The NR4A2/VGF pathway fuels inflammation-induced neurodegeneration via promoting neuronal glycolysis

Abstract

A disturbed balance between excitation and inhibition (E/I balance) is increasingly recognized as a key driver of neurodegeneration in multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. To understand how chronic hyperexcitability contributes to neuronal loss in MS, we transcriptionally profiled neurons from mice lacking inhibitory metabotropic glutamate signaling with shifted E/I balance and increased vulnerability to inflammation-induced neurodegeneration. This revealed a prominent induction of the nuclear receptor NR4A2 in neurons. Mechanistically, NR4A2 increased susceptibility to excitotoxicity by stimulating continuous VGF secretion leading to glycolysis-dependent neuronal cell death. Extending these findings to people with MS (pwMS), we observed increased VGF levels in serum and brain biopsies. Notably, neuron-specific deletion of Vgf in a mouse model of MS ameliorated neurodegeneration. These findings underscore the detrimental effect of a persistent metabolic shift driven by excitatory activity as a fundamental mechanism in inflammation-induced neurodegeneration.

Keywords: Inflammation; Multiple sclerosis; Neurodegeneration; Neuroscience.

Figure 1. NR4A2 is induced in Grm8-deficient neurons.

(A) The number of Grm8⁺ neurons assessed by RNAScope in cervical spinal cords of healthy and EAE mice 15 days after immunization (n = 5). Scale bar: 20 μm. (B) Heatmap of top 15 up and downregulated genes in NeuN⁺ cortical nuclei of Grm8^–/– compared with WT mice (n = 3). (C) Relative Nr4a2 expression in NeuN⁺ cortical nuclei of Grm8^–/– and WT mice in relative units (RU). (D) Nr4a2 expression (RU) in WT and Grm8^–/– primary cortical neurons (PCNs; n = 5). Paired t test was performed. (E) NR4A2 mean fluorescence intensity (MFI) in cortical neuronal nuclei estimated by flow cytometry of WT (n = 4) and Grm8^–/– mice (n = 5). (F and G) Nr4a2 expression (RU) in WT (F) and Grm8^–/– (G) PCNs after glutamate stimulation (n = 5). Paired t test was performed. (H) NR4A2 MFI in PCNs that were treated with glutamate and vehicle, 2 mM EDTA or 50 μM 2-APB (n = 6). Scale bar: 20 μm. (I) Dead cells per field of view (FOV) of control (mScarlet) or Nr4a2-overexpressing PCNs 7 days after transduction (D.A.T.; left) and 14 D.A.T. (right). PCNs were treated with vehicle or 1 μM AZ12216052 every other day (n = 5). (J and K) Viability (RU) of control (mScarlet) or Nr4a2-overexpressing PCNs that were exposed to glutamate and vehicle or 1 μM AZ12216052 (n = 5, J) or 50 nM Ip7e (n = 5; K). (L) Calcium traces and somatic calcium accumulation in glutamate-treated control (mScarlet) or Nr4a2-overexpressing PCNs (n = 4). Points represent individual experiments, additionally, the mean is shown. If not stated otherwise, unpaired t test with FDR correction for multiple comparisons was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2. NR4A2-induced VGF mediates neuronal susceptibility to excitotoxicity.

(A) Nr4a2 mRNA expression in NeuN⁺ nuclei sorted from spinal cords of healthy mice (n = 3) and acute EAE mice (n = 5) in relative units (RU). (B) Eigengene correlation matrix and dendrogram of weighted gene correlated network analysis (WGCNA) with 502 neuron-specific transcriptome data sets. (C) Enrichment analysis of the inflamed neuronal signature (ranked gene list retrieved from ref. 9) in module “darkgreen”. (D) GO term biological process enrichment analysis of the module “darkgreen”. Size shows number of genes of GO terms, color shows significance. (E) Heatmap of top 10 genes from the module “darkgreen,” which are differently expressed in neurons during EAE. (F) Chromatin immunoprecipitation (ChIP) was performed using an antibody against NR4A2 and an IgG control from 3 pooled mouse cortices. PCR primers were designed to amplify approximately 100 nucleotides flanking the canonical nuclear receptor binding (NRB) motif in the mouse Vgf promoter (left) or the 3′ untranslated region of Vgf as control (right). (G) VGF MFI in neuronal cultures after vehicle or glutamate stimulation (n = 4 per group). Scale bar: 50 μm. (H and I) Relative VGF MFI in neurons that overexpress mScarlet (controls), Nr4a2 or were exposed to 50 nM Ip7e (H; n = 4), and neurons that overexpress Nr4a1 or Nr4a3 (I; n = 6). Data is normalized to mScarlet-overexpressing control neurons. Scale bar: 20 μm. (J) VGF⁺ neurons in cortices of WT and Grm8^–/– mice (n = 5). Scale bar: 300 μm. (K) Cell viability (RU) of WT and Vgf^–/– neurons that overexpress Nr4a2 and were exposed to glutamate (n = 5). Points represent individual experiments, additionally mean is shown. If not stated otherwise, unpaired t test with FDR correction for multiple comparisons was used. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. VGF is increased in the CNS and blood of EAE animals and pwMS.

(A) Vgf expression in relative units (RU) in spinal cords and motoneurons of healthy mice and mice undergoing EAE (data was retrieved from ref. ; n = 5 per group). (B) VGF protein levels in the spinal cords of healthy mice (n = 14) and mice undergoing EAE 10 days (onset, n = 6), 15 days (acute, n = 5), and 30 days (chronic, n = 5) after immunization. (C) Neuronal VGF MFI in cervical spinal cords of healthy mice and mice undergoing acute and chronic EAE (n = 5 per group). Scale bar: 5 μm. (D) Relative fold change of total VGF in the plasma of healthy mice (n = 6) and mice undergoing EAE 10 days (onset, n = 6), 15 days (acute, n = 5), and 30 days (chronic, n = 6) after immunization. (E) Relative fold change of total VGF in the sera of healthy controls and with pwRRMS (n = 20 per group). Controls were age and sex matched. (F) Relative fold change of total VGF in sera of pwRRMS during stable disease and acute relapse (n = 9). Paired t test was used for statistical comparison. (G) Relative fold change of total VGF in the sera of pwRRMS before and after treatment with natalizumab (n = 6 per group). Paired t test was used for statistical comparison. (H) Neuronal VGF MFI in brain biopsies of noninflammatory CNS disease controls (controls) and pwMS (n = 4 per group). Scale bar: 5 μm. Points represent individual experiments, additionally mean is shown. If not stated otherwise, unpaired t test with FDR correction for multiple comparisons was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4. Persistent VGF exposure exacerbates excitotoxicity by inducing glycolysis.

(A) Relative cell viability (RU, relative units) of neuronal cultures that overexpress EGFP (controls) or VGF and were exposed to glutamate (n = 6 per group). (B) Volcano plot showing all differentially expressed genes (DEGs) between control (EGFP) and VGF-overexpressing neurons. (C) GSEA of all DEG between control (EGFP) and VGF-overexpressing neurons. Size shows number of genes of GO terms, color shows significance. (D) Baseline ATP levels in control (EGFP) and VGF-overexpressing neurons (n = 6 per group). (E and F) Mitochondrial respiration (E) and glycolytic index (F) of control (EGFP) and VGF-overexpressing neurons (n = 6 per group). (G) Glycolytic index of control (mScarlet) and NR4A2-overexpressing neurons (n = 5 per group). (H) Glycolytic index of WT and Vgf-deficient neurons (n = 5 per group). (I) Glycolytic index of WT and Vgf-deficient neurons that overexpress NR4A2 (n = 5 per group). (J) Relative cell viability (RU) VGF-overexpressing neurons that were exposed to glutamate and were pretreated with vehicle (control) or 5 mM 2-DG (n = 5 per group). Data was normalized to controls without glutamate stimulation. (K) Relative cell viability (RU) of neuronal cultures that overexpress mScarlet (control) or NR4A2 and were exposed to glutamate and pretreated with vehicle or 5 mM 2-DG (n = 5 per group). Data was normalized to controls without glutamate stimulation. Points represent individual experiments, additionally, mean is shown. If not stated otherwise, unpaired t test with FDR correction for multiple comparisons was used. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Neuronal VGF deficiency protects from neuroaxonal damage in EAE.

(A) EAE disease course of Snap25-Cre × Vgf^fl/fl (Vgf-cKO; n = 10) animals and respective Vgf^fl/fl (n = 11) control littermates. Points represent mean per group per day, additionally, SEM is shown. (B) Cumulative clinical score using AUC during acute (days 11–20), recovery (days 21–30), and chronic (days 31–40) disease stages of EAE. (C and D) Disease onset (C) and maximal disease score (D) of Vgf-cKO and control mice. (E) Number of HuC/D-positive neurons in the ventral horn spinal cords of WT (n = 5) and Vgf-cKO (n = 7) EAE mice 40 days after immunization. Scale bar: 50 μm. (F) Number of axons in dorsal columns of the cervical spinal cord assessed by SMI staining in Vgf-cKO (n = 9) and control mice (n = 10) undergoing EAE 40 days after immunization. Scale bar: 50 μm. (G) Quantification of luxol fast blue-positive axonal area per 100 μm² in the dorsal columns of the cervical spinal cords of WT (n = 6) and Vgf-cKO (n = 8) EAE mice 40 days after immunization. Scale bar: 500 μm. Points represent individual experiments, additionally mean is shown. For comparing EAE phenotypes nonparametric Kolmogorov-Smirnov test was used. For IHC analyses unpaired t test was used. *P < 0.05.