion disease in male, but not female NG2-glia-depleted (Pdgfrα^iDTR) mice compared to NG2 glia intact (Ctrl) mice after prion inoculation (median survival: 180 days for male Pdgfrα^iDTR mice; 187.5 days for male control mice; 174 days for female Pdgfrα^iDTR mice; 177 days for female control mice). Male mice: n = 8 for Ctrl; n = 9 for Pdgfrα^iDTR. Female mice: n = 8 for Ctrl; n = 5 for Pdgfrα^iDTR. Log-rank test: P = 0.0123 for male: P = 0.3571 for female.

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Extended Data Fig. 4 NG2 glia depletion does not influence brain vacuolation in prion-inoculated mice.

HE staining of brain tissues of NG2-glia-depleted (Pdgfrα^iDTR) and NG2 glia intact (Ctrl) mice after prion inoculation indicating similar vacuolation in different brain regions. NG2 glia depletion was induced at 16 wpi. Same staining was repeated independently in more than 3 mice for each group with similar results.

Extended Data Fig. 5 Loss of NG2 glia does not alter PrP^C expression, prion replication, and prion-induced neuroinflammation.

a-b, Western blots (a) and quantification (b) showing no changes of PrP^C level in NG2-glia-depleted (CP673451) Tga20 COCS compared to NG2 glia intact (DMSO) Tga20 COCS. n = 5 samples for DMSO and n = 4 samples for CP673451. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.1231. c-d, Western blots (c) and quantification (d) showing no changes of PK-resistant PrP^Sc levels in NG2-glia-depleted (CP673451) Tga20 COCS compared to NG2 glia intact (DMSO) Tga20 COCS after prion infection. n = 5 samples for DMSO and n = 4 samples for CP673451. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.3190. e-f, Western blots (e) and quantification (f) showing no changes of PrP^C levels in the brains of NG2-glia-depleted (Pdgfrα^iDTR) mice compared to NG2 glia intact (Ctrl) mice. n = 4 mice for Ctrl and n = 5 mice for Pdgfrα^iDTR. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.1274. g-h, Western blots (g) and quantification (h) showing no changes of PK-resistant PrP^Sc levels in the brains of NG2-glia-depleted (Pdgfrα^iDTR) mice compared to NG2 glia intact (Ctrl) mice after prion infection. NG2 glia depletion was induced at 16 wpi. n = 4 mice for Ctrl and n = 5 mice for Pdgfrα^iDTR. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.8643. i-j, Cd68 immunofluorescence (i) and quantification (j) showing similar levels of microglia activation in NG2-glia-depleted (CP673451) Tga20 COCS compared to NG2 glia intact (DMSO) Tga20 COCS after prion infection. Nuclei were stained with DAPI (blue). n = 20 slices for NBH + DMSO and Prion+CP673451; n = 17 slices for Prion+DMSO; n = 19 slices for NBH + CP673451. Data are presented as mean ± SEM. One-way ANOVA with Benjamini-Hochberg FDR adjustment for multiple comparisons: P = 0.9795 (NBH + DMSO vs. NBH + CP673451); P < 0.0001 (NBH + DMSO vs. Prion+DMSO); P = 0.5088 (Prion+DMSO vs. Prion+CP673451). k-l, Iba1 and Cd68 immunofluorescence (k) and quantification (l) showing similar levels of microglia activation in the hippocampi of NG2-glia-depleted (Pdgfrα^iDTR) mice compared to NG2 glia intact (Ctrl) mice after prion infection. NG2 glia depletion was induced at 16 wpi. Nuclei were stained with DAPI (blue). n = 6 mice/group. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.7170 (Iba1); P = 0.8422 (Cd68). m, qRT-PCR results showing minimal changes of proinflammatory factors TNFα, IL1β and IL12β in NG2-glia-depleted (CP673451) Tga20 COCS compared to NG2 glia intact (DMSO) Tga20 COCS after prion infection. n = 6 samples/condition. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.0022 (TNFα); P = 0.3561 (IL1β); P = 0.7870 (IL12β). n, qRT-PCR results showing no changes of proinflammatory factors TNFα, IL1β and IL12β in the brains of NG2-glia-depleted (Pdgfrα^iDTR) mice compared to NG2 glia intact (Ctrl) mice after prion inoculation. n = 5 mice/group. NG2 glia depletion was induced at 16 wpi. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.1904 (TNFα); P = 0.8378 (IL1β); P = 0.8608 (IL12β).

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Extended Data Fig. 6 NG2 glia depletion does not influence Cox1 and Ptgds expression.

qRT-PCR results showing no changes of Cox1 and Ptgds in NG2-glia-depleted (CP673451) Tga20 COCS compared to NG2 glia intact (DMSO) Tga20 COCS in the presence or absence of prions. n = 7 samples/condition. Data are presented as mean ± SEM. Multiple unpaired t tests with Benjamini-Hochberg FDR adjustment for multiple comparisons. Cox1: P = 0.1426 (NBH + DMSO vs. NBH + CP673451); P = 0.3797 (Prion+DMSO vs. Prion+CP673451). Ptgds: P = 0.6933 (NBH + DMSO vs. NBH + CP673451); P = 0.2202 (Prion+DMSO vs. Prion+CP673451).

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Extended Data Fig. 7 Cell-cell communications from NG2 glia to microglia in the hippocampus of prion-inoculated mice.

a, UMAP of single-cell RNA-seq data showing Cox2⁺ and Ptges⁺ microglia among total microglia in the hippocampus of prion- or NBH-inoculated mice. b, Quantification of Cox2⁺ and Ptges⁺ microglia fractions against total microglia in the hippocampus of prion- or NBH-inoculated mice shown in a. n = 2 mice for NBH and n = 7 mice for Prion. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.0216 (Cox2⁺ microglia); P = 0.0116 (Ptges⁺ microglia). c, Venn diagram showing numbers of shared and distinct DEGs of Cox2⁺ and Ptges⁺ microglia in the hippocampus of prion-inoculated mice. d, Heatmap showing downregulation of homeostatic microglia signature genes and upregulation of DAM and MHC-II microglia signature genes as well as inflammatory genes in Cox2⁺ and Ptges⁺ microglia in the hippocampus of prion-inoculated mice. e, CellChat analysis of cell-cell communications showing unaltered number of interactions (plotted as the thickness of the edges) but reduced strength of interactions (plotted as the thickness of the edges) from NG2 glia to Cox2⁺ microglia in the hippocampus of prion-inoculated mice. f, Heatmaps showing significantly weakened NG2 glia to Cox2⁺ microglia interaction pathways in the hippocampus of prion-inoculated mice.

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Extended Data Fig. 8 Purity of primary microglia cultures.

a, Iba1 immunofluorescence showing high purity of primary microglia cultures isolated by Cd11b immunopanning. Nuclei were stained with DAPI (blue). b, Quantification of Iba1+ cell percentage shown in a. n = 5 independent experiments. Data are presented as mean ± SEM.

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Extended Data Fig. 9 Expression of PGE2 receptors in neurons.

a, Immunofluorescence of NeuN and PGE2 receptors in the cerebral cortex showing neuronal expression of Ptger1, Ptger2, Ptger3 and Ptger4 in the adult mouse brain. Scale bar: 50 μm. Same staining was repeated independently in more than 3 mice with similar results. b, Immunofluorescence showing expression of Ptger1, Ptger2, Ptger3 and Ptger4 in primary neuronal cultures (3 weeks in vitro). Ptger2 is mainly located in the neuronal cell body; Ptger1, Ptger3 and Ptger4 are located in both the neuronal cell body and neuronal processes. Scale bar: 50 μm. Same staining was repeated independently in more than 3 batches of primary neuron preparations with similar results.

Extended Data Fig. 10 Activation of Ptger1 does not influence prion neurotoxicity in primary neuronal cultures and Tga20 COCS.

a, Immunofluorescence of NeuN, Map2 and Tau showing cellular damage of primary neurons treated with high concentration of Ptger4 agonist L902688. b, Quantification of neuronal density as well as Map2 positive and Tau positive areas shown in a. n = 6 independent experiments. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.0001 (NeuN); P < 0.0001 (Map2); P < 0.0001 (Tau). c, Immunofluorescence of NeuN, Map2 and Tau showing no damage of prion-infected primary neurons treated with different concentrations of Ptger1 agonist 17-pt-PGE2. d, Quantification of neuronal density as well as Map2 positive and Tau positive areas shown in c. n = 6 independent experiments. Data are presented as mean ± SEM. One-way ANOVA with Benjamini-Hochberg FDR adjustment for multiple comparisons. NeuN: P = 0.9792 for 1 μM vs. 0 μM, 5 μM vs. 0 μM, and 10 μM vs. 0 μM. Map2: P = 0.9445 for 1 μM vs. 0 μM, 5 μM vs. 0 μM, and 10 μM vs. 0 μM. Tau: P = 0.9165 for 1 μM vs. 0 μM, 5 μM vs. 0 μM, and 10 μM vs. 0 μM. e-f, NeuN immunofluorescence (e) and quantification (f) showing no change of prion-induced neurodegeneration in 17-pt-PGE2-treated Tga20 COCS. n = 10 slices/condition. Data are presented as mean ± SEM. One-way ANOVA with Benjamini-Hochberg FDR adjustment for multiple comparisons: P = 0.9799 for NBH + 0 μM vs. NBH + 1 μM, NBH + 0 μM vs. NBH + 5 μM, Prion+0 μM vs. Prion+1 μM, and Prion+0 μM vs. Prion+5 μM. P < 0.0001 for NBH + 0 μM vs. Prion+0 μM.

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Supplementary information

Reporting Summary

Supplementary Tables 1–4.

Table 1, Differentially expressed genes (Cox2⁺ versus Cox2⁻ microglia in the cerebral cortex of prion-inoculated mice). Table 2, Differentially expressed genes (Ptges⁺ versus Ptges⁻ microglia in the cerebral cortex of prion-inoculated mice). Table 3, Differentially expressed genes (Cox2⁺ versus Cox2⁻ microglia in the hippocampus of prion-inoculated mice). Table 4, Differentially expressed genes (Ptges⁺ versus Ptges⁻ microglia in the hippocampus of prion-inoculated mice).

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Liu, Y., Guo, J., Matoga, M. et al. NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling. Nat Neurosci (2024). https://doi.org/10.1038/s41593-024-01663-x

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Received: 10 April 2023
Accepted: 22 April 2024
Published: 27 May 2024
DOI: https://doi.org/10.1038/s41593-024-01663-x

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Abstract

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Main

Results

NG2 glia activation in prion disease models

Loss of NG2 glia accelerates prion disease

Unaltered prion replication and overall neuroinflammation

Loss of NG2 glia enhances microglial PGE2 biosynthesis

NG2 glia regulate microglial PGE2 via several mechanisms

Inhibition of PGE2 biosynthesis decelerates prion disease

PGE2 inhibition rescues accelerated prion disease

PGE2 enhances prion neurotoxicity via the EP4 receptor

Discussion

Methods

Mouse experiments

Prion inoculation

Cerebellar organotypic cultured slices

Treatment of brain slice cultures

NG2 glia depletion

Lentiviral production

Primary microglia culture

Primary neuronal culture

HovS cell culture

Western blotting

Immunofluorescence

Quantitative real-time PCR

Single-cell RNA sequencing analysis

Statistical analyses

Reporting summary

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