A study of the impact of MIA on the transcript isoforms was undertaken in recognition that neuropeptides and their receptors play an essential role in various physiological functions. Yet, the activity of these molecules depends on the transcribed isoform. Transcript isoform profiles from neuropeptide, corresponding receptors and genes connected through protein-protein interactions were studied in previously unpublished hippocampus samples and complemented with published amygdala samples [15] from pigs exposed to MIA relative to controls. This porcine model is suitable for MIA due to the many similarities to human physiology and the immune system [22]. The Porcine Reproductive and Respiratory Syndrome virus used to expose pigs to MIA belongs to the Arteriviridae family within the Nidovirales order, and this order includes the Coronaviridae family [23]. Viruses from these families and order have high incidence, causing colds and severe respiratory tract infections in human populations. These viruses can elicit immune activation in gestating females, and can cause significant financial cost to the swine industry due to reproductive failure and respiratory disease [24].

The experimental design and animal care protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois and were previously described [15, 25]. Briefly, pregnant gilts were intranasally inoculated with the Porcine Reproductive and Respiratory Syndrome virus (5 mL of 1 $\times$ 10${}^5$ TCID${}_{50}$, strain P129-BV, Purdue University, West Lafayette, IN, USA) on day 76 of gestation to induce MIA. Control gilts were inoculated with sterile Dulbecco’s modified Eagle medium on the same day. The viral inoculation protocol elicits MIA characterized by elevated body temperature and reduced feed intake and recovery two weeks after inoculation [25]. The viral-inoculated gilts received maximum daily feeding daily, and the control gilts were fed an amount equal to that consumed by immune-challenged gilts the day before. After farrowing at approximate gestation day 113, the offspring remained with their mothers and nursed, until an equal number of female and male pigs were anesthetized and euthanized at 22 days of age as previously described [15]. The hippocampus and amygdala were recognized following anatomical markers in the pig brain atlas [26] and removed, and immediately flash frozen on dry ice.

For each brain region, RNA was isolated using EZNA isolation kit following the manufacturer’s instructions (Omega Biotek, Norcross, GA, United States). The hippocampus and amygdala RNA integrity was at least 7.5 and the RNA-Seq libraries were prepared with TruSeq Stranded mRNAseq Sample Prep kit’ (Illumina Inc, San Diego, CA). The individual pig libraries of the hippocampus (n = 48) and amygdala (n = 24) were sequenced in two lanes on a NovaSeq 6000 resulting in 150 nt long paired-end reads. Prior analysis of the amygdala expression patterns at the gene level identified differential abundance on a handful of neuropeptide and receptor genes, yet insufficient to support the enrichment of the Gene Ontology neuropeptide signaling pathways [15]. The present study broadens our previous gene-level analysis into the transcript isoform profiles in the amygdala and the hippocampus, another brain region that modulates behavior. We found that transcript isoforms provided more accurate gene networks than gene-level abundance [27].

The hippocampus and amygdala RNA-seq sequence reads were mapped to the Sus scofa genome v11.1 genome using Kallisto v0.43.0 with default settings [28]. Previously unpublished transcript isoform from neuropeptide and neuropeptide receptor genes from the hippocampus and amygdala were identified and analyzed. For each region, the count of each transcript was analyzed using a negative binomial generalized linear model of MIA, sex and the interaction between MIA and sex. The statistical analyses were conducted using SAS v9.4 (SAS Institute Inc., Cary, NC, United States). A preliminary analysis of the transcript isoforms corresponding to 185 neuropeptide and receptor genes demonstrated that less than 5% presented significant interaction effects, and therefore interaction effects are not reported. Of interest was the effect of MIA on the transcript isoform profiles, irrespectively of the sex; therefore, only the estimates of the effect of MIA, adjusted for sex effects, are presented.

Protein-protein and other molecular interaction databases were searched for genes known to connect neuropeptides and receptors that had transcript isoforms impacted by MIA in each brain region using the BisoGenet plugin [29] in the Cytoscape environment [30]. The visualization of the molecular relationships employed a maximum of two connections between neuropeptide-related genes based on the human gene and protein interactions identified by BisoGenet. Transcript isoforms of connecting pathway genes were analyzed for MIA effects using the same neuropeptide and receptor genes.

The hippocampus study yielded 207 transcript isoforms identified from 97 neuropeptide genes, 196 transcript isoforms from 87 neuropeptide receptor genes, and 211 transcript isoforms from 41 connecting pathway genes. Significant MIA effects (P-value 0.01) were detected in 12, 9, and 8 neuropeptide, neuropeptide receptors, and connecting pathway gene transcript isoforms. Only IGF1 and phospholipase C gamma 1 (PLCG1) had two significant transcript isoforms in the hippocampus. Supplementary Table 1 lists the differential expression between treatments of all transcript isoforms and brain regions.

The amygdala study yielded 207 transcript isoforms identified from 97 neuropeptide genes, 196 transcript isoforms from 87 neuropeptide receptor genes, and 178 transcript isoforms from 49 connecting pathway genes. Significant MIA effect (P-value 0.01) were detected in 3, 4, and 2 neuropeptides, neuropeptide receptors, and connecting pathway transcript isoforms, respectively, in the amygdala. No gene had more than one significant transcript isoform in the amygdala.

Table 1 summarizes the number of transcript isoforms and genes differentially expressed (P-value 0.01) between MIA and control pigs within the brain region. Transcript isoforms from oxidized low-density lipoprotein receptor 1 (OLR1) and peptide YY (PYY) were significantly differentially expressed in MIA in both brain regions. Fig. 1 depicts the network of related neuropeptide, neuropeptide receptor, and connecting pathway genes in the hippocampus and amygdala, respectively.

Fig. 1.

Pathway of significantly differently expressed neuropeptide (triangle), receptor (square) and connecting (circle) genes in the A) amygdala and B) hippocampus. Gene symbol size represents the transcript isoform fold change between MIA and control pigs, and gene symbols with darker colors more significantly differential expression of transcript isoform than gene symbols with lighter colors.

The evaluation of the patterns of transcript isoform expression offered further insights into the impact of MIA. Tables 2 and 3 summarize the transcript’s hippocampus profiles isoforms over- and under-expressed in MIA relative to control pigs (P-value 0.01), while Table 4 reports matching information amygdala profiles.

In the hippocampus, 19 transcript isoforms were over-expressed in MIA relative to control pigs at (Table 2), whereas 8 transcript isoforms were under-expressed in MIA relative to control pigs at P-value 0.01 (Table 3). In the amygdala (Table 4), 4 transcript isoforms that were over-expressed in MIA relative to control pigs, whereas 5 transcript isoforms were under-expressed in MIA relative to control pigs at P-value 0.01.

In general, differentially expressed transcript isoforms (P-value 0.01) from the same gene followed the same pattern between treatment groups, and this pattern was consistent with that of other non-significant transcript isoforms from the same gene (Supplementary Table 1). Transcript isoforms that presented the same pattern in both brain regions but varying significance (P-value 0.10) included calcitonin-related polypeptide 1 (CRSP1), gonadotropin-releasing hormone (type 2) receptor 2 (GNRHR2), neuropeptide Y receptor Y1 (NPY1R), OLR1, pro-melanin concentrating hormone (PMCH), PYY, solute carrier family 40 member 1 (SLC40A1), and vasoactive intestinal peptide receptor 1 (VIPR1). Transcript isoforms from arginine vasopressin receptor 1B (AVPR1B), chromogranin B (CHGB), and dipeptidyl peptidase 4 (DPP4) presented the different expression profiles across brain regions but varying significance (P-value 0.10).

The present study validated our working hypothesis that a significant mode of action of MIA is through disruption of the levels of transcript isoform expression, which in turn influences the function and activity of molecules in the neuropeptide system. Our systematic study of over 200 transcript isoforms produced by genes in the neuropeptide system enabled us to expose various MIA effects within gene and across brain regions that are subsequently exemplified. Transcript isoforms were identified in the hippocampus and hypothalamus for most neuropeptide and receptor genes known in mammals [16, 17, 38, 39]. Transcript isoforms from neuropeptide Y receptor Y6 (NPY6R), insulin (INS) and urocortin 2 (UCN2) were identified in fewer than 5% of the pigs in both regions. Transcript isoforms from neuropeptides glucagon (GCG), urocortin 3 (UCN3), and urotensin 2B (UTS2B) were identified in the hippocampus of fewer than 5% of the pigs but were identified in a higher percentage of the amygdala samples. Similarly, transcript isoform from insulin-like 3 (INSL3), secretin receptor (SCTR) and urotensin 2 (UTS2) were identified in the amygdala of fewer than 5% of the pigs. Still, it was identified in a higher percentage of the hippocampus samples. These genes with low transcript isoform numbers also have very low or no expression in the human brain [40].

In addition to our transcript isoform-level discoveries, we confirmed reports of an association between numerous genes and MIA or MIA-related disorders. Many of the transcript isoforms presenting significant MIA effects are annotated to genes in Tables 2-4 that have differentially expressed genes between MIA and control pigs have been associated with neurological disorders. Some MIA associations with genes detected in the present study were also established at the DNA variant level. For example, single nucleotide polymorphism in or near ADCYAP receptor type I (ADCYAP1R1), NPS receptor (NPSR1), estrogen receptor 1 (ESR1), SRSF protein kinase 2 (SRPK2) genes have been associated with autism spectrum disorders, Alzheimer’s disease behavioral traits, anxiety, impulsivity and attention deficit-related traits [21, 54, 55, 56, 57, 58, 59].

Among the MIA effects bridging the hippocampus and amygdala, our results offer original evidence that MIA can disrupt the vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP). Significant effects of MIA were detected on transcript isoforms of the neuropeptide receptor ADCYAP1R1 and vasoactive intestinal peptide receptor 1 (VIPR1) receptors and the adenylate cyclase-activating polypeptide 1 (ADCYAP1) gene that produces PACAP27 and PACAP38 neuropeptides. However, no differential expression was detected with the VIP ligand and VIPR2. While the ADCYAP1R1, VPAC1, and VPAC2 interact with receptor-activity-modifying proteins (RAMPs) [60, 61], none of the RAMPs were differentially expressed. The VIP/PACAP signaling pathway is important in brain development, psychiatric illness and behavior [62, 63]. Altered behavior was observed in mouse lines knockout for ADCYAP1 and VIP prohormone genes [64, 65], and abnormal social behaviors have been recorded in ADCYAP1R1 deficient mice [66, 67]. ADCYAP1R1 is a member of the circadian entrainment pathway with multiple transcript isoforms presenting different ligand binding properties and signal transduction, therefore modulating the response to a vasoactive intestinal peptide (VIP) and PACAP [68, 69]. Comparison of the pig ADCYAP1R1 protein sequences identified that the different transcripts corresponded to the third intercellular loop that generate differential responses to PACAP-27 [70]. Compared to normal zebrafish (Danio renio), larvae lacking the hop exon had increased anxiety-like behavior [71, 72]. ADCYAP1 is a member of the circadian entrainment pathway and considered a potential target for therapies to ameliorate drug abuse, migraine, stress-related psychopathologies and inflammatory diseases [69, 73, 74, 75, 76, 77, 78]. In addition to the anti-inflammatory effect of VIPR1 [64], mutant mouse strains with inactivated VIPR1 showed a hippocampus-dependent deficit in fear conditions amygdala amygdala-dependent fear conditioning [79].

Peptidylglycine alpha-amidating monooxygenase (PAM) and ESR1 are genes connecting MIA-dysregulated neuropeptides and receptor transcript isoforms in the networks depicted in Fig. 1. PAM is responsible for the post-translational amidation of neuropeptides that can affect bioactivity, and this monooxygenase [80, 81] was over-expressed in MIA compared to control pigs in the hippocampus and amygdala. Our results are consistent with a report that PAM was over-expressed in the neocortex of 56-day old mice prenatally exposed to influenza [82]. ESR1 served as a connector between neuropeptides genes natriuretic peptide A (NPPA) and platelet-derived growth factor subunit A (PDGFA) and receptors RXFP1, VIPR1, and neuropeptide Y receptor Y2 (NPY2R) and was over-expressed in MIA relative to control pigs in the hippocampus and amygdala. Sequence variants of ESR1 have been linked to impaired social interaction [58, 83].

A review of the impact of MIA in the hippocampus indicates significant effects on the transcript isoforms from genes, including neuropeptide arginine vasopressin (AVP), calcitonins, hypocretin neuropeptide precursor (HCRT), IGF1, MAS related GPR family member X2 (MRGPRX2), PLCG1, relaxin 3 (RLN3), and SLC40A1. The detection of MIA associations with the previously enumerated transcript isoforms agrees with findings on behavior disorders, including reports that HCRT regulates sleep, feeding behavior, wakefulness, emotion, and stress response [41]. Similarly, RLN3 influences feeding behavior, stress responses, anxiety and memory [84], and AVP is a regulator of social behavior [85, 86]. IGF1 has been associated with autism spectrum disorder symptoms [87, 88, 89], with IGF1-treatment proposed as an autism therapeutic intervention [88].

The over-expression of SLC40A1 in the hippocampus of MIA relative to control pigs suggests that transcript isoform dysregulation may be a neuro-inflammatory response to the MIA signaling [89]. SLC40A1 has been implicated in the incidence of autism spectrum disorders [90]. MRGPRX2 is a receptor with multiple neuropeptide ligands, including cortistatin-14, and this receptor’s role was noted in a review of molecular mechanisms of autism spectrum disorder [91].

Whereas two calcitonin genes, calcitonin related polypeptide alpha (CALCA) and calcitonin related polypeptide beta (CALCB), have been uncovered in human and rodents, four calcitonin genes (CRSP1, CRSP2, CRSP3 and CALCB) are known in pigs [39]. The associated transcript isoforms can code for numerous neuropeptides. The distinct evolutionary path of the CALCA gene family between pigs and other biomedical species challenges the direct alignment of published associations. Nevertheless, an overarching profile can be identified between the over-expression of the calcitonin transcript isoforms in the hippocampus of MIA relative to control pigs and published work in other species and MIA-related phenotypes. The N-terminal procalcitonin (NPCT) neuropeptide, cleaved from calcitonin producing CALCA transcript isoform, participates in an inflammatory response and is a potential human biomarker and therapeutic target for Alzheimer disease [92]. Also, calcitonin gene-related peptide 1 (alpha-CGRP), a neuropeptide produced from a different CALCA transcript isoform that produces NPCT, plays an important role in social interactions with autism spectrum disorders [92, 93, 94].

Significant effects of MIA amygdala transcript isoforms were detected on AVPR1B, DPP4, GNRHR2, growth hormone-releasing hormone receptor (GHRHR), islet amyloid polypeptide (IAPP), OLR1, and POMC. Neuropeptide IAPP is associated with motivated behaviors [85]. Participants in the neuroactive ligand-receptor interaction pathway also detected an association with MIA at the gene level [15]. Similar to PYY, this neuropeptide has been linked to changes in feeding patterns that are also observed in autism spectrum disorder patients with the over-expression of IAPP transcript isoforms in the amygdala of MIA pigs, this protein aggregates in amyloid deposits and the detection of IAPP in the brain has been associated with cognitive decline. Also over-expressed in the amygdala of MIA relative to control pigs was PMCH. This result substantiates reports that dysregulation of PMCH is associated with the development of affective disorders [95].

Among other transcript isoforms dysregulated by MIA in the amygdala, the detection of POMC is in agreement with our analysis at the gene level [15] and with reports of pre- and post-translational bioactive forms associated with behaviors observed in autism spectrum disorders [96]. AVPR1B has been associated with autism spectrum disorders and is a receptor involved in the AVP-mediated activation of the hypothalamic-pituitary-adrenal-axis [97, 98, 99]. Consistent with this known function, and AVPR1B transcript isoform was over-expressed in the amygdala of MIA relative to control pigs. The impact of MIA on the expression of a GHRH transcript isoform could relate to the established influence of this hormone neuropeptide in animal cognition [100, 101].

OLR1 (also known as lectin-like oxidized low-density lipoprotein receptor-1, LOX-1) participates in inflammatory responses [102] and agrees with these results OLR1 transcript isoform was over-expressed in the amygdala of MIA relative to control pigs. In the present study, DPP4 is a connecting pathway gene with a transcript isoform under-expressed in the amygdala of MIA relative to control pigs. The observed pattern agrees with the role of DPP4 in processing the neuropeptides substance P and NPY and immune response [103] and the proposed association with autism spectrum disorders [104]. In agreement with the under-expression of the neuropeptide insulin-like growth factor 2 (IGF2) transcript isoform in the amygdala of MIA relative to control pigs, IGF2 treatment can reverse behavior disorders such as anxiety-like phenotypes [105]. IGF2 has also been associated with autism spectrum disorders and similar phenotypes [105, 106].

Bioactive neuropeptides result from multi-tiered pre- and post-transcriptional and translational processes that can influence the profile of neuropeptides across brain regions and should be considered in understanding the impact of MIA on neuropeptide systems. This study focused on exposing the effect of MIA at the post-transcriptional stage. However, pre-transcriptional effects such as epigenetics and post-transcriptional effects such as post-translational modifications can further alter the neuropeptide and receptor system’s profile and activity. Methylation can alter glucocorticoid receptors’ expression in a tissue-specific manner [107]. High-throughput or candidate proteomic detection of neuropeptides is complex. The small size and complex post-translational modifications and differential cleavage in different tissues result in some neuropeptides’ differential occurrence, such as glucagon [108]. Our results demonstrated the impact of MIA on multiple isoform profiles corresponding to genes in the neuropeptide system that can modulate behavior disorders.