Tempo and mode of gene expression evolution in the brain across primates.

Primate evolution has led to a remarkable diversity of behavioral specializations and pronounced brain size variation among species (Barton, 2012; DeCasien and Higham, 2019; Powell et al., 2017). Gene expression provides a promising opportunity for studying the molecular basis of brain evolution, but it has been explored in very few primate species to date (e.g. Khaitovich et al., 2005; Khrameeva et al., 2020; Ma et al., 2022; Somel et al., 2009). To understand the landscape of gene expression evolution across the primate lineage, we generated and analyzed RNA-seq data from four brain regions in an unprecedented eighteen species. Here, we show a remarkable level of variation in gene expression among hominid species, including humans and chimpanzees, despite their relatively recent divergence time from other primates. We found that individual genes display a wide range of expression dynamics across evolutionary time reflective of the diverse selection pressures acting on genes within primate brain tissue. Using our samples that represent a 190-fold difference in primate brain size, we identified genes with variation in expression most correlated with brain size. Our study extensively broadens the phylogenetic context of what is known about the molecular evolution of the brain across primates and identifies novel candidate genes for the study of genetic regulation of brain evolution.

Human-specific features and developmental dynamics of the brain N-glycome.

Comparative "omics" studies have revealed unique aspects of human neurobiology, yet an evolutionary perspective of the brain N-glycome is lacking. We performed multiregional characterization of rat, macaque, chimpanzee, and human brain N-glycomes using chromatography and mass spectrometry and then integrated these data with complementary glycotranscriptomic data. We found that, in primates, the brain N-glycome has diverged more rapidly than the underlying transcriptomic framework, providing a means for rapidly generating additional interspecies diversity. Our data suggest that brain N-glycome evolution in hominids has been characterized by an overall increase in complexity coupled with a shift toward increased usage of α(2-6)-linked N-acetylneuraminic acid. Moreover, interspecies differences in the cell type expression pattern of key glycogenes were identified, including some human-specific differences, which may underpin this evolutionary divergence. Last, by comparing the prenatal and adult human brain N-glycomes, we uncovered region-specific neurodevelopmental pathways that lead to distinct spatial N-glycosylation profiles in the mature brain.

Demography and epidemiology of captive former biomedical research chimpanzees (Pan troglodytes) 1: Survival and mortality.

Accurate and up-to-date data on longevity and mortality are essential for describing, analyzing, and managing animal populations in captivity. We assembled a comprehensive demography data set and analyzed survival and mortality patterns in a population of captive former biomedical research chimpanzees. The study synthesized over 51,000 life-years of demographic data collected on 2349 individuals between 1923 and 2014. Our goal was to assess the population's current age-sex composition, estimate rates of survivorship, mortality and life expectancy, and compare findings with other chimpanzee populations of interest. Results indicated an increasingly geriatric contemporary population declining in size. The median life expectancy (MLE) of the entire population was 32.6 years (males 29.1, females 36.1). For chimpanzees who reached 1 year of age, the MLE increased to 34.9 years (males 31.0, females 38.8). Survival probability was influenced by both sex and birth type. Females exhibited greater survivorship than males (ß1 = -0.34, z = -5.74, p < 0.001) and wild-born individuals exhibited greater survivorship than captive-born individuals (ß2 = -0.55, z = -5.89, p < 0.001). There was also a seasonal trend in mortality, wherein more individuals died during the winter months (December-February) compared with other seasons. Analyses of life expectancy over time showed continual increases in both median age of living individuals and median age at death, suggesting that these chimpanzees have yet to reach their full aging potential in a postresearch environment. As they continue to age, ongoing monitoring and analysis of demographic changes will be necessary for science-based population and program management until extinction occurs some decades in the future.

Human-specific features and developmental dynamics of the brain N-glycome.

Comparative "omics" studies have revealed unique aspects of human neurobiology, yet an evolutionary perspective of the brain N-glycome is lacking. Here, we performed multi-regional characterization of rat, macaque, chimpanzee, and human brain N-glycomes using chromatography and mass spectrometry, then integrated these data with complementary glycotranscriptomic data. We found that in primates the brain N-glycome has evolved more rapidly than the underlying transcriptomic framework, providing a mechanism for generating additional diversity. We show that brain N-glycome evolution in hominids has been characterized by an increase in complexity and α(2-6)-linked N-acetylneuraminic acid along with human-specific cell-type expression of key glycogenes. Finally, by comparing the prenatal and adult human brain N-glycome, we identify region-specific neurodevelopmental pathways that lead to distinct spatial N-glycosylation profiles in the mature brain. One-Sentence Summary: Evolution of the human brain N-glycome has been marked by an increase in complexity and a shift in sialic acid linkage.

Molecular and cellular evolution of the primate dorsolateral prefrontal cortex.

The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2, which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

Epigenetic ageing of the prefrontal cortex and cerebellum in humans and chimpanzees.

Epigenetic age has emerged as an important biomarker of biological ageing. It has revealed that some tissues age faster than others, which is vital to understanding the complex phenomenon of ageing and developing effective interventions. Previous studies have demonstrated that humans exhibit heterogeneity in pace of epigenetic ageing among brain structures that are consistent with differences in structural and microanatomical deterioration. Here, we add comparative data on epigenetic brain ageing for chimpanzees, humans' closest relatives. Such comparisons can further our understanding of which aspects of human ageing are evolutionarily conserved or specific to our species, especially given that humans are distinguished by a long lifespan, large brain, and, potentially, more severe neurodegeneration with age. Specifically, we investigated epigenetic ageing of the dorsolateral prefrontal cortex and cerebellum, of humans and chimpanzees by generating genome-wide CpG methylation data and applying established epigenetic clock algorithms to produce estimates of biological age for these tissues. We found that both species exhibit relatively slow epigenetic ageing in the brain relative to blood. Between brain structures, humans show a faster rate of epigenetic ageing in the dorsolateral prefrontal cortex compared to the cerebellum, which is consistent with previous findings. Chimpanzees, in contrast, show comparable rates of epigenetic ageing in the two brain structures. Greater epigenetic change in the human dorsolateral prefrontal cortex compared to the cerebellum may reflect both the protracted development of this structure in humans and its greater age-related vulnerability to neurodegenerative pathology.

Comparative analysis reveals distinctive epigenetic features of the human cerebellum.

Identifying the molecular underpinnings of the neural specializations that underlie human cognitive and behavioral traits has long been of considerable interest. Much research on human-specific changes in gene expression and epigenetic marks has focused on the prefrontal cortex, a brain structure distinguished by its role in executive functions. The cerebellum shows expansion in great apes and is gaining increasing attention for its role in motor skills and cognitive processing, including language. However, relatively few molecular studies of the cerebellum in a comparative evolutionary context have been conducted. Here, we identify human-specific methylation in the lateral cerebellum relative to the dorsolateral prefrontal cortex, in a comparative study with chimpanzees (Pan troglodytes) and rhesus macaques (Macaca mulatta). Specifically, we profiled genome-wide methylation levels in the three species for each of the two brain structures and identified human-specific differentially methylated genomic regions unique to each structure. We further identified which differentially methylated regions (DMRs) overlap likely regulatory elements and determined whether associated genes show corresponding species differences in gene expression. We found greater human-specific methylation in the cerebellum than the dorsolateral prefrontal cortex, with differentially methylated regions overlapping genes involved in several conditions or processes relevant to human neurobiology, including synaptic plasticity, lipid metabolism, neuroinflammation and neurodegeneration, and neurodevelopment, including developmental disorders. Moreover, our results show some overlap with those of previous studies focused on the neocortex, indicating that such results may be common to multiple brain structures. These findings further our understanding of the cerebellum in human brain evolution.

Evolution of regulatory signatures in primate cortical neurons at cell-type resolution.

The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell-type-specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee, and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype-specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder, and drug addiction. We observe preferential evolutionary divergence in neuron subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergoes subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type-dependent gene-expression programs, and provides a resource for further exploration of human brain evolution and function.

Neuron loss associated with age but not Alzheimer's disease pathology in the chimpanzee brain.

In the absence of disease, ageing in the human brain is accompanied by mild cognitive dysfunction, gradual volumetric atrophy, a lack of significant cell loss, moderate neuroinflammation, and an increase in the amyloid beta (Aß) and tau proteins. Conversely, pathologic age-related conditions, particularly Alzheimer's disease (AD), result in extensive neocortical and hippocampal atrophy, neuron death, substantial Aß plaque and tau-associated neurofibrillary tangle pathologies, glial activation and severe cognitive decline. Humans are considered uniquely susceptible to neurodegenerative disorders, although recent studies have revealed Aß and tau pathology in non-human primate brains. Here, we investigate the effect of age and AD-like pathology on cell density in a large sample of postmortem chimpanzee brains (n = 28, ages 12-62 years). Using a stereologic, unbiased design, we quantified neuron density, glia density and glia:neuron ratio in the dorsolateral prefrontal cortex, middle temporal gyrus, and CA1 and CA3 hippocampal subfields. Ageing was associated with decreased CA1 and CA3 neuron densities, while AD pathologies were not correlated with changes in neuron or glia densities. Differing from cerebral ageing and AD in humans, these data indicate that chimpanzees exhibit regional neuron loss with ageing but appear protected from the severe cell death found in AD. This article is part of the theme issue 'Evolution of the primate ageing process'.

Age-associated epigenetic change in chimpanzees and humans.

Methylation levels have been shown to change with age at sites across the human genome. Change at some of these sites is so consistent across individuals that it can be used as an 'epigenetic clock' to predict an individual's chronological age to within a few years. Here, we examined how the pattern of epigenetic ageing in chimpanzees compares with humans. We profiled genome-wide blood methylation levels by microarray for 113 samples from 83 chimpanzees aged 1-58 years (26 chimpanzees were sampled at multiple ages during their lifespan). Many sites (greater than 65 000) showed significant change in methylation with age and around one-third (32%) of these overlap with sites showing significant age-related change in humans. At over 80% of sites showing age-related change in both species, chimpanzees displayed a significantly faster rate of age-related change in methylation than humans. We also built a chimpanzee-specific epigenetic clock that predicted age in our test dataset with a median absolute deviation from known age of only 2.4 years. However, our chimpanzee clock showed little overlap with previously constructed human clocks. Methylation at CpGs comprising our chimpanzee clock showed moderate heritability. Although the use of a human microarray for profiling chimpanzees biases our results towards regions with shared genomic sequence between the species, nevertheless, our results indicate that there is considerable conservation in epigenetic ageing between chimpanzees and humans, but also substantial divergence in both rate and genomic distribution of ageing-associated sites. This article is part of the theme issue 'Evolution of the primate ageing process'.

Evolution of ASPM coding variation in apes and associations with brain structure in chimpanzees.

Studying genetic mechanisms underlying primate brain morphology can provide insight into the evolution of human brain structure and cognition. In humans, loss-of-function mutations in the gene coding for ASPM (Abnormal Spindle Microtubule Assembly) have been associated with primary microcephaly, which is defined by a significantly reduced brain volume, intellectual disability and delayed development. However, less is known about the effects of common ASPM variation in humans and other primates. In this study, we characterized the degree of coding variation at ASPM in a large sample of chimpanzees (N = 241), and examined potential associations between genotype and various measures of brain morphology. We identified and genotyped five non-synonymous polymorphisms in exons 3 (V588G), 18 (Q2772K, K2796E, C2811Y) and 27 (I3427V). Using T1-weighted magnetic resonance imaging of brains, we measured total brain volume, cerebral gray and white matter volume, cerebral ventricular volume, and cortical surface area in the same chimpanzees. We found a potential association between ASPM V588G genotype and cerebral ventricular volume but not with the other measures. Additionally, we found that chimpanzee, bonobo, and human lineages each independently show a signature of accelerated ASPM protein evolution. Overall, our results suggest the potential effects of ASPM variation on cerebral cortical development, and emphasize the need for further functional studies. These results are the first evidence suggesting ASPM variation might play a role in shaping natural variation in brain structure in nonhuman primates.

Astrocytic changes with aging and Alzheimer's disease-type pathology in chimpanzees.

Astrocytes are the main homeostatic cell of the central nervous system. In addition, astrocytes mediate an inflammatory response when reactive to injury or disease known as astrogliosis. Astrogliosis is marked by an increased expression of glial fibrillary acidic protein (GFAP) and cellular hypertrophy. Some degree of astrogliosis is associated with normal aging and degenerative conditions such as Alzheimer's disease (AD) and other dementing illnesses in humans. The recent observation of pathological markers of AD (amyloid plaques and neurofibrillary tangles) in aged chimpanzee brains provided an opportunity to examine the relationships among aging, AD-type pathology, and astrocyte activation in our closest living relatives. Stereologic methods were used to quantify GFAP-immunoreactive astrocyte density and soma volume in layers I, III, and V of the prefrontal and middle temporal cortex, as well as in hippocampal fields CA1 and CA3. We found that the patterns of astrocyte activation in the aged chimpanzee brain are distinct from humans. GFAP expression does not increase with age in chimpanzees, possibly indicative of lower oxidative stress loads. Similar to humans, chimpanzee layer I astrocytes in the prefrontal cortex are susceptible to AD-like changes. Both prefrontal cortex layer I and hippocampal astrocytes exhibit a high degree of astrogliosis that is positively correlated with accumulation of amyloid beta and tau proteins. However, unlike humans, chimpanzees do not display astrogliosis in other cortical layers. These results demonstrate a unique pattern of cortical aging in chimpanzees and suggest that inflammatory processes may differ between humans and chimpanzees in response to pathology.

Microglia changes associated to Alzheimer's disease pathology in aged chimpanzees.

In Alzheimer's disease (AD), the brain's primary immune cells, microglia, become activated and are found in close apposition to amyloid beta (Aß) protein plaques and neurofibrillary tangles (NFT). The present study evaluated microglia density and morphology in a large group of aged chimpanzees (n = 20, ages 37-62 years) with varying degrees of AD-like pathology. Using immunohistochemical and stereological techniques, we quantified the density of activated microglia and morphological variants (ramified, intermediate, and amoeboid) in postmortem chimpanzee brain samples from prefrontal cortex, middle temporal gyrus, and hippocampus, areas that show a high degree of AD pathology in humans. Microglia measurements were compared to pathological markers of AD in these cases. Activated microglia were consistently present across brain areas. In the hippocampus, CA3 displayed a higher density than CA1. Aß42 plaque volume was positively correlated with higher microglial activation and with an intermediate morphology in the hippocampus. Aß42-positive vessel volume was associated with increased hippocampal microglial activation. Activated microglia density and morphology were not associated with age, sex, pretangle density, NFT density, or tau neuritic cluster density. Aged chimpanzees displayed comparable patterns of activated microglia phenotypes as well as an association of increased microglial activation and morphological changes with Aß deposition similar to AD patients. In contrast to human AD brains, activated microglia density was not significantly correlated with tau lesions. This evidence suggests that the chimpanzee brain may be relatively preserved during normal aging processes but not entirely protected from neurodegeneration as previously assumed.

FOXP2 variation in great ape populations offers insight into the evolution of communication skills.

The gene coding for the forkhead box protein P2 (FOXP2) is associated with human language disorders. Evolutionary changes in this gene are hypothesized to have contributed to the emergence of speech and language in the human lineage. Although FOXP2 is highly conserved across most mammals, humans differ at two functional amino acid substitutions from chimpanzees, bonobos and gorillas, with an additional fixed substitution found in orangutans. However, FOXP2 has been characterized in only a small number of apes and no publication to date has examined the degree of natural variation in large samples of unrelated great apes. Here, we analyzed the genetic variation in the FOXP2 coding sequence in 63 chimpanzees, 11 bonobos, 48 gorillas, 37 orangutans and 2 gibbons and observed undescribed variation in great apes. We identified two variable polyglutamine microsatellites in chimpanzees and orangutans and found three nonsynonymous single nucleotide polymorphisms, one in chimpanzees, one in gorillas and one in orangutans with derived allele frequencies of 0.01, 0.26 and 0.29, respectively. Structural and functional protein modeling indicate a biochemical effect of the substitution in orangutans, and because of its presence solely in the Sumatran orangutan species, the mutation may be associated with reported population differences in vocalizations.

Molecular and cellular reorganization of neural circuits in the human lineage.

To better understand the molecular and cellular differences in brain organization between human and nonhuman primates, we performed transcriptome sequencing of 16 regions of adult human, chimpanzee, and macaque brains. Integration with human single-cell transcriptomic data revealed global, regional, and cell-type-specific species expression differences in genes representing distinct functional categories. We validated and further characterized the human specificity of genes enriched in distinct cell types through histological and functional analyses, including rare subpallial-derived interneurons expressing dopamine biosynthesis genes enriched in the human striatum and absent in the nonhuman African ape neocortex. Our integrated analysis of the generated data revealed diverse molecular and cellular features of the phylogenetic reorganization of the human brain across multiple levels, with relevance for brain function and disease.

X-linked adrenoleukodystrophy in a chimpanzee due to an ABCD1 mutation reported in multiple unrelated humans.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder leading to the accumulation of very long chain fatty acids (VLCFA) due to a mutation in the ABCD1 gene. ABCD1 mutations lead to a variety of phenotypes, including cerebral X-ALD and adrenomyeloneuropathy (AMN) in affected males and 80% of carrier females. There is no definite genotype-phenotype correlation with intrafamilial variability. Cerebral X-ALD typically presents in childhood, but can also present in juveniles and adults. The most affected tissues are the white matter of the brain and adrenal cortex. MRI demonstrates a characteristic imaging appearance in cerebral X-ALD that is used as a diagnostic tool. OBJECTIVES: We aim to correlate a mutation in the ABCD1 gene in a chimpanzee to the human disease X-ALD based on MRI features, neurologic symptoms, and plasma levels of VLCFA. METHODS: Diagnosis of X-ALD made using MRI, blood lipid profiling, and DNA sequencing. RESULTS: An 11-year-old chimpanzee showed remarkably similar features to juvenile onset cerebral X-ALD in humans including demyelination of frontal lobes and corpus callosum on MRI, elevated plasma levels of C24:0 and C26:0, and identification of the c.1661G>A ABCD1 variant. CONCLUSIONS: This case study presents the first reported case of a leukodystrophy in a great ape, and underscores the fidelity of MRI pattern recognition in this disorder across species.

Aged chimpanzees exhibit pathologic hallmarks of Alzheimer's disease.

Alzheimer's disease (AD) is a uniquely human brain disorder characterized by the accumulation of amyloid-beta protein (Aß) into extracellular plaques, neurofibrillary tangles (NFT) made from intracellular, abnormally phosphorylated tau, and selective neuronal loss. We analyzed a large group of aged chimpanzees (n = 20, age 37-62 years) for evidence of Aß and tau lesions in brain regions affected by AD in humans. Aß was observed in plaques and blood vessels, and tau lesions were found in the form of pretangles, NFT, and tau-immunoreactive neuritic clusters. Aß deposition was higher in vessels than in plaques and correlated with increases in tau lesions, suggesting that amyloid build-up in the brain's microvasculature precedes plaque formation in chimpanzees. Age was correlated to greater volumes of Aß plaques and vessels. Tangle pathology was observed in individuals that exhibited plaques and moderate or severe cerebral amyloid angiopathy, a condition in which amyloid accumulates in the brain's vasculature. Amyloid and tau pathology in aged chimpanzees suggests these AD lesions are not specific to the human brain.

Divergent lactate dehydrogenase isoenzyme profile in cellular compartments of primate forebrain structures.

The compartmentalization and association of lactate dehydrogenase (LDH) with specific cellular structures (e.g., synaptosomal, sarcoplasmic or mitochondrial) may play an important role in brain energy metabolism. Our previous research revealed that LDH in the synaptosomal fraction shifts toward the aerobic isoforms (LDH-B) among the large-brained haplorhine primates compared to strepsirrhines. Here, we further analyzed the subcellular localization of LDH in primate forebrain structures using quantitative Western blotting and ELISA. We show that, in cytosolic and mitochondrial subfractions, LDH-B expression level was relatively elevated and LDH-A declined in haplorhines compared to strepsirrhines. LDH-B expression in mitochondrial fractions of the neocortex was preferentially increased, showing a particularly significant rise in the ratio of LDH-B to LDH-A in chimpanzees and humans. We also found a significant correlation between the protein levels of LDH-B in mitochondrial fractions from haplorhine neocortex and the synaptosomal LDH-B that suggests LDH isoforms shift from a predominance of A-subunits toward B-subunits as part of a system that spatially buffers dynamic energy requirements of brain cells. Our results indicate that there is differential subcellular compartmentalization of LDH isoenzymes that evolved among different primate lineages to meet the energy requirements in neocortical and striatal cells.

Changes in Lipidome Composition during Brain Development in Humans, Chimpanzees, and Macaque Monkeys.

Lipids are essential components of the brain. Here, we conducted a comprehensive mass spectrometry-based analysis of lipidome composition in the prefrontal cortex of 40 humans, 40 chimpanzees, and 40 rhesus monkeys over postnatal development and adulthood. Of the 11,772 quantified lipid peaks, 7,589 change significantly along the lifespan. More than 60% of these changes occur prior to adulthood, with less than a quarter associated with myelination progression. Evolutionarily, 36% of the age-dependent lipids exhibit concentration profiles distinct to one of the three species; 488 (18%) of them were unique to humans. In both humans and chimpanzees, the greatest extent of species-specific differences occurs in early development. Human-specific lipidome differences, however, persist over most of the lifespan and reach their peak from 20 to 35 years of age, when compared with chimpanzee-specific ones.

Disruption of an Evolutionarily Novel Synaptic Expression Pattern in Autism.

Cognitive defects in autism spectrum disorder (ASD) include socialization and communication: key behavioral capacities that separate humans from other species. Here, we analyze gene expression in the prefrontal cortex of 63 autism patients and control individuals, as well as 62 chimpanzees and macaques, from natal to adult age. We show that among all aberrant expression changes seen in ASD brains, a single aberrant expression pattern overrepresented in genes involved synaptic-related pathways is enriched in nucleotide variants linked to autism. Furthermore, only this pattern contains an excess of developmental expression features unique to humans, thus resulting in the disruption of human-specific developmental programs in autism. Several members of the early growth response (EGR) transcription factor family can be implicated in regulation of this aberrant developmental change. Our study draws a connection between the genetic risk architecture of autism and molecular features of cortical development unique to humans.