Leah Catherine Solberg Woods

Background: Rats are a powerful model organism for genome-wide association studies (GWAS) of physiological and behavioral traits. Heterogeneous Stock (HS) rats, which were created by intercrossing 8 inbred rat strains, are heavily used for this purpose because of their genetic diversity and accumulated recombinations due to over 100 generations of outbreeding, which improves mapping precision. However, functional genomics resources for post-GWAS interpretation of genomic findings have been limited. Results: We processed 3,160 RNA-seq samples, 1,932 of which were previously unpublished, from 13 tissues as part of RatGTEx. We used Pantry to quantify six modalities of transcriptome regulation (expression, isoform ratio, intron excision ratio, alternative transcription start site (TSS), alternative poly(A), and RNA stability) in each sample, which allowed us to identify 144,876 independent quantitative trait loci (QTL) for 22,599 genes. We performed a transcriptome-wide association study (TWAS) with 19,796 unique genes across tissues and transcriptional modalities and 308 traits collected from 14 studies using FUSION, which identified 7,473 significant gene-trait associations. Conclusions: This work presents a significant expansion of the RatGTEx resource in terms of samples, modes of transcriptome regulation, and genetic analyses. Our findings provide molecular evidence for the involvement of specific genes in mediating GWAS signals through a range of transcriptome regulatory mechanisms. These gene-to-phenotype relationships can be easily applied to genetic analyses in humans and other species. Further experiments can use this evidence to confirm these molecular mediators and target them for the treatment of behavioral and physiological traits and disorders. To facilitate use of these results, we have updated results on RatGTEx.org and provided a new interactive TWAS web portal ( twas.ratgtex.org ).

Cocaine use disorder (CUD) is a major public health crisis. The specific genes mediating CUD remain largely unknown. We conducted a genome-wide association study (GWAS) using outbred N/NIH Heterogeneous Stock (HS; n = 836, female = 415, male = 421) rats. We examined CUD-related phenotypes including acquisition of self-administration, escalation of intake, and compulsive-like responding. These traits were phenotypically correlated and exhibited modest SNP heritability (h ² = 0.07 – 0.16). We identified six genome-wide significant associations (>-log ₁₀ (p)=5.58; α = 0.05 by permutation). One locus on chromosome 19 was associated with variable time between cocaine infusions (post infusion interval) and contains several carboxylesterase genes that are orthologous to the human CES1 gene. Notably, carboxylesterases metabolize cocaine. Three non-synonymous coding variants in Ces1c and Ces1d were in perfect linkage disequilibrium with this locus. The other five loci contained promising coding and expression variants, including Trak2 , a gene previously associated with CUD in human GWAS and Slc10a7 , Plcl1 , and Satb2 which have been associated with alcohol and tobacco use disorder. This is the largest genetic study of cocaine self-administration ever conducted in rats. Our results replicate previous loci associated with CUD in humans and provide several novel biological insights including the potential of pharmacological strategies targeting carboxylesterases.

The intestinal microbiome influences health and disease. Its composition is affected by host genetics and environmental exposures. Understanding host genetic effects is critical but challenging in humans, due to the difficulty of detecting, mapping and interpreting them. To address this, we analyse host genetic effects in four cohorts of outbred laboratory rats exposed to distinct but controlled environments. We show that polygenic host genetic effects are consistent across cohort environments. We identify three replicated microbiome-associated loci, one of which involves the sialyltransferase gene St6galnac1 and Paraprevotella . We find a similar association in a human cohort, between ST6GAL1 and Paraprevotella , both of which have been linked with immune and infectious diseases. Moreover, we find indirect (i.e. social) genetic effects on microbiome phenotypes, which substantially increase the total genetic variance. Finally, we identify a novel mechanism whereby indirect genetic effects can contribute to “missing heritability”.

Background: Cocaine use disorder (CUD) is a major public health crisis with detrimental individual and societal effects. The specific genes mediating CUD remain largely unknown. Methods: We conducted a genome-wide association study (GWAS) using outbred N/NIH Heterogeneous Stock (HS; n = 836, female = 415, male = 421) rats. We examined multiple CUD-related phenotypes that captured acquisition of self-administration, escalation of intake, and compulsive-like responding. Results: Consistent with the existing literature, these traits were phenotypically and genetically correlated and exhibited modest heritability (h ² = 0.07 – 0.16). We identified six genome-wide significant associations. One locus on chromosome 19 was associated with the variable time between cocaine infusions (post infusion interval) and contains several carboxylesterase genes that are orthologous to the human CES1 gene; notably, carboxylesterases metabolize cocaine. Three non-synonymous coding variants in the genes Ces1c and Ces1d were in perfect linkage disequilibrium with this locus, suggesting that one or more of them might be the causal SNP. The other 5 loci also contained promising coding and expression variants, including Trak2, a gene previously associated with CUD in human GWAS and Slc10a7 , Plcl1 , and Satb2 which have been associated with alcohol and tobacco use disorder. Conclusions: This is the largest genetic study of cocaine self-administration ever conducted in any species. Our results replicate previous loci associated with CUD in humans and provide several novel biological insights including the potential of pharmacological strategies targeting carboxylesterases for the treatment of CUD.

G protein-coupled receptor kinase 5 (GRK5) is implicated in the pathogenesis of obesity in both humans and rodent models. Our previous work demonstrated that genetic deletion or pharmacological inhibition of GRK5 suppresses 3T3-L1 adipocyte differentiation. Here, we assessed the small-molecule GRK5 inhibitor, GRK5-IN-2, for its effects on metabolic tissues and therapeutic potential in a diet-induced obesity mouse model. Mice were fed a high-fat diet for 8 weeks to induce obesity, followed by continued a high-fat diet with oral administration of GRK5-IN-2 (25 or 50 mg/kg) or water vehicle, five days per week for an additional 16 weeks. GRK5-IN-2 treatment had no effect on body weight, fat/lean mass, insulin tolerance, food intake, or energy expenditure but significantly reduced hepatic triglyceride accumulation and de novo lipogenesis. A follow-up study using 25 mg/kg of GRK5-IN-2 confirmed no effect on adiposity but reduced hepatic triglycerides. GRK5-IN-2 treatment decreased expression of the lipogenic gene Acc2 while upregulating lipid utilization proteins COXIV and ACSL1 in the liver, likely contributing to lower triglyceride levels. Together, these findings suggest that GRK5 inhibition selectively modulates hepatic lipid metabolism without altering systemic metabolic parameters, highlighting GRK5 as a potential therapeutic target for fatty liver disease.

We have previously demonstrated that a transmembrane domain mutation in Adenylate cyclase 3 (Adcy3) causes increased adiposity and negative emotion‐like behaviors in a rat model. We set out to replicate and expand upon our previous study by conducting comprehensive behavioral testing, and we also investigated the molecular changes that result from this mutation. Rats with a mutation in the second transmembrane helix of ADCY3 (Adcy3^mut/mut) and wild‐type rats were fed a high‐fat diet for 12 weeks. We measured body weight, body composition, and depression‐like and anxiety‐like behaviors using the following tests: sucrose splash test, sucrose preference test, forced swim test, open field test, elevated plus maze, successive alleys test, and novelty‐suppressed feeding. We also measured serum leptin levels, hypothalamic cyclic AMP (cAMP) production, and membrane fraction ADCY3 content. Adcy3^mut/mut male and female rats had increased adiposity. Adcy3^mut/mut males showed increased despair‐ and anxiety‐like behaviors, food seeking, and higher leptin levels relative to wild‐type males. Adcy3^mut/mut females showed only mildly increased anxiety‐like behaviors relative to wild‐type females. Adcy3^mut/mut rats of both sexes had decreased cAMP production in the hypothalamus, with no changes in ADCY3 content in the membrane fraction. We conclude that the transmembrane domain of ADCY3 plays a critical role in regulating adiposity and behavior, as well as cAMP production. There were key differences between males and females for the observed phenotypes. This study supports the idea that Adcy3 contributes to emotion‐like behaviors and potentially mental health disorders, and that the transmembrane domain of ADCY3 is important for protein function.

We have previously demonstrated that a transmembrane domain mutation in Adenylate cyclase 3 ( Adcy3 ) causes increased adiposity and negative emotion-like behaviors in a rat model. We set out to replicate and expand upon our previous study by conducting comprehensive behavioral testing, and we also investigated the molecular changes that result from this mutation. Rats with a mutation in the second transmembrane helix of ADCY3 (Adcy3 ^mut/mut ) and wild-type rats were fed a high-fat diet for 12 weeks. We measured body weight, body composition, and depression-like and anxiety-like behaviors using the following tests: sucrose splash test, sucrose preference test, forced swim test, open field test, elevated plus maze, successive alleys test, and novelty-suppressed feeding. We also measured serum leptin levels, hypothalamic cyclic AMP (cAMP) production, and membrane fraction ADCY3 content. Adcy3 ^mut/mut male and female rats had increased adiposity. Adcy3 ^mut/mut males showed increased despair- and anxiety-like behaviors, food seeking, and higher leptin levels relative to wild-type males. Adcy3 ^mut/mut females showed only mildly increased anxiety-like behaviors relative to wild-type females. Adcy3 ^mut/mut rats of both sexes had decreased cAMP production in the hypothalamus, with no changes in ADCY3 content in the membrane fraction. We conclude that the transmembrane domain of ADCY3 plays a critical role regulating adiposity and behavior, as well as cAMP production. There were key differences between males and females for the observed phenotypes. This study supports the idea that Adcy3 contributes to emotion-like behaviors and potentially mental health disorders, and that the transmembrane domain of ADCY3 is important for protein function.

The intestinal microbiome influences health and disease. Its composition is affected by host genetics and environmental exposures. Understanding host genetic effects is critical but challenging in humans, due to the difficulty of detecting, mapping and interpreting them. To address this, we analysed host genetic effects in four cohorts of outbred laboratory rats exposed to distinct but controlled environments. We found that polygenic host genetic effects were consistent across environments. We identified three replicated microbiome-associated loci, one of which involved a sialyltransferase gene and Paraprevotella . We found a similar association in a human cohort, between ST6GAL1 and Paraprevotella , both of which have been linked with immune and infectious diseases. Moreover, we found evidence of indirect genetic effects on microbiome phenotypes, which substantially increased their total genetic variance. Finally, we identified a novel mechanism whereby indirect genetic effects can contribute to “missing heritability”.

Addiction vulnerability is associated with the tendency to attribute incentive salience to reward predictive cues. Both addiction and the attribution of incentive salience are influenced by environmental and genetic factors. To characterize the genetic contributions to incentive salience attribution, we performed a genome‐wide association study (GWAS) in a cohort of 1596 heterogeneous stock (HS) rats. Rats underwent a Pavlovian conditioned approach task that characterized the responses to food‐associated stimuli (“cues”). Responses ranged from cue‐directed “sign‐tracking” behavior to food‐cup directed “goal‐tracking” behavior (12 measures, SNP heritability: 0.051–0.215). Next, rats performed novel operant responses for unrewarded presentations of the cue using the conditioned reinforcement procedure. GWAS identified 14 quantitative trait loci (QTLs) for 11 of the 12 traits across both tasks. Interval sizes of these QTLs varied widely. Seven traits shared a QTL on chromosome 1 that contained a few genes (e.g., Tenm4, Mir708) that have been associated with substance use disorders and other psychiatric disorders in humans. Other candidate genes (e.g., Wnt11, Pak1) in this region had coding variants and expression‐QTLs in mesocorticolimbic regions of the brain. We also conducted a Phenome‐Wide Association Study (PheWAS) on addiction‐related behaviors in HS rats and found that the QTL on chromosome 1 was also associated with nicotine self‐administration in a separate cohort of HS rats. These results provide a starting point for the molecular genetic dissection of incentive motivational processes and provide further support for a relationship between the attribution of incentive salience and drug abuse‐related traits.

Previous studies have identified G protein-coupled receptor (GPCR) kinase 5 (GRK5) as a genetic factor contributing to obesity pathogenesis, but the underlying mechanism remains unclear. We demonstrate here that Grk5 mRNA is more abundant in stromal vascular fractions of mouse white adipose tissue, the fraction that contains adipose progenitor cells, or committed preadipocytes, than in adipocyte fractions. Thus, we generated a GRK5 knockout (KO) 3T3-L1 preadipocyte to further investigate the mechanistic role of GRK5 in regulating adipocyte differentiation. During adipogenic stimulation, GRK5 KO preadipocytes had decreased lipid accumulation and delayed mature adipocyte development compared to wildtype cells coupled with suppressed adipogenic and lipogenic gene expression. Beside GPCR signaling, RNA sequencing and pathway analysis identified insulin-like growth factor 1 (IGF-1) signaling to be one of the top 5 significantly dysregulated pathways in GRK5 KO cells. GRK5 KO cells also displayed decreased insulin-stimulated ERK phosphorylation, a downstream target of insulin-stimulated IGF-1 receptor activation, suggesting that GRK5 acts through this critical pathway to impact 3T3-L1 adipocyte differentiation. To find a more translational approach, we identified a new small molecule GRK5 inhibitor that was able to reduce 3T3-L1 adipogenesis. These data suggest that GRK5 is required for adipocyte differentiation through IGF-1 receptor/ERK activation and may be a promising translational target for obesity.