Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids

Neural tube defects(NTDs)are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure.Although folate supplementation has been shown to mitigate the incidence of NTDs,some cases,often attributable to genetic factors,remain unpreventable.The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation;at present,however,the underlying mechanism remains unclear.Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate.To determine the role of SHROOM3 in early developmental morphogenesis,we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase.Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei.These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins,namely fibrous actin(F-actin),myosin II,and phospho-myosin light chain(PMLC),to the apical side of the neuroepithelial cells.Notably,these defects were not rescued by folate supplementation.RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis.In summary,we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

Neuroprotectants attenuate hypobaric hypoxia-induced brain injuries in cynomolgus monkeys

Hypobaric hypoxia (HH) exposure can cause serious brain injury as well as life-threatening cerebral edema in severe cases. Previous studies on the mechanisms of HH-induced brain injury have been conducted primarily using non-primate animal models that are genetically distant to humans, thus hindering the development of disease treatment. Here, we report that cynomolgus monkeys (Macaca fascicularis) exposed to acute HH developed human-like HH syndrome involving severe brain injury and abnormal behavior. Transcriptome profiling of white blood cells and brain tissue from monkeys exposed to increasing altitude revealed the central role of the HIF-1 and other novel signaling pathways, such as the vitamin D receptor (VDR) signaling pathway, in co-regulating HH-induced inflammation processes. We also observed profound transcriptomic alterations in brains after exposure to acute HH, including the activation of angiogenesis and impairment of aerobic respiration and protein folding processes, which likely underlie the pathological effects of HH-induced brain injury. Administration of progesterone (PROG) and steroid neuroprotectant 5α-androst-3β,5,6β-triol (TRIOL) significantly attenuated brain injuries and rescued the transcriptomic changes induced by acute HH. Functional investigation of the affected genes suggested that these two neuroprotectants protect the brain by targeting different pathways, with PROG enhancing erythropoiesis and TRIOL suppressing glutamate-induced excitotoxicity. Thus, this study advances our understanding of the pathology induced by acute HH and provides potential compounds for the development of neuroprotectant drugs for therapeutic treatment.

A natural model of behavioral depression in postpartum adult female cynomolgus monkeys (Macaca fascicularis)

Postpartum depression （PPD） is a modified form of major depressive disorders （MDD） that can exert profound negative effects on both mothers and infants than MDD. Within the postpartum period, both mothers and infants are susceptible; but because PPD typically occurs for short durations and has moderate symptoms, there exists challenges in exploring and addressing the underlying cause of the depression. This fact highlights the need for relevant animal models. In the present study, postpartum adult female cynomolgus monkeys （Macaca fascicularis） living in breeding groups were observed for typical depressive behavior. The huddle posture behavior was utilized as an indicator of behavioral depression postpartum （BDP） as it has been established as the core depressive-like behavior in primates. Monkeys were divided into two groups： A t3DP group （n=6）, which were found to spend more time huddling over the first two weeks postpartum than other individuals that formed a non-depression control group （n=4）. The two groups were then further analyzed for locomotive activity, stressful events, hair cortisol levels and for maternal interactive behaviors. No differences were found between the BDP and control groups in locomotive activity, in the frequencies of stressful events experienced and in hair cortisol levels. These findings suggested that the postpartum depression witnessed in the monkeys was not related to external factors other than puerperium period. Interestingly, the BDP monkeys displayed an abnormal maternal relationship consisting of increased infant grooming. Taken together, these findings suggest that the adult female cynomolgus monkeys provide a natural model of behavioral postpartum depression that holds a number of advantages over commonly used rodent systems in PPD modeling. The cynomolgus monkeys have a highly-organized social hierarchy and reproductive characteristics without seasonal restriction--similar to humans--as well as much greater homology to humans than rodents. As such, this model may provide a greater translational efficiency and research platform for systematically investigating the etiology, treatment, prevention of PPD.

Novel D-galactosamine-induced cynomolgus monkey model of acute liver failure

AIM To establish a simplified, reproducible D-galactosamineinduced cynomolgus monkey model of acute liver failure having an appropriate treatment window. METHODS Sixteen cynomolgus monkeys were randomly dividedinto four groups(A, B, C and D) after intracranial pressure(ICP) sensor implantation. D-galactosamine at 0.3, 0.25, 0.20 + 0.05(24 h interval), and 0.20 g/kg body weight, respectively, was injected via the small saphenous vein. Vital signs, ICP, biochemical indices, and inflammatory factors were recorded at 0, 12, 24, 36, 48, 72, 96, and 120 h after D-galactosamine administration. Progression of clinical manifestations, survival times, and results of H&E staining, TUNEL, and Masson staining were recorded. RESULTS Cynomolgus monkeys developed different degrees of debilitation, loss of appetite, and jaundice after D-galactosamine administration. Survival times of groups A, B, and C were 56 ± 8.7 h, 95 ± 5.5 h, and 99 ± 2.2 h, respectively, and in group D all monkeys survived the 144-h observation period except for one, which died at 136 h. Blood levels of ALT, AST, CK, LDH, TBi L, Cr, BUN, and ammonia, prothrombin time, ICP, endotoxin, and inflammatory markers [(tumor necrosis factor(TNF)-α, interleukin(IL)-1β, and IL-6)] significantly increased compared with baseline values in different groups(P < 0.05). Pathological results showed obvious liver cell necrosis that was positively correlated with the dose of D-galactosamine.CONCLUSION We successfully established a simplified, reproducible D-galactosamine-induced cynomolgus monkey model of acute liver failure, and the single or divided dosage of 0.25 g/kg is optimal for creating this model.

Stress-relevant social behaviors of middle-class male cynomolgus monkeys （Macaca fascicularis）

Stress from dominance ranks in human societies, or that of other social animals, especially nonhuman primates, can have negative influences on health. Individuals holding different social status may be burdened with various stress levels. The middle class experiences a special stress situation within the dominance hierarchy due to its position between the higher and lower classes. Behaviorally, questions about where middle-class stress comes from and how individuals adapt to middle-class stress remain poorly understood in nonhuman primates. In the present study, social interactions, including aggression, avoidance, grooming and mounting behaviors, between beta males, as well as among group members holding higher or lower social status, were analyzed in captive male-only cynomolgus monkey groups. We found that aggressive tension from the higher hierarchy members was the main origin of stress for middle- class individuals. However, behaviors such as attacking lower hierarchy members immediately after being the recipient of aggression, as well as increased avoidance, grooming and mounting toward both higher and lower hierarchy members helped alleviate middle-class stress and were particular adaptations to middle-class social status.

MPTP Induces Systemic Parkinsonism in Middle-Aged Cynomolgus Monkeys:Clinical Evolution and Outcomes

In this study, we developed a systemic PD model in middle-aged cynomolgus monkeys using individualized low-dose MPTP, to explore effective indicators for the early prediction of clinical outcomes. MPTP was not stopped until the animals showed typical PD motor symptoms on days 10 to 13 after MPTP administration when the Kurlan score reached 10; this abrogated the dif- ferences in individual susceptibility to MPTP. The clinical symptoms persisted, peaking on days 3 to 12 after MPTP withdrawal （rapid progress stage）, and then the Kurlan score plateaued. A Kurlan score at the end of the rapid progress stage 〉15 reflected stable or slowly-progressive PD, while a score 〈15 indicated spontaneous recovery. The entire clinical evolution and outcome of the systemic PD model was characterized in this study, thus providing options for therapeutic and translational research.

Modeling Rett syndrome using TALEN-edited MECP2 mutant cynomolgus monkeys

Subject Code:H09With the support by the National Natural Science Foundation of China,a collaborative study by the research group led by Prof.Chen Yongchang(陈永昌)and Ji Weizhi from the Yunnan Key Laboratory of Primate Biomedicine Research&Institute of Primate Translational Medicine,Kunming University

In Vivo Development of Fetal Pig Kidneys in Mature Monkeys under Clinically Approved Immunosuppressant Drugs

Controlling the immune response with only clinically approved immunosuppressant drugs is difficult in renal heterotra ns plantation from pigs to nonhuman primates.Moreover,to the best of our knowledge,no reports exist on the use of fetal pigs as kidney donors.This study aimed to compare the degree of transplant rejection between neonatal and fetal kidneys,with genetically unmodified pigs as donors and cynomolgus monkeys as recipients.The left kidneys of the recipient monkeys were removed,followed by transplantation of neonatal as well as fetal pig kidneys,which had undergone vascular anastomosis at the same site,into the retroperitoneum.Immunosuppression was performed with only US Food and Drug Administration-approved drugs.The fetal kidneys were transplanted into the omentum and paraaortic regions of cynomolgus monkeys.Consequently,the engraftment and development of the transplanted tissues were pathologically examined by sampling over time(twice in each experiment).An acute rejection was observed after a few weeks in neonatal renal grafts with vascular anastomosis.However,fetal pig kidneys were spared from rejection despite the administration of the same immunosuppressive protocol to the monkeys and the recipient blood vessels flowing into the fetal kidneys.The immunogenicity of fetal kidneys in pig-monkey renal heterotransplantation was lower than that of neonatal kidneys.

Choroid changes in vortex vein-occluded monkeys

AIM：To determine acute and chronic choroidal vascular changes after vortex vein occlusion in monkeys.METHODS：One or two temporal vortex veins were occluded in 8 cynomolgus monkeys.Fluorescein angiography（FA）,indocyanine green angiogram（ICGA）,and enhanced-depth imaging optical coherence tomography（EDI-OCT）were performed preoperatively and at 1d,1,4,8 and 12wk after occlusion.EDI-OCT images were binarized to calculate the choroid vascular index（CVI）.RESULTS：ICGA showed delayed filling of choroidal arteries in occluded quadrants in eyes with two occluded temporal vortex veins within 1wk.The thickness of the superotemporal choroid increased 1d and 4wk after occlusion,the thickness of the superonasal and inferonasal choroid increased 12wk after occlusion,and the CVI of the superonasal quadrant increased 8wk after occlusion in eyes with 2 occluded vortex veins. CONCLUSION：Occlusion of two vortex veins leads to hemodynamic and structural changes in choroidal layers in the acute phase,while autoregulation may play the main role in the long term.Occlusion of one vortex vein has little influence on the hemodynamic and structural status of the choroid.

Morphological evaluation of retinal ganglion cells expressing the L132C/T159C ChR2 mutant transgene in young adult cynomolgus monkeys

To characterize recombinant AAV2(rAAV2)-mediated expression of L132 C/T159 C ChR2 mutant in retinal ganglion cells(RGCs)of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP(ChR2-GFP) were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing(RBPMS) was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS+and GFP+ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 μm from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 μm from the edge. On average,the ratio of the double-labeled RGCs versus RBPMS+ RGCs approached 0.32±0.15(n=14 fields) at the central foveal region(0.1 to 0.53 mm). We observed that the ratio reached 0.78±0.16(n=21 fields) at peripheral retinal locations(eccentricity >7 mm). This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.