Fig 2: Grm5 ablation protects against GDL and prion-induced neurotoxicity in slice cultures.(A-B) scPOM1 induced CGL profound neurotoxicity in Grm5+/+ slices. However, toxicity was much less pronounced in Grm5+/- and Grm5-/- slices. (B) NeuN morphometry of Grm5+/- and Grm5-/- and Grm5+/+ slices exposed to scPOM1 or scPOM1 blocked with recPrP from 14–22 dpe. (C-D) CGL ablation induced by RML6 infection in control Grm5+/+ slices, and amelioration in Grm5+/- and Grm5-/- slices. Slices were maintained in culture for 60 dpi. (E-F) Genetic ablation of Grm5 rescued prion-induced neurodegeneration in HOCS. (E) Representative images of HOCS, showing ablation of the hippocampal neuronal layer induced by RML6 infection in control Grm5+/+ slices, that is significantly ameliorated by the genetic deletion of Grm5 (Grm5-/- slices). Slices were maintained in culture for 60 dpi. (F) NeuN morphometry of Grm5+/- and Grm5-/- and Grm5+/+ slices exposed to RML6 or NBH. RML6-induced neurodegeneration is rescued in the Grm5+/- and Grm5-/- HOCS. All scale bars: 500 μm. (B), (D) and (F): NeuN relative signal intensity as percentage of control samples (Grm5+/+, NBH or POM1+recPrP); each dot corresponds to a pool of 7–10 cerebellar slices or 4–6 hippocampal slices cultured in the same well; Data are presented as mean ± s.d.; One-way ANOVA followed by Dunnett’s post-hoc test. *: P < 0.05, **: P < 0.01, ****: P < 0.0001.

Fig 3: mGluR1/5 inhibition rescues prion neurotoxicity in organotypic slice cultures.(A-B) Treatment with the mGluR5 inhibitor (MPEP) rescued neurodegeneration in tga20 RML6-treated COCS. (A) Fluorescence micrographs of tga20 COCS. RML6-induced ablation of the cerebellar granular layer (CGL) was significantly ameliorated by the mGluR5 inhibitor, MPEP. All scale bars: 500μm. (B) NeuN coverage in tga20 COCS exposed to RML6 or NBH and treated with MPEP at 21–45 days post inoculation (dpi), expressed as percentage of NBH samples. Each dot represents a pool of 4–10 slices cultured in the same well. Data points are mean ± s.d.; one-way ANOVA followed by Dunnett’s post-hoc test. (C-D) Treatment with the mGluR5 inhibitor AFQ056 (mavoglurant) also rescued neurodegeneration in tga20 RML6-treated COCS (experimental conditions as in panels A-B). (E-F) Treatment with the mGluR5 inhibitor (MPEP) rescued neurodegeneration in tga20 RML6-treated HOCS. (E) Fluorescence micrographs of tga20 HOCS, showing ablation of hippocampal neurons induced by RML6 infection (middle), that is significantly ameliorated by addition of the IC50 concentration of MPEP (36nM, 21–45 dpi, right). (F) Morphometry of the experiment shown in panel E. (G) Treatment with the mGluR1 inhibitor (YM202074) rescued neurodegeneration in tga20 RML6-treated COCS. Experimental conditions were the same as in the panels above. (H) Morphometry of the experiment shown in panel G; *: P < 0.05, **: P < 0.01, ***: P < 0.001; For (A), (C), (E) and (G) panels: Scale bar is 500μm.

Fig 4: Group-I mGluR inhibition abolishes GDL toxicity in organotypic slice cultures.(A-B) Treatment with the mGluR5 inhibitor (MPEP) rescued neurodegeneration in scPOM1-treated COCS from tga20 mice. (A) Ablation of the cerebellar granular layer (CGL) after exposure of tga20 COCS to scPOM1, and amelioration by MPEP. (B) NeuN morphometry of tga20 slices exposed to scPOM1 or control (scPOM1 blocked with recPrP) and treated with MPEP at 14–28 days post exposure (dpe). (C) CGL ablation after exposure to scPOM1, and amelioration by AFQ056. (D) NeuN morphometry of tga20 slices exposed to scPOM1 or control scPOM1 blocked with recPrP and treated with MPEP from 14–22 dpe. (E-F) Treatment with MPEP rescued neurodegeneration in tga20 scPOM1-treated COCS. (E) Ablation of the hippocampal neuronal layer induced by exposure of HOCS to scPOM1 (middle), and amelioration by MPEP. (F) NeuN morphometry of tga20 slices exposed to scPOM1 or control (scPOM1 blocked with recPrP) and treated with MPEP from 14–22 dpe. (G-H) Treatment with the mGluR1 inhibitor (YM202074) rescued neurodegeneration in tga20 scPOM1-treated COCS. (G) Ablation of the CGL in COCS by exposure to scPOM1, and suppression of toxicity by the mGluR1 antagonist, YM202074. (H) NeuN morphometry of tga20 slices as in panel F, but treated with YM202074 (14–22 dpe). All scale bars: 500μm. For (B), (D), (F) and (H): NeuN relative signal intensity as percentage of scPOM1+recPrP control samples. Each dot represents a pool of 7–10 cerebellar slices or 4–6 hippocampal slices cultured in the same well; Data are presented as mean ± s.d.; One-way ANOVA followed by Dunnett’s post-hoc test; **: P < 0.01, ***: P < 0.001.

Fig 5: Model of the interactions between mGluR5, PrPC, and anti-PrP antibodies.(A) In untreated neurons, mGluR5-PrPC complexes are distributed within and outside spines. Upon exposure to prion-mimetic antibodies (B), mGluR5 translocates to the spine, where it may enhance neurotoxicity by contributing to a Ca2+ overload. (C) Exposure to POM2, in contrast, engages the N-terminal “flexible tail” of PrPC, thereby making it unavailable to mGluR5. Consequently, mGluR5-PrPC (and possibly also mGluR1-PrPC) complexes do not translocate to spines. As a result, POM2 affords functional neuroprotection similarly to mGluR5 antagonists. (D) We speculate that prion infection may trigger topological rearrangements similar to those observed after POM1 exposure.