Mice Carrying a Human GLUD2 Gene Recapitulate Aspects of Human Transcriptome and Metabolome Development
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Mice carrying a human GLUD2 gene recapitulate aspects of human transcriptome and metabolome development Qian Lia,b,1, Song Guoa,1, Xi Jianga, Jaroslaw Brykc,2, Ronald Naumannd, Wolfgang Enardc,3, Masaru Tomitae, Masahiro Sugimotoe, Philipp Khaitovicha,c,f,4, and Svante Pääboc,4 aChinese Academy of Sciences Key Laboratory of Computational Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200031 Shanghai, China; bUniversity of Chinese Academy of Sciences, 100049 Beijing, China; cMax Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany; dMax Planck Institute of Molecular Cell Biology and Genetics, D-01307 Dresden, Germany; eInstitute for Advanced Biosciences, Keio University, 997-0035 Tsuruoka, Yamagata, Japan; and fSkolkovo Institute for Science and Technology, 143025 Skolkovo, Russia Edited by Joshua M. Akey, University of Washington, Seattle, WA, and accepted by the Editorial Board April 1, 2016 (received for review September 28, 2015) Whereas all mammals have one glutamate dehydrogenase gene metabolic flux from glucose and glutamine to lipids by way of the (GLUD1), humans and apes carry an additional gene (GLUD2), TCA cycle (12). which encodes an enzyme with distinct biochemical properties. To investigate the physiological role the GLUD2 gene may We inserted a bacterial artificial chromosome containing the human play in human and ape brains, we generated mice transgenic for GLUD2. GLUD2 gene into mice and analyzed the resulting changes in the a genomic region containing human We compared effects transcriptome and metabolome during postnatal brain development. on gene expression and metabolism during postnatal development Effects were most pronounced early postnatally, and predominantly of the frontal cortex of the brain in these mice and their wild-type genes involved in neuronal development were affected. Remarkably, littermates, with similar data obtained from humans and rhesus the effects in the transgenic mice partially parallel the transcriptome macaques, the closest relative of humans and apes, which lack the GLUD2 gene. and metabolome differences seen between humans and macaques GLUD2 analyzed. Notably, the introduction of did not affect gluta- Results mate levels in mice, consistent with observations in the primates. A Mouse Model of GLUD2. We constructed transgenic mice carrying Instead, the metabolic effects of GLUD2 center on the tricarboxylic GLUD2 GLUD2 a 176-kb-long human genomic region containing the gene, acid cycle, suggesting that affects carbon flux during early as well as 43 kb of upstream and 131 kb of downstream DNA se- brain development, possibly supporting lipid biosynthesis. quences (SI Appendix,Fig.S1). To account for effects potentially caused by stochastic insertion of GLUD2 sequence into the mouse human evolution | GLUD2 | brain metabolism Significance lutamate dehydrogenase (GDH) is a metabolic enzyme cat- α Galyzing the conversion of glutamate to -ketoglutarate and A novel version of the glutamate dehydrogenase gene, GLUD2, ammonia (1). Whereas the ammonia is metabolized via the urea α evolved in the common ancestors of humans and apes. Based on cycle, the -ketoglutarate enters the tricarboxylic acid (TCA) sequence and expression pattern, GLUD2 has been suggested to cycle in mitochondria. Besides its metabolic role, glutamate also playaroleinglutamatemetabolisminhumanandapebrains.We functions as a major excitatory neurotransmitter (2, 3). have generated transgenic mice carrying a human GLUD2 gene. Whereas most organisms contain one copy of the GLUD gene GLUD1 Analysis of transcriptome and metabolome changes induced by encoding the GDH enzyme, humans and apes have two: GLUD2 and an additional gene, GLUD2, which originated by retroposition in the cerebral cortex revealed no changes in glutamate of the GLUD1 transcript after the split from apes and old world concentration but instead changes to metabolic pathways center- monkeys (4). Sequence analysis suggests that it is highly unlikely ing on the TCA cycle during early postnatal development. These changes mirrored differences seen between human and macaque that GLUD2 would have contained an intact ORF and a Ka/Ks <1 throughout the evolution of apes without being functional (5). during cortex development, suggesting that GLUD2 mayplaya Moreover, positive selection has affected GLUD2 (4), including role during brain development in apes and humans, possibly by changes in amino acid residues that make it less sensitive to low pH providing precursors for the biosynthesis of lipids. and GTP inhibition, and resulting in a requirement for high ADP levels for allosteric activation (4, 6, 7). GLUD2 mRNA expression Author contributions: W.E., P.K., and S.P. designed research; X.J., J.B., R.N., and M.S. levels in tissues are lower than those of GLUD1, but similarly performed research; W.E., M.T., and M.S. contributed reagents/analytic tools; Q.L. and distributed across tissues. However, whereas the ancestral version S.G. analyzed data; and Q.L., P.K., and S.P. wrote the paper. of the GLUD enzyme occurs both in mitochondria and the cyto- The authors declare no conflict of interest. plasm, GLUD2 is specifically targeted to mitochondria (8, 9). This article is a PNAS Direct Submission. J.M.A. is a guest editor invited by the Editorial Changes in GLUD2 properties have been suggested to reflect Board. functional adaptation to the metabolism of the neurotransmitter Freely available online through the PNAS open access option. glutamate in the brain (4, 10), and the fact that GLUD2 has been Data deposition: The data reported in this paper have been deposited in the Gene Ex- positively selected and maintained during ape and human evo- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE80122). lution suggests that it may have physiological effects important 1Q.L. and S.G. contributed equally to this work. for the function of ape and human brains. However, the con- 2Present address: School of Applied Sciences, University of Huddersfield, HD1 3DH nection between the emergence of the GLUD2 gene in the an- Huddersfield, UK. cestors of apes and humans and changes in brain function 3Present address: Department of Biology II, Ludwig Maximilians University, 82152 remains elusive. To date, the only direct insights into GLUD2 Martinsried, Germany. function come from a rare GLUD2 mutation linked to the onset of 4To whom correspondence may be addressed. Email: [email protected] or paabo@ Parkinson’s disease (11) and from glioma cells carrying a mutated eva.mpg.de. isocitrate dehydrogenase 1 gene (IDH1)whereGLUD2 expression This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. reverses the effects of the IDH1 mutation by reactivation of the 1073/pnas.1519261113/-/DCSupplemental. 5358–5363 | PNAS | May 10, 2016 | vol. 113 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1519261113 development in humans (Pearson correlation, r = 0.86, P < 0.0001; SI Appendix,Fig.S4). We also detected expression of a long noncoding A (transgenic mice) (control mice) (human) (rhesus macaque) RNA (lncRNA) originating from the opposite strand of the trans- fected human genomic region upstream of the GLUD2 transcription age start site (SI Appendix,Fig.S5). Expression profile of this lncRNA closely resembled the expression profile of GLUD2 (SI Appendix,Fig. B S6), suggesting that it is expressed from the same bidirectional pro- moter as GLUD2 (SI Appendix,TableS4). age An effect of the GLUD2 genotype, albeit small, was de- tectable: using analysis of covariance (ANCOVA) with linear, quadratic, and cubic models, we identified 12 protein-coding age0 6 16 38 90 215 0 6 16 38 90 215 0 2.5 10 22 40 61 90 0 0.6 2.5 5.51016 22 genes and one lncRNA showing significant expression differ- (d) (d) (y) (y) ences between the two transgenic lines and control littermates Fig. 1. Sample information. (A) Schematic representation of age, sex, and (F-test, BH corrected P < 0.05, permutations P < 0.05, FDR < transgenic line information for mouse and primate samples used for tran- 0.01, SI Appendix,TableS5). Strikingly, the effect of the scriptome (RNA-seq) measurements. (B) Schematic representation of age, sex, GLUD2 genotype was observed only during early postnatal and transgenic line information for mouse and primate samples used for development and disappeared at approximately 2 wk of age metabolome (CE-MS) measurements. Each symbol represents an individual (Fig. 2B). By contrast, other genes expressed in the mouse sample. The colors indicate genotype/species information: orange, transgenic brain did not show any increased divergence between trans- mice; green, control mice; red, humans; blue, rhesus macaques. Lighter shades genic and control mice during early development (Fig. 2C). of color indicate younger ages. The bars show the corresponding age intervals: Eleven of the 13 genes differentially expressed between trans- d, days; y, years. The symbols indicate mice lines: circles, line a; triangles, line b. genic and control mice fell into the same coexpressed module in Filled symbols indicate males; empty symbols indicate females. an unsupervised hierarchical clustering analysis (Fig. 3A and SI Appendix, Table S5 and Fig. S7), an observation not expected by chance (permutations, P < 0.001) (SI Appendix, Fig. S8). The genome, we constructed two independent transgenic lines (a and b). GLUD2 Anatomical, neurophysiological, and