Eric J. Vallender

Accelerated Evolution of Nervous System Genes in the Origin of Homo sapiens

Human evolution is characterized by a dramatic increase in brain size and complexity. To probe its genetic basis, we examined the evolution of genes involved in diverse aspects of nervous system biology. We found that these genes display significantly higher rates of protein evolution in primates than in rodents. Importantly, this trend is most pronounced for the subset of genes implicated in nervous system development. Moreover, within primates, the acceleration of protein evolution is most prominent in the lineage leading from ancestral primates to humans. Thus, the remarkable phenotypic evolution of the human nervous system has a salient molecular correlate, i.e., accelerated evolution of the underlying genes, particularly those linked to nervous system development. In addition to uncovering broad evolutionary trends, our study also identified many candidate genes--most of which are implicated in regulating brain size and behavior--that might have played important roles in the evolution of the human brain.

Evidence that the adaptive allele of the brain size gene microcephalin introgressed into Homo sapiens from an archaic Homo lineage

At the center of the debate on the emergence of modern humans and their spread throughout the globe is the question of whether archaic Homo lineages contributed to the modern human gene pool, and more importantly, whether such contributions impacted the evolutionary adaptation of our species. A major obstacle to answering this question is that low levels of admixture with archaic lineages are not expected to leave extensive traces in the modern human gene pool because of genetic drift. Loci that have undergone strong positive selection, however, offer a unique opportunity to identify low-level admixture with archaic lineages, provided that the introgressed archaic allele has risen to high frequency under positive selection. The gene microcephalin (MCPH1) regulates brain size during development and has experienced positive selection in the lineage leading to Homo sapiens. Within modern humans, a group of closely related haplotypes at this locus, known as haplogroup D, rose from a single copy ≈37,000 years ago and swept to exceptionally high frequency (≈70% worldwide today) because of positive selection. Here, we examine the origin of haplogroup D. By using the interhaplogroup divergence test, we show that haplogroup D likely originated from a lineage separated from modern humans ≈1.1 million years ago and introgressed into humans by ≈37,000 years ago. This finding supports the possibility of admixture between modern humans and archaic Homo populations (Neanderthals being one possibility). Furthermore, it buttresses the important notion that, through such adminture, our species has benefited evolutionarily by gaining new advantageous alleles. The interhaplogroup divergence test developed here may be broadly applicable to the detection of introgression at other loci in the human genome or in genomes of other species.

Functional characterization of the human TPH2 5' regulatory region: untranslated region and polymorphisms modulate gene expression in vitro.

Tryptophan hydroxylase-2 (TPH2) is a recently identified TPH isoform responsible for neuronal serotonin (5-HT) synthesis, and TPH2 polymorphisms are associated with a range of behavioral traits and psychiatric disorders. This study characterized cis-acting elements and three common polymorphisms (−703G/T, −473T/A, and 90A/G) in the 5′ regulatory region of human TPH2 by using luciferase reporter assay, quantitative real-time PCR, and electrophoretic mobility shift assay (EMSA). The core promoter of human TPH2 was localized to the region between −107 and +7, and the segment of +8 to +53 within the 5′-UTR was found to exert a potent inhibitory effect on gene expression at both transcriptional and post-transcriptional levels. In both RN46A and HEK-293 cell lines, the TTA (−703T/−473T/90A) haplotype of the three polymorphisms showed the lowest gene expression compared with other haplotypes, and the −703G/T and −473T/A polymorphisms tended to exert a synergic effect on gene expression dependent upon the sequence of the 5′-UTR. In RN46A, the 90A/G polymorphism significantly increased luciferase activity and mRNA level irrespective of the other two polymorphisms, while in HEK-293 cells the effect of 90A/G was dependent on the alleles at loci −703 and −473. EMSA showed that all the three polymorphisms potentially alter DNA–protein interactions, while the 90A/G polymorphism predictably alters the 5′-UTR secondary structure of mRNA and influences RNA–protein interactions. In conclusion, our present study demonstrates that both the 5′-UTR and common polymorphisms (especially the 90A/G) in the 5′ regulatory region of human TPH2 have a significant impact on gene expression.

Analysis of copy number variation in the rhesus macaque genome identifies candidate loci for evolutionary and human disease studies

Copy number variants (CNVs) are heritable gains and losses of genomic DNA in normal individuals. While copy number variation is widely studied in humans, our knowledge of CNVs in other mammalian species is more limited. We have designed a custom array-based comparative genomic hybridization (aCGH) platform with 385 000 oligonucleotide probes based on the reference genome sequence of the rhesus macaque (Macaca mulatta), the most widely studied non-human primate in biomedical research. We used this platform to identify 123 CNVs among 10 unrelated macaque individuals, with 24% of the CNVs observed in multiple individuals. We found that segmental duplications were significantly enriched at macaque CNV loci. We also observed significant overlap between rhesus macaque and human CNVs, suggesting that certain genomic regions are prone to recurrent CNV formation and instability, even across a total of approximately 50 million years of primate evolution ( approximately 25 million years in each lineage). Furthermore, for eight of the CNVs that were observed in both humans and macaques, previous human studies have reported a relationship between copy number and gene expression or disease susceptibility. Therefore, the rhesus macaque offers an intriguing, non-human primate outbred model organism with which hypotheses concerning the specific functions of phenotypically relevant human CNVs can be tested.

Rhesus Monkey Trace Amine-Associated Receptor 1 Signaling: Enhancement by Monoamine Transporters and Attenuation by the D2 Autoreceptor in Vitro

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor that directly responds to endogenous monoamines as well as amphetamine-related psychostimulants, including methamphetamine. In the present study, we demonstrate TAAR1 mRNA and protein expression in rhesus monkey brain regions associated with monoaminergic systems, variable cellular distribution of TAAR1 in rhesus monkey brain, and TAAR1 coexpression with the dopamine transporter (DAT) in a subset of dopamine neurons in both rhesus monkey and mouse substantia nigra. On this basis, we evaluated rhesus monkey TAAR1 activation by different compounds and its functional relation with monoamine transporters and the dopamine D2 receptor (D2) short isoform (D2s) autoreceptor in vitro using a cAMP response element-luciferase assay. TAAR1 activation by monoamines and amphetamine-related compounds was greatly enhanced by coexpression of dopamine, norepinephrine, or serotonin transporters, and the activation enhancement was blocked by monoamine transporter inhibitors. This enhancement did not occur in control experiments in which the dopamine D1 receptor (D1) was substituted for TAAR1. Furthermore, activation of TAAR1 by dopamine was completely inhibited by D2s when coexpressed with TAAR1, and this inhibition was blocked by the D2 antagonist raclopride. Last, dopamine activation of TAAR1 could induce c-FOS-luciferase expression but only in the presence of DAT, whereas dopamine activation of D1 resulted in equivalent c-FOS expression in the presence or absence of DAT. Together, these data reveal a broad agonist spectrum for TAAR1, a functional relation of TAAR1 with monoamine transporters and D2s, and a mechanism by which D2 receptor drugs can influence brain monoaminergic function and have efficacy through affecting TAAR1 signaling.

Genetic basis of human brain evolution

Human evolution is characterized by a rapid increase in brain size and complexity. Decades of research have made important strides in identifying anatomical and physiological substrates underlying the unique features of the human brain. By contrast, it has become possible only very recently to examine the genetic basis of human brain evolution. Through comparative genomics, tantalizing insights regarding human brain evolution have emerged. The genetic changes that potentially underlie human brain evolution span a wide range from single-nucleotide substitutions to large-scale structural alterations of the genome. Similarly, the functional consequences of these genetic changes vary greatly, including protein-sequence alterations, cis-regulatory changes and even the emergence of new genes and the extinction of existing ones. Here, we provide a general review of recent findings into the genetic basis of human brain evolution, highlight the most notable trends that have emerged and caution against over-interpretation of current data.

Normal thermoregulatory responses to 3‐iodothyronamine, trace amines and amphetamine‐like psychostimulants in trace amine associated receptor 1 knockout mice

3‐Iodothyronamine (T1AM) is a metabolite of thyroid hormone. It is an agonist at trace amine‐associated receptor 1 (TAAR1), a recently identified receptor involved in monoaminergic regulation and a potential novel therapeutic target. Here, T1AM was studied using rhesus monkey TAAR1 and/or human dopamine transporter (DAT) co‐transfected cells, and wild‐type (WT) and TAAR1 knock‐out (KO) mice. The IC50 of T1AM competition for binding of the DAT‐specific radio‐ligand [3H]CFT was highly similar in DAT cells, WT striatal synaptosomes and KO striatal synaptosomes (0.72–0.81 μM). T1AM inhibition of 10 nM [3H]dopamine uptake (IC50: WT, 1.4 ± 0.5 μM; KO, 1.2 ± 0.4 μM) or 50 nM [3H]serotonin uptake (IC50: WT, 4.5 ± 0.6 μM; KO, 4.7 ± 1.1 μM) in WT and KO synaptosomes was also highly similar. Unlike other TAAR1 agonists that are DAT substrates, TAAR1 signaling in response to T1AM was not enhanced in the presence of DAT as determined by CRE‐luciferase assay. In vivo, T1AM induced robust hypothermia in WT and KO mice equivalently and dose dependently (maximum change degrees Celsius: 50 mg/kg at 60 min: WT −6.0 ± 0.4, KO −5.6 ± 1.0; and 25 mg/kg at 30 min: WT −2.7 ± 0.4, KO −3.0 ± 0.2). Other TAAR1 agonists including beta–phenylethylamine (β‐PEA), MDMA (3,4‐methylenedioxymethamphetamine) and methamphetamine also induced significant, time‐dependent thermoregulatory responses that were alike in WT and KO mice. Therefore, TAAR1 co‐expression does not alter T1AM binding to DAT in vitro nor T1AM inhibition of [3H]monoamine uptake ex vivo, and TAAR1 agonist‐induced thermoregulatory responses are TAAR1‐independent. Accordingly, TAAR1‐directed compounds will likely not affect thermoregulation nor are they likely to be cryogens.

Biogeography of the Intestinal Mucosal and Lumenal Microbiome in the Rhesus Macaque

The gut microbiome is widely studied by fecal sampling, but the extent to which stool reflects the commensal composition at intestinal sites is poorly understood. We investigated this relationship in rhesus macaques by 16S sequencing feces and paired lumenal and mucosal samples from ten sites distal to the jejunum. Stool composition correlated highly with the colonic lumen and mucosa and moderately with the distal small intestine. The mucosal microbiota varied most based on location and was enriched in oxygen-tolerant taxa (e.g., Helicobacter and Treponema), while the lumenal microbiota showed inter-individual variation and obligate anaerobe enrichment (e.g., Firmicutes). This mucosal and lumenal community variability corresponded to functional differences, such as nutrient availability. Additionally, Helicobacter, Faecalibacterium, and Lactobacillus levels in stool were highly predictive of their abundance at most other gut sites. These results quantify the composition and biogeographic relationships between gut microbial communities in macaques and support fecal sampling for translational studies.

Augmentation of methamphetamine-induced behaviors in transgenic mice lacking the trace amine-associated receptor 1.

The trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor that is functionally activated by amphetamine-based psychostimulants, including amphetamine, methamphetamine and MDMA. Previous studies have shown that in transgenic mice lacking the TAAR1 gene (TAAR1 knockout; KO) a single injection of amphetamine can produce enhanced behavioral responses compared to responses evoked in wild-type (WT) mice. Further, the psychostimulant effects of cocaine can be diminished by selective activation of TAAR1. These findings suggest that TAAR1 might be implicated in the rewarding properties of psychostimulants. To investigate the role of TAAR1 in the rewarding effects of drugs of abuse, the psychomotor stimulating effects of amphetamine and methamphetamine and the conditioned rewarding effects of methamphetamine and morphine were compared between WT and TAAR1 KO mice. In locomotor activity studies, both single and repeated exposure to d-amphetamine or methamphetamine generated significantly higher levels of total distance traveled in TAAR1 KO mice compared to WT mice. In conditioned place preference (CPP) studies, TAAR1 KO mice acquired methamphetamine-induced CPP earlier than WT mice and retained CPP longer during extinction training. In morphine-induced CPP, both WT and KO genotypes displayed similar levels of CPP. Results from locomotor activity studies suggest that TAAR1 may have a modulatory role in the behavioral sensitization to amphetamine-based psychostimulants. That methamphetamine-but not morphine-induced CPP was augmented in TAAR1 KO mice suggests a selective role of TAAR1 in the conditioned reinforcing effects of methamphetamine. Collectively, these findings provide support for a regulatory role of TAAR1 in methamphetamine signaling.

Trace Amine Associated Receptor 1 Signaling in Activated Lymphocytes

Although most research to date on Trace Amine Associated Receptor 1 (TAAR1) has focused on its role in the brain, it has been recognized since its discovery in 2001 that TAAR1 mRNA is expressed in peripheral tissues as well, suggesting that this receptor may play a role in non-neurological pathways. This study reports TAAR1 expression, signaling and functionality in rhesus monkey lymphocytes. We detected a high level of TAAR1 protein in immortalized rhesus monkey B cell lines and a significant upregulation of TAAR1 protein expression in rhesus monkey lymphocytes following PHA treatment. Through screening a wide range of signaling pathways for their upregulation following TAAR1 activation by its potent agonist methamphetamine, we identified two transcription factors, CREB and NFAT, which are commonly associated with immune activation. Furthermore, we observed a TAAR1-dependent phosphorylation of PKA and PKC following treatment with methamphetamine in transfected HEK293 cells, immortalized rhesus monkey B cells and PHA-activated rhesus monkey lymphocytes. Accordingly, the high levels of TAAR1 that we observed on lymphocytes are inducible and fully functional, capable of transmitting a signal likely via PKA and PKC activation following ligand binding. More importantly, an increase in TAAR1 receptor expression is concomitant with lymphocyte immune activation, suggesting a possible role for TAAR1 in the generation or regulation of an immune response. TAAR1 is emerging as a potential therapeutic target, with regard to its ability to modulate brain monoamines. The current data raises the possibility that TAAR1-targeted drugs may also alter immune function.