Supplementary Methods and Results from Cannabinoid receptor 2 evolutionary gene loss makes parrots more susceptible to neuroinflammation

In vertebrates, cannabinoids modulate neuroimmune interactions through two cannabinoid receptors (CNRs) conservatively expressed in the brain (CNR1, syn. CB1) and in the periphery (CNR2, syn. CB2). Our comparative genomic analysis indicates several evolutionary losses in the CNR2 gene that is involved in immune regulation. Notably, we show that the CNR2 gene pseudogenised in all parrots (Psittaciformes). This CNR2 gene loss occurred due to chromosomal rearrangements. Our positive selection analysis suggests the absence of any specific molecular adaptations in parrot CNR1 that would compensate for the CNR2 loss in the modulation of the neuroimmune interactions. Using transcriptomic data from the brains of birds with experimentally induced sterile inflammation we highlight possible functional effects of such a CNR2 gene loss. We compare the expression patterns of CNR and neuroinflammatory markers in CNR2-deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2-intact passerines (represented by the zebra finch, Taeniopygia guttata). Unlike in passerines, stimulation with lipopolysaccharide resulted in the parrots in neuroinflammation linked with a significant upregulation of expression in proinflammatory cytokines (including interleukin 1 beta, IL1B and IL6) in the brain. Our results indicate the functional importance of the CNR2 gene loss for increased sensitivity to brain inflammation

Supplementary Data from Cannabinoid receptor 2 evolutionary gene loss makes parrots more susceptible to neuroinflammation

Table S21: Putative presence or absence of given immune gene (based on GO term: “negative regulation of inflammatory response“, GO:0050728) based on Avian Immunome database (AVIMM, Mueller et. al. 2020). Chicken gene annotation is used. Gene – gene name abbreviation, Description – full gene name. Putative presence or absence of given gene is shown for each species by the name of database where sequence was retrieved or NA, respectively. Data in AVIMM were retrieved from the following public repositories: ENSEMBL, Uniprot or The Bird 10,000 Genomes project (b10k). The following avian species were included: parrots (Melopsittacus undulatus, Eolophus roseicapillus, Probosciger aterrimus, Amazona guildingii, Agapornis roseicollis, Nestor notabilis) and passerines (Corvus moneduloides, Ficedula albicollis, Hirundo rustica, Lepidothrix coronata, Lonchura striata, Molothrus ater, Parus major, Passer domesticus, Serinus canaria, Sturnus vulgaris, Taeniopygia guttata, Zonotrichia albicollis, Zosterops hypoxanthus). Reference: Mueller RC, Mallig N, Smith J et al. Avian Immunome DB: an example of a user-friendly interface for extracting genetic information. BMC Bioinformatics 2020;21:1–16.. Table S22: Gene expression data. RT-qPCR Cp values for individual genes investigated; E1-3 = experiment 1-3