The purpose of this study was to determine the relationship between mitochondrial DNA (mtDNA) deletions, mtDNA content and aging in rhesus monkeys. Using 2 sets of specific primers, we amplified an 8 kb mtDNA fragment covering a common 5.7 kb deletion and the entire 16.5 kb mitochondrial genome in the brain and buffy-coats of young and aged monkeys. We studied a total of 66 DNA samples: 39 were prepared from a buffy-coat and 27 were prepared from occipital cortex tissues. The mtDNA data were assessed using a permutation test to identify differences in mtDNA, in the different monkey groups. Using real-time RT-PCR strategy, we also assessed both mtDNA and nuclear DNA levels for young, aged and male and female monkeys. We found a 5.7 kb mtDNA deletion in 81.8% (54 of 66) of the total tested samples. In the young group of buffy-coat DNA, we found 5.7 kb deletions in 7 of 17 (41%), and in the aged group, we found 5.7 kb deletions in 12 of 22 (54%), suggesting that the prevalence of mtDNA deletions is related to age. We found decreased mRNA levels of mtDNA in aged monkeys relative to young monkeys. The increases in mtDNA deletions and mtDNA levels in aged rhesus monkeys suggest that damaged DNA accumulates as rhesus monkeys age and these altered mtDNA changes may have physiological relevance to compensate decreased mitochondrial function.

Of the acetylcholine muscarinic receptors, the type 1 (M1) and type 2 (M2) receptors are expressed at the highest levels in the prefrontal cortex (PFC) and hippocampus, brain regions important for cognition. As equivocal findings of age-related changes of M1 and M2 in the nonhuman primate brain have been reported, we first assessed age-related changes in M1 and M2 in the PFC and hippocampus using saturation binding assays. Maximum M1 receptor binding, but not affinity of M1 receptor binding, decreased with age. In contrast, the affinity of M2 receptor binding, but not maximum M2 receptor binding, increased with age. To determine if in the elderly cognitive performance is associated with M1 or M2 function, we assessed muscarinic function in elderly female rhesus macaques in vivo using a scopolamine challenge pharmacological magnetic resonance imaging and in vitro using saturation binding assays. Based on their performance in a spatial maze, the animals were classified as good spatial performers (GSP) or poor spatial performers (PSP). In the hippocampus, but not PFC, the GSP group showed a greater change in T2*-weighted signal intensity after scopolamine challenge than the PSP group. The maximum M1 receptor binding and receptor binding affinity was greater in the GSP than the PSP group, but no group difference was found in M2 receptor binding. Parameters of circadian activity positively correlated with the difference in T2*-weighted signal intensity before and after the challenge, the maximum M1 receptor binding, and the M1 receptor binding affinity. Thus, while in rhesus macaques, there are age-related decreases in M1 and M2 receptor binding, in aged females, hippocampal M1, but not M2, receptor function is associated with spatial learning and memory and circadian activity.

Aging is associated with a general dysregulation in immune function, commonly referred to as “immune senescence”. Several studies have shown that female sex steroids can modulate the immune response. However, the impact of menopause-associated loss of estrogen and progestins on immune senescence remains poorly understood. To help answer this question, we examined the effect of ovariectomy on T-cell homeostasis and function in adult and aged female rhesus macaques. Our data show that in adult female rhesus macaques, ovariectomy increased the frequency of naïve CD4 T cells. In contrast, ovariectomized (ovx) aged female rhesus macaques had increased frequency of terminally differentiated CD4 effector memory T cells and inflammatory cytokine-secreting memory T cells. Moreover, ovariectomy reduced the immune response (T-cell cytokine and IgG production) following vaccination with modified vaccinia ankara in both adult and aged female rhesus macaques compared to ovary-intact age-matched controls. Interestingly, hormone therapy (estradiol alone or in conjunction with progesterone) partially improved the T-cell response to vaccination in aged ovariectomized female rhesus macaques. These data suggest that the loss of ovarian steroids, notably estradiol and progesterone, may contribute to reduced immune function in post-menopausal women and that hormone therapy may improve immune response to vaccination in this growing segment of the population.

Objective

To describe Japanese macaque encephalomyelitis (JME), a spontaneous inflammatory demyelinating disease occurring in the Oregon National Primate Research Center’s (ONPRC) colony of Japanese macaques (JM, Macaca fuscata).

Methods

JM with neurologic impairment were removed from the colony, evaluated and treated with supportive care. Animals were humanely euthanized and their central nervous system (CNS) examined.

Results

ONPRC’s JM colony was established in 1965 and no cases of JME occurred until 1986. Since 1986, 56 JM spontaneously developed a disease characterized clinically by paresis of one or more limbs, ataxia or ocular motor paresis. Most animals were humanely euthanized during their initial episode. Three recovered, later relapsed and were then euthanized. There was no gender predilection and the median age for disease was 4 years. Magnetic resonance imaging of eight cases of JME revealed multiple gadolinium enhancing T1-weighted hyperintensities in the white matter of the cerebral hemispheres, brainstem, cerebellum and cervical spinal cord. The CNS of monkeys with JME contained multifocal plaque-like demyelinated lesions of varying ages, including acute and chronic, active demyelinating lesions with macrophages and lymphocytic periventricular infiltrates, and chronic, inactive demyelinated lesions. A previously undescribed gamma-herpesvirus was cultured from acute JME white matter lesions. Cases of JME continue to affect 1–3% of the ONPRC colony per year.

Interpretation

JME is a unique spontaneous disease in a nonhuman primate that has similarities with multiple sclerosis (MS) and is associated with a novel simian herpesvirus. Elucidating the pathogenesis of JME may shed new light on MS and other human demyelinating diseases.

Cerebral ischemic injury is a significant portion of the burden of disease in developed countries; rates of mortality are high and the costs associated with morbidity are enormous. Recent therapeutic approaches have aimed at mitigating the extent of damage and/or promoting repair once injury has occurred. Often, patients at high risk of ischemic injury can be identified in advance and targeted for antecedent neuroprotective therapy. Agents that stimulate the innate pattern recognition receptor, Toll-like receptor 9, have been shown to induce tolerance (precondition) to ischemic brain injury in a mouse model of stroke. Here, we demonstrate for the first time that pharmacological preconditioning against cerebrovascular ischemic injury is also possible in a nonhuman primate model of stroke in the rhesus macaque. The model of stroke used is a minimally invasive transient vascular occlusion, resulting in brain damage that is primarily localized to the cortex and as such, represents a model with substantial clinical relevance. Finally, K-type (also referred to as B-type) cytosine-guanine-rich DNA oligonucleotides, the class of agents employed in this study, are currently in use in human clinical trials, underscoring the feasibility of this treatment in patients at risk of cerebral ischemia.

We recently reported that in aged female rhesus macaques, spatial learning and memory correlates with circadian sleep-wake measures and hippocampal muscarinic type 1 (M1) receptor binding. To investigate if spatial memory also correlates with measures of immune function, we now assessed the magnitude of the adaptive immune response to vaccination in the same old female rhesus macaques. Cognitively characterized animals were classified as good spatial performers (GSP) or poor spatial performers (PSP) based on performance in the Spatial Foodport maze. The GSP group had higher frequency of CD8, but not CD4, interferon-γ (IFN-γ) producing cells following vaccination compared to the PSP group, suggesting a stronger CD8 T cell response in the GSP group. In addition, the number of CD-8 IFN-γ positive cells correlated with measures of sleep quality. Interestingly, the PSP group had a significantly higher antibody titer compared to the GSP group, and antibody titer negatively correlated with day-time activity. Thus, in aged female rhesus macaques, superior cognitive performance is correlated with a more robust CD8 T cell response but a reduced antibody response to vaccination.

Loss of synaptic integrity in the hippocampus and prefrontal cortex (PFC) may play an integral role in age-related cognitive decline. Previously, we showed age-related increases in the dendritic marker microtubule associated protein 2 (MAP-2) and the synaptic marker synaptophysin (SYN) in mice. Similarly, apolipoprotein E (apoE), involved in lipid transport and metabolism, and glial fibrillary acidic protein (GFAP), a glia specific marker, increase with age in rodents. In this study, we assessed whether these four proteins show similar age-related changes in a nonhuman primate, the rhesus macaque. Free-floating sections from the PFC and hippocampus from adult, middle-aged, and aged rhesus macaques were immunohistochemically labeled for MAP-2, SYN, apoE, and GFAP. Protein levels were measured as area occupied by fluorescence using confocal microscopy as well as by Western blot. In the PFC and hippocampus of adult and middle-aged animals, the levels of SYN, apoE, and GFAP immunoreactivity were comparable but there was a trend towards higher MAP-2 levels in middle-aged than adult animals. There was significantly less SYN and more MAP-2, apoE, and GFAP immunoreactivity in the PFC and hippocampus of aged animals compared to adult or middle-aged animals. Thus, the age-related changes in MAP-2, apoE, and GFAP levels were similar to those previously observed in rodents. On the other hand, the age-related changes in SYN levels were not, but were similar to those previously observed in the aging human brain. Taken together, these data emphasize the value of the rhesus macaque as a pragmatic translational model for human brain aging.

Apyrimidinic/apurinic endonuclease (APE) is a key protein involved in the base excision DNA repair (BER) pathway of oxidative DNA lesions. Using a novel oligonucleotide substrate, we demonstrate that APE activity in the frontal/parietal cortex (F/PCTX), cerebellum, brainstem, midbrain and hypothalamus declined with age in rats on an ad libitum (AL) diet. In contrast, APE activity for these brain regions was ~1.5-3 times higher in young, caloric restricted (CR) rats. Despite continuous CR treatment in all animals since six weeks of age, APE activity in the CR group started to decline by middle-age and continued into old age. However, CR maintained APE activity at a level that was significantly higher than that in AL rats across age and in the brain regions examined. Because Western analysis of APE, DNA polymerase β and DNA ligase III levels in the F/PCTX of both CR and AL rats remained unchanged with age, this suggests that the increased APE activity in CR rats is the result of differential post-translational modification of APE.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, and the responsiveness of neurons to GABA can be modulated by sex steroids. To better understand how ovarian steroids influence GABAergic system in the primate brain, we evaluated the expression of genes encoding GABA receptor subunits, glutamic acid decarboxylase (GAD) and a GABA transporter in the brains of female rhesus macaques. Ovariectomized adults were subjected to a hormone replacement paradigm involving either 17β-estradiol (E), or E plus progesterone (E+P). Untreated animals served as controls. Using GeneChip® microarray analysis and real-time RT-PCR (qPCR), we examined gene expression differences within and between the amygdala (AMD), hippocampus (HPC) and arcuate nuclei of the medial basal hypothalamus (MBH). The results from PCR corresponded with results from representative GeneChip® probesets, and showed similar effects of sex steroids on GABA receptor subunit gene expression in the AMD and HPC, and a more pronounced expression than in the MBH. Exposure to E+P attenuated GAD1, GAD2 and SLC32A1 gene expression in the AMD and HPC, but not in the MBH. GABA receptor subunit gene expression was generally higher in the AMD and HPC than in the MBH, with the exception of receptor subunits ε and γ2. Taken together, the data demonstrate differential regulation of GABA receptor subunits and GABAergic system components in the MBH compared to the AMD and HPC of rhesus macaques. Elevated ε and reduced δ subunit expression in the MBH supports the hypothesis that the hypothalamic GABAergic system is resistant to the modulatory effects of sex steroids.

Nonhuman primates (NHPs) are an invaluable resource for the study of genetic regulation of disease mechanisms. The main disadvantage of using NHPs as a preclinical model of human disease is the difficulty of manipulating the monkey genome using conventional gene modifying strategies. Lentiviruses offer the possibility of circumventing this difficulty because they can infect and transduce either dividing or nondividing cells, without producing an immune response. In addition, lentiviruses can permanently integrate into the genome of host cells, and are able to maintain long-term expression. In this article we describe the lentiviral vectors that we use to both express transgenes and suppress expression of endogenous genes via RNA interference (RNAi) in NHPs. We also discuss the safety features of currently available vectors that are especially important when lentiviral vectors are used in a species as closely related to humans as NHPs. Finally, we describe in detail the lentiviral vector production protocol we use and provide examples of how the vector can be employed to target peripheral tissues and the brain.

The development of species-specific gene microarrays has greatly facilitated gene expression profiling in nonhuman primates. However, to obtain accurate and physiologically meaningful data from these microarrays, one needs to consider several factors when designing the studies. This article focuses on effective experimental design while the companion article focuses on methodology and data analysis. Biological cycles have a major influence on gene expression, and at least 10% of the expressed genes are likely to show a 24-hour expression pattern. Consequently, the time of day when RNA samples are collected can influence detection of significant changes in gene expression levels. Similarly, when photoperiodic species such as the rhesus macaque are housed outdoors, some of their genes show differential expression according to the time of year. In addition, the sex-steroid environment of humans and many nonhuman primates changes markedly across the menstrual cycle, and so phase of the cycle needs to be considered when studying gene expression in adult females.

Gene microarray analyses represent potentially effective means for high-throughput gene expression profiling in nonhuman primates. In the companion article we emphasize effective experimental design based on the in vivo physiology of the rhesus macaque, whereas this article emphasizes considerations for gene annotation and data interpretation using gene microarray platforms from Affymetrix®. Initial annotation of the rhesus genome array was based on Affymetrix® human GeneChips®. However, annotation revisions improve the precision with which rhesus transcripts are identified. Annotation of the rhesus GeneChip® is under continuous revision with large percentages of probesets under multiple annotation systems having undergone multiple reassignments between March 2007 and November 2008. It is also important to consider that quantitation and comparison of gene expression levels across multiple chips requires appropriate normalization. External corroboration of microarray results using PCR-based methodology also requires validation of appropriate internal reference genes for normalization of expression values. Many tools are now freely available to aid investigators with microarray normalization and selection of internal reference genes to be used for independent corroboration of microarray results.

Background

Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods.

We recently demonstrated that relative gene expression from qRT-PCR data normalized using GAPDH, ALG9 and RPL13A expression values mirrored relative expression using quantile normalization in Robust Multichip Analysis (RMA) on the Affymetrix® GeneChip® rhesus Macaque Genome Array.

Having shown that qRT-PCR and Affymetrix® GeneChip® data from the same hormone replacement therapy (HRT) study yielded concordant results, we used quantile-normalized gene microarray data to identify the most stably expressed among probe sets for prospective internal reference genes across three brain regions from the HRT study and an additional study of normally menstruating rhesus macaques (cycle study). Gene selection was limited to 575 previously published human "housekeeping" genes. Twelve animals were used per study, and three brain regions were analyzed from each animal. Gene expression stabilities were determined using geNorm, NormFinder and BestKeeper software packages.

Results

Sequences co-annotated for ribosomal protein S27a (RPS27A), and ubiquitin were among the most stably expressed under all conditions and selection criteria used for both studies. Higher annotation quality on the human GeneChip® facilitated more targeted analysis than could be accomplished using the rhesus GeneChip®. In the cycle study, multiple probe sets annotated for actin, gamma 1 (ACTG1) showed high signal intensity and were among the most stably expressed.

Conclusions

Using gene microarray analysis, we identified genes showing high expression stability under various sex-steroid environments in different regions of the rhesus macaque brain. Use of quantile-normalized microarray gene expression values represents an improvement over traditional methods of selecting internal reference genes for PCR analysis.

Magnetic resonance imaging (MRI) studies of non-human primates are becoming increasingly common; however, the well-developed voxel-based methodologies used in human studies are not readily applied to non-human primates. In the present study, we create a population-average MRI-based atlas collection for the rhesus macaque (Macaca mulatta) that can be used with common brain mapping packages such as SPM or FSL. In addition to creating a publicly available T1-weighted atlas (http://www.brainmap.wisc.edu/monkey.html), probabilistic tissue classification maps and T2-weighted atlases were also created. Theses atlases are aligned to the MRI volume from the Saleem-Logothetis (2006) atlas providing an explicit link to histological sections. Additionally, we have created a transform to integrate these atlases with the F99 surface-based atlas in CARET. It is anticipated that these tools will help facilitate voxel-based imaging methodologies in non-human primate species, which in turn may increase our understanding of brain function, development, and evolution.

Primate models are essential tools for translational research in stroke, but are reportedly inconsistent in their ability to produce cortical infarcts of reproducible size. Here we report a new stroke model using a transorbital, reversible, two-vessel occlusion approach in male rhesus macaques that produces consistent and reproducible cortical infarcts. The right middle cerebral artery (MCA) (distal to the orbitofrontal branch), and both anterior cerebral arteries (ACA) were occluded with vascular clips. Bilateral occlusion of the ACA was critical for reducing collateral flow to the ipsilateral cortex. Reversible ischemia were induced for 45, 60, or 90 minutes (n=2/timepoint) and infarct volume and neurological outcome were evaluated. The infarcts were located predominantly in the cortex and increased in size with extended duration of ischemia determined by T2-weighted MRI. Infarct volume measured by 2,3,5-triphenyl tetrazolium chloride (TTC) and cresyl violet staining corroborated MRI results. Neurological deficit scores worsened gradually with longer occlusion times. A subset of animals (n=5) underwent 60 minutes of ischemia resulting in consistent infarct volumes primarily located to the cortex which correlated well with neurological deficit scores. This approach offers promise for evaluating therapeutic interventions in stroke.

Evidence supports a pathogenic role for free radical injury to brain regions in Alzheimer's disease (AD); however, clinical trial results are only mildly encouraging. Examining brains from The Adult Changes in Thought study offers a unique perspective. Selectively increased free radical damage to cerebral cortex was associated with AD, microvascular brain injury (μ-VBI), and current smoking, but not with antioxidant supplement usage. Our results support suppression of free radical injury to brain as a therapeutic target for AD and μ-VBI; however, future clinical trials should consider other antioxidants or doses than those identified in our study.

The locus coeruleus (LC) is a major noradrenergic brain nucleus that regulates states of arousal, optimizes task-oriented decision-making, and may also play an important role in modulating the activity of the reproductive neuroendocrine axis. Rodent studies have shown that the LC is responsive to glutamate receptor agonists, and that it expresses various glutamate receptor subunits. However, glutamate receptor subunit expression has not been extensively examined in the primate LC. We previously demonstrated expression of the NR1 NMDA glutamate receptor subunit in the rhesus macaque LC, and now extend this work by also examining the expression of non-NMDA (AMPA and kainate) ionotropic glutamate receptor subunits. Using in situ hybridization histochemistry and immunohistochemistry, we confirmed the presence of the obligatory NR1 subunit in the LC. In addition, we demonstrated expression of the AMPA glutamate receptor subunits GluR1, GluR2 and GluR3. More extensive receptor profiling, using rhesus monkey gene microarrays (Affymetrix GeneChip®), further corroborated the histological findings and showed expression of mRNA encoding ionotropic glutamate receptor subunits NR2A, NR2D, GluR4, and GluR6, as well as the metabotropic glutamate receptor subunits mGluR1, mGluR3, mGluR4, mGluR5 and mGluR7. These data provide a foundation for future examination of how changes in glutamate receptor composition contribute to the control of primate physiology.