Aged Monkeys Research Articles

Innate immunity stimulation via CpG oligodeoxynucleotides ameliorates Alzheimer's disease pathology in aged squirrel monkeys.

Alzheimer's disease is the most common cause of dementia and the only illness among the top 10 causes of death for which there is no disease-modifying therapy. The failure rate of clinical trials is very high, in part due to the premature translation of successful results in transgenic mouse models to patients. Extensive evidence suggests that dysregulation of innate immunity and microglia/macrophages plays a key role in Alzheimer's disease pathogenesis. Activated resident microglia and peripheral macrophages can display protective or detrimental phenotypes depending on the stimulus and environment. Toll-like receptors (TLRs) are a family of innate immune regulators known to play an important role in governing the phenotypic status of microglia. We have shown in multiple transgenic Alzheimer's disease mouse models that harnessing innate immunity via TLR9 agonist CpG oligodeoxynucleotides (ODNs) modulates age-related defects associated with immune cells and safely reduces amyloid plaques, oligomeric amyloid-β, tau pathology, and cerebral amyloid angiopathy (CAA) while promoting cognitive benefits. In the current study we have used a non-human primate model of sporadic Alzheimer's disease pathology that develops extensive CAA-elderly squirrel monkeys. The major complications in current immunotherapeutic trials for Alzheimer's disease are amyloid-related imaging abnormalities, which are linked to the presence and extent of CAA; hence, the prominence of CAA in elderly squirrel monkeys makes them a valuable model for studying the safety of the CpG ODN-based concept of immunomodulation. We demonstrate that long-term use of Class B CpG ODN 2006 induces a favourable degree of innate immunity stimulation without producing excessive or sustained inflammation, resulting in efficient amelioration of both CAA and tau Alzheimer's disease-related pathologies in association with behavioural improvements and in the absence of microhaemorrhages in aged elderly squirrel monkeys. CpG ODN 2006 has been well established in numerous human trials for a variety of diseases. The present evidence together with our earlier, extensive preclinical research, validates the beneficial therapeutic outcomes and safety of this innovative immunomodulatory approach, increasing the likelihood of CpG ODN therapeutic efficacy in future clinical trials.

Neural presbycusis at ultra-high frequency in aged common marmosets and rhesus monkeys.

The aging of the population and environmental noise have contributed to high rates of presbycusis, also known as age-related hearing loss (ARHL). Because mice have a relatively short life span, murine models have not been suitable for determining the mechanism of presbycusis development and methods of diagnosis. Although the common marmoset, a non-human primate (NHP), is an ideal animal model for studying age-related diseases, its auditory spectrum has not been systematically studied. Auditory brainstem responses (ABRs) from 38 marmosets of different ages demonstrated that auditory function correlated with age. Hearing loss in geriatric common marmosets started at ultra-high frequency (>16 kHz), then extended to lower frequencies. Despite age-related deterioration of ABR threshold and amplitude in marmosets, outer hair cell (OHC) function remained stable at all ages. Spiral ganglion neurons (SGNs), which are the first auditory neurons in the auditory system, were found to degenerate distinctly in aged common marmosets, indicating that neural degeneration caused presbycusis in these animals. Similarly, age-associated ABR deterioration without loss of OHC function was observed in another NHP, rhesus monkeys. Audiometry results from these two species of NHP suggested that NHPs were ideal for studying ARHL and that neural presbycusis at high frequency may be prevalent in primates.

Extensive Sub-RPE Complement Deposition in a Nonhuman Primate Model of Early-Stage Diabetic Retinopathy.

PurposeThis study aims to reveal retinal abnormities in a spontaneous diabetic nonhuman primate model and explore the mechanism of featured injuries.MethodsTwenty-eight cynomolgus monkeys were identified to suffer from spontaneous type 2 diabetes from a colony of more than eight-hundred aged monkeys, and twenty-six age-matched ones were chosen as controls. Their blood biochemistry profiles were determined and retinal changes were examined by multimodal imaging, hematoxylin and eosin staining, and immunofluorescence. Retinal pigment epithelium (RPE) cells were further investigated by RNA sequencing and computational analyses.ResultsThese diabetic monkeys were characterized by early retinal vascular and neural damage and dyslipidemia. The typical acellular capillaries and pericyte ghost were found in the diabetic retina, which also exhibited reduced retinal nerve fiber layer thickness compared to controls (all P < 0.05). Of note, distinct sub-RPE drusenoid lesions were extensively observed in these diabetic monkeys (46.43% vs. 7.69%), and complements including C3 and C5b-9 were deposited in these lesions. RNA-seq analysis revealed complement activation, AGE/RAGE activation and inflammatory response in diabetic RPE cells. Consistently, the plasma C3 and C4 were particularly increased in the diabetic monkeys with drusenoid lesions (P = 0.028 and 0.029).ConclusionsThe spontaneous type 2 diabetic monkeys featured with early-stage retinopathy including not only typical vascular and neural damage but also a distinct sub-RPE deposition. The complement activation of RPE cells in response to hyperglycemia might contribute to the deposition, revealing an unrecognized role of RPE cells in the early-stage pathological process of diabetic retinopathy.

Aged Monkeys Fed a High-Fat/High-Sugar Diet Recapitulate Metabolic Disorders and Cardiac Contractile Dysfunction.

Aged nonhuman primate (NHP) models are of great value for studying the pathology of metabolic heart diseases and developing therapeutic strategies. In this study, aged male cynomolgus monkeys were fed a regular diet or a high-fat/high-sugar diet (HFSD) for 8 months. Metabolic disorders were diagnosed by 1H-NMR and serum biochemistry, and cardiac function was evaluated by echocardiography. Our results showed that serum metabolic profiles were altered in aged monkeys fed a HFSD, in line with aortic tissue damage, cardiac remodeling, and contractile dysfunction. This aged monkey model significantly increased expression of proinflammatory cytokines and altered expression and phosphorylation of intracellular signaling proteins in the heart, as compared to aged monkeys on a regular diet. Furthermore, the animals demonstrated increased phosphorylation of cardiac myofilament proteins which are causatively associated with decreased myofilament contractility. We conclude that the aged monkey model fed a HFSD exhibits metabolic disorders and cardiac contractile dysfunction.

T Cells Actively Infiltrate the White Matter of the Aging Monkey Brain in Relation to Increased Microglial Reactivity and Cognitive Decline

Normal aging is characterized by declines in processing speed, learning, memory, and executive function even in the absence of neurodegenerative diseases such as Alzheimer's Disease (AD). In normal aging monkeys and humans, neuronal loss does not account for cognitive impairment. Instead, loss of white matter volume and an accumulation of myelin sheath pathology begins in middle age and is associated with cognitive decline. It is unknown what causes this myelin pathology, but it likely involves increased neuroinflammation in white matter and failures in oligodendrocyte function (maturation and repair). In frontal white matter tracts vulnerable to myelin damage, microglia become chronically reactive and secrete harmful pro-inflammatory cytokines. Despite being in a phagocytic state, these microglia are ineffective at phagocytosing accruing myelin debris, which directly inhibits myelin sheath repair. Here, we asked whether reported age-related increases in pro-inflammatory markers were accompanied by an adaptive immune response involving T cells. We quantified T cells with immunohistochemistry in the brains of 34 cognitively characterized monkeys and found an age-related increase in perivascular T cells that surround CNS vasculature. We found a surprising age-related increase in T cells that infiltrate the white matter parenchyma. In the cingulum bundle the percentage of these parenchymal T cells increased with age relative to those in the perivascular space. In contrast, infiltrating T cells were rarely found in surrounding gray matter regions. We assessed whether T cell infiltration correlated with fibrinogen extravasation from the vasculature as a measure of BBB leakiness and found no correlation, suggesting that T cell infiltration is not a result of passive extravasation. Importantly, the density of T cells in the cingulum bundle correlated with microglial reactivity and with cognitive impairment. This is the first demonstration that T cell infiltration of white matter is associated with cognitive decline in the normal aging monkey.

Extracellular vesicles derived from bone marrow mesenchymal stem cells enhance myelin maintenance after cortical injury in aged rhesus monkeys

Cortical injury, such as stroke, causes neurotoxic cascades that lead to rapid death and/or damage to neurons and glia. Axonal and myelin damage in particular, are critical factors that lead to neuronal dysfunction and impair recovery of function after injury. These factors can be exacerbated in the aged brain where white matter damage is prevalent. Therapies that can ameliorate myelin damage and promote repair by targeting oligodendroglia, the cells that produce and maintain myelin, may facilitate recovery after injury, especially in the aged brain where these processes are already compromised. We previously reported that a novel therapeutic, Mesenchymal Stem Cell derived extracellular vesicles (MSC-EVs), administered intravenously at both 24 h and 14 days after cortical injury, reduced microgliosis (Go et al. 2019), reduced neuronal pathology (Medalla et al. 2020), and improved motor recovery (Moore et al. 2019) in aged female rhesus monkeys. Here, we evaluated the effect of MSC-EV treatment on changes in oligodendrocyte maturation and associated myelin markers in the sublesional white matter using immunohistochemistry, confocal microscopy, stereology, qRT-PCR, and ELISA. Compared to vehicle control monkeys, EV-treated monkeys showed a reduction in the density of damaged oligodendrocytes. Further, EV-treatment was associated with enhanced myelin maintenance, evidenced by upregulation of myelin-related genes and increases in actively myelinating oligodendrocytes in sublesional white matter. These changes in myelination correlate with the rate of motor recovery, suggesting that improved myelin maintenance facilitates this recovery. Overall, our results suggest that EVs act on oligodendrocytes to support myelination and improves functional recovery after injury in the aged brain. SignificanceWe previously reported that EVs facilitate recovery of function after cortical injury in the aged monkey brain, while also reducing neuronal pathology (Medalla et al. 2020) and microgliosis (Go et al. 2019). However, the effect of injury and EVs on oligodendrocytes and myelination has not been characterized in the primate brain (Dewar et al. 1999; Sozmen et al. 2012; Zhang et al. 2013). In the present study, we assessed changes in myelination after cortical injury in aged monkeys. Our results show, for the first time, that MSC-EVs support recovery of function after cortical injury by enhancing myelin maintenance in the aged primate brain.

Acute Respiratory Distress in Aged, SARS-CoV-2–Infected African Green Monkeys but Not Rhesus Macaques

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a wide range of disease severity, ranging from asymptomatic infection to a life-threating illness, particularly in the elderly population and individuals with comorbid conditions. Among individuals with serious coronavirus 2019 (COVID-19) disease, acute respiratory distress syndrome (ARDS) is a common and often fatal presentation. Animal models of SARS-CoV-2 infection that manifest severe disease are needed to investigate the pathogenesis of COVID-19–induced ARDS and evaluate therapeutic strategies. We report two cases of ARDS in two aged African green monkeys (AGMs) infected with SARS-CoV-2 that had pathological lesions and disease similar to severe COVID-19 in humans. We also report a comparatively mild COVID-19 phenotype characterized by minor clinical, radiographic, and histopathologic changes in the two surviving, aged AGMs and four rhesus macaques (RMs) infected with SARS-CoV-2. Notable increases in circulating cytokines were observed in three of four infected, aged AGMs but not in infected RMs. All the AGMs had increased levels of plasma IL-6 compared with baseline, a predictive marker and presumptive therapeutic target in humans infected with SARS-CoV-2. Together, our results indicate that both RMs and AGMs are capable of modeling SARS-CoV-2 infection and suggest that aged AGMs may be useful for modeling severe disease manifestations, including ARDS.

Hypothesis: Tau pathology is an initiating factor in sporadic Alzheimer's disease.

The etiology of the common, sporadic form of Alzheimer's disease (sAD) is unknown. We hypothesize that tau pathology within select projection neurons with susceptible microenvironments can initiate sAD. This postulate rests on extensive data demonstrating that in human brains tau pathology appears about a decade before the formation of Aβ plaques (Aβps), especially targeting glutamate projection neurons in the association cortex. Data from aging rhesus monkeys show abnormal tau phosphorylation within vulnerable neurons, associated with calcium dysregulation. Abnormally phosphorylated tau (pTau) on microtubules traps APP‐containing endosomes, which can increase Aβ production. As Aβ oligomers increase abnormal phosphorylation of tau, this would drive vicious cycles leading to sAD pathology over a long lifespan, with genetic and environmental factors that may accelerate pathological events. This hypothesis could be testable in the aged monkey association cortex that naturally expresses characteristics capable of promoting and sustaining abnormal tau phosphorylation and Aβ production.

Auditory and Visual System White Matter Is Differentially Impacted by Normative Aging in Macaques.

Deficits in auditory and visual processing are commonly encountered by older individuals. In addition to the relatively well described age-associated pathologies that reduce sensory processing at the level of the cochlea and eye, multiple changes occur along the ascending auditory and visual pathways that further reduce sensory function in each domain. One fundamental question that remains to be directly addressed is whether the structure and function of the central auditory and visual systems follow similar trajectories across the lifespan or sustain the impacts of brain aging independently. The present study used diffusion magnetic resonance imaging and electrophysiological assessments of auditory and visual system function in adult and aged macaques to better understand how age-related changes in white matter connectivity at multiple levels of each sensory system might impact auditory and visual function. In particular, the fractional anisotropy (FA) of auditory and visual system thalamocortical and interhemispheric corticocortical connections was estimated using probabilistic tractography analyses. Sensory processing and sensory system FA were both reduced in older animals compared with younger adults. Corticocortical FA was significantly reduced only in white matter of the auditory system of aged monkeys, while thalamocortical FA was lower only in visual system white matter of the same animals. Importantly, these structural alterations were significantly associated with sensory function within each domain. Together, these results indicate that age-associated deficits in auditory and visual processing emerge in part from microstructural alterations to specific sensory white matter tracts, and not from general differences in white matter condition across the aging brain.SIGNIFICANCE STATEMENT Age-associated deficits in sensory processing arise from structural and functional alterations to both peripheral sensory organs and central brain regions. It remains unclear whether different sensory systems undergo similar or distinct trajectories in function across the lifespan. To provide novel insights into this question, this study combines electrophysiological assessments of auditory and visual function with diffusion MRI in aged macaques. The results suggest that age-related sensory processing deficits in part result from factors that impact the condition of specific white matter tracts, and not from general decreases in connectivity between sensory brain regions. Such anatomic specificity argues for a framework aimed at understanding vulnerabilities with relatively local influence and brain region specificity.

Amyloidosis increase is not attenuated by long-term calorie restriction or related to neuron density in the prefrontal cortex of extremely aged rhesus macaques.

As human lifespan increases and the population ages, diseases of aging such as Alzheimer's disease (AD) are a major cause for concern. Although calorie restriction (CR) as an intervention has been shown to increase healthspan in many species, few studies have examined the effects of CR on brain aging in primates. Using postmortem tissue from a cohort of extremely aged rhesus monkeys (22-44years old, average age 31.8years) from a longitudinal CR study, we measured immunohistochemically labeled amyloid beta plaques in Brodmann areas 32 and 46 of the prefrontal cortex, areas that play key roles in cognitive processing, are sensitive to aging and, in humans, are also susceptible to AD pathogenesis. We also evaluated these areas for cortical neuron loss, which has not been observed in younger cohorts of aged monkeys. We found a significant increase in plaque density with age, but this was unaffected by diet. Moreover, there was no change in neuron density with age or treatment. These data suggest that even in the oldest-old rhesus macaques, amyloid beta plaques do not lead to overt neuron loss. Hence, the rhesus macaque serves as a pragmatic animal model for normative human aging but is not a complete model of the neurodegeneration of AD. This model of aging may instead prove most useful for determining how even the oldest monkeys are protected from AD, and this information may therefore yield valuable information for clinical AD treatments.

A single-cell transcriptomic atlas of primate pancreatic islet aging.

Aging-related degeneration of pancreatic islet cells contributes to impaired glucose tolerance and diabetes. Endocrine cells age heterogeneously, complicating the efforts to unravel the molecular drivers underlying endocrine aging. To overcome these obstacles, we undertook single-cell RNA sequencing of pancreatic islet cells obtained from young and aged non-diabetic cynomolgus monkeys. Despite sex differences and increased transcriptional variations, aged β-cells showed increased unfolded protein response (UPR) along with the accumulation of protein aggregates. We observed transcriptomic dysregulation of UPR components linked to canonical ATF6 and IRE1 signaling pathways, comprising adaptive UPR during pancreatic aging. Notably, we found aging-related β-cell-specific upregulation of HSP90B1, an endoplasmic reticulum-located chaperone, impeded high glucose-induced insulin secretion. Our work decodes aging-associated transcriptomic changes that underlie pancreatic islet functional decay at single-cell resolution and indicates that targeting UPR components may prevent loss of proteostasis, suggesting an avenue to delaying β-cell aging and preventing aging-related diabetes.

Aging Affects Fine and Coarse Coding of Orientation Information in Macaque Primary Visual Cortex

Human visual function degrades with age. Previous studies of visual perception have shown that aged people have worse performance in the coding of orientation information. However, the neuronal mechanism still remains elusive. In this study, we performed in vivo extracellular single-unit recording in the primary visual cortex of senescent and young monkeys, and we used the Chernoff distance to quantify the encoded information of neurons for fine and coarse orientation difference. Our results showed that the Chernoff distance for fine orientation difference in senescent monkeys is significantly smaller than that in young monkeys. In contrast, the Chernoff distance for the coarse coding was comparable in young and old groups. Meanwhile, increased spontaneous response and maximum evoked response was also observed. Further investigation of neuronal correlation showed higher noise and signal correlations in aging monkeys than that in young monkeys. These correlation changes predicted a detrimental effect on the efficiency of population coding of orientation information. Taken together, our results suggest that the information coding efficiency of orientation information is impaired during aging and might account for the degradation of performance in human fine orientation discrimination task.

Changes of the end plate cartilage are associated with intervertebral disc degeneration: A quantitative magnetic resonance imaging study in rhesus monkeys and humans.

SummaryBackgroundThe end plate plays an important role in intervertebral disc degeneration progression. The aim of the study was to examine the compositional and structural changes of the end plate with age and to investigate the correlation between end plate and disc degeneration by T1ρ and T2 map magnetic resonance imaging.MethodsThere were 12 young monkeys (6-7 years old), 20 aged monkeys (14-17 years old) and 12 human participants (30-50 years old) in this study. T1ρ or T2 map values of the nucleus pulposus and end plate cartilage were analyzed according to Pfirrmann grades and age. Afterwards, micro computed tomography and histological analysis were used to confirm the end plate changes in monkeys. Pearson’s correlation was performed to investigate the relationship between end plate and disc degeneration.ResultsIn monkeys, T1ρ (r=-0.794, P<0.001) and T2 map values (r=-0.8, P<0.001) of the nucleus pulposus were negatively associated with Pfirrmann grades. Moreover, the T2 map was more suitable than T1ρ for the evaluation of end plate degeneration. Age was an important influence factor of end plate and disc degeneration, which was confirmed by microcomputed tomography, Safranin O/fast green staining, and collagen II staining. The T2 map value of lower end plate degeneration positively correlated with that of the intervertebral discs in monkeys (R2=0.3133, P<0.001) and humans (R2=0.2092, P<0.001).ConclusionThis study suggests that the compositional and structural changes of the end plate can be quantitatively evaluated by T2 map. Furthermore, cartilage end plate degeneration is associated with disc degeneration during ageing.The translational potential of this articleA better understanding of how the cartilage end plate affects disc degeneration is needed, which may propose a new clinical application using T2 map to evaluate end plate degeneration.

A single-cell transcriptomic landscape of primate arterial aging

Our understanding of how aging affects the cellular and molecular components of the vasculature and contributes to cardiovascular diseases is still limited. Here we report a single-cell transcriptomic survey of aortas and coronary arteries in young and old cynomolgus monkeys. Our data define the molecular signatures of specialized arteries and identify eight markers discriminating aortic and coronary vasculatures. Gene network analyses characterize transcriptional landmarks that regulate vascular senility and position FOXO3A, a longevity-associated transcription factor, as a master regulator gene that is downregulated in six subtypes of monkey vascular cells during aging. Targeted inactivation of FOXO3A in human vascular endothelial cells recapitulates the major phenotypic defects observed in aged monkey arteries, verifying FOXO3A loss as a key driver for arterial endothelial aging. Our study provides a critical resource for understanding the principles underlying primate arterial aging and contributes important clues to future treatment of age-associated vascular disorders.

Plasmacytoid dendritic cells (pDCs) and IFN-α production in aged squirrel monkeys (Saimiri boliviensis boliviensis) stimulated with Class C CpG Oligodeoxynucleotides

Abstract Synthetic CpG oligodeoxynucleotides (ODNs) with defined CpG motifs and immunomodulatory properties signal through an endosomal membrane-based type of pattern recognition receptor (PRR), the Toll-like receptor 9 (TLR9), that has been identified in dendritic cells (DCs), B cells, and other human immune cell types. CpG-ODNs influence several signaling pathways, leading to cytokine production in many mammalian species that make CpG ODNs suitable as therapeutic interventions in a variety of human disease conditions. The squirrel monkey (SQM), a well-established New World non-human primate (NHP) model is known to be used for studying various diseases such as Alzheimer’s disease (AD), malaria, and transmissible spongiform encephalopathies (Creutzfeldt-Jacob disease). In the present study, we generated the first report of immunoregulatory activity of class C CpG-ODN in aged squirrel monkey. CpG-ODN exhibits very potent effects on squirrel monkey immune cells by inducing IFN-γ/IFN-α from peripheral blood mononuclear cells and demonstrates accelerated kinetics of activity. In addition, class C CpG-ODN specifically activates pDCs to undergo maturation and secrete cytokines, including high levels of IFN-α. To best to our knowledge, this is the first documentation describing immune status in squirrel monkeys immunized with C class CpG-ODN by concentrating on lymphocyte surface antigen expression and function. Overall, our findings provide critical data regarding the immunomodulatory potential of CpG-ODN in this NHP model allowing for future therapeutic trials of innate immunity stimulation via TLR9 agonists for diverse indications including AD therapeutics.

Treatment with Mesenchymal-Derived Extracellular Vesicles Reduces Injury-Related Pathology in Pyramidal Neurons of Monkey Perilesional Ventral Premotor Cortex.

Functional recovery after cortical injury, such as stroke, is associated with neural circuit reorganization, but the underlying mechanisms and efficacy of therapeutic interventions promoting neural plasticity in primates are not well understood. Bone marrow mesenchymal stem cell-derived extracellular vesicles (MSC-EVs), which mediate cell-to-cell inflammatory and trophic signaling, are thought be viable therapeutic targets. We recently showed, in aged female rhesus monkeys, that systemic administration of MSC-EVs enhances recovery of function after injury of the primary motor cortex, likely through enhancing plasticity in perilesional motor and premotor cortices. Here, using in vitro whole-cell patch-clamp recording and intracellular filling in acute slices of ventral premotor cortex (vPMC) from rhesus monkeys (Macaca mulatta) of either sex, we demonstrate that MSC-EVs reduce injury-related physiological and morphologic changes in perilesional layer 3 pyramidal neurons. At 14-16 weeks after injury, vPMC neurons from both vehicle- and EV-treated lesioned monkeys exhibited significant hyperexcitability and predominance of inhibitory synaptic currents, compared with neurons from nonlesioned control brains. However, compared with vehicle-treated monkeys, neurons from EV-treated monkeys showed lower firing rates, greater spike frequency adaptation, and excitatory:inhibitory ratio. Further, EV treatment was associated with greater apical dendritic branching complexity, spine density, and inhibition, indicative of enhanced dendritic plasticity and filtering of signals integrated at the soma. Importantly, the degree of EV-mediated reduction of injury-related pathology in vPMC was significantly correlated with measures of behavioral recovery. These data show that EV treatment dampens injury-related hyperexcitability and restores excitatory:inhibitory balance in vPMC, thereby normalizing activity within cortical networks for motor function.SIGNIFICANCE STATEMENT Neuronal plasticity can facilitate recovery of function after cortical injury, but the underlying mechanisms and efficacy of therapeutic interventions promoting this plasticity in primates are not well understood. Our recent work has shown that intravenous infusions of mesenchymal-derived extracellular vesicles (EVs) that are involved in cell-to-cell inflammatory and trophic signaling can enhance recovery of motor function after injury in monkey primary motor cortex. This study shows that this EV-mediated enhancement of recovery is associated with amelioration of injury-related hyperexcitability and restoration of excitatory-inhibitory balance in perilesional ventral premotor cortex. These findings demonstrate the efficacy of mesenchymal EVs as a therapeutic to reduce injury-related pathologic changes in the physiology and structure of premotor pyramidal neurons and support recovery of function.

Pancreatic Islets Aging in Old Rhesus Monkey

BackgroundType 2 diabetes is an age associated disease which characterized by insulin resistance and loss of β cell mass and function. Accumulation of senescent β cells with age probably lead to compromised capacity of insulin synthesis and secretion. Nevertheless, other studies also suggested systemic aging may impair glucose tolerance via compromised peripheral insulin sensitivity and microcirculation. So, the pathophysiological process of pancreatic β cell senescence, and the relationship between senescent β cells and diabetes are not clarified. Rhesus monkey is one of the most ideal non‐human primate models of human aging. To date, the researches on islets function of rhesus monkeys, particularly aged monkeys are so limited. So, in this study, we are aiming to explore the link between islets senescence and diabetes in aged rhesus monkeys of approximately 20 years old that may reflect the characters of humans around 80 years old.MethodsSix middle age rhesus monkeys (average 10 years old) and six old rhesus monkeys (average 20 years old) were used in this study. Blood chemistry was detected and the intravenous glucose tolerance tests (IVGTT) were carried out. After sacrifice, the pancreatic islets were isolated to evaluate β cell function, and the histopathological analysis was done on the pancreatic tissues.ResultsIn the blood chemistry, no significant differences in kidney and liver functions were observed between the two groups. The old monkeys show slight increase on the fasting blood glucose, while their fasting insulin levels were increased distinctly. In addition, the IVGTT results showed that the glucose tolerance of the old group was slightly impaired, indicating the possible occurrence of peripheral insulin resistance. In respect to pancreatic histology, the immunohistochemistry staining with anti‐insulin antibody showed that both the total area of insulin producing β cells in pancreatic sections and the single islet mass decreased a lot in old rhesus monkeys, but interestingly, a notable elevated expression of pancreatic/duodenal homeobox‐1(PDX1), an important biomarker for β cell function, was observed. In respect to isolated islets, no significant difference was found in either glucose‐stimulated insulin secretion test(GSIS) or β‐Gal staining, suggesting the islets may function well in these old monkeys.ConclusionIn our 20 years old rhesus monkeys, the insulin resistance has appeared, and their islets area decreased obviously but not accompany significantly compromise of islets function, suggesting that the islets of old rhesus monkey may still have a strong compensatory ability.Support or Funding InformationThis study was supported by the Program of National Natural Science Foundation of China (81870609, 31571474, and 81571808), and the National Key Clinical Project.A, B, C) The fasting blood glucose, fasting blood insulin and intravenous glucose tolerance tests of middle age and old rhesus monkey. D) The glucose‐stimulated insulin secretion test (GSIS) of isolated isletsFigure 1E) The pancreatic sections immunohistochemistry staining with anti‐insulin antibody of middle age(left) and old(right) rhesus monkeyFigure 2

The Effects of the M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator VU0486846 on Cognitive Performance in Aged Nonhuman Primates

It is estimated that in the United States alone there will be nearly 70 million people over the age of 65 by the year 2030. Yet, there remains a critical unmet need to understand and identify novel therapeutic strategies to address age‐related impairments in arousal, cognitive function, and sleep architecture. Agents such as acetylcholinesterase inhibitors (AChEIs) that enhance overall cholinergic neurotransmission are approved to ameliorate the loss of cognitive function in normal aging and Alzheimer’s disease (AD) populations. However, AChEIs are associated with dose‐limiting side effect due to nonselective activation of peripheral muscarinic acetylcholine receptors (mAChRs). Accumulating evidence indicates that enhancement of central cholinergic neurotransmission through selective activation of the M1 mAChR subtype represents an exciting alternative approach for the treatment of impairments in cognition, arousal and sleep observed in normal aging and AD. Recently we successfully optimized a novel series of M1 positive allosteric modulators (PAMs) as an alternative strategy for the selective activation of M1 receptors, represented by VU0486846. The goal of the present study was to provide the first characterization of the effects of an M1 PAM on cognitive performance in aged nonhuman primates. For these studies, 16 aged (&gt; 20 years old) female and male (N=8/sex) cynomolgus monkeys were trained under two different cognitive tasks using CANTAB touchscreens. Eight monkeys (N=4/sex) were trained under a delayed match‐to‐sample (DMS) task using three delays and 2–3 distractors. The other monkeys were trained under an attention task, the multiple‐choice serial reaction time (MCSRT) task. All monkeys were tested with the following compounds, administered 60‐min before the session: donepezil (0.1–0.56 mg/kg, IM); VU0486846 (1.0–5.6 mg/kg, PO) and d‐amphetamine (0.01–0.056 mg/kg, IM). With no published data on training of aged monkeys in a DMS task, we found that the eight aged monkeys required approximately 1 year of training prior to pharmacologic challenge. The effects of donepezil were variable across monkeys, up to doses that produced adverse effects. In contrast, VU0486846 improved working memory, at doses that did not result in any side effects. As a positive control, d‐amphetamine also improved cognitive performance. In preliminary studies, testing ineffective doses of donepezil + VU0486846 also resulted in improved DMS performance. Regarding MCSRT, effects of both donepezil and VU0486846 were determined on a reaction time task. At doses that improved working memory, VU0486846 did not affect reaction time while in some but not all monkeys, donepezil slowed reaction times. These promising preliminary data will be extended in order to examine sex‐specific and age‐related effects of chronic VU0486846 and the consequences of the M1 PAM and donepezil on sleep architecture in aged nonhuman primates.Support or Funding InformationNIH grant AG054622

Mesenchymal Stem Cell Derived Extracellular Vesicles Enhance Myelin Plasticity in Rhesus Monkeys

Cortical injury, such as injury from stroke, results in a cascade of events that includes cell death, inflammation and disruption of myelin. To date, there are no highly effective treatments for reducing the deficits that occur after injury. Recently, we have demonstrated that extracellular vesicles (EVs) harvested from rhesus monkey bone marrow derived cells when given 1 day and 14 days following injury facilitate recovery of function in aged rhesus monkeys within the first 3–5 weeks after cortical injury. Based on these findings and current proteomic literature of MSC‐EVs, we hypothesized that MSC‐EVs enhance myelin plasticity by limiting damage to oligodendrocytes and stimulating remyelination.To assess general myelin integrity after injury, we used Spectral Confocal Reflectance Microscopy (SCoRe) to image myelinated axons and found an increase in the density of myelinated axons in the EV group (p &lt; 0.05). To assess whether the difference was due to reduced damage or remyelination, in sublesional white matter we assessed immunohistochemical labeling of Olig2, a general oligodendrocyte marker, and 8OHdG, a marker for DNA damage. We found reduced densities of Olig2 colocalized with 8OHdG in the EV group (p&lt;0.05). As a marker of active demyelination and myelin debris clearance, we measured Myelin Basic Protein (MBP) concentrations in CSF and found a longitudinal reduction in the EV animals. To assess remyelination, we measured expression of MBP, a gene for myelination in mature oligodendrocytes, Myelin Regulatory Factor (MyRF), a gene for oligodendrocyte differentiation and maintenance, and Breast Carcinoma Amplified Sequence 1 (BCAS1), a gene for newly myelinating oligodendrocytes. Interestingly, we found a 4 fold increase in MyRF expression, and a 1.5 fold increase in MBP and BCAS1 in the EV animals relative to the vehicle control animals in perilesional brain tissue. Consistent with these gene expression differences associated with re‐myelination, we found that the densities of newly‐myelinating oligodendrocytes immune‐labeled with BCAS1, as well as mature oligodendrocytes expressing CC1, exhibited a trend towards an increase in the EV group (p = 0.09). These results suggest that EV treatment reduces myelin damage, while also stimulating myelin repair. Finally, these results correlated with enhanced motor recovery, suggesting that EV‐mediated white matter plasticity is a critical component for recovery after cortical injury.Support or Funding InformationSupported by NIH grant R21NS102991‐02.

Muscarinic M1 Receptors Modulate Working Memory Performance and Activity via KCNQ Potassium Channels in the Primate Prefrontal Cortex

Working memory relies on the dorsolateral prefrontal cortex (dlPFC), where microcircuits of pyramidal neurons enable persistent firing in the absence of sensory input, maintaining information through recurrent excitation. This activity relies on acetylcholine, although the molecular mechanisms for this dependence are not thoroughly understood. This study investigated the role of muscarinic M1 receptors (M1Rs) in the dlPFC using iontophoresis coupled with single-unit recordings from aging monkeys with naturally occurring cholinergic depletion. We found that M1R stimulation produced an inverted-U dose response on cell firing and behavioral performance when given systemically to aged monkeys. Immunoelectron microscopy localized KCNQ isoforms (Kv7.2, Kv7.3, and Kv7.5) on layer III dendrites and spines, similar to M1Rs. Iontophoretic manipulation of KCNQ channels altered cell firing and reversed the effects of M1R compounds, suggesting that KCNQ channels are one mechanism for M1R actions in the dlPFC. These results indicate that M1Rs may be an appropriate target to treat cognitive disorders with cholinergic alterations.