Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys.

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

Human antimicrobial peptide LL-37 contributes to Alzheimer's disease progression.

As a prime mover in Alzheimer's disease (AD), microglial activation requires membrane translocation, integration, and activation of the metamorphic protein chloride intracellular channel 1 (CLIC1), which is primarily cytoplasmic under physiological conditions. However, the formation and activation mechanisms of functional CLIC1 are unknown. Here, we found that the human antimicrobial peptide (AMP) LL-37 promoted CLIC1 membrane translocation and integration. It also activates CLIC1 to cause microglial hyperactivation, neuroinflammation, and excitotoxicity. In mouse and monkey models, LL-37 caused significant pathological phenotypes linked to AD, including elevated amyloid-ß, increased neurofibrillary tangles, enhanced neuronal death and brain atrophy, enlargement of lateral ventricles, and impairment of synaptic plasticity and cognition, while Clic1 knockout and blockade of LL-37-CLIC1 interactions inhibited these phenotypes. Given AD's association with infection and that overloading AMP may exacerbate AD, this study suggests that LL-37, which is up-regulated upon infection, may be a driving force behind AD by acting as an endogenous agonist of CLIC1.

EGF/bFGF promotes survival, migration and differentiation into neurons of GFP-labeled rhesus monkey neural stem cells xenografted into the rat brain.

Stem cell replacement therapy is considered a promising treatment for diseases of the central nervous system. Improving the ratio of surviving transplanted cells and the efficiency of differentiation into functional neuronal cells are the most important issues related to research on neuroregenerative medicine. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have been reported to promote the proliferation and differentiation of neural stem cells (NSCs) in vitro, but whether they have the same effect in vivo is unclear. In this study, NSCs derived from rhesus monkey embryonic stem cells (ESCs) were resuspended in medium with or without EGF/bFGF and xenotransplanted into the rat striatum. No behavioral abnormalities or teratoma formation were observed in the recipient engrafted rats. GFP-labeled cells exhibited a higher survival rate and longer migration in the EGF/bFGF group than control group at 2 months after transplantation. Moreover, the percentages of Tuj1+ neurons and Map2+ neurons in the EGF/bFGF group were significantly higher than those in the control group, while the percentages of astrocytes and oligodendrocytes were significantly lower in the EGF/bGFG group than control group. These findings indicated that EGF/bFGF can promote protrusion of nerve fibers and the survival and neuronal differentiation of transplanted NSCs in the recipient brain, suggesting that EGF/bFGF has a potential application for stem cell therapy.

Developmental trajectory of magnetic susceptibility in the healthy rhesus macaque brain.

Quantitative susceptibility mapping (QSM) is used to quantify iron deposition in non-human primates in our study. Although QSM has many applications in detecting iron deposits in the human brain, including the distribution of iron deposits in specific brain regions, the change of iron deposition with aging, and the comparison of iron deposits between diseased groups and healthy controls, few studies have applied QSM to non-human primates, while most animal brain experiments focus on biochemical and anatomical results instead of non-invasive experiments. Additionally, brain imaging in children's research is difficult, but can be substituted using young rhesus monkeys, which are very similar to humans, as research animals. Therefore, understanding the relationship between iron deposition and age in rhesus macaques' brains can offer insights into both the developmental trajectory of magnetic susceptibility in the animal model and the correlated evidence in children's research. Twenty-three healthy rhesus macaque monkeys (23 ± 7.85 years, range 2-29 years) were included in this research. Seven regions of interest (ROIs-globus pallidus, substantia nigra, dentate nucleus, caudate nucleus, putamen, thalamus, red nucleus) have been analyzed in terms of QSM and R2 * (apparent relaxation rate). Susceptibility in most ROIs correlated significantly with the growth of age, similarly to the results for R2 *, but showed different trends in the thalamus and red nucleus, which may be caused by the different sensitivities of myelination and iron deposition in R2 * and QSM analysis. By assessing the correlation between iron content and age in healthy rhesus macaques' brains using QSM, we provide a piece of pilot information on normality for advanced animal disease models. Meanwhile, this study also could serve as the normative basis for further clinical studies using QSM for iron content quantification. Due to the comparison of the susceptibility on the same experimental objects, this research can also provide practical support for future research on characteristics for QSM and R2 *.

Normative Analysis of Individual Brain Differences Based on a Population MRI-Based Atlas of Cynomolgus Macaques.

The developmental trajectory of the primate brain varies substantially with aging across subjects. However, this ubiquitous variability between individuals in brain structure is difficult to quantify and has thus essentially been ignored. Based on a large-scale structural magnetic resonance imaging dataset acquired from 162 cynomolgus macaques, we create a species-specific 3D template atlas of the macaque brain, and deploy normative modeling to characterize individual variations of cortical thickness (CT) and regional gray matter volume (GMV). We observed an overall decrease in total GMV and mean CT, and an increase in white matter volume from juvenile to early adult. Specifically, CT and regional GMV were greater in prefrontal and temporal cortices relative to early unimodal areas. Age-dependent trajectories of thickness and volume for each cortical region revealed an increase in the medial temporal lobe, and decreases in all other regions. A low percentage of highly individualized deviations of CT and GMV were identified (0.0021%, 0.0043%, respectively, P < 0.05, false discovery rate [FDR]-corrected). Our approach provides a natural framework to parse individual neuroanatomical differences for use as a reference standard in macaque brain research, potentially enabling inferences regarding the degree to which behavioral or symptomatic variables map onto brain structure in future disease studies.

Annotation and cluster analysis of spatiotemporal- and sex-related lncRNA expression in rhesus macaque brain.

Long noncoding RNAs (lncRNAs) mediate important epigenetic regulation in a wide range of biological processes and diseases. We applied comprehensive analyses of RNA-seq and CAGE-seq (cap analysis of gene expression and sequencing) to characterize the dynamic changes in lncRNA expression in rhesus macaque (Macaca mulatta) brain in four representative age groups. We identified 18 anatomically diverse lncRNA modules and 14 mRNA modules representing spatial, age, and sex specificities. Spatiotemporal- and sex-biased changes in lncRNA expression were generally higher than those observed in mRNA expression. A negative correlation between lncRNA and mRNA expression in cerebral cortex was observed and functionally validated. Our findings offer a fresh insight into spatial-, age-, and sex-biased changes in lncRNA expression in macaque brain and suggest that the changes represent a previously unappreciated regulatory system that potentially contributes to brain development and aging.

Sr2+ has low efficiency in regulating spontaneous release at the Calyx of Held synapses.

It has been known that Ca2+ plays an essential role in mediating different modes of neurotransmitter release via different sensing mechanisms. Synaptotagmin 1, 2, and 9 were found to act as the Ca2+ sensors for synchronous release and synaptotagmin 7 and Doc-2 were proposed as the Ca2+ sensors for asynchronous release. Comparatively, the Ca2+ sensor for spontaneous release remains a mystery. At the Calyx of Held synapse, the Ca2+ sensor for spontaneous release was found not identical to the sensor for synchronous release, synaptotagmin 2. As Ca2+ sensors have different sensitivity to Sr2+ and Ca2+ and induce significantly different rate of vesicle release, Sr2+ is traditionally used as a tool to examine the intrinsic properties of different Ca2+ sensors. Here, we employed cell-attached patch recording and presynaptic/postsynaptic whole-cell recording at the Calyx of Held synapses of synaptotagmin 2 knock-out mice to assay the Sr2+ and Ca2+ influx into the nerve terminal at resting potential and observed the effects of Ca2+ and Sr2+ on spontaneous neurotransmitter release. We found that the dwell time of single voltage gated Ca2+ channel opening increased around threefold for Sr2+ than Ca2+ with the channel conductance unchanged; the divalent cation sensing machinery in regulating spontaneous release has much lower sensitivity to Sr2+ than Ca2+ . Thus, our study reveals some of the intrinsic properties of Ca2+ sensor(s) of spontaneous transmitter release and provided an insight into the underlying mechanisms.

Mutant alpha-synuclein causes age-dependent neuropathology in monkey brain.

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2-3, 7-8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD.

A new method for piercing the tentorium cerebelli for implanting fragile electrodes into the brain stem in the rhesus monkey (Macaca mulatta).

Recent developments in neuron recording techniques include the invention of some fragile electrodes. The fragility of these electrodes impedes their successful use in deep brain recordings because it is difficult to penetrate the electrodes through the dura mater, especially the tentorium cerebelli (TC) enclosing the cerebellum and brain stem. This paper reports a new method to pierce the TC for inserting fragile electrodes into the inferior colliculus of rhesus monkeys. Briefly, a unique tool kit, consisting of needles with sharp tips, a guide tube and an "impactor," was used in a multistep protocol to pierce the TC. The impactor provided a brief force that quickly thrusts the needles through the meninges without causing significant damage to the brain tissue under the TC. Using this novel approach, tetrodes were successfully implanted into the inferior colliculus of a rhesus monkey and neuronal discharge signals were recorded. This method, which is simple, convenient and economical, allows neurophysiologists to study the electrophysiological characteristics of deep brain structures under the TC with advanced, albeit fragile, electrodes.

Maternal separation produces lasting changes in cortisol and behavior in rhesus monkeys.

Maternal separation (MS), which can lead to hypothalamic pituitary adrenal axis dysfunction and behavioral abnormalities in rhesus monkeys, is frequently used to model early adversity. Whether this deleterious effect on monkeys is reversible by later experience is unknown. In this study, we assessed the basal hair cortisol in rhesus monkeys after 1.5 and 3 y of normal social life following an early separation. These results showed that peer-reared monkeys had significantly lower basal hair cortisol levels than the mother-reared monkeys at both years examined. The plasma cortisol was assessed in the monkeys after 1.5 y of normal social life, and the results indicated that the peak in the peer-reared cortisol response to acute stressors was substantially delayed. In addition, after 3 y of normal social life, abnormal behavioral patterns were identified in the peer-reared monkeys. They showed decreases in locomotion and initiated sitting together, as well as increases in stereotypical behaviors compared with the mother-reared monkeys. These results demonstrate that the deleterious effects of MS on rhesus monkeys cannot be compensated by a later normal social life, suggesting that the effects of MS are long-lasting and that the maternal-separated rhesus monkeys are a good animal model to study early adversity and to investigate the development of psychiatric disorders induced by exposure to early adversity.

Heavy traffic feasible hybrid intracycle and cyclic sleep for power saving in 10G-EPON.

Energy consumption in optical access networks costs carriers substantial operational expense (OPEX) every year and is one of contributing factors for the global warming. To reduce energy consumption in the 10-gigabit Ethernet passive optical network (10G-EPON), a hybrid intracycle and cyclic sleep mechanism is proposed in this paper. Under heavy traffic load, optical network units (ONUs) can utilize short idle slots within each scheduling cycle to enter intracycle sleep without postponing data transmission. In this way, energy conservation is achieved even under heavy traffic load with quality of service (QoS) guarantee. Under light traffic load, ONUs perform long cyclic sleep for several scheduling cycles. The adoption of cyclic sleep instead of intracycle sleep under light traffic load can reduce unnecessary frequent transitions between sleep and full active work caused by using intracycle sleep. Further, the Markov chain of the proposed mechanism is established. The performances of the proposed mechanism and existing approaches are analyzed quantitatively based on the chain. For the proposed mechanism, power saving ability with QoS guarantee even under heavy traffic and better power saving performance than existing approaches are verified by the quantitative analysis. Moreover, simulations validate the above conclusions based on the chain.

Lumbar puncture increases Alzheimer's disease biomarker levels in cerebrospinal fluid of rhesus monkeys.

Cerebrospinal fluid (CSF) samples are commonly collected via lumbar puncture (LP) in both clinical and research settings for measurement of biomarkers of Alzheimer's disease (AD). To determine the effects of LP on CSF AD biomarkers, we collected CSF samples at seven different time points after an LP in rhesus monkeys. We find that amyloid-beta (Aß) and Tau levels increased significantly on day 1, peaked on day 3, and returned to baseline on day 10 after LP. The NFL levels increased significantly on day 5, peaked on day 10, and returned to baseline after day 30. The increased AD biomarker levels were mainly due to CSF outflow and deep intrathecal invasion during LP. Therefore, if LPs are repeated within a short period of time, prior LP can affect Aß and Tau levels within 10 days and NFL levels within 30 days, which may lead to clinical misdiagnosis or incorrect scientific conclusions.

Kisspeptin-10 modulates the proliferation and differentiation of the rhesus monkey derived stem cell line: R366.4.

The rhesus monkey embryonic stem cell line R366.4 has been identified to differentiate into a number of cell types. However, it has not been well characterized for its response to drugs affecting reproductive endocrinology. Kisspeptins (KPs) are ligands for the GPR-54, which is known to modulate reproductive function. The current study was designed to determine the effect of the KP-10 peptide on R366.4 cells and to investigate the role of KP-GPR54 in the cell proliferation process. Four different doses (0.1, 1, 10, and 100 nM) of KP-10 and control were selected to evaluate cell growth parameters and cellular morphological changes over a 72 hr period. The cells were treated with kisspeptin-10 during the early rosette stage. Proliferation rates, analyzed by flow cytometry and cell count methods, were found to be decreased after treatment. Moreover, the number of rosettes was found to decrease following KP-10 treatments. Morphological changes consisting of neuronal projections were also witnessed. This suggested that KP-10 had an antiproliferative effect on R366.4 cells leading to a differentiation state and morphological changes consistent with neuronal stem cell development. The R366.4 stem cell line differentiates based on kisspeptin signaling and may be used to investigate reproductive cell endocrinology in vitro.

Abnormality of anxious behaviors and functional connectivity between the amygdala and the frontal lobe in maternally deprived monkeys.

OBJECTIVE: Anxious behaviors often occur in individuals who have experienced early adversity. Anxious behaviors can bring many hazards, such as social withdrawal, eating disorders, negative self-efficacy, self-injurious thoughts and behaviors, anxiety disorders, and even depression. Abnormal behavior are is closely related to changes in corresponding circuit functions in the brain. This study investigated the relationship between brain circuits and anxious behaviors in maternal-deprived rhesus monkey animal model, which mimic early adversity in human. METHODS: Twenty-five rhesus monkeys (Macaca mulatta) were grouped by two different rearing conditions: 11 normal control and mother-reared (MR) monkeys and 14 maternally deprived and peer-reared (MD) monkeys. After obtaining images of the brain areas with significant differences in maternal separation and normal control macaque function, the relationship between functional junction intensity and stereotypical behaviors was determined by correlation analysis. RESULTS: The correlation analysis revealed that stereotypical behaviors were negatively correlated with the coupling between the left lateral amygdala subregion and the left inferior frontal gyrus in both MD and MR macaques. CONCLUSION: This study suggests that early adversity-induced anxious behaviors are associated with changes in the strength of the amygdala-prefrontal connection. The normalization of the regions involved in the functional connection might reverse the behavioral abnormality. It provides a solid foundation for effective intervention in human early adversity. SIGNIFICANCE STATEMENT: This study suggests that early adversity-induced anxious behaviors are associated with changes in the strength of the amygdala-prefrontal connection. The higher the amygdala-prefrontal connection strength, the less stereotyped behaviors exhibited by monkeys experiencing early adversity. Thus, in the future, changing the strength of the amygdala-prefrontal connection may reverse the behavioral abnormalities of individuals who experience early adversity. This study provides a solid foundation for effective intervention in humans' early adversity.

Transient neurogenesis in ischemic cortex from Sox2+ astrocytes.

The adult cortex has long been regarded as non-neurogenic. Whether injury can induce neurogenesis in the adult cortex is still controversial. Here, we report that focal ischemia stimulates a transient wave of local neurogenesis. Using 5'-bromo-2'-deoxyuridine labeling, we demonstrated a rapid generation of doublecortin-positive neuroblasts that died quickly in mouse cerebral cortex following ischemia. Nestin-CreER-based cell ablation and fate mapping showed a small contribution of neuroblasts by subventricular zone neural stem cells. Using a mini-photothrombotic ischemia mouse model and retrovirus expressing green fluorescent protein labeling, we observed maturation of locally generated new neurons. Furthermore, fate tracing analyses using PDGFRα-, GFAP-, and Sox2-CreER mice showed a transient wave of neuroblast generation in mild ischemic cortex and identified that Sox2-positive astrocytes were the major neurogenic cells in adult cortex. In addition, a similar upregulation of Sox2 and appearance of neuroblasts were observed in the focal ischemic cortex of Macaca mulatta. Our findings demonstrated a transient neurogenic response of Sox2-positive astrocytes in ischemic cortex, which suggests the possibility of inducing neuronal regeneration by amplifying this intrinsic response in the future.

Effect of morphine on conditioned place preference in rhesus monkeys.

In rodents, a conditioned place preference (CPP) can be induced by morphine. In the current study, we designed a biased place conditioning paradigm to test the rewarding effects of morphine in freely moving rhesus monkeys. Five monkeys were first placed in three serial rooms with the doors open between them for three days. After this habituation period, during which baseline preference for each of the two end rooms was measured, CPP conditioning occurred when the monkeys were injected intramuscularly with morphine at an increasing dose (1.5, 3, 4.5 mg/kg) before they entered the non-preferred room and on alternate days, with saline before they entered the preferred room. Morphine and saline treatment lasted for six days, respectively. CPP was tested 24 hours after the end of CPP training. The result showed that in all five monkeys, CPP was induced by the morphine treatment. The preference lasted for at least 15.3 ± 1.7 months.

Ultrasonic-assisted extraction of polysaccharides from coix seeds: Optimization, purification, and in vitro digestibility.

To optimize the extraction of polysaccharides from coix seeds (CSP), an auxiliary method of ultrasound was developed by response surface methodology (RSM). The maximum extraction yield (8.340%) was obtained under 480 W power, 16 min ultrasound extraction (UE) time and 21.00 mL/g water to raw material ratio. Compared to hot water extraction (HE), UE-treated CSP led to a higher extraction efficiency and decreased average CSP molecular weight. FT-IR indicated that CSP extracted by UE and HE were neutral polysaccharides, and linkages between sugar units were mainly in the α-conformation. Furthermore, NMR spectra indicated that UE-treated CSP was a neutral polysaccharide with (1 â†’ 6)-linked α-d-glucopyranose in the main chain. Two polysaccharide components (CSP-A and CSP-B) were purified by anion exchange chromatography, therein, CSP-A was more resistant to the digestion in stomach and intestine. These results suggest that CSP-A has the potential to be a functional agent utilized by gut microbes.

Structural characterization of exopolysaccharides from Weissella cibaria NC516.11 in distiller grains and its improvement in gluten-free dough.

In this study, an exopolysaccharide (EPS) was produced by Weissella cibaria NC516.11 isolated from distiller grains of Chinese Baijiu. The structural characterization of EPS determined using fourier transform infrared spectra and nuclear magnetic resonance spectra demonstrated that W. cibaria NC516.11 had α-(1 â†’ 6) (93.46%) d-glucose linkages with a few α-(1 â†’ 3) (6.54%) d-glucose linked branches. The monosaccharide composition of the EPS was glucose, and its molecular weight was 2.82 × 106 Da. Scanning electron microscopy showed that the microstructure of EPS had a three-dimensional structure at low magnification and a particle structure that protruded from the surface at high magnification. The addition of EPS into dough can promote the cross-linking of starch molecules and increase the water-holding capacity. Dynamic rheology indicated that the aqueous solution of EPS is a pseudoplastic fluid, and the higher the concentration of EPS, the greater the viscosity. The addition of EPS to the gluten-free dough showed G' > G″, which could increase the viscoelastic properties of the dough and enhance the gluten network.

Bimetal heterointerfaces towards enhanced electro-activation of O2 under room condition.

Efficient catalysts for oxygen (O2) activation under room condition are required for effective wet air oxidation (WAO) technology. Here, we report a novel manganese-cobalt-based composite (MnO-CoO@Co) fabricated on a graphite felt (GF) support for catalyzing the electro-activation of O2 under room condition. Abundant Co-MnO and CoO-MnO heterointerfaces are formed in the composite. In comparison to the single-metal counterparts, i.e. CoO@Co/GF (16.99 wt% Co) and MnO/GF (26.83 wt% Mn), the bimetal MnO-CoO@Co/GF (5.29 wt% Co and 8.79 wt% Mn) displays an improved oxygen storage capacity and provides more active sites to accommodate surface adsorbed oxygen species. Notably, the strong synergy derived from bimetal heterointerfaces enhances the electron transfer and oxygen mobilization during the electro-activation of O2, thereby significantly reducing the reaction barrier. MnO-CoO@Co/GF exhibits excellent efficiency and stability in electrocatalytic WAO (ECWAO) towards the removal of pharmaceuticals and personal care products (PPCPs) over a wide pH range from 4.0 to 10.0. A model pollutant sulfamethoxazole (SMX) acquires mineralization efficiency of 78.4 ± 2.1% and mineralization current efficiency of 157.89% at +1.0 V of electrode potential. The toxicity of PPCPs can be totally eliminated after the ECWAO treatment. This work highlights the synergy derived from bimetal heterointerfaces in O2 electrocatalysis, and provides a promising approach for advanced WAO catalysts in PPCPs pollution control.