RNA-Seq investigations of human post-mortem trigeminal ganglia

Abstract

Background The trigeminal ganglion contains neurons that relay sensations of pain, touch, pressure, and many other somatosensory modalities to the central nervous system. The ganglion is also a reservoir for latent herpes virus 1 infection. To gain a better understanding of molecular factors contributing to migraine and headache, transcriptome analyses were performed on postmortem human trigeminal ganglia. Methods RNA-Seq measurements of gene expression were conducted on small sub-regions of 16 human trigeminal ganglia. The samples were also characterized for transcripts derived from viral and microbial genomes. Herpes simplex virus 1 (HSV-1) antibodies in blood were measured using the luciferase immunoprecipitation assay. Results Observed molecular heterogeneity could be explained by sampling of anatomically distinct sub-regions of the excised ganglia consistent with neurally-enriched and non-neural, i.e. Schwann cell, enriched subregions. The levels of HSV-1 transcripts detected in trigeminal ganglia correlated with blood levels of HSV-1 antibodies. Multiple migraine susceptibility genes were strongly expressed in neurally-enriched trigeminal samples, while others were expressed in blood vessels. Conclusions These data provide a comprehensive human trigeminal transcriptome and a framework for evaluation of inhomogeneous post-mortem tissues through extensive quality control and refined downstream analyses for RNA-Seq methodologies. Expression profiling of migraine susceptibility genes identified by genetic association appears to emphasize the blood vessel component of the trigeminovascular system. Other genes displayed enriched expression in the trigeminal compared to dorsal root ganglion, and in-depth transcriptomic analysis of the KCNK18 gene underlying familial migraine shows selective neural expression within two specific populations of ganglionic neurons. These data suggest that expression profiling of migraine-associated genes can extend and amplify the underlying neurobiological insights obtained from genetic association studies.

Keywords: RNA-Seq; herpes simplex virus; migraine; post-mortem; transcriptome; trigeminal ganglion.

Figure 1.

Experimental design and alignment to bacterial and viral genomes in human trigeminal ganglia. (a) Overall schema of experimental design. Trigeminal ganglia were sequenced, and these data were integrated with existing datasets to analyze migraine-related genes. (b) Initial alignment to the standard human genomic target sequence showed a high incidence of reads that were of good quality but failed to align to any queried sequence. The incidence of these high quality unaligned reads was inversely correlated with RIN, indicating the presence of contamination in the low RIN samples from sequences not contributed by the human genome. After the addition of additional target sequences, including microbial and viral genomes, the correlation was abolished and the percentage of these reads was substantially reduced. (c) The percentage of reads aligning to bacterial genomes inversely correlates with RIN, with as much as ~ 12% of RNA in some samples contributed by bacteria. (d) Approximately 76% of bacterial reads were contributed by E. coli, which is normally present within the human enteric gut. (e) A small number of reads aligning to viral genomes were detected, with 14.9% contributed by human endogenous retrovirus, and 80.3% contributed by Human herpesvirus 1.

Figure 2.

Human herpes virus 1 (HSV-1) antibody levels from whole blood and transcript levels of HSV-1 from human post-mortem trigeminal ganglia. Antibody levels for gG-1, a serological HSV-1 target, were measured using the supernatants from whole cadaveric blood obtained from 13 patients. Transcript levels for HSV-1 were determined from trigeminal RNA-Seq data for each subject. (a) The dotted line represents the cutoff value for HSV-1 seropositivity based on our previously published study (26). Six of the 13 were HSV-1 seropositive, and five of those six had detectable HSV-1 reads via RNA-Seq. The limit of quantitation was 1 read. ND = not detectable for HSV-1 reads. There was a significant correlation between HSV-1 reads and antibody levels from LIPS (rs = 0.833, p < 0.001, Spearman Correlation). (b) There was a significant difference in average antibody levels between the HSV-1-negative and HSV-1-positive groups as determined by RNA-Seq (p = 0.002, Mann Whitney U Test). Error bars represent standard error of the mean. The reads aligning to the HSV-1 genome come almost exclusively from the LAT transcript (c) which is the main transcript produced while HSV-1 is latent in the trigeminal ganglia. This suggests that none of the tissue donors had actively replicating HSV-1 at the time of death.

Figure 3.

Segregation of trigeminal ganglion samples containing primarily neuronal, Schwann cell/axonal, or connective tissues based on marker gene expression and correlation analysis. (a) Human TG was stained for Neurofilament (scale bar represents 5 mm). There are three distinct cell types present in the tissue section: (b) neuronal, (c) Schwann, and (d) other (scale bar represents 100 μm). This represents the high inhomogeneity of trigeminal ganglion and presents a clear problem when extracting RNA from only a few small pieces of the larger tissue. (e) Differential covariance analysis was performed using all genes expressed over 1 sFPKM, using the MAGIC pipeline to generate correlation coefficients, subsequently sorted using heatmap.2 in R, resulting in two well-separated clusters of samples with one outlier (TG8). (f) A panel of selective marker genes were chosen to identify the cell types within each cluster of samples. All of the neuronal marker genes were enriched in the eight samples in the first cluster (Neural cluster), while the non-neural markers of Schwann cells (such as MPZ and MBP) were present in both clusters (Non-neural cluster). TG9 and TG14 are highly correlated, and both express high levels of neuronal marker genes.

Figure 4.

Differential expression of genes in neural enriched trigeminal ganglia vs. trigeminal containing mostly non-neural cells. The eight most neurally-enriched samples (Neural: TG1, TG3, TG5, TG9, TG11, TG13, TG14, TG16) were compared to the samples that showed enrichment for Schwann cell markers without a strong neural transcriptomic signature (Non-neural: TG2, TG4, TG6, TG7, TG10, TG12, TG22). Differential expression was plotted for strongly differentially expressed neural genes with scores ≥83, as well as significantly differential non-neural genes with scores > 105 (a) showing several known markers of neurons enriched in the neural sample subset relative to the non-neural subset. TRPVI is highlighted as one such strongly differential gene (score = 134). Staining for the TRPV1 protein shows strong expression in a subset of neurons, with no expression in non-neural cells ((b); scale bar represents 250 μm). Dense staining is observed in small diameter neurons, which are likely thermosensitive C-fibers ((c); scale bar represents 100 μm). Based on the observation that the five selected “non-neural” containing trigeminal samples expressed high levels of Schwann cell markers, we compared the top 125 genes in neural and non-neural subsets to expression in DRG tissue homogenate and sciatic nerve tissue homogenate as described by Sapio, et al. 2016 (d). Genes highly enriched in the DRG relative to the sciatic nerve are mostly neural genes, whereas sciatic nerve enriched genes are largely markers of Schwann cells and connective tissue. The majority of genes enriched in the neural subset were also enriched in the DRG relative to the sciatic nerve. Conversely, the majority of genes enriched in the non-neural subset were also enriched in the sciatic nerve relative to the DRG.

Figure 5.

Expression patterns and enrichment of migraine and trigeminal pain genes. Gene expression from trigeminal datasets (sFPKMs) was compared to the data available in the GTEx database (RPKMs, divided by vertical white space). Data were normalized so that each value in a row is expressed as a fraction of the maximum in that row, and colored according to the flame scale (bottom). Enrichment of migraine genes in tissues was ranked from left to right. Subsequently, datasets were reordered so that the vascular and brain datasets were grouped together. The top 32 enriched datasets are plotted, along with skeletal muscle and whole blood, which are included for comparison. Whole blood shows very little enrichment for any migraine gene. Genes were loosely categorized into clusters based on enrichment in trigeminal, vascular tissue, or brain (right labels). A literature review was performed to categorize the genes as directly acting on neural cells (green) versus those genes that, when mutated, cause vascular defects or abnormalities (purple).

Figure 6.

Selected differentially expressed genes between rat trigeminal and dorsal root ganglia. Trigeminal (TG) and dorsal root ganglia (DRG) transcriptomic datasets were compared to look for highly enriched genes in each. Several neural ion channels responsible for conducting nociceptive inputs are differentially enriched in trigeminal ganglia relative to DRG (top row), while several neuropeptides, including the mRNA encoding the Calcitonin Gene-related Peptide precursor, are enriched in DRG (Calca, Calcb, Sst). Several other proteins are equal in both datasets (Tac1, Trpv1).

Figure 7.

Expression profiling of the migraine susceptibility gene KCNK18 in human, rat and mouse sensory ganglia. The trigeminally-enriched potassium channel, KCNK18 is more highly expressed in the neural-enriched human trigeminal samples (a), and in the rat DRG relative to the sciatic nerve (b), suggesting a highly neural distribution with little expression in non-neural ganglia cells. In the mouse DRG, Kcnk18 is expressed in several populations of neurons, previously classified in Usoskin et al. (2015). Two subclasses of neurons express both Kcnk18 and Calca, which encodes the precursor to the calcitonin gene related peptide (CGRP)(top panel, (c); subclasses demarked by bracket). These cells, which encode both the potassium channel mutated in some patients with familial migraine, and which release CGRP peptide, which has been implicated in migraine, are potentially the subpopulation of cells by which this mutation causes migraine. Mrgprd and Mrgpra3 are additional markers of the cells co-expressing these two genes. These cells also contain broader non-specific markers (middle panel, (c)) such as Trpa1, Scn9a, and P2rx3 whereas Trpv1, Tac1 and Oprm1 are largely in a separate population of cells (bottom panel, (c)). Using t-distributed stochastic neighbor embedding (t-SNE) plots (D-F), we show the colocalization of Kcnk18 and Calca transcripts in mouse DRG neurons sequenced in Li et al. (2016). Points represent cells in the database, and cells with similar gene expression are clustered together in the plot. Gene expression for Calca is high across many cells (d), compared to the more restricted expression of Kcnk18 (e). A cluster of cells ((f), gold cells) have high expression of Calca (100 FPKM) and also express Knck18 (5 FPKM), further implicating this population of neurons in migraine.