Zhengbo Wang

Mutant Alpha-Synuclein Causes Age-Dependent Neuropathology in Monkey Brain

Parkinsons 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.

Alzheimer's Disease and Methanol Toxicity (Part 2): Lessons from Four Rhesus Macaques (Macaca mulatta) Chronically Fed Methanol

A recently established link between formaldehyde, a methanol metabolite, and Alzheimers disease (AD) pathology has provided a new impetus to investigate the chronic effects of methanol exposure. This paper expands this investigation to the non-human primate, rhesus macaque, through the chronic feeding of young male monkeys with 3% methanol ad libitum. Variable Spatial Delay Response Tasks of the monkeys found that the methanol feeding led to persistent memory decline in the monkeys that lasted 6 months beyond the feeding regimen. This change coincided with increases in tau protein phosphorylation at residues T181 and S396 in cerebrospinal fluid during feeding as well as with increases in tau phosphorylated aggregates and amyloid plaques in four brain regions postmortem: the frontal lobe, parietal lobe, temporal lobe, and the hippocampus. Tau phosphorylation in cerebrospinal fluid was found to be dependent on methanol feeding status, but phosphorylation changes in the brain were found to be persistent 6 months after the methanol feeding stopped. This suggested the methanol feeding caused long-lasting and persistent pathological changes that were related to AD development in the monkey. Most notably, the presence of amyloid plaque formations in the monkeys highlighted a marked difference in animal systems used in AD investigations, suggesting that the innate defenses in mice against methanol toxicity may have limited previous investigations into AD pathology. Nonetheless, these findings support a growing body of evidence that links methanol and its metabolite formaldehyde to AD pathology.

The temporary and accumulated effects of transcranial direct current stimulation for the treatment of advanced Parkinson’s disease monkeys

Transcranial direct current stimulation (tDCS) is a useful noninvasive technique of cortical brain stimulation for the treatment of neurological disorders. Clinical research has demonstrated tDCS with anodal stimulation of primary motor cortex (M1) in Parkinson’s disease (PD) patients significantly improved their motor function. However, few studies have been focused on the optimization of parameters which contributed significantly to the treatment effects of tDCS and exploration of the underline neuronal mechanisms. Here, we used different stimulation parameters of anodal tDCS on M1 for the treatment of aged advanced PD monkeys induced with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) administration, and then analyzed the temporary and accumulated effects of tDCS treatment. The results indicated anodal tDCS on M1 very significantly improved motor ability temporarily; importantly, the treatment effects of anodal tDCS on M1 were quantitatively correlated to the accumulated stimulation instead of the stimuli intensity or duration respectively. In addition, c-fos staining showed tDCS treatment effects activated the neurons both in M1 and substantia nigra (SN). Therefore, we propose that long time and continue anodal tDCS on M1 is a better strategy to improve the motor symptoms of PD than individual manipulation of stimuli intensity or duration.

Resveratrol Attenuates Formaldehyde Induced Hyperphosphorylation of Tau Protein and Cytotoxicity in N2a Cells

Recent studies have demonstrated that formaldehyde (FA)—induced neurotoxicity is important in the pathogenesis of Alzheimers disease (AD). Elevated levels of FA have been associated with memory impairments and the main hallmarks of AD pathology, including β-amyloid plaques, tau protein hyperphosphorylation, and neuronal loss. Resveratrol (Res), as a polyphenol anti-oxidant, has been considered to have therapeutic potential for the treatment of AD. However, it has not been elucidated whether Res can exert its neuroprotective effects against FA-induced neuronal damages related to AD pathology. To answer this question, the effects of Res were investigated on Neuro-2a (N2a) cells prior to and after FA exposure. The experiments found that pre-treatment with Res significantly decreased FA-induced cytotoxicity, reduced cell apoptosis rates, and inhibited the hyperphosphorylation of tau protein at Thr181 in a dose-dependent manner. Further tests revealed that this effect was associated with the suppression of glycogen synthase kinase (GSK-3β) and calmodulin-dependent protein kinase II (CaMKII) activities, both of which are important kinases for tau protein hyperphosphorylation. In addition, Res was found to increase the activity of phosphoseryl/phosphothreonyl protein phosphatase-2A (PP2A). In summary, these findings provide evidence that Res protects N2a cells from FA-induced damages and suggests that inhibition of GSK-3β and CaMKII and the activation of PP2A by Res protect against the hyperphosphorylation and/or mediates the dephosphorylation of tau protein, respectively. These possible mechanisms underlying the neuroprotective effects of Res against FA-induced damages provide another perspective on AD treatment via inhibition of tau protein hyperhosphorylation.

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.

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.

Glaucoma model for stem cell transplantation research in New Zealand white rabbits

Glaucoma is a typical irreversible blind neurodegenerative disease for which there is no effective treatment for halting visual deterioration. The recent development of neural stem cells studies sheds light on a potential resolution for this disease. As a result, an appropriate glaucoma modeling method for stem cell transplantation study is needed. In the present study, Dexamethasone was injected unilaterally into the conjunctiva of New Zealand rabbit at the dose of 2.5 mg (5 mg/mL), three times a week. After eight weeks, the eye ground photography showed that the optic nerve head of the treated eye was expanded, and the blood vessel was geniculate compared to the control eye, while the ocular media remained transparent. The hematoxylin-eosin (HE) stain of the retinal nerve fiber layer (RNFL) sections showed optic neuron death in the treated eye. The Heidelberg Retina Tomography (HRT) results showed optic disk morphological changes consistent with the pathophysiology of glaucoma in the treated eye, including a decrease in the rim area (1.10±0.88) mm(2) and mean RNFL thickness (0.44±0.31) mm, and an increase in the cup/disk ratio 0.17±0.13. Then neural stem cells were injected into the vitreous body of the treated eye. After five months, surviving transplanted cells were observed. These results suggest a simple and reproducible chronic glaucoma model, which is appropriate for neural stem cell transplant research, has been successfully developed.

Phosphorylated α-Synuclein Accumulations and Lewy Body-like Pathology Distributed in Parkinson’s Disease-Related Brain Areas of Aged Rhesus Monkeys Treated with MPTP

Phosphorylation of α-synuclein at serine 129 (P-Ser 129 α-syn) is involved in the pathogenesis of Parkinsons disease (PD) and Lewy body (LB) formation. However, there is no clear evidence indicates the quantitative relation of P-Ser 129 α-syn accumulation and dopaminergic cell loss, LBs pathology and the affected brain areas in PD monkeys. Here, pathological changes in the substantia nigra (SN) and PD-related brain areas were measured in aged monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) utilizing a modeling-recovery-remodeling strategy. Compared to age-matched controls, the MPTP-treated monkeys showed significantly reduced tyrosine hydroxylase (TH)-positive neurons and increased P-Ser 129 α-syn-positive aggregations in the SN. Double-labeling Immunofluorescence found some TH-positive neurons to be co-localized with P-Ser129 α-syn in the SN, suggesting the inverse correlation between P-Ser 129 α-syn aggregations and dopaminergic cell loss in the SN may represent an interactive association related to the progression of the PD symptoms in the model. P-Ser 129 α-syn aggregations or LB-like pathology was also found in the midbrain and the neocortex, specifically in the oculomotor nucleus (CN III), temporal cortex (TC), prefrontal cortex (PFC) and in cells surrounding the third ventricle. Notably, the occipital cortex (OC) was P-Ser 129 α-syn negative. The findings of LB-like pathologies, dopaminergic cell loss and the stability of the PD symptoms in this model suggest that the modeling-recovery-remodeling strategy in aged monkeys may provide a new platform for biomedical investigations into the pathogenesis of PD and potential therapeutic development.

Kisspeptin-10 treatment generated specific GnRH expression in cells differentiated from rhesus monkey derived Lyon NSCs

Embryonic stem cells (ESCs) have enormous potential as novel cell-based therapies, but their effectiveness depends on stem cell differentiation and specific signaling regulators, which remain poorly understood. In this study, a kisspeptin peptide (KP-10) was used at different dosages to determine whether rhesus macaque-derived tau GFP-Lyon ES cells underwent kisspeptin-specific neuronal differentiation. It was found that KP-10 exhibited an anti-proliferative effect on the cells and led to morphological changes and cellular differentiation consistent with neuronal stem cell (NSC) development. The cells differentiated into Gonadotrophin Releasing Hormone (GnRH) neuronal-like cell types in response to the KP-10 treatment. There has been a previously observed connection between kisspeptin signaling, GnRH neurons and their dysfunction found in congenital disorders like idiopathic hypogonadotropic hypogonadism (IHH). Although therapeutics are a still a far-off goal, the formation and development of GnRH-positive neuronal-like cells following the application of KP-10 to Lyon NSC cells opens the door for future NSC-based therapies to treat specific reproductive disorders.

Rhesus monkey embryonic stem cells differentiation,proliferation and allotransplantation

To investigate the characteristics of rhesus monkey embryonic stem cells and to promote their clinical application, the differentiation and proliferation of rosettes neural stem cells from GFP marked rhesus monkey embryonic stem cells were studied The results showed that: 1) A stable and high-efficient neural differentiation system was established. More than 95% of the embryonic stem cells were differentiated into neural stem cells on the 12(th) days of differentiation; 2) the rosettes neural stem cells differentiated from the rhesus monkey embryonic stem cells could maintain their rosettes-shape by proliferating with bFGF/EGF; 3) the neural stem cells could differentiate into neurons after transplanted into the rhesus monkey brain. In conclusion, the rosettes neural stem cells differentiated from rhesus monkey embryonic stem cells could maintain their characteristics after proliferation with bFGF/EGF and they could survive and differentiate into neurons after transplanted into the rhesus monkey brain, which strongly supports the clinical application of neural stem cells in the future.