At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau (MAPT) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD-MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n = 2 WT, n = 2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau ( MAPT ) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD -MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored cell surface protein that regulates leukocyte adhesion, migration, and activation, thereby contributing to inflammation and tissue remodeling. However, its role in Alzheimer's disease (AD), particularly in relation to glial dysfunction, remains poorly defined. Here, we investigated the temporal and spatial regulation of uPAR expression across AD mouse models with intact or deficient adaptive immunity. Using immunohistochemistry, we assessed uPAR expression in Rag2/Il2rg-/- (Rag), Rag2/Il2rg-/--5xFAD (Rag-5xFAD), C57BL/6 (WT), and 5xFAD mice across multiple brain regions. uPAR expression increased with age and was significantly elevated in 5xFAD mice, with robust upregulation evident by 6 months irrespective of immune status. Immunofluorescence revealed that uPAR localized predominantly to Iba1+ microglia clustered around Aβ plaques, with limited neuronal expression. Bulk RNA sequencing of Rag-5xFAD brain tissue demonstrated enrichment of disease-associated microglia (DAM) and senescence-related transcriptional programs. These findings indicate that uPAR marks a subset of plaque-associated glial cells undergoing functional and transcriptional remodeling in AD, independent of peripheral adaptive immune signaling. Collectively, our results identify uPAR as a marker of dysfunctional, DAM-like microglia and implicate it in senescence-associated neuroinflammatory pathways. This work provides a framework for future studies targeting uPAR-expressing glial populations as a potential therapeutic strategy in AD.

IntroductionAging is accompanied by systemic metabolic changes that contribute to disease susceptibility and functional decline. Sex differences in aging have been reported in humans, yet their mechanistic basis remains poorly understood. Due to their physiological similarity to humans, rhesus macaques are a powerful translational model to investigate sex-specific metabolomic aging under controlled conditions.MethodsTargeted serum metabolomics were conducted in 58 rhesus (35 females, 23 males), ranging from 1.66 to 25.71 years of age, quantifying 513 metabolites spanning lipids, amino acids, and related compounds. Multivariate, univariate, and generalized additive model (GAM) analyses were performed to evaluate age-associated trajectories and test for sex differences.ResultsAge-related changes in both sexes were identified in metabolites related to hormones (e.g., DHEAS), amino acid biosynthesis and catabolism (e.g., beta-alanine, sarcosine, t4-OH-pro), and energy metabolism (e.g., hexose). Sex affected age-related metabolic trajectories in lipids, amino acids and related compounds, and gut microbial species. Females demonstrated a profound increase in serum triglycerides (TGs), amino acids, and other small molecules, while males exhibited a heterogenous profile with changes in lipids, but no TGs were affected. Males also exhibited altered levels of amino acids and related metabolites, hormones, gut microbial metabolites, and energy-associated metabolites.ConclusionThese results highlight pronounced sex differences in metabolomic aging trajectories in rhesus macaques, particularly in lipid and amino acid metabolism. These findings underscore the importance of incorporating sex as a biological variable in aging studies and support the utility of rhesus macaques for identifying conserved, sex-specific biomarkers of biological aging.

Transgenic pigs are emerging as large animal models for biomedical research. Green fluorescent protein (GFP)-expressing transgenic reporter pigs (GFP-Tg; NSRRC:0016 GFP NT92) express GFP via the ubiquitous pCAGG promoter. Their level of GFP expression across brain regions has not yet been evaluated. Brain tissue sections from GFP-Tg animals were immunostained against GFP and the neuronal marker NeuN, or against GFP, NeuN, and the dopaminergic neuronal marker tyrosine hydroxylase (TH). The presence and intensity of neuronal GFP expression was evaluated in the amygdala, caudate, hippocampus, primary motor cortex (M1), putamen, and substantia nigra pars compacta (SNpc). Images from each region were captured, and individual neurons were counted and rated based on their intensity of GFP expression as being GFP positive (GFP+), equal (GFP=) to neuropil, dim (GFPd), or negative (GFP-). Nearly all neurons in the amygdala (99.2%), caudate (99.0%), hippocampus (99.7%), M1 (99.4%), and putamen, (98.8%) showed high GFP-immunoreactivity (combined GFP+ and GFP=), in contrast to the SNpc, which had significantly less (61.4%; p=0.0266). The number of GFP- neurons was significantly higher in the SNpc than in the amygdala (p =0.0342), hippocampus (p=0.0366), and M1 (p=0.0437). Overall, this study demonstrates high neuronal GFP expression in the GFP-Tg reporter pig, but with variability between brain regions, particularly decreased GFP expression in dopaminergic nigral neurons. Future research utilizing this model, such as studies to evaluate the efficacy of genome-editors for Parkinson's disease through their ability to turn off GFP expression in the nigrostriatal system, should account for this neuronal GFP-expression variability.

The PD-AGE international task force underscores the pivotal role that non-human primate (NHP) models play in advancing our understanding of Parkinson’s disease (PD) and ageing. Due to their close genetic, anatomical, and behavioural similarity to humans, NHPs uniquely enable translational research to bridge basic science towards clinical application. They are indispensable for modelling the complex motor and non-motor symptoms of PD, as well as age-related neurodegeneration. This paper outlines the scientific rationale, methodological strengths, and ethical considerations surrounding NHP use in PD research. We highlight the need for standardised models, innovative tools, and long-term collaborative infrastructure to enhance the translational value of NHP studies. We propose a three-phase roadmap to develop a global research consortium to optimise resource use, improve model fidelity, and accelerate therapeutic development for PD and related neurodegenerative disorders.

Intracerebral grafting of dopamine-producing cells is proposed as a strategy to replace the typical neurons lost to Parkinson’s disease (PD) and improve PD motor symptoms. Non-human primate studies have provided clues on the relationship between the host’s immune response and grafting success. Herein, we discuss how the host’s immune system differentially affects the graft depending on the origin of the cells and reflect on the advantages and limitations of the immune paradigms utilized to assess graft-related outcomes. We also consider new strategies to minimize or circumvent the host’s immunological response and related preclinical research needed to identify the most promising new approaches to be translated into the clinic.

As the global human population rapidly ages and diseases of aging become more prevalent, preclinical models of age-related neurodegenerative disorders are increasingly important for identifying early diagnostic biomarkers, monitoring disease progression, and evaluating treatment responsiveness. Rhesus macaques are an ideal species for studies on neurodegeneration due to their phylogenetic relatedness to humans and their complex brain anatomy and physiology. Technological advances in assay sensitivity have facilitated the identification of blood-based biomarkers of neurodegeneration and inflammation in human populations. The aim of this study was to translate these methods for use in male and female rhesus macaques across adulthood. We collected plasma samples from 47 rhesus macaques representing pre-adult (1-5 years, n = 6 female, n = 5 male), young (5-7 years, n = 5 female, n = 7 male), middle (8-16 years, n = 7 female, n = 7 male), and older adult (17-22 years, n = 6 female, n = 4 male) subjects. Quantified biomarkers included neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), amyloid beta (Aβ42, Aβ40, and their ratio), total tau, phosphorylated tau (pTau181), interleukin (IL) 2, IL-6, IL-8, and IL-10. Plasma NfL and IL-6 levels were significantly increased with age in both sexes, with a marked rise during middle adulthood. The ratio of Aβ42/Aβ40 was significantly lower in the middle and older aged females compared to the youngest group. There was no effect of age or sex on total tau or pTau181 levels. Overall, these results demonstrate the feasibility of evaluating blood biomarkers of neurodegeneration and inflammation in rhesus macaques during adulthood.

Tauopathies are neurodegenerative disorders characterized by the abnormal accumulation and aggregation of hyperphosphorylated tau protein. They can be primary or secondary depending on whether tau inclusions are the predominant pathology (e.g.: frontotemporal dementia related to tau) or are found with other proteinopathies (e.g.: Alzheimer’s disease), respectively. Currently, there are no effective treatments to prevent or slow down progressive tau accumulation. Animal models play a critical role in the efforts to unravel the mechanisms leading to tauopathies and identifying therapeutic targets. Nonhuman primates (NHPs) present several advantages for the study of tauopathies, as they have complex neuroanatomy and behavior that resembles human traits, and their tau gene and protein are highly conserved. Moreover, aged NHPs, like humans, can present various tau inclusions in their brains, although whether NHPs can develop human-like tau-related neurodegenerative disorders is currently debated. The main goal of this review is to analyze available reports on tau pathologies and models of tauopathies in NHPs considering the complexity of the tau protein and associated tau pathologies. Here, we first summarize current available information on human and NHP tau under physiological conditions in order to highlight species differences and gaps in knowledge. We then analyze reports on tau pathologies in aged NHPs compared to human aging and tauopathy, followed by an evaluation of current and emerging NHP models of tauopathy. Lastly, we discuss the practical and ethical challenges of doing tauopathy research in NHPs, and how to best leverage it to ultimately find solutions for patients with these disorders.

Successful cell replacement strategies for brain repair depend on graft integration into the neural network, which is affected by the immune response to the grafted cells. Using Parkinson disease as an example, in this chapter, we consider the immune system interaction and its role in autologous vs heterologous graft survival and integration, as well as past and emerging strategies to overcome the immunologic response. We also reflect on the role of nonhuman primate research to assess novel approaches and consider the role of different stakeholders on advancing the most promising new approaches into the clinic.

Cardiac ventricular pressure overload affects patients with congenital heart defects and can cause cardiac insufficiency. Grafts of stem cell–derived cardiomyocytes are proposed as a complementary treatment to surgical repair of the cardiac defect, aiming to support ventricular function. Here, we report successful engraftment of human induced pluripotent stem cell–derived cardiac lineage cells into the heart of immunosuppressed rhesus macaques with a novel surgical model of right ventricular pressure overload. The human troponin+ grafts were detected in low-dose (2 × 10 6 cells/kg) and high-dose (10 × 10 6 cells/kg) treatment groups up to 12 weeks post-injection. Transplanted cells integrated and progressively matched the organization of the surrounding host myocardium. Ventricular tachycardia occurred in five out of 16 animals receiving cells, with episodes of incessant tachycardia observed in two animals; ventricular tachycardia events resolved within 19 days. Our results demonstrate that grafted cardiomyocytes mature and integrate into the myocardium of nonhuman primates modeling right ventricular pressure overload.

OBJECTIVE:The objective of this study was to develop and evaluate the feasibility and safety of a novel transaxial surgical approach for the delivery of human induced pluripotent stem cell-derived dopaminergic neuroprogenitor cells (DANPCs) into the putamen nucleus using nonhuman primates and surgical techniques and tools relevant to human clinical translation. METHODS:Nine immunosuppressed, unlesioned adult cynomolgus macaques (4 females, 5 males) received intraputaminal injections of vehicle or DANPCs (0.9 × 105 to 1.1 × 105 cells/µL) under real-time intraoperative MRI guidance. The infusates were combined with 1-mM gadoteridol (for intraoperative MRI visualization) and delivered via two tracks per hemisphere (ventral and dorsal) using a transaxial approach. The total volumes of infusion were 25 µL and 50 µL for the right and left putamen, respectively (infusion rate 2.5 µL/min). Animals were evaluated with a battery of clinical and behavioral outcome measures and euthanized 7 or 30 days postsurgery; full necropsies were performed by a board-certified veterinary pathologist. Brain tissues were collected and processed for immunohistochemistry, including against the human-specific marker STEM121. RESULTS:The optimized surgical technique and tools produced successful targeting of the putamen via the transaxial approach. Intraoperative MR images confirmed on-target intraputaminal injections in all animals. All animals survived to scheduled termination without clinical evidence of neurological deficits. The first 4 animals to undergo surgery had mild brain swelling noted at the end of surgery, of which 3 had transient reduced vision; administration of mannitol therapy and reduced intravenous fluid during the surgical procedure addressed these complications. Immunostaining against STEM121 confirmed the presence of grafted cells along the injection track within the targeted putamen area of DANPC-treated animals. All adverse histological findings were limited in scope and consistent with surgical manipulation, injection procedure, and postsurgical inflammatory response to the mechanical disruption caused by the cannula insertion. CONCLUSIONS:The delivery system, injection procedure, and DANPCs were well tolerated in all animals. Prevention of mild brain swelling by mannitol dosing and reduction of intravenous fluids during surgery allowed visual effects to be avoided. The results of the study established that this novel transaxial approach can be used to correctly and safely target cell injections to the postcommissural putamen and support clinical investigation.

Negative results can be a source of disappointment for scientists, yet their publication is needed for scientific progress, in particular for cutting-edge translational research of novel therapeutics. This manuscript is directed to scientists, junior and senior, that produce and review data for publication. It discusses the difference between ‘negative’ or ‘unexpected’ data and ‘useless’ data, re-evaluates the importance of the experimental design to generate valuable data and proposes strategies to work with and report negative results. Overall, it aims to reframe the perception of working with, reporting and reviewing unexpected data as an opportunity to provide rationale for innovative ideas, prevent the misuse of limited resources and, ultimately, strengthen the reputation of a scientist.

Depression and anxiety are some of the most prevalent and debilitating mental health conditions in humans. They can present on their own or as co-morbidities with other disorders. Like humans, non-human primates (NHPs) can develop depression- and anxiety-like signs. Here, we first define human depression and anxiety, examine equivalent species-specific behaviors in NHPs, and consider models and current methods to identify and evaluate these behaviors. We also discuss knowledge gaps, as well as the importance of evaluating the co-occurrence of depression- and anxiety-like behaviors in animal models of human disease. Lastly, we consider ethical challenges in depression and anxiety research on NHPs in order to ultimately advance the understanding and the personalized treatment of these disorders.

Introduction Targeted gene editing is proposed as a therapeutic approach for numerous disorders, including neurological diseases. As the brain is organized into neural networks, it is critical to understand how anatomically connected structures are affected by genome editing. For example, neurons in the substantia nigra pars compacta (SNpc) project to the striatum, and the striatum contains neurons that project to the substantia nigra pars reticulata (SNpr). Methods Here, we report the effect of injecting genome editors into the striatum of Ai14 reporter mice, which have a LoxP-flanked stop cassette that prevents expression of the red fluorescent protein tdTomato. Two weeks following intracerebral delivery of either synthetic nanocapsules (NCs) containing CRISPR ribonucleoprotein targeting the tdTomato stop cassette or adeno-associated virus (AAV) vectors expressing Cre recombinase, the brains were collected, and the presence of tdTomato was assessed in both the striatum and SN. Results TdTomato expression was observed at the injection site in both the NC- and AAV-treated groups and typically colocalized with the neuronal marker NeuN. In the SN, tdTomato-positive fibers were present in the pars reticulata, and SNpr area expressing tdTomato correlated with the size of the striatal genome edited area. Conclusion These results demonstrate in vivo anterograde axonal transport of reporter gene protein products to the SNpr following neuronal genome editing in the striatum.

Tau is a neuronal protein involved in microtubule stabilization and intracellular vesicle transport in axons. In neurodegenerative disorders termed "tauopathies," like Alzheimer's and Parkinson's disease, tau becomes hyperphosphorylated and forms intracellular inclusions. Rhesus macaques are widely used for studying ageing processes and modeling neurodegenerative disorders, yet little is known about endogenous tau expression in their brains. In this study, immunohistochemical methods were used to map and characterize total tau, 3R- and 4R-tau isoforms, and phosphorylated tau (pThr231-tau and pSer202/Thr205-tau/AT8) expression bilaterally in 16 brain regions of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced hemiparkinsonian adult rhesus macaques. Tau-immunoreactivity (-ir), including both 3R and 4R isoforms, was observed throughout the brain, with varying regional intensities. The anterior cingulate cortex, entorhinal cortex, and hippocampus displayed the most robust tau-ir, while the subthalamic nucleus and white matter regions had minimal expression. Tau was present in neurons of gray matter regions; it was preferentially observed in fibers of the globus pallidus and substantia nigra and in cell bodies of the thalamus and subthalamic nucleus. In white matter regions, tau was abundantly present in oligodendrocytes. Additionally, neuronal pThr231-tau-ir was abundant in all brain regions, but not AT8-ir. Differences in regional and intracellular protein expression were not detected between control subjects and both brain hemispheres of MPTP-treated animals. Specifically, tau-ir in the substantia nigra of all subjects colocalized with GABAergic neurons. Overall, this report provides an in-depth characterization of tau expression in the rhesus macaque brain to facilitate future investigations for understanding and modeling tau pathology in this species.

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the healthy mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo.