Large-scale analyses of CAV1 and CAV2 suggest their expression is higher in post-mortem ALS brain tissue and affects survival

Abstract

Introduction: Caveolin-1 and Caveolin-2 (CAV1 and CAV2) are proteins associated with intercellular neurotrophic signalling. There is converging evidence that CAV1 and CAV2 (CAV1/2) genes have a role in amyotrophic lateral sclerosis (ALS). Disease-associated variants have been identified within CAV1/2 enhancers, which reduce gene expression and lead to disruption of membrane lipid rafts. Methods: Using large ALS whole-genome sequencing and post-mortem RNA sequencing datasets (5,987 and 365 tissue samples, respectively), and iPSC-derived motor neurons from 55 individuals, we investigated the role of CAV1/2 expression and enhancer variants in the ALS phenotype. Results: We report a differential expression analysis between ALS cases and controls for CAV1 and CAV2 genes across various post-mortem brain tissues and three independent datasets. CAV1 and CAV2 expression was consistently higher in ALS patients compared to controls, with significant results across the primary motor cortex, lateral motor cortex, and cerebellum. We also identify increased survival among carriers of CAV1/2 enhancer mutations compared to non-carriers within Project MinE and slower progression as measured by the ALSFRS. Carriers showed a median increase in survival of 345 days. Discussion: These results add to an increasing body of evidence linking CAV1 and CAV2 genes to ALS. We propose that carriers of CAV1/2 enhancer mutations may be conceptualised as an ALS subtype who present a less severe ALS phenotype with a longer survival duration and slower progression. Upregulation of CAV1/2 genes in ALS cases may indicate a causal pathway or a compensatory mechanism. Given prior research supporting the beneficial role of CAV1/2 expression in ALS patients, we consider a compensatory mechanism to better fit the available evidence, although further investigation into the biological pathways associated with CAV1/2 is needed to support this conclusion.

Keywords: ALS (Amyotrophic lateral sclerosis); CAV1 and CAV2; Cav; caveolin; differential expression analysis (DEA); enhancer variant; neurodegeneration; survival analysis.

Figure 1

Diagrammatic representation of RNA-seq differential expression pipeline. Each RNA-seq step is shown in the blue circles, with the tool used at each step given beside each red circle.

Figure 2

Sample overview across the RNAseq datasets used in the differential expression analyses. Datasets were obtained from the KCL Brain Bank (green), Mayo Clinic (orange), and TargetALS (NYGC; blue).

Figure 3

Sample sizes for each Project MinE survival analysis. Samples are divided by those with CAV1/2 enhancer mutations (orange) and without (blue). Analyses are: Full dataset including all rare mutations; excluding C9orf72 samples; and with CAV1 and CAV- samples only with CAV2 and CAV- samples only. CAV- refers to patients who do not carry CAV1/2 enhancer mutations and CAV+ refers to those who do.

Figure 4

This figure shows violin plots of significant gene expression for CAV1 and CAV2 between cases and controls. The X-axis indicates tissue/dataset combination and case/control status. The Y-axis is normalised gene expression. Coloured dots inside violin plots are jittered gene expressions for each sample. Boxplots inside each violin plot show gene expression for each category. Violin plot colour: Condition (case: red; control: blue). Note that CAV1 differential expression in the lateral motor cortex is significant only to p < 0.1. Violin plots for all analyses are available in Supplementary Figure 1.

Figure 5

Scatter plot showing normalised CAV2 gene expression against rate of change in the ALSFRS.

Figure 6

Survival Curves comparing survival of patients with vs without any CAV1/2 mutation. The left graph is based upon data from Analysis 1, inclusive of patients with C9orf72 repeat expansion. The right graph is from Analysis 2, with C9orf72 samples removed. Patients with CAV1/2 mutations have a longer survival time (C9orf72-inclusive analysis: median survival difference of 345 days. See Table 5 for a full descriptive summary). Y-axis is the fraction of surviving sample. X-axis is time in days. Dashed lines indicate 95% confidence intervals. Orthogonal lines indicate death or censoring event. Graphs exclude 22 samples from patients surviving over 10,000 days to improve scaling. Complete graphs are available in Supplementary Figure 2.