Functional dissection of two amino acid substitutions unique to the human FOXP2 protein

doi:10.21203/rs.3.rs-1917670/v1

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Functional dissection of two amino acid substitutions unique to the human FOXP2 protein

https://doi.org/10.21203/rs.3.rs-1917670/v1

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published 06 Mar, 2023

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The transcription factor forkhead box P2 (FOXP2) is involved in the development of language and speech in humans. Two amino acid substitutions (T303N, N325S) occurred in the human FOXP2 after the divergence from the chimpanzee lineage. It has previously been shown that when they are introduced into the FOXP2 protein of mice they alter striatal synaptic plasticity by increasing long-term depression in medium spiny neurons. Here we introduce each of these amino acid substitutions individually into mice and analyze their effects in the striatum. We find that long-term depression in medium spiny neurons is increased in mice carrying only the T303N substitution to the same extent as in mice carrying both amino acid substitutions. In contrast, the N325S substitution has no discernable effects.

medium spiny neurons

striatum

synaptic plasticity

evolution

language

speech

In humans, mutations that inactivate one allele of the FOXP2 gene lead to difficulties in learning and performing complex orofacial movements, including those used for speech, as well as deficits in receptive and expressive language (Vargha-Khadem, et al. 2005; Liegeois, et al. 2011). Although the transcription factor FOXP2 is thus involved in a trait unique to humans, only three out of the 715 amino acids in the FOXP2 protein differ between humans and mice, making it among the 5% most conserved proteins between the two species (Enard 2011). Remarkably, two of these amino acid substitutions (T303N and N325S) occurred on the human evolutionary lineage since its divergence from the chimpanzee lineage (Fig. 1). They have therefore been suggested to be of relevance for the evolution of speech and language (Enard, et al. 2002; Zhang, et al. 2002; Enard 2011). Mice where these two human substitutions have been introduced into the endogenous Foxp2 gene (Foxp2^hum mice) are generally healthy but show enhanced synaptic plasticity in the form of stronger long-term depression (LTD) in the medium spiny neurons (MSN) of the striatum, suggesting that cortico-basal ganglia circuits are affected (Enard, et al. 2009; Reimers-Kipping, et al. 2011; Schreiweis et al., 2014). However, it is unclear whether one or both of the two amino acid changes cause these effects.

To better understand the contributions of the two amino acid substitutions, we have generated one mouse line which carries only the threonine-to-asparagine substitution at position 302 (T302N; N303 in humans) and one mouse line which carries only the asparagine-to-serine at position 324 (N324S; S325 in humans) in their endogenous Foxp2 genes. Here, we analyze how these Foxp2 alleles (Foxp2^T302N, Foxp2^N324S) affect LTD in MSNs.

Generation of Foxp2 mice. Mice carrying Foxp2 alleles encoding each of the two single amino acid substitutions (Foxp2^N324S, Foxp2^T302N) were generated (Fig. 1) as previously described for the Foxp2^hum mice (Enard, et al. 2009). Mutations were confirmed by Southern analysis as well as PCR and sequencing of genomic DNA and cDNA from brain (data not shown). We compared animals homozygous for each of the two alleles (Foxp2T302N, Foxp2N324S) with wildtype littermates obtained from crossings of heterozygous parents or to animals of generated by crossing of homozygous animals in the next generation.

Synaptic plasticity in Foxp2^T302N and Foxp2^N324S mice. We have previously shown that LTD in neurons of the central (Enard, et al. 2009) and dorsolateral parts (Reimers-Kipping, et al. 2011; Schreiweis, et al. 2014) of the striatum of Foxp2^hum mice is enhanced. Using identical protocols, we made dorsolateral recordings from Foxp2^T302N (N = 8) and Foxp2^N324S (N = 12) and their wildtype littermates (N = 3 and N = 8, respectively) and compared them to previously published data of Foxp2^hum mice (Reimers-Kipping, et al. 2011; Schreiweis, et al. 2014). At 30–40 minutes after high-frequency stimulation, LTD of Foxp2^T302N cells is increased compared to Foxp2^wt cells (Mann Whitney U test (MWU): P = 0.011, Fig. 2A, C). When compared to the previously published data for Foxp2^hum cells, no significant differences are seen (MWU: P = 0.48). In contrast, LTD in Foxp2^N324S cells differs from Foxp2^T302N cells (MWU: P = 0.028, Fig. 2C) and is not detectably different from Foxp2^wt cells (MWU: P = 0.93; Fig. 2B, C).

A major question regarding the two amino acid substitutions that affected the FOXP2 protein during human evolution is if they both have physiological consequences or if only one of them has effects. Here we show that mice homozygous for the T302N substitution exhibit increased LTD in the dorsolateral striatum to an extent similar to mice carrying both amino acid substitutions. In contrast, LTD in mice homozygous for N324S substitution does not differ from LTD in their wildtype littermates (Fig. 2). Thus, whereas the T302N substitution has clear effects in terms of synaptic plasticity, N324S substitution does not. More work is needed to understand how these observations relate to phenotypic effects that the two amino acid substitutions have in the mouse, notably subtle differences in vocalization (Enard, et al. 2002) and enhanced transition from declarative to procedural performance during certain learning tasks (Schreiweis et al. 2014).

However, the variation in FOXP2 amino acid sequences among and within species can give indications about how important the two amino acid substitutions may be. Among vertebrates, the N324S substitution occurs also in carnivores and birds and is thus not unique to humans. Furthermore, 26% of western gorillas carry a substitution two amino acids away at position 326 (A326S) (Staes et al. 2017). In contrast, the T302N substitution has not been observed in any other organism than humans. Among 125,642 humans for which exome sequences are available (gnomAD, v2.1.1), no individual carry the ancestral amino acid variants at either position. However, two individuals carry a serine to arginine substitution at the position corresponding to position 324 in the mouse (S325R). Thus, whereas the T303N substitution in humans affects a position that is extremely conserved and not known to vary among humans today, the N325S substitution has occurred also among other mammals and the positions varies among humans, albeit very rarely.

It should be noted that the two amino acid substitutions analyzed here are shared with Neandertals and Denisovans (Krause, et al. 2007) - hominin forms that diverged from the ancestors of present-day humans 550,000-765,000 years ago (Pruefer et al. 2014). They thus occurred so early on the human evolutionary lineage that the presence or absence of signals of positive selective among present-day humans cannot be informative about if they were selected when they occurred (Enard, et al. 2002; Coop, et al. 2008; Atkinson et al. 2018). Nevertheless, other functional changes may have affected the FOXP2 gene on the human evolutionary lineage more recently. For example, a substitution in a transcription factor-binding site in intron 8 of the FOXP2 gene is unique to modern humans and thus occurred during the past 500,000 years. The transcription factor that binds to this site, POU3F2 (also called BRN2), is expressed uniquely in the nervous system and is involved in neuronal differentiation and the substitution reduces the efficiency with which POU3F2 dimers bind to the binding site and drive transcription (Maricic, et al. 2013). It is thus possible that multiple changes that affect both the function of the FOXP2 protein and the expression of the FOXP2 gene have occurred on the human evolutionary lineage. However, in terms of the effects of the protein on synaptic plasticity, the T303N rather than the N325S substitution is of importance.

Generation of Foxp2 ^N324S and Foxp2^T302N expressing mice. The vectors for generating the Foxp2^N324S and Foxp2^T302N allele were created by mutagenesis of the targeting vector used for the Foxp2^hum allele (details see (Enard, et al. 2009)) and mice were generated from Bruce4 C57BL/6 ES cells by Ozgene (Bentley, Australia) as described (Enard, et al. 2009). Founder animals were confirmed to have the correct mutation in the Foxp2 gene as well as in Foxp2 cDNA in the brain (data not shown). They were crossed to mice transgenic for the recombinase FLPe under the control of the human ACTB promoter (Jackson Laboratory, Stock Number 003800; C57BL/6J; (Rodriguez, et al. 2000) to generate Foxp2^N324S or Foxp2^T302N alleles in which the FRT-flanked neomycin resistance cassettes have been removed as described (Enard, et al. 2009). The recombinase transgenes were outcrossed using C57BL/6J mice in the next generation and further crossings were made in C57BL/6J mice (C57BL/6J@Rj; Janvier, St. Berthevin, France). Hence, all alleles are on the same genomic background and lack the neomycin resistance cassette of the targeting vector. Mice used for recordings were homozygous for the wildtype (Foxp2^wt) or the FoxP2 locus (Foxp2^N324S or Foxp2^T302N). They were either derived directly from the first generation of crossings of heterozygous animals or from the following second generation where homozygous wildtype or FoxP2 siblings from the first generation were crossed. Thus, mice compared with each other were either matched littermates or second generation offspring of such littermates. Genotyping was as described (Enard, et al. 2009). All mouse experiments were overseen and approved by the Institutional Animal Welfare Officer of the Max Planck Institute for Evolutionary Anthropology (Dr. Gerd Möbius, Fac. of Veterinary Medicine, Univ. Leipzig). They were performed in accordance to the German Animal Welfare Legislation (“Tierschutzgesetz”), and approved and registered with the Federal State Authority Landesdirektion Sachsen (No. 24-9162.11 (T 38/12)).

We recently discovered that a wildtype deletion as described for the Harlan line C57BL/6JOlaHsd (365 kb between pos. 60.976 and 61.341 Mb of Chr. 6, including the Snca and Mmrn1 locus; Specht and Schoepfer, 2001, Specht and Schoepfer, 2004) occurred in some of our lines (incl. the FLPe line). Hence, we tested all animals used in these experiments with PCR protocols as given in (Specht and Schoepfer, 2004). No animals carrying this deletion were found.

Slice electrophysiology. Brains of slightly anesthetized mice (P21 – P53; isoflurane) were prepared into ice-cold sucrose-based cutting solution (in mM: 85 sucrose, 60 NaCl, 3.5 KCl, 6 MgCl₂, 0.5 CaCl₂, 38 NaHCO₃, 1.25 NaH₂PO₄, 10 HEPES, 25 glucose). Coronal slices (250 µm) were cut (Vibroslice 7000smz, Campden Instruments, UK), incubated in artificial cerebrospinal fluid (aCSF; in mM: 120 NaCl, 3.5 KCl, 1 MgCl₂, 2 CaCl₂, 30 NaHCO₃, 1.25 NaH₂PO₄, 15 glucose) supplemented with 5 mM HEPES, 1 MgCl₂ for 30 min at 35°C and allowed to recover at room temperature for at least 40 min.

MSN were identified as in (Pawlak and Kerr 2008). They were recorded in the current clamp configuration with the bridge mode enabled (EPC-10 amplifier, Patch- and Fitmaster software; HEKA, Lambrecht, Germany). The internal solution contained (in mM): 150 Kgluconate, 10 NaCl, 3 Mg-ATP, 0.5 GTP, 10 HEPES and 0.05 EGTA adjusted to pH = 7.3 and 310 mOsm with the liquid junction potential (15 mV) corrected online. Slices were perfused (2–3 ml/min, aCSF, 21–24°C) in presence of the GABA_AR antagonist gabazine (SR-95531, 10 µM, Sigma). All solutions were continuously oxygenated with 95% O₂, 5% CO₂ gas.

Glutamatergic excitatory afferents where stimulated intrastriatally with aCSF-filled theta-glass electrodes typically ~ 100–150 µm away from the MSN soma (position of stimulation electrode between MSN and corpus callosum). A bipolar voltage pulse (0.1 ms, ± 5 to ± 30 V) at 0.2 Hz induced subthreshold excitatory postsynaptic potentials (EPSPs; 4–10 mV). Following 10–15 min baseline recording synaptic plasticity was induced by a high frequency protocol (four 100Hz tetani, 3 s long, separated by 30 s; holding potential − 70 mV). Recordings were rejected if the membrane potential was more positive than − 80 mV or the input resistance changed by more than 30%. We verified that no background long-term potentiation was present as APV ((2R)-amino-5-phosphonovaleric acid), a specific blocker of a subtype of glutamate receptors, did not alter the effect in wildtype mice (Schreiweis, et al. 2014).

Statistical analysis. EPSP amplitudes were normalized to a mean baseline level at t = -10 to 0 min. LTD magnitude of individual cells was calculated by averaging amplitudes 30–40 min after induction with the high frequency protocol. For comparisons we used previously published data for Foxp2^wt and Foxp2^hum obtained under identical conditions (Reimers-Kipping, et al. 2011; Schreiweis, et al. 2014).

The experimenters and initial evaluators were blinded towards the genotype. All methods are reported in accordance to the ARRIVE guidelines.

Acknowledgments

We are grateful to K. Bruns and R. Voigtländer for animal care and to I. Bliesener and V. Wiebe for technical assistance. This work was supported by the Max Planck Society and the NOMIS Foundation.

Author contributions

U.B. and W.H. conducted all electrophysiological recordings; U.B., W.E. & W.H. designed the experiments, performed data analyses, and wrote the manuscript; W.E., W.H. & S.P. supervised experiments and H.Z. & S.P. edited the manuscript.

Data availability

All datasets recorded and analyzed during the current study are available from the corresponding author on request.

Additional Information

Competing interests

The author(s) declare no competing interests.

Atkinson EG, Audesse AJ, Palacios JA, Bobo DM, Webb AE, Ramachandran S, Henn BM. 2018. No Evidence for Recent Selection at FOXP2 among Diverse Human Populations. Cell 174(6): 1424-1435 e1415.
Coop G, Bullaughey K, Luca F, Przeworski M. 2008. The timing of selection at the human FOXP2 gene. Mol Biol Evol 25:1257-1259.
Enard W. 2011. FOXP2 and the role of cortico-basal ganglia circuits in speech and language evolution. Curr Opin Neurobiol 21:415-424.
Enard W, Gehre S, Hammerschmidt K, Holter SM, Blass T, Somel M, Bruckner MK, Schreiweis C, Winter C, Sohr R, et al. 2009. A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice. Cell 137:961-971.
Enard W, Przeworski M, Fisher SE, Lai CS, Wiebe V, Kitano T, Monaco AP, Paabo S. 2002. Molecular evolution of FOXP2, a gene involved in speech and language. Nature 418:869-872.
Krause J, Lalueza-Fox C, Orlando L, Enard W, Green RE, Burbano HA, Hublin JJ, Hanni C, Fortea J, de la Rasilla M, et al. 2007. The derived FOXP2 variant of modern humans was shared with Neandertals. Curr Biol 17:1908-1912.
Liegeois F, Morgan AT, Connelly A, Vargha-Khadem F. 2011. Endophenotypes of FOXP2: dysfunction within the human articulatory network. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society 15:283-288.
Maricic T, Gunther V, Georgiev O, Gehre S, Curlin M, Schreiweis C, Naumann R, Burbano HA, Meyer M, Lalueza-Fox C, et al. 2013. A recent evolutionary change affects a regulatory element in the human FOXP2 gene. Mol Biol Evol 30:844-852.
Meyer M, Kircher M, Gansauge MT, Li H, Racimo F, Mallick S, Schraiber JG, Jay F, Prufer K, de Filippo C, et al. 2012. A high-coverage genome sequence from an archaic Denisovan individual. Science 338:222-226.
Pawlak V, Kerr JN. 2008. Dopamine receptor activation is required for corticostriatal spike-timing-dependent plasticity. J Neurosci 28:2435-2446.
Reimers-Kipping S, Hevers W, Paabo S, Enard W. 2011. Humanized Foxp2 specifically affects cortico-basal ganglia circuits. Neuroscience 175:75-84.
Rodriguez CI, Buchholz F, Galloway J, Sequerra R, Kasper J, Ayala R, Stewart AF, Dymecki SM. 2000. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat Genet 25:139-140.
Schreiweis C, Bornschein U, Burguiere E, Kerimoglu C, Schreiter S, Dannemann M, Goyal S, Rea E, French CA, Puliyadi R, Groszer M, Fisher SE, Mundry R, Winter C, Hevers W, Paabo S, Enard W, Graybiel AM. 2014. Humanized Foxp2 accelerates learning by enhancing transitions from declarative to procedural performance. Proc Natl Acad Sci U S A 111: 14253-14258.
Specht, C. G. and R. Schoepfer. 2001. Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice. BMC Neurosci 2: 11.
Specht, C. G. and R. Schoepfer. 2004. Deletion of multimerin-1 in alpha-synuclein-deficient mice. Genomics 83(6): 1176-1178.
Staes N, Sherwood CC, Wright K, de Manuel M, Guevara EE, Marques-Bonet T, Krützen M, Massiah M, Hopkins WD, Ely JJ, et al. 2017. FOXP2 variation in great ape populations offers insight into the evolution of communication skills. Scientific Reports 7:16866.
Vargha-Khadem F, Gadian DG, Copp A, Mishkin M. 2005. FOXP2 and the neuroanatomy of speech and language. Nat Rev Neurosci 6:131-138.
Zerbino DR, Achuthan P, Akanni W, Amode M R, Barrell D, Bhai J, Billis K, Cummins C, Gall A, Girón CG, et al. 2018. Ensembl 2018. Nucleic Acids Research 46:D754-D761.
Zhang J, Webb DM, Podlaha O. 2002. Accelerated protein evolution and origins of human-specific features: Foxp2 as an example. Genetics 162:1825-1835.

No competing interests reported.

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Functional dissection of two amino acid substitutions unique to the human FOXP2 protein

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