Striatonigral distribution of a fluorescent reporter following intracerebral delivery of genome editors.

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.

Expression of tau and phosphorylated tau in the brain of normal and hemiparkinsonian rhesus macaques.

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.

Quantification of early gait development: Expanding the application of Catwalk technology to an infant rhesus macaque model.

BACKGROUND: Understanding gait development is essential for identifying motor impairments in neurodevelopmental disorders. Defining typical gait development in a rhesus macaque model is critical prior to characterizing abnormal gait. The goal of this study was to 1) explore the feasibility of using the Noldus Catwalk to assess gait in infant rhesus macaques and 2) provide preliminary normative data of gait development during the first month of life. NEW METHOD: The Noldus Catwalk was used to assess gait speed, dynamic and static paw measurements, and interlimb coordination in twelve infant rhesus macaques at 14, 21, and 28 days of age. All macaque runs were labeled as a diagonal or non-diagonal walking pattern. RESULTS: Infant rhesus macaques primarily used a diagonal (mature) walking pattern as early as 14 days of life. Ten infant rhesus macaques (83.3%) were able to successfully walk across the Noldus Catwalk at 28 days of life. Limited differences in gait parameters were observed between timepoints because of the variability within the group at 14, 21, and 28 days. COMPARISON WITH EXISTING METHODS: No prior gait analysis system has been used to provide objective quantification of gait parameters for infant macaques. CONCLUSIONS: The Catwalk system can be utilized to quantify gait in infant rhesus macaques less than 28 days old. Future applications to infant rhesus macaques could provide a better understanding of gait development and early differences within various neurodevelopmental disorders.

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins.

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.

Reframing the perception of outliers and negative data in translational research.

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.

Real-time trajectory guide tracking for intraoperative MRI-guided neurosurgery.

PURPOSE: In current intraoperative MRI (IMRI) methods, an iterative approach is used to aim trajectory guides at intracerebral targets: image MR-visible features, determine current aim by fitting model to image, manipulate device, repeat. Infrequent updates are produced by such methods, compared to rapid optically tracked stereotaxy used in the operating room. Our goal was to develop a real-time interactive IMRI method for aiming. METHODS: The current trajectory was computed from two points along the guide's central axis, rather than by imaging the entire device. These points were determined by correlating one-dimensional spokes from a radial sequence with the known cross-sectional projection of the guide. The real-time platform RTHawk was utilized to control MR sequences and data acquisition. On-screen updates were viewed by the operator while simultaneously manipulating the guide to align it with the planned trajectory. Accuracy was quantitated in a phantom, and in vivo validation was demonstrated in nonhuman primates undergoing preclinical gene ( n = 5 $$ n=5 $$ ) and cell ( n = 4 $$ n=4 $$ ) delivery surgeries. RESULTS: Updates were produced at 5 Hz In 10 phantom experiments at a depth of 48 mm, the cannula tip was placed with radial error of (min, mean, max) = (0.16, 0.29, 0.68) mm. Successful in vivo delivery of payloads to all 14 targets was demonstrated across nine surgeries with depths of (min, mean, max) = (33.3, 37.9, 42.5) mm. CONCLUSION: A real-time interactive update rate was achieved, reducing operator fatigue without compromising accuracy. Qualitative interpretation of images during aiming was rendered unnecessary by objectively computing device alignment.

Alpha-synuclein and tau are abundantly expressed in the ENS of the human appendix and monkey cecum.

α-Synuclein (α-syn) proteinopathy in the neurons of the Enteric Nervous System (ENS) is proposed to have a critical role in Parkinson's disease (PD) onset and progression. Interestingly, the ENS of the human appendix harbors abundant α-syn and appendectomy has been linked to a decreased risk and delayed onset of PD, suggesting that the appendix may influence PD pathology. Common marmosets and rhesus macaques lack a distinct appendix (a narrow closed-end appendage with a distinct change in diameter at the junction with the cecum), yet the cecal microanatomy of these monkeys is similar to the human appendix. Sections of human appendix (n = 3) and ceca from common marmosets (n = 4) and rhesus macaques (n = 3) were evaluated to shed light on the microanatomy and the expression of PD-related proteins. Analysis confirmed that the human appendix and marmoset and rhesus ceca present thick walls comprised of serosa, muscularis externa, submucosa, and mucosa plus abundant lymphoid tissue. Across all three species, the myenteric plexus of the ENS was located within the muscularis externa with nerve fibers innervating all layers of the appendix/ceca. Expression of α-syn and tau in the appendix/cecum was present within myenteric ganglia and along nerve fibers of the muscularis externa and mucosa in all species. In the myenteric ganglia α-syn, p-α-syn, tau and p-tau immunoreactivities (ir) were not significantly different across species. The percent area above threshold of α-syn-ir and tau-ir in the nerve fibers of the muscularis externa and mucosa were greater in the human appendix than in the NHP ceca (α-syn-ir p<0.05; tau-ir p<0.05). Overall, this study provides critical translational evidence that the common marmoset and rhesus macaque ceca are remarkably similar to the human appendix and, thus, that these NHP species are suitable for studying the development of PD linked to α-syn and tau pathological changes in the ENS.

Modeling genetic diseases in nonhuman primates through embryonic and germline modification: Considerations and challenges.

Genetic modification of the embryo or germ line of nonhuman primates is envisioned as a method to develop improved models of human disease, yet the promise of such animal models remains unfulfilled. Here, we discuss current methods and their limitations for producing nonhuman primate genetic models that faithfully genocopy and phenocopy human disease. We reflect on how to ethically maximize the translational relevance of such models in the search for new therapeutic strategies to treat human disease.

Evaluating depression- and anxiety-like behaviors in non-human primates.

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.

Myelin Basic Protein and Cardiac Sympathetic Neurodegeneration in Nonhuman Primates.

Minimal myelination is proposed to be a contributing factor to the preferential nigral neuronal loss in Parkinson's disease (PD). Similar to nigral dopaminergic neurons, sympathetic neurons innervating the heart have long, thin axons which are unmyelinated or minimally myelinated. Interestingly, cardiac sympathetic loss in PD is heterogeneous across the heart, yet the spatial relationship between myelination and neurodegeneration is unknown. Here, we report the mapping of myelin basic protein (MBP) expression across the left ventricle of normal rhesus macaques (n = 5) and animals intoxicated with systemic 6-OHDA (50 mg/kg iv) to model parkinsonian cardiac neurodegeneration (n = 10). A subset of 6-OHDA-treated rhesus received daily dosing of pioglitazone (5 mg/kg po; n = 5), a PPARγ agonist with neuroprotective properties. In normal animals, MBP-immunoreactivity (-ir) was identified surrounding approximately 14% of axonal fibers within nerve bundles of the left ventricle, with more myelinated nerve fibers at the base level of the left ventricle than the apex (p < 0.014). Greater MBP-ir at the base was related to a greater number of nerve bundles at that level relative to the apex (p < 0.05), as the percent of myelinated nerve fibers in bundles was not significantly different between levels of the heart. Cardiac sympathetic loss following 6-OHDA was associated with decreased MBP-ir in cardiac nerve bundles, with the percent decrease of MBP-ir greater in the apex (84.5%) than the base (52.0%). Interestingly, cardiac regions and levels with more MBP-ir in normal animals showed attenuated sympathetic loss relative to areas with less MBP-ir in 6-OHDA + placebo (r = -0.7, p < 0.014), but not in 6-OHDA + pioglitazone (r = -0.1) subjects. Our results demonstrate that myelination is present around a minority of left ventricle nerve bundle fibers, is heterogeneously distributed in the heart of rhesus macaques, and has a complex relationship with cardiac sympathetic neurodegeneration and neuroprotection.

The NIH Somatic Cell Genome Editing program.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Autologous transplant therapy alleviates motor and depressive behaviors in parkinsonian monkeys.

Degeneration of dopamine (DA) neurons in the midbrain underlies the pathogenesis of Parkinson's disease (PD). Supplement of DA via L-DOPA alleviates motor symptoms but does not prevent the progressive loss of DA neurons. A large body of experimental studies, including those in nonhuman primates, demonstrates that transplantation of fetal mesencephalic tissues improves motor symptoms in animals, which culminated in open-label and double-blinded clinical trials of fetal tissue transplantation for PD1. Unfortunately, the outcomes are mixed, primarily due to the undefined and unstandardized donor tissues1,2. Generation of induced pluripotent stem cells enables standardized and autologous transplantation therapy for PD. However, its efficacy, especially in primates, remains unclear. Here we show that over a 2-year period without immunosuppression, PD monkeys receiving autologous, but not allogenic, transplantation exhibited recovery from motor and depressive signs. These behavioral improvements were accompanied by robust grafts with extensive DA neuron axon growth as well as strong DA activity in positron emission tomography (PET). Mathematical modeling reveals correlations between the number of surviving DA neurons with PET signal intensity and behavior recovery regardless autologous or allogeneic transplant, suggesting a predictive power of PET and motor behaviors for surviving DA neuron number.

Acute Exposure to the Food-Borne Pathogen Listeria monocytogenes Does Not Induce α-Synuclein Pathology in the Colonic ENS of Nonhuman Primates.

INTRODUCTION: Gastrointestinal (GI) inflammation elicited by environmental factors is proposed to trigger Parkinson's disease (PD) by stimulating accumulation of pathological α-synuclein (α-syn) in the enteric nervous system (ENS), which then propagates to the central nervous system via the vagus nerve. The goal of this study was to model, in nonhuman primates, an acute exposure to a common food-borne pathogen in order to assess whether the related acute GI inflammation could initiate persistent α-syn pathology in the ENS, ultimately leading to PD. METHODS: Adult female cynomolgus macaques were inoculated by oral gavage with 1×108 colony-forming units (CFUs) Listeria monocytogenes (LM, n=10) or vehicle (mock, n=3) and euthanized 2 weeks later. Evaluations included clinical monitoring, blood and fecal shedding of LM, and postmortem pathological analysis of colonic and cecal tissues. RESULTS: LM inoculation of healthy adult cynomolgus macaques induced minimal to mild clinical signs of infection; LM shedding in feces was not seen in any of the animals nor was bacteremia detected. Colitis varied from none to moderate in LM-treated subjects and none to minimal in mock-treated subjects. Expression of inflammatory markers (HLA-DR, CD3, CD20), oxidative stress (8-OHDG), α-syn, and phosphorylated-α-syn in the enteric ganglia was not significantly different between treatment groups. DISCUSSION: Our results demonstrate that cynomolgus macaques orally inoculated with LM present with a clinical response that resembles human LM exposure. They also suggest that acute exposure to food-borne pathogens is not sufficient to induce significant and persistent α-syn changes in healthy adult female subjects. Based on the results of this limited experimental setting, we propose that, if LM has a role in PD pathology, other underlying factors or conditions, such as male sex, inflammatory bowel disease, exposure to toxins, dysbiosis, and/or aging, are needed to be present.

[18F]FEPPA PET imaging for monitoring CD68-positive microglia/macrophage neuroinflammation in nonhuman primates.

PURPOSE: The aim of this study was to examine whether the translocator protein 18-kDa (TSPO) PET ligand [18F]FEPPA has the sensitivity for detecting changes in CD68-positive microglial/macrophage activation in hemiparkinsonian rhesus macaques treated with allogeneic grafts of induced pluripotent stem cell-derived midbrain dopaminergic neurons (iPSC-mDA). METHODS: In vivo positron emission tomography (PET) imaging with [18F]FEPPA was used in conjunction with postmortem CD68 immunostaining to evaluate neuroinflammation in the brains of hemiparkinsonian rhesus macaques (n = 6) that received allogeneic iPSC-mDA grafts in the putamen ipsilateral to MPTP administration. RESULTS: Based on assessment of radiotracer uptake and confirmed by visual inspection of the imaging data, nonhuman primates with allogeneic grafts showed increased [18F]FEPPA binding at the graft sites relative to the contralateral putamen. From PET asymmetry analysis of the images, the mean asymmetry index of the monkeys was AI = - 0.085 ± 0.018. Evaluation and scoring of CD68 immunoreactivity by an investigator blind to the treatment identified significantly more neuroinflammation in the grafted areas of the putamen compared to the contralateral putamen (p = 0.0004). [18F]FEPPA PET AI showed a positive correlation with CD68 immunoreactivity AI ratings in the monkeys (Spearman's ρ = 0.94; p = 0.005). CONCLUSION: These findings reveal that [18F]FEPPA PET is an effective marker for detecting increased CD68-positive microglial/macrophage activation and demonstrates sufficient sensitivity to detect changes in neuroinflammation in vivo following allogeneic cell engraftment.

Effects of Cardiac Sympathetic Neurodegeneration and PPARγ Activation on Rhesus Macaque Whole Blood miRNA and mRNA Expression Profiles.

Degeneration of sympathetic innervation of the heart occurs in numerous diseases, including diabetes, idiopathic REM sleep disorder, and Parkinson's disease (PD). In PD, cardiac sympathetic denervation occurs in 80-90% of patients and can begin before the onset of motor symptoms. Today, there are no disease-modifying therapies for cardiac sympathetic neurodegeneration, and biomarkers are limited to radioimaging techniques. Analysis of expression levels of coding mRNA and noncoding RNAs, such as microRNAs (miRNAs), can uncover pathways involved in disease, leading to the discovery of biomarkers, pathological mechanisms, and potential drug targets. Whole blood in particular is a clinically relevant source of biomarkers, as blood sampling is inexpensive and simple to perform. Our research group has previously developed a nonhuman primate model of cardiac sympathetic denervation by intravenous administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). In this rhesus macaque (Macaca mulatta) model, imaging with positron emission tomography showed that oral administration of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone (n = 5; 5 mg/kg daily) significantly decreased cardiac inflammation and oxidative stress compared to placebo (n = 5). Here, we report our analysis of miRNA and mRNA expression levels over time in the whole blood of these monkeys. Differential expression of three miRNAs was induced by 6-OHDA (mml-miR-16-2-3p, mml-miR-133d-3p, and mml-miR-1262-5p) and two miRNAs by pioglitazone (mml-miR-204-5p and mml-miR-146b-5p) at 12 weeks posttoxin, while expression of mRNAs involved in inflammatory cytokines and receptors was not significantly affected. Overall, this study contributes to the characterization of rhesus coding and noncoding RNA profiles in normal and disease-like conditions, which may facilitate the identification and clinical translation of biomarkers of cardiac neurodegeneration and neuroprotection.

In Vitro CRISPR/Cas9-Directed Gene Editing to Model LRRK2 G2019S Parkinson's Disease in Common Marmosets.

Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1-3% of Parkinson's disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.

Post mortem evaluation of inflammation, oxidative stress, and PPARγ activation in a nonhuman primate model of cardiac sympathetic neurodegeneration.

Cardiac dysautonomia is a common nonmotor symptom of Parkinson's disease (PD) associated with loss of sympathetic innervation to the heart and decreased plasma catecholamines. Disease-modifying strategies for PD cardiac neurodegeneration are not available, and biomarkers of target engagement are lacking. Systemic administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) recapitulates PD cardiac dysautonomia pathology. We recently used positron emission tomography (PET) to visualize and quantify cardiac sympathetic innervation, oxidative stress, and inflammation in adult male rhesus macaques (Macaca mulatta; n = 10) challenged with 6-OHDA (50mg/kg; i.v.). Twenty-four hours post-intoxication, the animals were blindly and randomly assigned to receive daily doses of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone (n = 5; 5mg/kg p.o.) or placebo (n = 5). Quantification of PET radioligand uptake showed increased oxidative stress and inflammation one week after 6-OHDA which resolved to baseline levels by twelve weeks, at which time pioglitazone-treated animals showed regionally preserved sympathetic innervation. Here we report post mortem characterization of heart and adrenal tissue in these animals compared to age and sex matched normal controls (n = 5). In the heart, 6-OHDA-treated animals showed a significant loss of sympathetic nerve fibers density (tyrosine hydroxylase (TH)-positive fibers). The anatomical distribution of markers of sympathetic innervation (TH) and inflammation (HLA-DR) significantly correlated with respective in vivo PET findings across left ventricle levels and regions. No changes were found in alpha-synuclein immunoreactivity. Additionally, CD36 protein expression was increased at the cardiomyocyte intercalated discs following PPARγ-activation compared to placebo and control groups. Systemic 6-OHDA decreased adrenal medulla expression of catecholamine producing enzymes (TH and aromatic L-amino acid decarboxylase) and circulating levels of norepinephrine, which were attenuated by PPARγ-activation. Overall, these results validate in vivo PET findings of cardiac sympathetic innervation, oxidative stress, and inflammation and illustrate cardiomyocyte CD36 upregulation as a marker of PPARγ target engagement.

Spatiotemporal quantification of gait in common marmosets.

BACKGROUND: Objective gait evaluation in humans is used as a predictive disability outcome measure as well as an indicator for intervention effectiveness. Parallel methods of gait analysis in nonhuman primate models are essential for clinical translation. The goal of this study was to first assess whether marmosets' gait data could be reliably collected in a Noldus CatWalk XT10.6 and second, establish a testing protocol to assess gait and the intraindividual variability during repeated testing. NEW METHOD: The CatWalk, originally developed for rodents, was modified and used to assess gait in eight adult common marmoset monkeys across multiple days and trials. Data was first analyzed to identify valid runs. Repeated measures ANOVA was completed for the following gait measures: mean base of support, average stride length, average swing time, and average stance time. RESULTS: Raters had a high level of concurrence of usable data across all trials with successful trials including four consecutive hindfoot footfalls, during a continuous, uninterrupted segment of walking. A significant main effect of time (p < 0.000) but not rater (p = 0.98) was present with significant interactions for time by subject (p < 0.000), but not rater per subject (p = 0.538), time (p = 0.186), or three-way interaction (p = 0.297). COMPARISON WITH EXISTING METHOD(S): Gait has been assessed using force-plate and video data. The CatWalk allowed reproducible, automated and translational locomotor data to be collected at multiple time points with detailed analyses that identified a diagonal gait pattern. CONCLUSIONS: The CatWalk system, similar to those used in humans, can be effectively used to quantify spatiotemporal characteristics of gait in the common marmoset.

Identification of novel rhesus macaque microRNAs from naïve whole blood.

MicroRNAs (miRNAs) are emerging as novel molecular tools for diagnosing and treating diseases. Rhesus monkeys (Macaca mulatta) are the most widely used nonhuman primate species for biomedical studies, yet only 912 mature miRNAs have been identified in this species compared to 2654 in humans and 1978 in mice. The aim of this project was to help bridge that gap in knowledge by evaluating circulating miRNA in naïve rhesus monkeys and comparing results with currently available databases in different species in order to identify novel, mature miRNAs. Total RNA was isolated from whole blood of ten healthy, adult rhesus macaques. After performing next generation sequencing (NGS), 475 novel, mature miRNAs were identified in rhesus macaques for the first time; of those, 423 were identified for the first time in any species. The most abundantly expressed novel rhesus macaque miRNA, hsa-miR-744-5p, has previously been described in humans. Database assessment of hsa-miR-744-5p potential gene targets showed that while the gene targets showed > 90% sequence similarity between rhesus and humans, many did not share the same consensus sequences. The identification of 475 novel miRNAs in the blood of rhesus macaque reflects the complexity and variety of miRNAs across species. Further NGS studies are needed to reveal novel miRNA that will inform on species-, tissue-, and condition-specific miRNAs.

Colonic inflammation affects myenteric alpha-synuclein in nonhuman primates.

Background: Parkinson's disease (PD) patients frequently present gastrointestinal (GI) dysfunction that, in many cases, predates the onset of motor symptoms. In PD, the presynaptic protein alpha-synuclein (α-syn) undergoes pathological changes, including phosphorylation and aggregation leading to the formation of Lewy bodies, which can be found in neurons of the enteric nervous system (ENS). Inflammation has been proposed as a possible trigger of α-syn pathology. Interestingly, patients with inflammatory bowel disease and irritable bowel syndrome, conditions associated with GI inflammation, are at higher risk of developing PD. Captive common marmosets (Callithrix jacchus) develop colitis, providing a natural platform to assess the relationship between α-syn pathology and GI inflammation. Materials and Methods: Sections of proximal colon from marmosets with colitis (n=5; 5.3±2.3 years old; 4 male) and normal controls (n=5; 4.1±1.6 years old; 1 male) were immunostained against protein gene product 9.5 (PGP9.5), human leukocyte antigen DR (HLA-DR), cluster of differentiation 3 (CD3), cluster of differentiation 20 (CD20), glial fibrillary acidic protein (GFAP), 8-hydroxy-2'-deoxyguanosine (8-OHdG), α-syn, and serine 129 phosphorylated α-syn (p-α-syn). Immunoreactivity of each staining in the myenteric plexus was quantified using NIH ImageJ software. Results: Marmosets with colitis had significantly increased expression of inflammatory markers (HLA-DR, p<0.02; CD3, p<0.008), oxidative stress (8-OHdG, p<0.05), and p-α-syn (p<0.02) and decreased expression of α-syn (p<0.04) in the colonic myenteric ganglia compared to normal, healthy controls. Conclusion: Colonic inflammation is associated with changes in α-syn expression and phosphorylation in the myenteric plexus of common marmosets. Future evaluation of the vagus nerve and brain of animals with colitis will be key to assess the contribution of colitis-induced ENS α-syn pathology to PD-like pathology in the brain.