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Perspective

Why Mice Are Neither Miniature Humans nor Small Rats: A Cautionary Tale Involving 5-Hydroxytryptamine-6 Serotonin Receptor Species Variants

Departments of Biochemistry, Psychiatry, and Neurosciences, Case Western Reserve University Medical School, Cleveland, Ohio

Received August 1, 2003; accepted August 25, 2003.

Serotonin [5-hydroxytryptamine (5-HT)] is a biogenic amine neurotransmitter that modulates a host of important biological processes, such as mood, cognition, perception, feeding behavior, smooth muscle contractility, and platelet aggregation. The myriad actions of 5-HT are mediated by one or more of at least seven subtypes of receptors, all but one of which (i.e., the 5-HT₃ receptor) are members of the G protein-coupled receptor super-family (Kroeze et al., 2002) (see phylogram, Fig. 1). Receptors that are closer on the phylogram are more likely to share pharmacological properties than receptors that are farther apart (distant relatives). One of the most enigmatic 5-HT receptors, the 5-HT₆ receptor, was cloned in 1993 from a rat striatal cDNA library taken from sequence homology with existing 5-HT receptors and found to be expressed in the striatum, olfactory tubercule, cortex, and hippocampus (Monsma et al., 1993; Ruat et al., 1993; Ward et al., 1995). The human (Kohen et al., 1996) and mouse (Kohen et al., 2001) 5-HT₆ receptors were subsequently cloned with the human version shown to be pharmacologically similar to the rat (Kohen et al., 1996). Shortly after the discovery of 5-HT₆ receptors, Roth et al. (1994) reported that a large number of typical and atypical antipsychotic drugs bound with unexpectedly high affinity (K_i values of <20 nM) to cloned 5-HT₆ receptors. The expression of 5-HT₆ receptors in limbic areas and the basal ganglia, and the selective labeling of 5-HT₆ receptors in rat brain with [³H]clozapine (Glatt et al., 1995), strongly implicated 5-HT₆ receptors in at least some of the actions of antipsychotic drugs.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Phylogram of mouse, rat, and human 5-HT G protein-coupled receptors shows that 5-HT6 receptors are most similar in amino acid sequence to 5-HT2 family receptors. Multiple pairwise alignments were performed, and the phylogram was produced using the AlignX module of the Vector NTI Suite (Informax, Frederick, MD).

More recently, 5-HT₆ receptors have been demonstrated to regulate central cholinergic neurotransmission. In this regard, the administration of the 5-HT₆ receptor-selective antagonist Ro 04-6790 reversed scopolamine-induced rotation in 6-hydroxydopamine-lesioned rats (Bourson et al., 1998). Additionally, Rogers and Hagan (2001) and Woolley et al. (2001) found that either 5-HT₆ receptor antisense oligonucleotides or 5-HT₆ receptor-selective inhibitors enhanced retention by rats of the learned platform position in the Morris water maze. These data suggest that 5-HT₆ receptor antagonists might boost cholinergic neurotransmission and reduce the cognitive impairments experienced by patients with dementia or schizophrenia. Intriguingly, Tsai et al. (1999) determined that the 267C allele of the 5-HT₆ receptor is a significant risk factor for Alzheimer's disease. Taken together, these findings indicate that 5-HT₆ antagonists might prove useful in treating a number of common illnesses, including dementia and schizophrenia.

The now-classic approach for validating 5-HT₆ receptors as molecular targets for therapeutics is to construct a 5-HT₆ knockout mouse and to characterize its phenotype. As Hirst et al. (2003) discovered, however, it is unlikely that 5-HT₆ knockout mice will be useful for validating the 5-HT₆ receptor as a therapeutic target because of pronounced and unexpected species differences in both receptor regional distribution and pharmacology. It is now widely appreciated that slight differences in rodent and human amino acid sequences can lead to unexpectedly large differences in the pharmacology of the receptors, with potentially disastrous effects for drug-discovery efforts. What has not been clearly documented until the Hirst et al. study (2003), however, is that mouse receptors could be significantly different from rat receptors.

In the article published in this issue of Molecular Pharmacology, Hirst et al. (2003) elegantly demonstrate that the mouse 5-HT₆ receptor is, in nearly every respect, distinct from rat and human 5-HT₆ receptors. They investigated these differences because of preliminary studies in which they were unable to quantify mouse 5-HT₆ receptors with a highly selective radioligand ([¹²⁵I]SB-258585) that labels both rat and human 5-HT₆ receptors. In addition to species differences in the binding of drugs to 5-HT₆ receptors, they found differences in the regional expression of 5-HT₆ receptors. Thus, quantitative polymerase chain reaction studies demonstrated that the mouse 5-HT₆ receptor mRNA was at least 10-fold less abundant than the rat or human 5-HT₆ receptor mRNAs in every brain region examined. Surprisingly, whereas 5-HT₆ receptor mRNA and radioligand binding activity was enriched in the basal ganglia of rat and human brain, there was no such enrichment in the mouse brain.

Additionally, via a combination of site-directed mutagenesis and molecular modeling studies, Hirst et al. (2003) describe the presumed molecular and atomic reasons for the peculiar mouse 5-HT₆ pharmacology. Two amino acids—Tyr188 (in helix 5, which is Phe188 in rats and humans) and Ser290 (in helix 6 which is Asn290 in rats and humans)—were found to account for the bulk of the differences in pharmacology. A nice feature of the study is the parallel inclusion of elegant modeling studies of the various ligands used. Hirst et al. (2003) use this model to present a plausible molecular rationale for the differential interactions of various 5-HT₆ receptor-selective ligands with human, rat, and mouse 5-HT₆ receptors.

The findings described by Hirst et al. (2003) have important implications for drug discovery. Because the mouse 5-HT₆ receptor is distinct in nearly every way from the human (and rat) 5-HT₆ receptor, the results force us to question the use of knockout mice in a wholesale fashion to provide validated molecular targets for drug discovery. Their studies strongly imply that before a knockout mouse is accepted as a validated model for a particular human disease, the molecular target needs to be demonstrated to have a pharmacology, regional tissue distribution, and abundance similar to the human homolog. Therefore, this study stands as an important reminder to us all that mice are not miniature humans and, sometimes, not even small rats.

	Footnotes

 
ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; Ro 04-6790, 4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulfonamide; SB-258585, 4-iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulfonamide.

Address correspondence to: Dr. Bryan Roth, Department of Biochemistry, Psychiatry, and Neurosciences, Case Western University, 10900 Euclid Avenue, Room RT-500, Cleveland, OH 44106-4936. E-mail: bryan.roth{at}case.edu

	References

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