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Microarray analysis of nonhuman primates: validation of experimental models in neurological disorders ¹

MARKÉTA MARVANOVÁ, JEAN MÉNAGER^*, ERWAN BEZARD, RONALD E. BONTROP, LAURENT PRADIER^* and GARRY WONG²

Functional Genomics and Bioinformatics Laboratory, Department of Neurobiology, A. I. Virtanen Institute, University of Kuopio, 70211 Kuopio, Finland;
^* Department of Neurodegenerative Diseases, Aventis Pharma, 94400 Vitry sur Seine, France;
Basal Gang, Department of Neurophysiology, CNRS UMR5543, 33076 Bordeaux, France; and
Biomedical Primate Research Centre, 2280 Rijswijk, The Netherlands

²Correspondence: A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland. E-mail: garry.wong{at}uku.fi

SPECIFIC AIMS

Nonhuman primates are used as experimental models to study a wide range of human neurodegenerative diseases. We used human microarrays to profile genes from brains of human, macaque, and marmosets and combined this with available data from chimpanzee and orangutan to create a data set that provide salient similarities and differences in expression of genes underlying Alzheimer’s, Huntington’s, and Parkinson’s diseases.

PRINCIPAL FINDINGS

1. A large number of genes are expressed in human prefrontal cortex; a significant percentage of these are also expressed in nonhuman primates
The arrays used in this study were U95A GeneChip probe arrays (Affymetrix, Inc., Santa Clara, CA, USA). This single array represents 12,000 sequences previously characterized in terms of function or disease association. RNA was isolated from a healthy 7-year-old male common chimpanzee (Pan troglodytes), 3- to 3.5-year-old female cynomolgus macaques (Macaca fascicularis, CRP, Port Louis, Mauritius) weighing 3 kg, and 2- to 2.5-year-old female common marmosets (Callithrix jacchus) weighing 350 g. Human tissue (19 h postmortem) of prefrontal cortex obtained from a frozen surgical specimen for the whole adult human brain RNA sample (Clontech, Palo Alto, CA, USA) was used. Downloaded data for additional human, chimpanzee, and orangutan were obtained from (http://email.eva.mpg.de/khaitovi/supplement1.html).

Of the 12,386 probe sets analyzed on the Affymetrix U95A chip, 5460 (45%) genes in whole human brain were "present." This indicates that at least 45% of the genes covered on the human arrays are detected in adult human brain. In prefrontal cortical tissues the number of genes present were: human, 4794 (39%); chimpanzee, 4173 (34%); orangutan, 3501 (28%); macaque, 3376 (27%); and marmoset, 2960 (24%) (Fig. 1 A, B). Comparisons of genes present among human and apes species (chimpanzee and orangutan) or human and monkey species (macaque and marmoset) were plotted with a Venn diagram. The proportion of genes present in humans and in both ape species (2954, 62%) was higher than in both monkey species (2145, 45%) (Fig. 1A, B ).

View larger version (39K): [in this window] [in a new window]   Figure 1. Comparison of genes present among human and nonhuman primates. Gene expression data were obtained from microarray hybridization of Affymetrix U95A human GeneChip probe arrays to RNAs isolated from frontal cortex of different primate species. Genes were determined to be "present" if they were scored as P (present) from the Affymetrix Suite perfect match/mismatch algorithm, had an intensity score >10, and there were no absent or marginal calls in any of the samples or replicates for that species. GeneSpring 4.2 was used to detect overlapping and nonoverlapping genes "present" among human, chimpanzee, orangutan, macaque, and marmoset. The genes present among human and apes species (A) and human and monkey species (B) are represented by Venn diagrams. Numerals indicate the number of individual genes.

2. Approximately 20% of present human genes had a different expression profile (>2-fold change) in chimpanzees and >25% of genes in orangutan, macaque, and marmoset had a different expression profile
Distribution plots were generated to identify genes and compare expression of human to nonhuman primates. More than 80% of genes had a similar expression level (<2-fold change) in human compared with chimpanzee and > 60% of the other species studied. The percentage of genes present in prefrontal cortex and displaying a different expression level (>2-fold change) was chimpanzee, 18%; orangutan, 37%; macaque, 26%; marmoset, 33%. The number of genes more highly expressed (>2-fold change) in humans/more highly expressed in NHPs, and ratio were chimpanzee (470/231 genes, 2.0), orangutan (910/249 genes, 3.7), macaque (528/280 genes, 1.9), and marmoset (539/311 genes, 1.7).

3. Genes involved in common neurodegenerative diseases AD, PD, and HD contained qualitative and quantitative differences in NHP PFCs
Some genes known to play a role in Alzheimer’s disease were concordant in humans and NHPs (PS1, AD amyloid, amyloid precursor, CHRM3, tau, ubiquitin) but others were not (apolipoprotein E, NMDAR2C, TNF-). The six genes related to dopaminergic system and thus possibly to Parkinson’s disease(ubiquitin, gamma synuclein, dopamine 1 receptor, MAOB, MAOA, COMT) displayed good concordance in human, chimpanzee, and orangutan (except dopamine 1 receptor), but absent scores for dopamine 1 receptor and COMT were seen in macaque and for dopamine 1 receptor, MAOA, and COMT in marmoset. Huntington’s-related gene HD was not detected in any species tested and HIP2 was detected only in PFC of profiled monkeys. Of the 12 genes related to basic mechanisms (growth factors and their receptors, transcription factors, cytokines, and apoptosis-related molecules), 9 were concordant and 3 were discordant, mostly in the case of marmoset PFCs, with the exception of Bcl-2 transcript, which was not detected in orangutan or macaque. Glutamate receptor 2 was up-regulated and four transcription-involved genes were down-regulated in all NHPs compared with humans.

CONCLUSIONS AND SIGNIFICANCE

The purpose of the study (Fig. 2 ) was to determine by applying microarrays whether primate models of human neurodegenerative diseases are valid according to genomic criteria. The presence and expression levels of selected genes known to play a role in these disorders were established. These results were combined with limited sequence information; together, this indicates that vast numbers of genes in NHPs share close nucleotide sequence as well as qualitative and quantitative expression levels similar to humans in the prefrontal cortex.

View larger version (42K): [in this window] [in a new window]   Figure 2. Schematic diagram. Our data indicate that information about the presence of genes in brain region of different primate species might help us to look more carefully at which nonhuman primate to use as models based on gene expression evidence. As an example of this approach, several genes known to play an important role in Alzheimer, Parkinson’s, and Huntington’s disease were studied for their presence in human and nonhuman primate prefrontal cortices using expression data generated from human U95A chips. PFC, prefrontal cortex; NHPs, nonhuman primates.

Many genes found in Alzheimer’s pathology (amyloid precursor, CHRM3, tau, and ubiquitin) were also found in NHPs while those absent in humans (presenilin 1, AD amyloid) were absent in NHPs. Several genes related to Huntington’s disease pathology, such as the HD gene, were detected in human PFC but not in other NHP PFCs; Huntingtin protein 2 (HIP2) was not detected in human and ape PFCs but was in monkey PFCs. Moreover, COMT, an enzyme with a role in dopamine degradation, was present in humans and apes but was not detected in any of the monkey species tested. It is proposed that pharmacological studies using selective antagonists of COMT might be more precise and relevant in apes than in monkeys, pending structural studies. Thus, the information about the qualitative nature (presence) of genes in brain regions of different primates might help to better plan pharmacological and behavior studies as well as determine which NHPs to use based on the gene expression evidence. Four genes related to transcription were down-regulated by at least twofold in all NHP species analyzed compared with humans. Whether this finding is a distinguishing feature that underlies many of the differences between humans and other NHPs requires further investigation.

Since entire genome sequences or even large clustered EST sequences from many of the NHPs used as experimental models in biomedical research are not currently available, interspecies microarray hybridization studies represent one possible way to identify genes within a transcriptome and profile the expression levels. The microarray technology platform used here is uniform and data can be normalized from different experiments. This makes it possible to facilitate comparison of microarray data generated by different laboratories (such as performed in this study) and to diminish the potential number of animals and work needed for similar expression studies, an advantage of this microarray platform over other currently available systems. This is relevant in primate studies due to high cost, supply limitation, and the ethical issue involved in such research. On the other hand, although this approach is useful scientifically, it will be desirable and perhaps necessary to eventually have chips with homologous sequence that can be used among species. Moreover, probes in this system represent only the coding sequence and it might be considered that noncoding sequences could have a real interest in terms of regulation. An admitted weakness in this system is that "absent" calls could be interpreted for a given gene as having a degenerated sequence, below detection, or highly variable in expression between individuals. Thus, these factors would all suggest that the current analysis likely underestimates the total number of genes present in humans and NHPs.

In conclusion, this study demonstrates use of the oligonucleotide-based microarrays to assess global expression profiles for NHP species (P. troglodytes, Pongo pygmaeus, M. fascicularis, and C. jacchus). The approach described here should validate and more accurately focus the use of apes and of Old World and New World monkeys in neurological models. The data are publically available for validation of other biomedically important models and to perform molecular evolution studies. Additional brain regions important in other neurological disorders could also be considered for profiling (e.g., hippocampus, entorhinal cortex, amygdala, striatum). Finally, our analyses of genes known to be important in neurological disorders validate some experimental models while indicating the context where these models are most appropriate.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0681fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0681fje.

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