Altered perivascular fibroblast activity precedes ALS disease onset

Abstract

Apart from well-defined factors in neuronal cells¹, only a few reports consider that the variability of sporadic amyotrophic lateral sclerosis (ALS) progression can depend on less-defined contributions from glia^2,3 and blood vessels⁴. In this study we use an expression-weighted cell-type enrichment method to infer cell activity in spinal cord samples from patients with sporadic ALS and mouse models of this disease. Here we report that patients with sporadic ALS present cell activity patterns consistent with two mouse models in which enrichments of vascular cell genes preceded microglial response. Notably, during the presymptomatic stage, perivascular fibroblast cells showed the strongest gene enrichments, and their marker proteins SPP1 and COL6A1 accumulated in enlarged perivascular spaces in patients with sporadic ALS. Moreover, in plasma of 574 patients with ALS from four independent cohorts, increased levels of SPP1 at disease diagnosis repeatedly predicted shorter survival with stronger effect than the established risk factors of bulbar onset or neurofilament levels in cerebrospinal fluid. We propose that the activity of the recently discovered perivascular fibroblast can predict survival of patients with ALS and provide a new conceptual framework to re-evaluate definitions of ALS etiology.

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Figure 1. ALS patients show increased transcriptional activity of perivascular fibroblast cell gene markers which occur at presymptomatic disease stage in SOD1^G93A and TARDBP^Q331K mice.

(A) Schematic of expression weighted cell type enrichment (EWCE) analysis. Cell type-specific gene rankings from single cell sequencing data allow to infer cell type activity in bulk tissue transcriptomes. (B) Enrichment z - scores for up and down-regulated genes in ten cell type classes in sALS patients, SOD1^G93A and TARDBP^Q331K mice. In the SOD1^G93A mice, onset of neuromuscular junction decoupling (8 weeks) and clinical symptoms (peak body weight - 16 weeks) are indicated with arrowheads. P-values are specified in Ext. data Fig. 2. (C) Cell type RNA specificity for genes enriched in perivascular fibroblasts. N number of cells per category is described in Ext. data Fig. 1. Bars show median count of RNA per cell ± SEM. (D) Perivascular fibroblast specific gene activity in sALS patient spinal cords. sALS n=12, Ctrl n=8. Boxplots show median, 2nd-3rd quartile and whiskers show +/-1.5 of the IQR. Two-tailed t-test p-values are indicated next to brackets. (E) Expression of perivascular fibroblast enriched genes in SOD1^G93A (p-value for ANOVA in early (4-10 weeks) and late (14-18 weeks) timepoints) and TARDBP^Q331K mice (p-values for two-tailed t-test). (F) Perivascular fibroblasts have distinct mRNA expression markers (median RNA molecule count with ±SEM). (G) Schematic illustration represents reported PVF location between astrocytes and mural/endothelial cells. (H) Transmission electron microscopy of mouse spinal cord tissue points to location of perivascular fibroblasts cells (PVF - shaded red) between basement membrane layers that delineate astrocyte endfeet (AC - blue) and mural cells (MC - brown), (EC - yellow: endothelial cell, aBM - astrocyte basement membrane, mBM - mural basement membrane). Scale bar 2μm.

Figure 2. Perivascular fibroblast marker proteins COL6A1 and SPP1 accumulate in enlarged perivascular spaces during ALS progression.

(A) Col6a1 and Spp1 mRNA specificity within CNS cell types. Bars represent relative count of RNA per cell ± SEM. (B) COL6A1 and SPP1 histochemistry in sALS and control spinal cords, bar: 10μm. (C) Quantifications of human tissue histochemistry from full frame 4x photos. sALS and Ctrl n=4 individuals (2-tailed t-test p-value). All boxplots show median, 2nd-3rd quartile and whiskers show +/-1.5 of the IQR. (D) Col6a1 and Spp1 accumulate around blood vessels (outlined with podocalyxin - cyan) in 14 week SOD1^G93A mouse spinal cords. Immunofluorescence z-stack renderings of 16μm thick sections, bars: 100μm (overview), 10μm (insert). (E) Quantifications of immunofluorescence stainings in mice from full-frame 20x photos. SOD1^G93A and BL/6 n=4, SOD1^wt n=3 mice (2-tailed t-test p-value). (F) Increased perivascular spaces appear in presymptomatic (8 weeks) SOD1^G93A mice spinal cords. Immunofluorescence for vascular (Col4a1) and astrocyte (Lama1) basement membranes, bar: 10μm. (G) Electron microscopy (tEM) of increased perivascular spaces in 14 week SOD1^G93A mice. Astrocyte (red) and vascular (blue) basement membranes are indicated with lines. Perivascular space is indicated with asterisk, bar: 5μm. (H) COL6A1 and SPP1 accumulate within increased perivascular spaces (outlined with COL4A1) in spinal cords of sALS patients. 2 color histochemistry, bar: 10μm. (I) Quantifications of COL6A1 and SPP1 immunostainings from panel H. sALS and Ctrl n=4 individuals (two-tailed t-test p-value). (J) Quantifications of perivascular space increase in SOD1^G93A mice from panel G (SOD1^G93A and BL/6 n=4 mice) and in sALS patients from panel H (sALS and Ctrl n=4 individuals) (two-tailed t-test p-value). (K) Schematic representation of perivascular fibroblast activity and enlarged perivascular spaces in ALS.

Figure 3. Prognostic value of SPP1 protein in plasma of ALS patients.

(A) Relative levels of SPP1 protein in plasma as measured by the HPA027541 antibody. Threshold selection and Kaplan-Meier survival estimates of ALS patients in discovery cohorts (Netherlands, Germany and Belgium, n=452) and in the replication cohort (Sweden, n=122). Red color indicates thresholded protein level. Thresholds are established using maximally selected log rank statistics. Boxplots show median, 2nd-3rd quartile and whiskers show +/-1.5 of the IQR. Survival probability graphs show proportion of censored patients within each arm and Kaplan-Meier logrank p-values. (B-C) Uni- and multivariate Cox proportional hazard models for continuous increase of plasma SPP1 relative to hazard ratios indicated by bulbar onset type, neurofilament light (NFL) in CSF (in the replication cohort), gender and plasma sampling age. Whiskers represent 95% CI. Cohort identity was additionally used as covariate in multivariate models.