Translational Utility of the Nonhuman Primate Model.

Nonhuman primates are essential for the study of human disease and to explore the safety of new diagnostics and therapies proposed for human use. They share similar genetic, physiologic, immunologic, reproductive, and developmental features with humans and thus have proven crucial for the study of embryonic/fetal development, organ system ontogeny, and the role of the maternal-placental-fetal interface in health and disease. The fetus may be exposed to a variety of inflammatory stimuli including infectious microbes as well as maternal inflammation, which can result from infections, obesity, or environmental exposures. Growing evidence supports that inflammation is a mediator of fetal programming and that the maternal immune system is tightly integrated with fetal-placental immune responses that may set a postnatal path for future health or disease. This review addresses some of the unique features of the nonhuman primate model system, specifically the rhesus monkey (Macaca mulatta), and importance of the species for studies focused on organ system ontogeny and the impact of viral teratogens in relation to development and congenital disorders.

Decreased number and increased activation state of astrocytes in gray and white matter of the prefrontal cortex in autism.

The cerebral cortex presents with alterations in the number of specific cell types in autism spectrum disorder (ASD). Astrocytes have many functions in the brain including a role in higher cognitive functions and in inflammatory brain processes. Therefore, an alteration in number, function, and/or activation state of astrocytes, could be present in ASD. We quantified astrocyte number in the gray and white matter of the prefrontal cortex-BA9, BA46, and BA47-in 15 ASD and 15 age- and sex-matched control cases. We labeled astrocytes with antibodies against the protein GFAP and S100ß, markers of astrocytes. We found a significant decrease in the number of astrocytes in the gray and white matter of all prefrontal areas of interest with both markers. We also found an increased state of activation of GFAP+ astrocytes in all areas. A reduced number of astrocytes in the cerebral cortex in ASD could lead to impaired synaptic function and disrupted connectivity. An increased astrocyte activation may indicate a chronic mild inflammatory state of the cerebral cortex in ASD. Overall, we found that astrocytes are disrupted in ASD.

Nonhuman Primates in Translational Research.

Nonhuman primates are critically important animal models in which to study complex human diseases, understand biological functions, and address the safety of new diagnostics and therapies proposed for human use. They have genetic, physiologic, immunologic, and developmental similarities when compared to humans and therefore provide important preclinical models of human health and disease. This review highlights select research areas that demonstrate the importance of nonhuman primates in translational research. These include pregnancy and developmental disorders, infectious diseases, gene therapy, somatic cell genome editing, and applications of in vivo imaging. The power of the immune system and our increasing understanding of the role it plays in acute and chronic illnesses are being leveraged to produce new treatments for a range of medical conditions. Given the importance of the human immune system in health and disease, detailed study of the immune system of nonhuman primates is essential to advance preclinical translational research. The need for nonhuman primates continues to remain a high priority, which has been acutely evident during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) global pandemic. Nonhuman primates will continue to address key questions and provide predictive models to identify the safety and efficiency of new diagnostics and therapies for human use across the lifespan.

Redefining varicose projection astrocytes in primates.

Varicose projection astrocytes (VP-As) are found in the cerebral cortex and have been described to be specific to humans and chimpanzees. To further examine the phylogenetic distribution of this cell type, we analyzed cortical tissue from several primates ranging from primitive primates to primates evolutionary closer to human such as apes. We specifically analyzed tissue from four strepsirrhine species, one tarsier, six species of platyrrhine monkeys, ten species of cercopithecoid monkeys, two hylobatid ape species, four to six cases each of chimpanzee, bonobo, gorilla, and orangutan, and thirteen human. We found that VP-As were present only in human and other apes (hominoids) and were absent in all other species. We showed that VP-As are localized to layer VI and the superficial white matter of the cortex. The presence of VP-As co-occured with interlaminar astrocytes that also had varicosities in their processes. Due to their location, their long tangential processes, and their irregular presence within species, we propose that VP-As are astrocytes that develop varicosities under specific conditions and that are not a distinct astrocyte type.

Greater Number of Microglia in Telencephalic Proliferative Zones of Human and Nonhuman Primate Compared with Other Vertebrate Species.

Microglial cells, the innate immune cells of the brain, are derived from yolk sac precursor cells, begin to colonize the telencephalon at the onset of cortical neurogenesis, and occupy specific layers including the telencephalic proliferative zones. Microglia are an intrinsic component of cortical germinal zones, establish extensive contacts with neural precursor cells (NPCs) and developing cortical vessels, and regulate the size of the NPC pool through mechanisms that include phagocytosis. Microglia exhibit notable differences in number and distribution in the prenatal neocortex between rat and old world nonhuman primate telencephalon, suggesting that microglia possess distinct properties across vertebrate species. To begin addressing this subject, we quantified the number of microglia and NPCs in proliferative zones of the fetal human, rhesus monkey, ferret, and rat, and the prehatch chick and turtle telencephalon. We show that the ratio of NPCs to microglia varies significantly across species. Few microglia populate the prehatch chick telencephalon, but the number of microglia approaches that of NPCs in fetal human and nonhuman primate telencephalon. These data demonstrate that microglia are in a position to perform important functions in a number of vertebrate species but more heavily colonize proliferative zones of fetal human and rhesus monkey telencephalon.

Neuronal and glial cell number is altered in a cortical layer-specific manner in autism.

LAY ABSTRACT: The cerebral cortex affected with autism spectrum disorder presents changes in the number of neurons and glia cells, possibly leading to a dysregulation of brain circuits and affecting behavior. However, little is known about cell number alteration in specific layers of the cortex in autism spectrum disorder. We found an increase in the number of neurons and a decrease in the number of astrocytes in specific layers of the prefrontal cortex in postmortem human brains from autism spectrum disorder cases. We hypothesize that this may be due to a failure in neural stem cells to shift differentiation from neurons to glial cells during prenatal brain development. These data provide key anatomical findings that contribute to the bases of autism spectrum disorder pathogenesis.

Cortical Interlaminar Astrocytes Are Generated Prenatally, Mature Postnatally, and Express Unique Markers in Human and Nonhuman Primates.

Interlaminar astrocytes (ILAs) are a subset of cortical astrocytes that reside in layer I, express GFAP, have a soma contacting the pia, and contain long interlaminar processes that extend through several cortical layers. We studied the prenatal and postnatal development of ILAs in three species of primates (rhesus macaque, chimpanzee, and human). We found that ILAs are generated prenatally likely from radial glial (RG) cells, that ILAs proliferate locally during gestation, and that ILAs extend interlaminar processes during postnatal stages of development. We showed that the density and morphological complexity of ILAs increase with age, and that ILAs express multiple markers that are expressed by RG cells (Pax6, Sox2, and Nestin), specific to inner and outer RG cells (Cryab and Hopx), and astrocyte markers (S100ß, Aqp4, and GLAST) in prenatal stages and in adult. Finally, we demonstrated that rudimentary ILAs in mouse also express the RG markers Pax6, Sox2, and Nestin, but do not express S100ß, Cryab, or Hopx, and that the density and morphological complexity of ILAs differ between primate species and mouse. Together these findings contribute new information on astrogenesis of this unique class of cells and suggest a lineal relationship between RG cells and ILAs.

Fetal Rhesus Monkey First Trimester Zika Virus Infection Impacts Cortical Development in the Second and Third Trimesters.

Zika virus is a teratogen similar to other neurotropic viruses, notably cytomegalovirus and rubella. The goal of these studies was to address the direct impact of Zika virus on fetal development by inoculating early gestation fetal rhesus monkeys using an ultrasound-guided approach (intraperitoneal vs. intraventricular). Growth and development were monitored across gestation, maternal samples collected, and fetal tissues obtained in the second trimester or near term. Although normal growth and anatomical development were observed, significant morphologic changes were noted in the cerebral cortex at 3-weeks post-Zika virus inoculation including massive alterations in the distribution, density, number, and morphology of microglial cells in proliferative regions of the fetal cerebral cortex; an altered distribution of Tbr2+ neural precursor cells; increased diameter and volume of blood vessels in the cortical proliferative zones; and a thinner cortical plate. At 3-months postinoculation, alterations in morphology, distribution, and density of microglial cells were also observed with an increase in blood vessel volume; and a thinner cortical plate. Only transient maternal viremia was observed but sustained maternal immune activation was detected. Overall, these studies suggest persistent changes in cortical structure result from early gestation Zika virus exposure with durable effects on microglial cells.

Development of the Neuro-Immune-Vascular Plexus in the Ventricular Zone of the Prenatal Rat Neocortex.

Microglial cells make extensive contacts with neural precursor cells (NPCs) and affiliate with vasculature in the developing cerebral cortex. But how vasculature contributes to cortical histogenesis is not yet fully understood. To better understand functional roles of developing vasculature in the embryonic rat cerebral cortex, we investigated the temporal and spatial relationships between vessels, microglia, and NPCs in the ventricular zone. Our results show that endothelial cells in developing cortical vessels extend numerous fine processes that directly contact mitotic NPCs and microglia; that these processes protrude from vessel walls and are distinct from tip cell processes; and that microglia, NPCs, and vessels are highly interconnected near the ventricle. These findings demonstrate the complex environment in which NPCs are embedded in cortical proliferative zones and suggest that developing vasculature represents a source of signaling with the potential to broadly influence cortical development. In summary, cortical histogenesis arises from the interplay among NPCs, microglia, and developing vasculature. Thus, factors that impinge on any single component have the potential to change the trajectory of cortical development and increase susceptibility for altered neurodevelopmental outcomes.

Microglia enhances proliferation of neural progenitor cells in an in vitro model of hypoxic-ischemic injury.

Microglial cells are the primary immune cells in the central nervous system. In the mature brain, microglia perform functions that include eliminating pathogens and clearing dead/dying cells and cellular debris through phagocytosis. In the immature brain, microglia perform functions that include synapse development and the regulation of cell production through extensive contact with and phagocytosis of neural progenitor cells (NPCs). However, the functional role of microglia in the proliferation and differentiation of NPCs under hypoxic-ischemic (HI) injury is not clear. Here, we tested the hypothesis that microglia enhance NPCs proliferation following HI insult. Primary NPCs cultures were divided into four treatment groups: 1) normoxic NPCs (NN); 2) normoxic NPCs cocultured with microglia (NN+M); 3) hypoxic NPCs (HN); and 4) hypoxic NPCs cocultured with microglia (HN+M). Hypoxic-ischemic injury was induced by pretreatment of the cell cultures with 100 µM deferoxamine mesylate (DFO). NPCs treated with 100 µM DFO (HN groups) for 24 hours had significantly increased expression of hypoxia-inducible factor 1 alpha (HIF-1α), a marker of hypoxic cells. Cell number, protein expression, mitosis, and cell cycle phase were examined, and the data were compared between the four groups. We found that the number of cells expressing the NPCs marker Sox2 increased significantly in the HN+M group and that the number of PH3-positive cells increased in the HN+M group; flow cytometry analysis showed a significant increase in the percentage of cells in the G2/M phase in the HN+M group. In summary, these results support the concept that microglia enhance the survival of NPCs under HI injury by increasing NPCs proliferation, survival, and differentiation. These results further suggest that microglia may induce neuroprotective effects after hypoxic injury that can be explored to develop novel therapeutic strategies for the treatment of HI injury in the immature brain.

GABAARα2 is Decreased in the Axon Initial Segment of Pyramidal Cells in Specific Areas of the Prefrontal Cortex in Autism.

Some forms of Autism Spectrum Disorder, a neurodevelopmental syndrome characterized by impaired communication and social skills as well as repetitive behaviors, are purportedly associated with dysregulation of the excitation/inhibition balance in the cerebral cortex. Through human postmortem tissue analysis, we previously found a significant decrease in the number of a gamma-aminobutyric acid (GABA)ergic interneuron subtype, the chandelier (Ch) cell, in the prefrontal cortex of subjects with autism. Ch cells exclusively target the axon initial segment (AIS) of excitatory pyramidal (Pyr) neurons, and a single Ch cell forms synapses on hundreds of Pyr cells, indicating a possible role in maintaining electrical balance. Thus, we herein investigated this crucial link between Ch and Pyr cells in the anatomy of autism neuropathology by examining GABA receptor protein expression in the Pyr cell AIS in subjects with autism. We collected tissue from the prefrontal cortex (Brodmann Areas (BA) 9, 46, and 47) of 20 subjects with autism and 20 age- and sex-matched control subjects. Immunohistochemical staining with antibodies against the GABAA receptor subunit α2 (GABAARα2) - the subunit most prevalent in the Pyr cell AIS - revealed a significantly decreased GABAARα2 protein in the Pyr cell AIS in supragranular layers of prefrontal cortical areas BA9 and BA47 in autism. Downregulated GABAARα2 protein in the Pyr cell AIS may result from decreased GABA synthesis in the prefrontal cortex of subjects with autism, and thereby contribute to an excitation/inhibition imbalance. Our findings support the potential for GABA receptor agonists asa therapeutic tool for autism.

Similar Microglial Cell Densities across Brain Structures and Mammalian Species: Implications for Brain Tissue Function.

Microglial cells play essential volume-related actions in the brain that contribute to the maturation and plasticity of neural circuits that ultimately shape behavior. Microglia can thus be expected to have similar cell sizes and even distribution both across brain structures and across species with different brain sizes. To test this hypothesis, we determined microglial cell densities (the inverse of cell size) using immunocytochemistry to Iba1 in samples of free cell nuclei prepared with the isotropic fractionator from brain structures of 33 mammalian species belonging to males and females of five different clades. We found that microglial cells constitute ∼7% of non-neuronal cells in different brain structures as well as in the whole brain of all mammalian species examined. Further, they vary little in cell density compared with neuronal cell densities within the cerebral cortex, across brain structures, across species within the same clade, and across mammalian clades. As a consequence, we find that one microglial cell services as few as one and as many as 100 neurons in different brain regions and species, depending on the local neuronal density. We thus conclude that the addition of microglial cells to mammalian brains is governed by mechanisms that constrain the size of these cells and have remained conserved over 200 million years of mammalian evolution. We discuss the probable consequences of such constrained size for brain function in health and disease.SIGNIFICANCE STATEMENT Microglial cells are resident macrophages of the CNS, with key functions in recycling synapses and maintaining the local environment in health and disease. We find that microglial cells occur in similar densities in the brains of different species and in the different structures of each individual brain, which indicates that these cells maintain a similar average size in mammalian evolution, suggesting in turn that the volume monitored by each microglial cell remains constant across mammals. Because the density of neurons is highly variable across the same brain structures and species, our finding implies that microglia-dependent functional recovery may be particularly difficult in those brain structures and species with high neuronal densities and therefore fewer microglial cells per neuron.

The fundamental building blocks of cortical development are established in human exencephaly.

BACKGROUND: The presence and status of progenitor/stem cells in excencephalic brain have not been previously examined. METHODS: Brain sections of excencephalic 17-week fetus were stained for specific stem and mature cell markers. RESULTS: The ventricles were open, the developing cerebral cortex was thin in the radial dimension, and the ventricular surface was undulated. There was a decreased ratio of subventricular/ventricular zone radial glia precursor cells (RGCs; PAX6+ and HOPX+ cells), a decreased number of intermediate progenitor cells (IPCs; TBR2+), a decreased number of neurons (MAP2+), and an increased number of astrocytes (S100b+), compared to the control. MAP2+ neurons, S100b+ astrocytes, and OLIG2+ oligodendrocytes were present within the subventricular zone. CONCLUSIONS: This indicates that the underlying condition did not initially preclude radial glial cells from undergoing asymmetric divisions that produce IPCs but halted the developmental progression. RGC and IPC presence in the developing cerebral cortex demonstrates that the fundamental building blocks of cortical formation had been established and that a normal sequence of developmental steps had been initiated in this case of exencephaly. These data expand our understanding of exencephaly etiology and highlight the status of cortical progenitor cells that may be linked to the disorder.

Microglia: An Intrinsic Component of the Proliferative Zones in the Fetal Rhesus Monkey (Macaca mulatta) Cerebral Cortex.

Microglial cells are increasingly recognized as modulators of brain development. We previously showed that microglia colonize the cortical proliferative zones in the prenatal brain and regulate the number of precursor cells through phagocytosis. To better define cellular interactions between microglia and proliferative cells, we performed lentiviral vector-mediated intraventricular gene transfer to induce enhanced green fluorescent protein expression in fetal cerebrocortical cells. Tissues were collected and counterstained with cell-specific markers to label microglial cells and identify other cortical cell types. We found that microglial cells intimately interact with the radial glial scaffold and make extensive contacts with neural precursor cells throughout the proliferative zones, particularly in the rhesus monkey fetus when compared to rodents. We also identify a subtype of microglia, which we term 'periventricular microglia', that interact closely with mitotic precursor cells in the ventricular zone. Our data suggest that microglia are structural modulators that facilitate remodeling of the proliferative zones as precursor cells migrate away from the ventricle and may facilitate the delamination of precursor cells. Taken together, these results indicate that microglial cells are an integral component of cortical proliferative zones and contribute to the interactive milieu in which cortical precursor cells function.

Periventricular microglial cells interact with dividing precursor cells in the nonhuman primate and rodent prenatal cerebral cortex.

Cortical proliferative zones have been studied for over 100 years, yet recent data have revealed that microglial cells constitute a sizeable proportion of ventricular zone cells during late stages of cortical neurogenesis. Microglia begin colonizing the forebrain after neural tube closure and during later stages of neurogenesis populate regions of the developing cortex that include the proliferative zones. We previously showed that microglia regulate the production of cortical cells by phagocytosing neural precursor cells (NPCs), but how microglia interact with NPCs remains poorly understood. Here we report on a distinct subset of microglial cells, which we term periventricular microglia, that are located near the lateral ventricle in the prenatal neocortex. Periventricular microglia exhibit a set of similar characteristics in embryonic rat and fetal rhesus monkey cortex. In both species, these cells occupy ~60 µm of the ventricular zone in the tangential axis and make contact with the soma and processes of NPCs dividing at the ventricle for over 50 µm along the radial axis. Periventricular microglia exhibit notable differences across species, including distinct morphological features such as terminal bouton-like structures that contact mitotic NPCs in the fetal rhesus monkey but not in rat. These morphological distinctions suggest differential functions of periventricular microglia in rat and rhesus monkey, yet are consistent with the concept that microglia regulate NPC function in the developing cerebral cortex of mammalian species.

Cortical interlaminar astrocytes across the therian mammal radiation.

Interlaminar astrocytes (ILA) in the cerebral cortex possess a soma in layer I and extend an interlaminar process that runs perpendicular to the pia into deeper cortical layers. We examined cerebral cortex from 46 species that encompassed most orders of therian mammalians, including 22 primate species. We described two distinct cell types with interlaminar processes that have been referred to as ILA, that we termed pial ILA and supial ILA. ILA subtypes differ in somatic morphology, position in layer I, and presence across species. We further described rudimentary ILA that have short GFAP+ processes that do not exit layer I, and "typical" ILA with longer GFAP+ processes that exit layer I. Pial ILA were present in all mammalian species analyzed, with typical ILA observed in Primates, Scandentia, Chiroptera, Carnivora, Artiodactyla, Hyracoidea, and Proboscidea. Subpial ILA were absent in Marsupialia, and typical subpial ILA were only found in Primate. We focused on the properties of pial ILA by investigating the molecular properties of pial ILA and confirming their astrocytic nature. We found that while the density of pial ILA somata only varied slightly, the complexity of ILA processes varied greatly across species. Primates, specifically bonobo, chimpanzee, orangutan, and human, exhibited pial ILA with the highest complexity. We showed that interlaminar processes contact neurons, pia, and capillaries, suggesting a potential role for ILA in the blood-brain barrier and facilitating communication among cortical neurons, astrocytes, capillaries, meninges, and cerebrospinal fluid.

Neural Progenitor Cell Terminology.

Since descriptions of neural precursor cells (NPCs) were published in the late 19th century, neuroanatomists have used a variety of terms to describe these cells, each term reflecting contemporary understanding of cellular characteristics and function. As the field gained knowledge through a combination of technical advance and individual insight, the terminology describing NPCs changed to incorporate new information. While there is a trend toward consensus and streamlining of terminology over time, to this day scientists use different terms for NPCs that reflect their field and perspective, i.e., terms arising from molecular, cellular, or anatomical sciences. Here we review past and current terminology used to refer to NPCs, including embryonic and adult precursor cells of the cerebral cortex and hippocampus.

Microglial cell activation and senescence are characteristic of the pathology FXTAS.

BACKGROUND: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder associated with premutation alleles of the FMR1 gene. Expansions of more than 200 CGG repeats give rise to fragile X syndrome, the most common inherited form of cognitive impairment. Fragile X-associated tremor/ataxia syndrome is characterized by cerebellar tremor and ataxia, and the presence of ubiquitin-positive inclusions in neurons and astrocytes. It has been previously suggested that fragile X-associated tremor/ataxia syndrome is associated with an inflammatory state based on signs of oxidative stress-mediated damage and iron deposition. OBJECTIVE: Determine whether the pathology of fragile X-associated tremor/ataxia syndrome involves microglial activation and an inflammatory state. METHODS: Using ionized calcium binding adaptor molecule 1 and cluster differentiation 68 antibodies to label microglia, we examined the number and state of activation of microglial cells in the putamen of 13 fragile X-associated tremor/ataxia syndrome and 9 control postmortem cases. RESULTS: Nearly half of fragile X-associated tremor/ataxia syndrome cases (6 of 13) presented with dystrophic senescent microglial cells. In the remaining fragile X-associated tremor/ataxia syndrome cases (7 of 13), the number of microglial cells and their activation state were increased compared to controls. CONCLUSIONS: The presence of senescent microglial cells in half of fragile X-associated tremor/ataxia syndrome cases suggests that this indicator could be used, together with the presence of intranuclear inclusions and the presence of iron deposits, as a biomarker to aid in the postmortem diagnosis of fragile X-associated tremor/ataxia syndrome. An increased number and activation indicate that microglial cells play a role in the inflammatory state present in the fragile X-associated tremor/ataxia syndrome brain. Anti-inflammatory treatment of patients with fragile X-associated tremor/ataxia syndrome may be indicated to slow neurodegeneration. © 2018 International Parkinson and Movement Disorder Society.