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.

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.

Continuous subcellular resolution three-dimensional imaging on intact macaque brain.

To decipher the organizational logic of complex brain circuits, it is important to chart long-distance pathways while preserving micron-level accuracy of local network. However, mapping the neuronal projections with individual-axon resolution in the large and complex primate brain is still challenging. Herein, we describe a highly efficient pipeline for three-dimensional mapping of the entire macaque brain with subcellular resolution. The pipeline includes a novel poly-N-acryloyl glycinamide (PNAGA)-based embedding method for long-term structure and fluorescence preservation, high-resolution and rapid whole-brain optical imaging, and image post-processing. The cytoarchitectonic information of the entire macaque brain was acquired with a voxel size of 0.32 µm × 0.32 µm × 10 µm, showing its anatomical structure with cell distribution, density, and shape. Furthermore, thanks to viral labeling, individual long-distance projection axons from the frontal cortex were for the first time reconstructed across the entire brain hemisphere with a voxel size of 0.65 µm × 0.65 µm × 3 µm. Our results show that individual cortical axons originating from the prefrontal cortex simultaneously target multiple brain regions, including the visual cortex, striatum, thalamus, and midbrain. This pipeline provides an efficient method for cellular and circuitry investigation of the whole macaque brain with individual-axon resolution, and can shed light on brain function and disorders.

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 *.

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.

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.

Gene editing in monogenic autism spectrum disorder: animal models and gene therapies.

Autism spectrum disorder (ASD) is a lifelong neurodevelopmental disease, and its diagnosis is dependent on behavioral manifestation, such as impaired reciprocal social interactions, stereotyped repetitive behaviors, as well as restricted interests. However, ASD etiology has eluded researchers to date. In the past decades, based on strong genetic evidence including mutations in a single gene, gene editing technology has become an essential tool for exploring the pathogenetic mechanisms of ASD via constructing genetically modified animal models which validates the casual relationship between genetic risk factors and the development of ASD, thus contributing to developing ideal candidates for gene therapies. The present review discusses the progress in gene editing techniques and genetic research, animal models established by gene editing, as well as gene therapies in ASD. Future research should focus on improving the validity of animal models, and reliable DNA diagnostics and accurate prediction of the functional effects of the mutation will likely be equally crucial for the safe application of gene therapies.

High-throughput mapping of a whole rhesus monkey brain at micrometer resolution.

Whole-brain mesoscale mapping in primates has been hindered by large brain sizes and the relatively low throughput of available microscopy methods. Here, we present an approach that combines primate-optimized tissue sectioning and clearing with ultrahigh-speed fluorescence microscopy implementing improved volumetric imaging with synchronized on-the-fly-scan and readout technique, and is capable of completing whole-brain imaging of a rhesus monkey at 1 × 1 × 2.5 µm3 voxel resolution within 100 h. We also developed a highly efficient method for long-range tracing of sparse axonal fibers in datasets numbering hundreds of terabytes. This pipeline, which we call serial sectioning and clearing, three-dimensional microscopy with semiautomated reconstruction and tracing (SMART), enables effective connectome-scale mapping of large primate brains. With SMART, we were able to construct a cortical projection map of the mediodorsal nucleus of the thalamus and identify distinct turning and routing patterns of individual axons in the cortical folds while approaching their arborization destinations.

Co-editing PINK1 and DJ-1 Genes Via Adeno-Associated Virus-Delivered CRISPR/Cas9 System in Adult Monkey Brain Elicits Classical Parkinsonian Phenotype.

Whether direct manipulation of Parkinson's disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6-10 months), and thus provides a practical transgenic monkey model for future PD studies.

Biological implications and limitations of a cynomolgus monkey with naturally occurring Parkinson's disease.

We recently identified a cynomolgus monkey with naturally occurring Parkinson's disease (PD), indicating that PD may not be a uniquely human disease (Li et al, 2020). In our previous study, four lines of evidence, including typical PD clinical symptoms, pharmacological responses, pathological hallmarks, and genetic mutations, strongly supported the identification of a monkey with spontaneous PD (Figure 1). To the best of our knowledge, this is the first reported case of naturally developed PD in animals. This suggests that PD is not a disease restricted to humans, with its existence in a non-human primate providing a novel evolutionary angle for understanding PD. As a close relative to humans (Buffalo et al, 2019; Phillips et al, 2014; Yan et al, 2011), this rare case of PD in another primate species provides solid evidence that monkeys are ideal candidates for the development of a genuine "animal version of PD", with conserved etiology and pathogenesis (Li et al, 2020). Furthermore, it allows us to compare similarities and differences in PD development between species and to understand PD pathogenesis from an evolutionary point of view.

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.