Cynomolgus Monkey Brain Research Articles

Correlation analysis of positron emission tomography/computed tomography-magnetic resonance imaging of cannabinoid type 1 receptor in the lumbar spine and brain of aged osteoporosis female cynomolgus monkeys.

Although cannabinoid receptor 1 (CB1R) antagonists can inhibit bone loss in osteoporosis mouse models, different strains of mice show different bone mass phenotypes after knock out the CB1R gene. The relationship between CB1R and bone metabolism is complex, and its regulatory role in bone metabolism and as a therapeutic target for osteoporosis requires further investigation. Based on lumbar spine volumetric bone mineral density (vBMD) data of healthy female cynomolgus monkeys aged 1-25 years, naturally aged postmenopausal female osteoporotic monkeys and normal young monkeys were screened by detecting lumbar vertebrae vBMD and estradiol levels in this study. Positron emission tomography-computed tomography (PET/CT) and magnetic resonance imaging (MRI) scans were performed on the lumbar spine and brain of the two groups of monkeys using the probe [11C]OMAR, which specifically targets CB1R, and the difference in the CB1R expression of osteoporotic monkeys was evaluated. The vBMD values of two standard deviations (SDs) below the peak bone value (428.1±53.8 g/cm3) were set as the reference standard for osteoporosis vBMD. Of the 49 healthy female cynomolgus monkeys, 4 postmenopausal older osteoporotic monkeys (18-26 years) and 5 young control monkeys (6-7 years) were selected, and the mean vBMD of the lumbar spine of the two groups was 295.07±19.11 and 419.72±16.14 g/cm3, respectively (P<0.0001). Radioactive uptake in the lumbar spine was linearly and negatively correlated with vBMD (r=-0.7977; P=0.01). Dynamic PET/MR imaging of the brains showed that CB1R was upregulated in the osteoporosis group, and there was a negative linear correlation between the vBMD and area under the time-radioactivity curve (AUC) of the thalamus (r=-0.8506; P=0.0153) and prefrontal cortex (r=-0.8306; P=0.0207). In this study, PET/CT-MRI molecular imaging technology revealed that CB1R was upregulated in the lumbar spine and brain of the osteoporosis monkeys and that CB1R may be regulated by the brain-bone axis. CB1R antagonist may be a potential drug for the treatment of osteoporosis.

Development of a Novel [11C]CO-Labeled Positron Emission Tomography Radioligand [11C]BIO-1819578 for the Detection of O-GlcNAcase Enzyme Activity.

Imaging O-GlcNAcase OGA by positron emission tomography (PET) could provide information on the pathophysiological pathway of neurodegenerative diseases and important information on drug-target engagement and be helpful in dose selection of therapeutic drugs. Our aim was to develop an efficient synthetic method for labeling BIO-1819578 with carbon-11 using 11CO for evaluation of its potential to measure levels of OGA enzyme in non-human primate (NHP) brain using PET. Radiolabeling was achieved in one-pot via a carbon-11 carbonylation reaction using [11C]CO. The detailed regional brain distribution of [11C]BIO-1819578 binding was evaluated using PET measurements in NHPs. Brain radioactivity was measured for 93 min using a high-resolution PET system, and radiometabolites were measured in monkey plasma using gradient radio HPLC. Radiolabeling of [11C]BIO-1819578 was successfully accomplished, and the product was found to be stable at 1 h after formulation. [11C]BIO-1819578 was characterized in the cynomolgus monkey brain where a high brain uptake was found (7 SUV at 4 min). A pronounced pretreatment effect was found, indicating specific binding to OGA enzyme. Radiolabeling of [11C]BIO-1819578 with [11C]CO was successfully accomplished. [11C]BIO-1819578 binds specifically to OGA enzyme. The results suggest that [11C]BIO-1819578 is a potential radioligand for imaging and for measuring target engagement of OGA in the human brain.

Characterization of a Novel M4 PAM PET Radioligand [11C]PF06885190 in Nonhuman Primates (NHP).

Muscarinic acetylcholine receptors (mAChR), including M4, draw attention as therapeutic targets for several neurodegenerative diseases including Alzheimer's disease (AD). PET imaging of M4 positive allosteric modulator (PAM) allows qualification of the distribution as well as the expression of this receptor under physiological conditions and thereby helps to assess the receptor occupancy (RO) of a drug candidate. In this study, our aims were (a) to synthesize a novel M4 PAM PET radioligand [11C]PF06885190 (b) to evaluate the brain distribution of [11C]PF06885190 in nonhuman primates (NHP) and (c) to analyze its radiometabolites in the blood plasma of NHP. Radiolabeling of [11C]PF06885190 was accomplished via N-methylation of the precursor. Six PET measurements were performed using two male cynomolgus monkeys, where three PET measurements were at baseline, two after pretreatment with a selective M4 PAM compound CVL-231 and one after pretreatment with donepezil. The total volume of distribution (VT) of [11C]PF06885190 was examined using Logan graphical analysis with arterial input function. Radiometabolites were analyzed in monkey blood plasma using gradient HPLC system. Radiolabeling of [11C]PF06885190 was successfully accomplished and the radioligand was found to be stable in the formulation, with radiochemical purity exceeding 99% 1 h after the end of the synthesis. [11C]PF06885190 was characterized in the cynomolgus monkey brain where a moderate brain uptake was found at the baseline condition. However, it showed fast wash-out as it dropped to half of the peak at around 10 min. Change of VT from baseline was around -10% after pretreatment with a M4 PAM, CVL-231. Radiometabolite studies showed relatively fast metabolism. Although sufficient brain uptake of [11C]PF06885190 was observed, these data suggest that [11C]PF06885190 might have too low specific binding in the NHP brain to be further applied in PET imaging.

Characterization of a HIR-Fab-IDS, Novel Iduronate 2-Sulfatase Fusion Protein for the Treatment of Neuropathic Mucopolysaccharidosis Type II (Hunter Syndrome)

Mucopolysaccharidosis type II is a severe lysosomal storage disease caused by deficient activity of the enzyme iduronate-2-sulfatase. The only medicinal product approved by the US Food and Drug Administration for enzyme replacement therapy, recombinant iduronate-2-sulfatase (idursulfase, Elaprase®), is a large molecule that is not able to cross the blood-brain barrier and neutralize progressive damage of the central nervous system caused by the accumulation of glycosaminoglycans. Novel chimeric protein HIR-Fab-IDS is an anti-human insulin receptor Fab fragment fused to recombinant modified iduronate-2-sulfatase. This modification provides a highly selective interaction with the human insulin receptor, which leads to the HIR-Fab-IDS crossing the blood-brain barrier owing to internalization of the hybrid molecule by transcytosis into endothelial cells adjacent to the nervous system by the principle of a 'molecular Trojan horse'. In this work, the physicochemical and biological characterization of a blood-brain barrier-penetrating fusion protein, HIR-Fab-IDS, is carried out. HIR-Fab-IDS consists of an anti-human insulin receptor Fab fragment fused to recombinant iduronate-2-sulfatase. Comprehensive analytical characterization utilizing modern techniques (including surface plasmon resonance and mass spectrometry) was performed using preclinical and clinical batches of HIR-Fab-IDS. Critical quality parameters that determine the therapeutic effect of iduronate-2-sulfatase, as well as IDS enzymatic activity and in vitro cell uptake activity were evaluated in comparison with the marketed IDS product Elaprase® (IDS RP). In vivo efficiency of HIR-Fab-IDS in reversing mucopolysaccharidosis type II pathology in IDS-deficient mice was also investigated. The affinity of the chimeric molecule for the INSR was also determined by both an enzyme-linked immunosorbent assay and surface plasmon resonance. We also compared the distribution of 125I-radiolabeled HIR-Fab-IDS and IDS RP in the tissues and brain of cynomolgus monkeys after intravenous administration. The HIR-Fab-IDS primary structure investigation showed no significant post-translational modifications that could affect IDS activity, except for the formylglycine content, which was significantly higher for HIR-Fab-IDS compared with that for IDS RP (~76.5 vs ~67.7%). Because of this fact, the specific enzyme activity of HIR-Fab-IDS was slightly higher than that of IDS RP (~2.73 × 106U/μmol vs ~2.16 × 106U/μmol). However, differences were found in the glycosylation patterns of the compared IDS products, causing a minor reduced in vitro cellular uptake of HIR-Fab-IDS by mucopolysaccharidosis type II fibroblasts compared with IDS RP (half-maximal effective concentration ~26.0 vs ~23.0nM). The efficacy of HIR-Fab-IDS in IDS-deficient mice has demonstrated a statistically significant reduction in the level of glycosaminoglycans in the urine and tissues of the main organs to the level of healthy animals. The HIR-Fab-IDS has revealed high in vitro affinity for human and monkey insulin receptors, and the radioactively labeled product has been shown to penetrate to all parts of the brain and peripheral tissues after intravenous administration to cynomolgus monkeys. These findings indicate that HIR-Fab-IDS, a novel iduronate-2-sulfatase fusion protein, is a promising candidate for the treatment of central nervous system manifestations in neurological mucopolysaccharidosis type II.

Age-related alterations in protein phosphatase 2A methylation levels in brains of cynomolgus monkeys: a pilot study.

The abnormal activity of PP2A, a dominant member of type 2A serine/threonine protein phosphatase, has been implicated in the development of Alzheimer's disease (AD) and dementia with Lewy bodies (DLB). PP2A is a holoenzyme, and protein methylation of the catalytic subunit, PP2Ac, alters the complex composition. A decrease in PP2Ac methylation levels has been reported in AD and DLB. Aging is the most common risk factor for AD and DLB, but the relationship between aging and PP2A has not been studied in detail. Cynomolgus monkey show increased phosphorylation levels of tau and α-synuclein with aging. In this study, we investigated the alterations in the PP2A activity regulation with aging in monkey brains from 2 to 43years of age using fractionated proteins. We found that type 2A protein phosphatase activity decreased with aging in cytoplasmic and nuclear-soluble fractions. PP2Ac methylation level was decreased in cytoplasmic and sarkosyl-insoluble fractions. A principal component analysis using PP2Ac, demethylated PP2Ac and PP2A methylesterase PME-1 levels in cytoplasmic and nuclear-soluble fractions as attributes showed that aged monkeys were in the same cluster. Our results show that brain aging in cynomolgus monkeys is closely related to changes in PP2A methylation.

Spatially resolved gene regulatory and disease-related vulnerability map of the adult Macaque cortex

Single cell approaches have increased our knowledge about the cell type composition of the non-human primate (NHP), but a detailed characterization of area-specific regulatory features remains outstanding. We generated single-cell transcriptomic and chromatin accessibility (single-cell ATAC) data of 358,237 cells from prefrontal cortex (PFC), primary motor cortex (M1) and primary visual cortex (V1) of adult female cynomolgus monkey brain, and integrated this dataset with Stereo-seq (spatial enhanced resolution omics-sequencing) of the corresponding cortical areas to assign topographic information to molecular states. We identified area-specific chromatin accessible sites and their targeted genes, including the cell type-specific transcriptional regulatory network associated with excitatory neurons heterogeneity. We reveal calcium ion transport and axon guidance genes related to specialized functions of PFC and M1, identified the similarities and differences between adult macaque and human oligodendrocyte trajectories, and mapped the genetic variants and gene perturbations of human diseases to NHP cortical cells. This resource establishes a transcriptomic and chromatin accessibility combinatory regulatory landscape at a single-cell and spatially resolved resolution in NHP cortex.

Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies

Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neurodegenerative disorders characterized by accumulation of hyperphosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we performed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 3′ UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.

The cerebral artery in cynomolgus monkeys (Macaca fascicularis).

Cerebral artery structure has not been extensively studied in primates. The aim of this study was to examine the cerebrovascular anatomy of cynomolgus monkeys (Macaca fascicularis), which are one of the most commonly used primates in medical research on human diseases, such as cerebral infarction and subarachnoid hemorrhage. In this study, we investigated the anatomy and diameter of cerebral arteries from 48 cynomolgus monkey brain specimens. We found three anatomical differences in the vascular structure of this species compared to that in humans. First, the distal anterior cerebral artery is single. Second, the pattern in which both the anterior inferior cerebellar artery and posterior inferior cerebellar artery branch from the basilar artery is the most common. Third, the basilar artery has the largest diameter among the major arteries. We expect that this anatomical information will aid in furthering research on cerebrovascular disease using cynomolgus monkeys.

Aging induces abnormal accumulation of Aβ in extracellular vesicle and/or intraluminal membrane vesicle-rich fractions in nonhuman primate brain

Aβ metabolism in the brain is mediated by endocytosis, one part of the intracellular membrane trafficking system. We previously showed that aging attenuates the interaction of dynein with dynactin, which disrupts the endosomal/lysosomal trafficking pathway involved in Aβ metabolism, resulting in intracellular accumulation of Aβ. Several studies have shown that in Alzheimer's disease (AD), intraneuronal accumulation of Aβ precedes extracellular Aβ depositions. However, it is unclear what accounts for this transition from intracellular to extracellular depositions. Accumulating evidence suggest that autophagy has an important role in AD pathology, and we observed that autophagy-related protein levels began to decrease before amyloid plaque formation in cynomolgus monkey brains. Surprisingly, experimental induction of autophagosome formation in Neuro2a cells significantly increased intracellular Aβ and decreased extracellular release of Aβ, accompanied by the prominent reduction of extracellular vesicle (EV) secretion. RNAi study confirmed that EV secretion affected intracellular and extracellular Aβ levels, and siRNA-induced downregulation of autophagosome formation enhanced EV secretion to ameliorate intracellular Aβ accumulation induced by dynein knockdown. In aged cynomolgus monkeys, Aβ levels in EV/intraluminal membrane vesicle (ILV)-rich fractions isolated from temporal lobe parenchyma were drastically increased. Moreover, EV/ILV marker proteins overlapped spatially with amyloid plaques. These findings suggest that EV would be an important carrier of Aβ in brain and abnormal accumulation of Aβ in EVs/ILVs may be involved in the transition of age-dependent Aβ pathology.

Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

A dedicated eight-channel receive RF coil array for monkey brain MRI at 9.4 T.

The neuroimaging of nonhuman primates (NHPs) realised with magnetic resonance imaging (MRI) plays an important role in understanding brain structures and functions, as well as neurodegenerative diseases and pathological disorders. Theoretically, an ultrahigh field MRI (≥7 T) is capable of providing a higher signal-to-noise ratio (SNR) for better resolution; however, the lack of appropriate radiofrequency (RF) coils for 9.4 T monkey MRI undermines the benefits provided by a higher field strength. In particular, the standard volume birdcage coil at 9.4 T generates typical destructive interferences in the periphery of the brain, which reduces the SNR in the neuroscience-focused cortex region. Also, the standard birdcage coil is not capable of performing parallel imaging. Consequently, extended scan durations may cause unnecessary damage due to overlong anaesthesia. In this work, assisted by numerical simulations, an eight-channel receive RF coil array was specially designed and manufactured for imaging NHPs at 9.4 T. The structure and geometry of the proposed receive array was optimised with numerical simulations, so that the SNR enhancement region was particularly focused on monkey brain. Validated with rhesus monkey and cynomolgus monkey brain images acquired from a 9.4 T MRI scanner, the proposed receive array outperformed standard birdcage coil with higher SNR, mean diffusivity and fractional anisotropy values, as well as providing better capability for parallel imaging.

Axonal Extensions along Corticospinal Tracts from Transplanted Human Cerebral Organoids.

SummaryThe reconstruction of lost neural circuits by cell replacement is a possible treatment for neurological deficits after cerebral cortex injury. Cerebral organoids can be a novel source for cell transplantation, but because the cellular composition of the organoids changes along the time course of the development, it remains unclear which developmental stage of the organoids is most suitable for reconstructing the corticospinal tract. Here, we transplanted human embryonic stem cell-derived cerebral organoids at 6 or 10 weeks after differentiation (6w- or 10w-organoids) into mouse cerebral cortices. 6w-organoids extended more axons along the corticospinal tract but caused graft overgrowth with a higher percentage of proliferative cells. Axonal extensions from 10w-organoids were smaller in number but were enhanced when the organoids were grafted 1 week after brain injury. Finally, 10w-organoids extended axons in cynomolgus monkey brains. These results contribute to the development of a cell-replacement therapy for brain injury and stroke.

In Vitro Induction of Human Embryonic Stem Cells into the Midbrain Dopaminergic Neurons and Transplantation in Cynomolgus Monkey.

A simple, rapid, efficient, and specialized culture system was successfully developed in this study to induce human embryonic stem cells into dopaminergic neurons in vitro. It only took 5 days to generate quickly and directly a large number of homogeneous neural stem cell (NSC) spheres by the introduction of small molecules LDN (inhibitor of BMP [bone morphogenetic protein] pathway that inhibits BMP type I receptors ALK2 and ALK3), SB431542 (inhibitor of TGF-β/Activin/Dodal pathway that inhibits ALK4, ALK5, and ALK7), CHIR99021 (inhibitors of GSK-3 [glycogen synthase kinase 3]), and basic fibroblast growth factor (bFGF). The dopaminergic neurons were successfully induced at day 25 (tyrosine hydroxylase [TH] expressed) and at day 32 (TH highly expressed) with high purity (TH/Tuj1: 84.14% and 93.15%, respectively) by the addition of FGF8 (fibroblast growth factor 8), sonic hedgehog (SHH), and Purmorphamine after the generation of NSC at day 5. And, the dopaminergic neurons induced by this system successfully survived and integrated into the striatum of cynomolgus monkey brain after transplantation, which verified the efficiency of the induction system developed in this study, suggesting the potential clinical application in cell therapy for neurological diseases.

Elevated Membrane Cholesterol Disrupts Lysosomal Degradation to Induce β-Amyloid Accumulation: The Potential Mechanism Underlying Augmentation of β-Amyloid Pathology by Type 2 Diabetes Mellitus

The endocytic membrane trafficking system is altered in the brains of early-stage Alzheimer disease (AD) patients, and endocytic disturbance affects the metabolism of β-amyloid (Aβ) protein, a key molecule in AD pathogenesis. It is widely accepted that type 2 diabetes mellitus (T2DM) is one of the strongest risk factors for development of AD. Supporting this link, experimentally induced T2DM enhances AD pathology in various animal models. Spontaneous T2DM also enhances Aβ pathology with severe endocytic pathology, even in nonhuman primate brains. However, it remains unclear how T2DM accelerates Aβ pathology. Herein, we demonstrate that cholesterol metabolism-related protein levels are increased and that membrane cholesterol level is elevated in spontaneous T2DM-affected cynomolgus monkey brains. Moreover, invitro studies that manipulate cellular cholesterol reveal that elevated membrane cholesterol disrupts lysosomal degradation and enhances chemical-induced endocytic disturbance, resulting in great accumulation of Aβ in Neuro2a cells. These findings suggest that an alteration of cerebral cholesterol metabolism may be responsible for augmentation of Aβ pathology in T2DM-affected brains, which, in turn, may increase the risk for developing AD.

Characterization of [11C]PXT012253 as a PET Radioligand for mGlu4 Allosteric Modulators in Nonhuman Primates.

Modulation of presynaptic metabotropic glutamate receptor 4 (mGlu4) by an allosteric ligand has been proposed as a promising therapeutic target in Parkinson's disease and levodopa-induced dyskinesia. A positron emission tomography (PET) ligand for an allosteric site of mGlu4 may provide evidence that a clinical drug candidate reaches and binds the target. A carbon-11-labeled PET radioligand binding an allosteric site of mGlu4, [11C]PXT012253, has been recently developed. Here, we describe the detailed characterization of this novel radiolabeled mGlu4 ligand in nonhuman primates. [11C]PXT012253 binding in the brain of cynomolgus monkeys, under the baseline and blocking conditions with the structurally different mGlu4 allosteric ligand PXT002331, currently in clinical trials for Parkinson's disease, was quantified with compartment and graphical modeling approaches using a radiometabolite-corrected plasma input function. Whole-body biodistribution of [11C]PXT012253 was then assessed using PET/x-ray computed tomography to estimate the human effective doses of [11C]PXT012253 for further clinical studies. [11C]PXT012253 displayed binding in mGlu4-expressing regions in the brain of cynomolgus monkeys. Brain regional time-activity curves of [11C]PXT012253 were well described in the two-tissue compartment model (2TC). Total distribution volume was stably estimated using Logan plot and multilinear analysis (MA1) although 2TC showed unstable values in some cases. Competition with PXT002331 showed high specific binding in the total distribution volume. Whole-body PET showed high accumulation of [11C]PXT012253 in the liver, kidney, heart, and brain in the initial phase. The radioligand was excreted through both the gastrointestinal and the urinary tracts. Effective dose of [11C]PXT012253 was estimated to be 0.0042mSv/MBq. [11C]PXT012253 was shown to be a promising PET radioligand for mGlu4 allosteric modulators in the monkey brain. MA1 would be the choice of quantitative method. Further development of [11C]PXT012253 in human subjects is warranted.

The brain-penetrant clinical ATM inhibitor AZD1390 radiosensitizes and improves survival of preclinical brain tumor models.

Poor survival rates of patients with tumors arising from or disseminating into the brain are attributed to an inability to excise all tumor tissue (if operable), a lack of blood-brain barrier (BBB) penetration of chemotherapies/targeted agents, and an intrinsic tumor radio-/chemo-resistance. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR. We report the primary pharmacology of the clinical-grade, exquisitely potent (cell IC50, 0.78 nM), highly selective [>10,000-fold over kinases within the same phosphatidylinositol 3-kinase-related kinase (PIKK) family], orally bioavailable ATM inhibitor AZD1390 specifically optimized for BBB penetration confirmed in cynomolgus monkey brain positron emission tomography (PET) imaging of microdosed 11C-labeled AZD1390 (Kp,uu, 0.33). AZD1390 blocks ATM-dependent DDR pathway activity and combines with radiation to induce G2 cell cycle phase accumulation, micronuclei, and apoptosis. AZD1390 radiosensitizes glioma and lung cancer cell lines, with p53 mutant glioma cells generally being more radiosensitized than wild type. In in vivo syngeneic and patient-derived glioma as well as orthotopic lung-brain metastatic models, AZD1390 dosed in combination with daily fractions of IR (whole-brain or stereotactic radiotherapy) significantly induced tumor regressions and increased animal survival compared to IR treatment alone. We established a pharmacokinetic-pharmacodynamic-efficacy relationship by correlating free brain concentrations, tumor phospho-ATM/phospho-Rad50 inhibition, apoptotic biomarker (cleaved caspase-3) induction, tumor regression, and survival. On the basis of the data presented here, AZD1390 is now in early clinical development for use as a radiosensitizer in central nervous system malignancies.

Age-dependent elevations of oligomeric and phosphorylated alpha-synuclein synchronously occurs in the brain and gastrointestinal tract of cynomolgus monkeys.

Fibrillary α-synuclein (α-syn), which constitutes the major component of Lewy pathology characterized for Parkinson's disease (PD), is found also in the aged enteric nervous system (ENS) and central nervous system (CNS). However, what happens to oligomeric α-syn (o-α-syn) in the aged ENS and CNS remains poorly understood. Here, by using ELISA methods specific for o-α-syn and phosphorylated α-syn (p-α-syn), we measured the levels of o-α-syn and p-α-syn in the gastrointestinal (GI) tract and brain of aging cynomolgus monkeys. We detected a synchronously occurred age-dependent elevation of o-α-syn in the GI tract and brain, which was accompanied by an age-dependent increase of α-syn phosphorylated at serine 129, a modification that promotes α-syn aggregation. Correlation analysis revealed that alterations in p-α-syn and o-α-syn in the colon were positively correlated to those in some brain regions such as the striatum, hippocampus, thalamus and temporal lobe. The results obtained suggest that age-dependent elevations of p-α-syn and o-α-syn occur synchronously in various regions of the ENS and CNS.

Tau pathology in aged cynomolgus monkeys is progressive supranuclear palsy/corticobasal degeneration- but not Alzheimer disease-like -Ultrastructural mapping of tau by EDX.

Concomitant deposition of amyloid -beta protein (Aβ) and neuronal tau as neurofibrillary tangles in the human brain is a hallmark of Alzheimer disease (AD). Because these deposits increase during normal aging, it has been proposed that aging brains may also undergo AD-like changes. To investigate the neuropathological changes that occur in the aging primate brain, we examined 21 brains of cynomolgus monkeys (7–36 years old) for Aβ- and tau-positive lesions. We found, 1) extensive deposition of Aβ in brains of cynomolgus monkeys over 25 years of age, 2) selective deposition of 4-repeat tau as pretangles in neurons, and as coiled body-like structures in oligodendroglia-like cells and astrocytes, 3) preferential distribution of tau in the basal ganglia and neocortex rather than the hippocampus, and 4) age-associated increases in 30–34 kDa AT8- and RD4-positive tau fragments in sarkosyl-insoluble fractions. We further labeled tau-positive structures using diaminobezidine enhanced with nickel, and visualized nickel-labeled structures by energy-dispersive X-ray (EDX) analysis of ultrathin sections. This allowed us to distinguish between nickel-labeled tau and background electron-dense structures, and we found that tau localized to 20–25 nm straight filaments in oligodendroglia-like cells and neurons. Our results indicate that the cytopathology and distribution of tau deposits in aged cynomolgus brains resemble those of progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) rather than AD. Thus, even in the presence of Aβ, age-associated deposition of tau in non-human primates likely does not occur through AD-associated mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0385-5) contains supplementary material, which is available to authorized users.

Precise and accurate assay of pregnenolone and five other neurosteroids in monkey brain tissue by LC–MS/MS

A series of steroids present in the brain have been named “neurosteroids” following the possibility of their role in the central nervous system impairments such as anxiety disorders, depression, premenstrual dysphoric disorder (PMDD), addiction, or even neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Study of their potential role requires a sensitive and accurate assay of their concentration in the monkey brain, the closest model to the human. We have thus developed a robust, precise and accurate liquid chromatography–tandem mass spectrometry method for the assay of pregnenolone, pregnanolone, epipregnanolone, allopregnanolone, epiallopregnanolone, and androsterone in the cynomolgus monkey brain. The extraction method includes a thorough sample cleanup using protein precipitation and phospholipid removal, followed by hexane liquid-liquid extraction and a Girard T ketone-specific derivatization. This method opens the possibility of investigating the potential implication of these six steroids in the most suitable animal model for neurosteroid-related research.

Dynein Dysfunction Reproduces Age-Dependent Retromer Deficiency: Concomitant Disruption of Retrograde Trafficking Is Required for Alteration in β-Amyloid Precursor Protein Metabolism

It is widely accepted that β-amyloid (Aβ) protein plays a pivotal role in Alzheimer disease pathogenesis, and accumulating evidence suggests that endocytic dysfunction is involved in Aβ pathology. Retromer, a conserved multisubunit complex, mediates the retrograde transport of numerous kinds of cargo from endosomes to the trans-Golgi network. Several studies have found that retromer deficiency enhances Aβ pathology both invitro and invivo. Cytoplasmic dynein, a microtubule-based motor protein, mediates minus-end-directed vesicle transport via interactions with dynactin, another microtubule-associated protein that also interacts with retromer. Aging attenuates the dynein-dynactin interaction, and dynein dysfunction reproduces age-dependent endocytic disturbance, resulting in the intracellular accumulation of beta-amyloid precursor protein (APP) and its β-cleavage products, including Aβ. Here, we report that aging itself affects retromer trafficking in cynomolgus monkey brains. In addition, dynein dysfunction reproduces this type of age-dependent retromer deficiency (ie, the endosomal accumulation of retromer-related proteins and APP. Moreover, we found that knockdown of Rab7, Rab9, or Rab11 did not alter endogenous APP metabolism, such as that observed in aged monkey brains and in dynein-depleted cells. These findings suggest that dynein dysfunction can cause retromer deficiency and that concomitant disruption of retrograde trafficking may be the key factor underlying age-dependent Aβ pathology.