Evaluation of Klotho gene expression and NGAL levels following acute kidney injury during pregnancy hypertensive disorders.

BACKGROUND: This study examined the relationship between hypertensive disorders and acute kidney injury (AKI) during pregnancy by evaluating Klotho (KL) gene expression and Neutrophil gelatinase-associated lipocalin (NGAL) levels in pregnant women. MATERIAL AND METHODS: Pregnant women were divided into 3 groups: (1) Pregnancy related hypertensive disorders with AKI (PR-AKI) (9 cases), (2) hypertensive disorders pregnancy (HDP) (40 cases), and (3) normal pregnancy (30 cases). For each group, Klotho gene transcription levels in the blood, Klotho and NGAL proteins levels, Malondialdehyde (MDA) and superoxide dismutase (SOD) activity levels were measured in serum. Statistical comparisons were made among the three groups. RESULTS: Klotho/ß-actin transcript levels and serum KL protein concentrations were significantly decreased in hypertensive disorder pregnancies with AKI complications. Serum NGAL protein levels were significantly increased in the hypertensive disorder pregnancies with AKI complications. Total serum Klotho protein was negatively correlated with creatinine, while serum NGAL was positively correlated with serum creatinine, urea nitrogen, uric acid, systolic blood pressure, diastolic blood pressure and 24 h urine protein levels. Serum levels of MDA and SOD were significantly increased in the hypertensive disorder pregnancy with AKI and the overall MDA concentration was negatively correlated with Klotho protein concentration. Klotho protein was found to have a direct effect on creatinine, and a mediating effect of MDA was found. CONCLUSION: Decreased expression of Klotho protein in correlation with increased levels of oxidative stress are found during of AKI complications in pregnancy hypertensive disorders.

Alzheimer's Disease-Like Pathologies and Cognitive Impairments Induced by Formaldehyde in Non-Human Primates.

BACKGROUND: Formaldehyde (FA) has been implicated in Alzheimer's disease (AD) pathology as an age-related factor and as a protein cross-linker known to aggregate Amyloid-Beta (Aß) and tau protein in vitro. Higher levels of FA have also been found in patients with greater cognitive impairment and in AD patient brains. OBJECTIVE: To directly evaluate the effect of chronically elevated FA levels on the primate brain with respect to AD pathological markers. METHOD: Young rhesus macaques (5-8 yrs, without AD related mutations) were given chronic intracerebroventricular (i.c.v.) injections of FA or vehicle over a 12-month period. Monkeys were monitored for changes in cognitive ability and evaluated post-mortem for common AD pathological markers. RESULTS: Monkeys injected with FA were found to have significant spatial working memory impairments. Histopathological analysis revealed the presence of amyloid-ß+ neuritic-like plaques, neurofibrillary tangle-like formations, increased tau protein phosphorylation, neuronal loss and reactive gliosis in three memory (and AD) related brain areas (the hippocampus, entorhinal cortex and prefrontal cortex (PFC)) of monkeys receiving i.c.v. injections of FA. ELISA assays revealed that the amounts of pT181 and Aß42 were markedly higher in the PFC and hippocampus of FA treated monkeys. CONCLUSION: FA was found to induce major AD-like pathological markers and cognitive impairments in young rhesus monkeys independent of genetic predispositions. This suggests FA may play a significant role in the initiation and progression of the disease.

Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains.

Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technologies are based on these systems. Although many viral vectors are used in rodents, their full application has been limited in non-human primates. To identify viral vectors that can stably and effectively express exogenous genes within non-human primates, eleven commonly used recombinant adeno-associated viral and lentiviral vectors, each carrying a gene to express green or red fluorescence, were injected into the parietal cortex of four rhesus monkeys. The expression of fluorescent cells was used to quantify transfection efficiency. Histological results revealed that recombinant adeno-associated viral vectors, especially the serotype 2/9 coupled with the cytomegalovirus, human synapsin I, or Ca2+/calmodulin-dependent protein kinase II promoters, and lentiviral vector coupled with the human ubiquitin C promoter, induced higher expression of fluorescent cells, representing high transfection efficiency. This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs). These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.

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.

Neurons Differentiated from Transplanted Stem Cells Respond Functionally to Acoustic Stimuli in the Awake Monkey Brain.

Here, we examine whether neurons differentiated from transplanted stem cells can integrate into the host neural network and function in awake animals, a goal of transplanted stem cell therapy in the brain. We have developed a technique in which a small "hole" is created in the inferior colliculus (IC) of rhesus monkeys, then stem cells are transplanted in situ to allow for investigation of their integration into the auditory neural network. We found that some transplanted cells differentiated into mature neurons and formed synaptic input/output connections with the host neurons. In addition, c-Fos expression increased significantly in the cells after acoustic stimulation, and multichannel recordings indicated IC specific tuning activities in response to auditory stimulation. These results suggest that the transplanted cells have the potential to functionally integrate into the host neural network.

Evidence for Conversion of Methanol to Formaldehyde in Nonhuman Primate Brain.

Many studies have reported that methanol toxicity to primates is mainly associated with its metabolites, formaldehyde (FA) and formic acid. While methanol metabolism and toxicology have been best studied in peripheral organs, little study has focused on the brain and no study has reported experimental evidence that demonstrates transformation of methanol into FA in the primate brain. In this study, three rhesus macaques were given a single intracerebroventricular injection of methanol to investigate whether a metabolic process of methanol to FA occurs in nonhuman primate brain. Levels of FA in cerebrospinal fluid (CSF) were then assessed at different time points. A significant increase of FA levels was found at the 18th hour following a methanol injection. Moreover, the FA level returned to a normal physiological level at the 30th hour after the injection. These findings provide direct evidence that methanol is oxidized to FA in nonhuman primate brain and that a portion of the FA generated is released out of the brain cells. This study suggests that FA is produced from methanol metabolic processes in the nonhuman primate brain and that FA may play a significant role in methanol neurotoxicology.

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 Alzheimer's disease (AD). Elevated levels of FA have been associated with memory impairments and the main hallmarks of AD pathology, including ß-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-3ß) 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-3ß 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.

A Robust Single Primate Neuroepithelial Cell Clonal Expansion System for Neural Tube Development and Disease Studies.

Developing a model of primate neural tube (NT) development is important to promote many NT disorder studies in model organisms. Here, we report a robust and stable system to allow for clonal expansion of single monkey neuroepithelial stem cells (NESCs) to develop into miniature NT-like structures. Single NESCs can produce functional neurons in vitro, survive, and extensively regenerate neuron axons in monkey brain. NT formation and NESC maintenance depend on high metabolism activity and Wnt signaling. NESCs are regionally restricted to a telencephalic fate. Moreover, single NESCs can turn into radial glial progenitors (RGPCs). The transition is accurately regulated by Wnt signaling through regulation of Notch signaling and adhesion molecules. Finally, using the "NESC-TO-NTs" system, we model the functions of folic acid (FA) on NT closure and demonstrate that FA can regulate multiple mechanisms to prevent NT defects. Our system is ideal for studying NT development and diseases.

Alzheimer's disease and methanol toxicity (part 1): chronic methanol feeding led to memory impairments and tau hyperphosphorylation in mice.

Although methanol toxicity is well known for acute neurological sequelae leading to blindness or death, there is a new impetus to investigate the chronic effects of methanol exposure. These include a recently established link between formaldehyde, a methanol metabolite, and Alzheimer's disease (AD) pathology. In the present study, mice were fed with methanol to revisit the chronic effects of methanol toxicity, especially as it pertains to AD progression. Three groups of mice (n = 9) were given either water as a control or a methanol solution (concentrations of 2% or 3.8%) over a 6-week period. The methanol-fed mice were found to have impaired spatial recognition and olfactory memory in Y-maze and olfactory memory paradigms. Immunohistochemical analysis of the mouse brains found increased neuronal tau phosphorylation in the hippocampus and an increased cellular apoptotic marker in hippocampal CA1 neurons (~10% of neurons displayed chromatin condensation) in the methanol-fed groups. Two additional in vitro experiments in mouse embryonic cerebral cortex neurons and mouse neuroblastoma N2a cells found that formaldehyde, but not methanol or the methanol end product formic acid, induced microtubule disintegration and tau protein hyperphosphorylation. The findings of the behavioral tests and immunohistochemical analysis suggested that the methanol-fed mice presented with partial AD-like symptoms. The in vitro experiments suggested that formaldehyde was most likely the detrimental component of methanol toxicity related to hippocampal tau phosphorylation and the subsequent impaired memory in the mice. These findings add to a growing body of evidence that links formaldehyde to AD pathology.

Is formaldehyde the missing link in AD pathology? The differential aggregation of amyloid-beta with APOE isoforms in vitro.

Apolipoprotein E (APOE) genetic variation and aging are the two most noted risk factors associated with the development of Alzheimer's disease (AD) related dementia. However, the relationship between these two pathological factors is not understood. Formaldehyde (FA) is an age related factor that has been found to be elevated in AD patients and is known to have protein cross-linking properties. FA forms cross-links with larger arginine, lysine and tryptophan residues but also has thiol reactivity. This study investigated the formation of protein aggregates between amyloid-beta (1-40) peptide (Aß), the main component of amyloid plaques in AD, with APOE isoforms in vitro. APOE4 protein, the isoform with arginines at residue 112 and 158, was found to form aggregates with more Aß (P < 0.001) and APOE (P < 0.05) protein content in 10 mM FA than aggregates formed with either APOE3 or APOE2 protein. This aggregation pattern reflected the trend of vulnerability conferred by the APOE genetic variation (APOE4 > APOE3 > APOE2) and suggested that FA may have a role in the differential pattern of amyloid plaque formation in people with differing APOE genetic backgrounds. All told, this finding adds to a growing body of evidence that FA has a role in AD progression as well as provides a novel link between aging and APOE risk factors; the cornerstones of one of the world's largest mental health concerns.

Establishment and partial characterization of a human tumor cell line, GBM-HSF, from a glioblastoma multiforme.

This paper outlines the establishment of a new and stable cell line, designated GBM-HSF, from a malignant glioblastoma multiforme (GBM) removed from a 65-year-old Chinese woman. This cell line has been grown for 1 year without disruption and has been passaged over 50 times. The cells were adherently cultured in RPMI-1640 media with 10% fetal bovine serum supplementation. Cells displayed spindle and polygonal morphology, and displayed multi-layered growth without evidence of contact inhibition. The cell line had a high growth rate with a doubling time of 51 h. The cells were able to grow without adhering to the culture plates, and 4.5% of the total cells formed colonies in soft agar. The cell line has also been found to form tumors in nude mice and to be of a highly invasive nature. The cells were also partially characterized with RT-PCR. The RT-PCR revealed that Nestin, ß-tubulin III, Map2, Klf4, Oct4, Sox2, Nanog, and CD26 were positively transcribed, whereas GFAP, Rex1, and CD133 were negatively transcribed in this cell line. These results suggest that the GBM-HSF cell line will provide a good model to study the properties of cancer stem cells and metastasis. It will also facilitate more detailed molecular and cellular studies of GBM cell division and pathology.

Melatonin treatment increases the transcription of cell proliferation-related genes prior to inducing cell death in C6 glioma cells in vitro.

A number of studies have suggested that melatonin possesses anticancer properties. However, conflicting data exists with regard to the role of melatonin in the treatment of cancer. In the present study, the effects of melatonin on the transcriptional regulation of three genes associated with cell proliferation (Nestin, Bmi-1 and Sox2), and on C6 glioma cell survival and viability, were investigated in vitro to evaluate the use of melatonin in cancer therapy. Melatonin was shown to increase the mRNA levels of Nestin, Bmi-1 and Sox2 in a similar pattern, with the highest mRNA levels noted at a concentration of 3 mM. At higher concentrations of melatonin (5 mM), the mRNA levels of Nestin, Bmi-1 and Sox2 were reduced from their peak levels, and were correlated with changes observed in immunofluorescence morphology studies, cell viability and survival assays. Immunofluorescence studies of Nestin-stained cells demonstrated that treatment with a higher concentration of melatonin (3 and 5 mM) led to the Nestin filaments condensing and rearranging around the cell nuclei, and an alteration in the cell morphology. C6 cell viability was also significantly decreased at 3 mM melatonin, and cell death was observed at 5 and 10 mM melatonin. These results suggested that Nestin, Bmi-1 and Sox2 were strongly correlated with the survival of C6 cells following treatment with melatonin, and that high therapeutic concentrations of melatonin (>5 mM) were required to induce cell death. These findings suggested that the implementation of melatonin in the treatment of glioma and other types of cancer may be inhibited by conflicting cell growth signals in cells. Therefore, adjunct therapy is required to improve the efficacy of melatonin in the treatment of cancer.

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.

Lesion of olfactory epithelium attenuates expression of morphine-induced behavioral sensitization and reinstatement of drug-primed conditioned place preference in mice.

Previous studies have shown that olfactory impairment by disrupting the olfactory epithelium prior to morphine administration attenuated the development addiction-related behaviors. However, it is unclear whether olfactory impairment will affect the expression of already established addiction-related behaviors. To address this issue, mice were conditioned with morphine to induce behavioral sensitization and condition placed preference (CPP). After an abstinence period, the animals were subjected to either an intranasal ZnSO(4) effusion (ZnE) or sham treatment with saline. Behavioral sensitization and CPP reinstatement were evaluated 24h later, as well as the expression of c-Fos protein, a marker of activated neural sites, in brain regions of interest. It was found that ZnE treatment attenuated morphine-induced behavioral sensitization and reinstatement of CPP. Compared to the saline-treated ones, the ZnE-treated animals showed reduced c-Fos expression in the nucleus accumbens (NAc) associated with behavioral sensitization, and in the NAc, cingulate cortex, dentate gyrus, amygdala, lateral hypothalamus and ventral tegmental area associated with CPP reinstatement. Together, these results demonstrated that acute olfactory impairment could attenuate already established addiction-related behaviors and expression of c-Fos in drug addiction related brain regions, perhaps by affecting the coordination between reward and motivational systems in the brain.

Deficits in water maze performance and oxidative stress in the hippocampus and striatum induced by extremely low frequency magnetic field exposure.

The exposures to extremely low frequency magnetic field (ELF-MF) in our environment have dramatically increased. Epidemiological studies suggest that there is a possible association between ELF-MF exposure and increased risks of cardiovascular disease, cancers and neurodegenerative disorders. Animal studies show that ELF-MF exposure may interfere with the activity of brain cells, generate behavioral and cognitive disturbances, and produce deficits in attention, perception and spatial learning. Although, many research efforts have been focused on the interaction between ELF-MF exposure and the central nervous system, the mechanism of interaction is still unknown. In this study, we examined the effects of ELF-MF exposure on learning in mice using two water maze tasks and on some parameters indicative of oxidative stress in the hippocampus and striatum. We found that ELF-MF exposure (1 mT, 50 Hz) induced serious oxidative stress in the hippocampus and striatum and impaired hippocampal-dependent spatial learning and striatum-dependent habit learning. This study provides evidence for the association between the impairment of learning and the oxidative stress in hippocampus and striatum induced by ELF-MF exposure.

The effects of lesion of the olfactory epithelium on morphine-induced sensitization and conditioned place preference in mice.

Animals attain information about their environment through different sense organs. For example, the dominant external resource about the environment for rodents is obtained through olfaction. Many environmental conditions (stress or enriched environment) are known to affect an animal's susceptibility to drug addiction. However, it is not known how external information is integrated and paired with drug stimuli to develop into addictive behavior. Here, we investigated the effects of olfactory epithelium lesions induced with ZnSO4 effusion (ZnE) on morphine-induced sensitization and conditioned place preference in mice. We found that the lesion of the olfactory epithelium attenuated the repeated morphine (40 mg/kg)-induced behavioral sensitization and morphine-induced conditioned place preference (CPP) behaviors, such as hyper-locomotion during morphine (40 mg/kg) conditioned training. Additionally, the expression of FosB-like proteins, transcription factors associated with behavioral alterations, in the nucleus accumbens of the brain was attenuated in morphine administered mice treated by ZnE. Taken together, these results indicated that lesion of the olfactory epithelium lead to a decrease in morphine sensitization and CPP behavior in mice as well as modulate specific molecular markers of neuroadaption to drugs of abuse. These findings also suggest that olfaction plays an important role in the development of addictive behaviors that can be modulated by external actions.