Multiple sclerosis (MS) is characterized by inflammatory insults that drive neuroaxonal injury. However, knowledge about neuron-intrinsic responses to inflammation is limited. By leveraging neuron-specific messenger RNA profiling, we found that neuroinflammation leads to induction and toxic accumulation of the synaptic protein bassoon (Bsn) in the neuronal somata of mice and patients with MS. Neuronal overexpression of Bsn in flies resulted in reduction of lifespan, while genetic disruption of Bsn protected mice from inflammation-induced neuroaxonal injury. Notably, pharmacological proteasome activation boosted the clearance of accumulated Bsn and enhanced neuronal survival. Our study demonstrates that neuroinflammation initiates toxic protein accumulation in neuronal somata and advocates proteasome activation as a potential remedy.
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Data generated for this study are available through the Gene Expression Omnibus under accession number GSE104899.
The routine for the single-cell data analysis can be assessed as an R Notebook file via github (github.com/INIMS/Bsn_scRNA-seq).
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We thank the UKE Mouse Pathology Facility for histopathology of EAE mice, N. Heintz for the Chat-bacTRAP mice, M. Heiman for advice on TRAP optimization, T. Dresbach for providing the Bsn DNA expression constructs, C.-L. Chien for providing the Map1a DNA expression construct and M. Binder and N. Akyüz for sequencing support. This work is supported by a Deutsche Forschungsgemeinschaft grant (grant no. FR 1720/11-1) and Oppenheim Förderpreis für Multiple Sklerose (Novartis) to M.A.F., as well as the Else Kröner-Fresenius-Stiftung (grant no. 2013_A217) to M.A.F. and K.E.D. D.M. is supported by the Swiss National Science Foundation (grant no. PP00P3_152928), Helmut Horten Foundation and Gebert-Rüf Foundation (grant no. GRS-049/13). U.T. and E.D.G. are supported by the Deutsche Forschungsgemeinschaft (grant no. CRC 854/B08).
The authors declare no competing interests.
Journal peer review information Nature Neuroscience thanks Joseph Dougherty, Hartmut Wekerle and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Integrated supplementary information
a, Representative immunohistochemical stainings of astrocytes (GFAP), vessels (CD31), microglia (Iba1), immune cells (CD45), oligodendrocytes (CNPase) and neurons (NeuN) co-localized with eGFP in cervical spinal cord sections of Chat-L10a-eGFP mice. Experiment was repeated two times with similar results. Scale bar, 50 µm. b, Number of motor neurons in cervical spinal cord sections of healthy (n = 3) and acute EAE (n = 4) Chat-L10a-eGFP mice with quantification. Student’s t-test, one-tailed: n = 3 healthy versus n = 4 EAE mice, t(5) = 2.440, P = 0.0293. Bars show mean values plus SEM. Scale bar, 100 µm. c, Principal component analysis including the top 500 variable genes. Experimental groups: spinal cord (SC), inflamed spinal cord (iSC), motor neurons (MN), inflamed motor neurons (iMN). d, Heatmap of Eeuclidean sample distances with unsupervised hierarchical clustering. Colors of experimental groups as in (c). *P < 0.05; **P < 0.01.
a, Comparison of inflamed spinal cords (iSC) versus healthy spinal cords (SC). Bar graph of differentially expressed (DE) genes. Red, up-regulated, Blue down-regulated. Darker tones of red and blue represent increasing absolute log2 fold changes (>1, >2, >4). Candidates were identified by DESeq2, negative binomial generalized linear models, adjusted for multiple comparisons by FDR: n = 5 biologically independent samples per group each pooled from 3 mice, log2 fold change > 1, FDR-adjusted P < 0.05. b, Percentage of DE genes annotated for the gene ontology (GO) term ‘immune system process’. c, Gene expression heatmap of top 20 up- and down-regulated genes. Genes annotated for ‘immune system process’ are marked in ocher on the left. d, Enrichment map of regulated biological process GO terms. Red, up-regulated. Blue, down-regulated. Node size, gene set size. Grey lines, gene set overlap.
Supplementary Fig. 3 Expression changes in inflamed motor neurons and candidate gene identification.
a, Comparison of inflamed motor neurons (iMN) versus healthy motor neurons (MN). Bar graph of differentially expressed (DE) genes. Red, up-regulated, Blue down-regulated. Darker tones of red and blue represent increasing absolute log2 fold changes (>1, >2, >4). Candidates were identified by DESeq2, negative binomial generalized linear models, adjusted for multiple comparisons by FDR: n = 5 biologically independent samples per group each pooled from 3 mice, log2 fold change > 1, FDR-adjusted P < 0.05. b, Percentage of DE genes annotated for the gene ontology (GO) term ‘immune system process’. c, Gene expression heatmap of top 20 up- and down-regulated genes. Genes annotated for ‘immune system process’ are marked in ocher on the left. d, Enrichment map of regulated biological process GO terms. Red, up-regulated. Blue, down-regulated. Node size, gene set size. Grey lines, gene set overlap. e, Venn diagrams summarizing candidate criteria. f, Bar graph of candidate genes. Red, up-regulated, Blue down-regulated. Darker tones of red and blue represent increasing absolute log2 fold changes (>1, >2, >4). g, Percentage of candidate genes annotated for the GO term ‘immune system process’.
a–b, Heatmap of candidate gene expression in healthy motor neurons (MN) and inflamed motor neurons (iMN) belonging to the regulated gene ontology (GO) terms ‘mitochondrion’ and ‘ubiquitin-dependent protein catabolic process’.
a, Analysis of BSN expression in human MS microarray data from Han, M. H. et al. J Exp Med 2012. b, Gene expression analysis of Bsn in an independent EAE TRAP cohort (n = 3 per group). MN, motor neurons. iMN, inflamed motor neurons. Student’s t-test, two-tailed: n = 4 MN versus n = 3 iMN samples each pooled from 3 mice, t(5) = 2.695, P = 0.0431. Bars show mean values plus SEM. c, Immunohistochemical stainings of Bsn with quantification in spinal cord sections of healthy and chronic EAE mice at day 30 post immunization. Co-staining for neurons (NeuN). Student’s t-test, two-tailed: n = 3 mice per group, t(4) = 4.062, P = 0.0153. Bars show mean values plus SEM. MFI, mean fluorescence intensity. Scale bar, 20 µm. d, Immunohistochemical staining of Bsn in spinal cord sections of animals immunized with complete Freund’s adjuvant but without MOG35–55 at day 15 post immunization. Co-staining for neurons (NeuN). Experiment was repeated two times with similar results. Scale bar, 20 µm. e, Immunohistochemical staining of Bsn in spinal cord sections of EAE animals at day 15 post immunization. Co-staining for neurons (NeuN). Experiment was repeated three times with similar results. Scale bar, 20 µm. f, Immunohistochemical stainings of synapsin 1/2 in cervical spinal cord sections of healthy and chronic EAE mice. Co-staining for neurons (NeuN) and bassoon (Bsn). Experiment was repeated two times with similar results. Scale bar, 20 µm. *P < 0.05.
a, Immunohistochemical stainings of exemplary MS lesions showing demyelination (loss of myelin basic protein, MBP) and varying degrees of inflammatory activity (CD3+ T cells, CD68+ activated phagocytes). Scale bar, 100 µm. b, Immunohistochemical staining of BSN in comparison to control stainings without BSN primary antibody in MS tissue. Co-staining for neurons (MAP2). Scale bar, 50 µm. c, Quantification of immunohistochemical stainings for BSN in spinal cord sections of control individuals and MS patients. 5–10 high power fields from 4 MS patients (n = 27 power fields) and 3 controls (n = 16 power fields). Median with interquartile range. d, Analysis of mouse Bsn and human BSN amino acid sequence for disordered residues by PONDR VSL2 algorithm. Red value indicates the percentage of disordered residues.
a, Somatic distribution of eGFP signal in N2a cells overexpressing eGFP, eGFP–Bsn or eGFP–Map1a. Experiment was repeated three times with similar results. Scale bar, 20 µm. b, Co-localization of punctate eGFP–Bsn accumulation with markers for Golgi (Golgin97), endosomes (Rab5), ER (PDI) and lysosomes (LAMP-2). Scale bar, 20 µm. c, Overlap of genes regulated in EAE motor neurons (EAE candidates) and eGFP–Bsn overexpressing N2a cells (Bsn overexpression). Statistical analysis was performed by hypergeometric testing. d, Overlap of GO terms regulated in EAE motor neurons (EAE candidates) and eGFP–Bsn overexpressing N2a cells (Bsn overexpression). Statistical analysis was performed by hypergeometric testing.
Supplementary Fig. 8 Neuroaxonal numbers and immune cell infiltration in Bsn−/−and IU1 treatment EAE.
a, Quantification of neurofilament-positive axons in the dorsal columns of cervical spinal cord sections of wildtype and Bsn−/− animals without EAE induction. Student’s t-test, two-tailed: n = 3 mice per group, t(4) = 1.165, P = 0.3086. Bars show mean values plus SEM. b, Quantification of NeuN-positive neurons in cervical spinal cord sections of wildtype and Bsn−/− animals without EAE induction. Student’s t-test, two-tailed: n = 3 mice per group, t(4) = 0.01243, P = 0.9907. Bars show mean values plus SEM. c, Gating strategy of CNS-infiltrating T cells. d, T cell subset composition in CNS infiltrates of wildtype and Bsn−/− EAE animals at day 15 after immunization. Two-way ANOVA with Sidak’s post hoc test: n = 3 mice per group, F(2,12) = 0.0060, P = 0.9940; Sidak’s post hoc test: CD8+ T cells, WT versus Bsn−/−: P = 0.9998; CD4+ T cells, WT versus Bsn−/−: P = 0.9998; CD4+Foxp3+ Treg, WT versus Bsn−/−: P > 0.9999. Bars show mean values plus SEM. e, T cell subset composition in CNS infiltrates of vehicle and IU1-treated EAE animals at day 15 after immunization. Two-way ANOVA with Sidak’s post hoc test: n = 3 mice per group, F(2,12) = 6.666, P = 0.0113; Sidak’s post hoc test: CD8+ T cells, vehicle-treated versus IU1-treated: P = 0.0778; CD4+ T cells, vehicle-treated versus IU1-treated: P = 0.0778; CD4+Foxp3+ Treg, vehicle-treated versus IU1-treated: P = 0.7514.Bars show mean values plus SEM.
To investigate the neuronal response to central nervous system (CNS) inflammation, ribosome-bound mRNA from spinal cord motor neurons was isolated by translating ribosome affinity purification (TRAP) comparing healthy mice to mice undergoing experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Samples were subjected to RNA-seq and regulated candidate genes and associated pathways were identified (top). Results indicate a cytoplasmic deposition of the strongly induced presynaptic protein bassoon (Bsn) in both EAE and MS that was accompanied by induction of protein catabolism and reduced energy metabolism. Bsn overexpression and genetic deletion established a toxic gain-of-function of Bsn that drives neurodegeneration during CNS inflammation. Pharmacological activation of the proteasome cleared Bsn deposits and enhanced neuronal survival (bottom).
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Schattling, B., Engler, J.B., Volkmann, C. et al. Bassoon proteinopathy drives neurodegeneration in multiple sclerosis. Nat Neurosci 22, 887–896 (2019). https://doi.org/10.1038/s41593-019-0385-4
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