[Mechanism of bilobalide promoting neuroprotection of macrophages].

This study aims to explore the neuroprotective effect of bilobalide(BB) and the mechanisms such as inhibiting inflammatory response in macrophage/microglia, promoting neurotrophic factor secretion, and interfering with the activation and differentiation of peripheral CD4~+ T cells. BB of different concentration(12.5, 25, 50, 100 µg·mL~(-1)) was used to treat the RAW264.7 and BV2 cells for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and cell counting kit-8(CCK-8) were employed to detect the cytotoxicity of BB and appropriate concentration was selected for further experiment. Lipopolysaccharide(LPS) was applied to elicit inflammation in RAW264.7 and BV2 cells, mouse bone marrow-derived macrophages(BMDMs), and primary microglia, respectively. The effect of BB on cell proliferation and secretion of inflammatory cytokines and neurotrophic factors was detected by enzyme-linked immunosorbent assay(ELISA). Spleen monocytes of C57BL/6 female mice(7-8 weeks old) were isolated, and CD4~+ T cells were separated by magnetic beads under sterile conditions. Th17 cells were induced by CD3/CD28 and the conditioned medium for eliciting the inflammation in BMDMs. The content of IL-17 cytokines in the supernatant was detected by ELISA to determine the effect on the activation and differentiation of CD4~+ T cells. In addition, PC12 cells were incubated with the conditioned medium for eliciting inflammation in BMDMs and primary microglia and the count and morphology of cells were observed. The cytoto-xicity was determined by lactate dehydrogenase(LDH) assay. The result showed that BB with the concentration of 12.5-100 µg·mL~(-1) had no toxicity to RAW264.7 and BV2 cells, and had no significant effect on the activity of cell model with low inflammation. The 50 µg·mL~(-1) BB was selected for further experiment, and the results indicated that BB inhibited LPS-induced secretion of inflammatory cytokines. The experiment on CD4~+ T cells showed that the conditioned medium for LPS-induced inflammation in BMDMs promoted the activation and differentiation of CD4~+ T cells, while the conditioned medium of the experimental group with BB intervention reduced the activation and differentiation of CD4~+ T cells. In addition, BB also enhanced the release of neurotrophic factors from BMDMs and primary microglia. The conditioned medium after BB intervention can significantly reduce the death of PC12 neurons, inhibit neuronal damage, and protect neurons. To sum up, BB plays a neuroprotective role by inhibiting macrophage and microglia-mediated inflammatory response and promoting neurotrophic factors.

The ethanolic extract of peanut shell attenuates the depressive-like behaviors of mice through modulation of inflammation and gut microbiota.

Peanut shell is an agricultural byproduct being wasted on a large scale, which is in urgent need to be recycled. To fully utilize its pharmacological ingredients, e.g. luteolin, eriodyctiol, and 5,7-dihydroxychromone, we evaluated the curative effect of ethanol extract deriving from peanut shell (PSE) in treating chronic unpredictable mild stress (CUMS)-induced depressive mice. The chronic stress lasted for 10 weeks, and PSE at 100-900 mg/kg/day was gavaged to mice in the last 2 weeks of modeling. The depressive behaviors were assessed by analyses of sucrose preference, tail suspension, and forced swimming. The brain injury was demonstrated by Hematoxylin and Eosin (H&E), Nissl body, and TdT-mediated dUTP nick end labeling (TUNEL) stainings in the mouse hippocampus. Biochemical indicators were analyzed, including levels of neurotrophic factors, neurotransmitters, stress hormones, and inflammatory mediators. The feces were collected for the 16S rDNA sequencing of gut microbiome. Administration of PSE improved the sucrose water consumption of depressive mice, while it decreased the immobile time in tail suspension and forced swimming tests. Meanwhile, the anti-depressive effect of PSE was supported by ameliorated histochemical staining, increased levels of neurotrophic factors and neurotransmitters, as well as down-regulated stress hormones. Furthermore, the treatment of PSE was able to mitigate the levels of inflammatory cytokines in brain, serum, and small intestine. Besides, the tight junction proteins, e.g., occludin and ZO-1, of gut showed elevated expressions, which coincided with the elevated abundance and diversity of gut microbiota upon PSE treatment. This study validated the therapeutic efficacy of PSE in fighting against depression, as well as its modulatory action on inflammation and gut microbiota, which promoted the recycling of this agricultural waste to be health supplements of added value.

Sesamol Attenuates Scopolamine-Induced Cholinergic Disorders, Neuroinflammation, and Cognitive Deficits in Mice.

Alzheimer's disease (AD) is a neurodegenerative disease, characterized by memory loss and cognitive deficits accompanied by neuronal damage and cholinergic disorders. Sesamol, a lignan component in sesame oil, has been proven to have neuroprotective effects. This research aimed to investigate the preventive effects of sesamol on scopolamine (SCOP)-induced cholinergic disorders in C57BL/6 mice. The mice were pretreated with sesamol (100 mg/kg/d, p.o.) for 30 days. Behavioral tests indicated that sesamol supplement prevented SCOP-induced cognitive deficits. Sesamol enhanced the expression of neurotrophic factors and postsynaptic density (PSD) in SCOP-treated mice, reversing neuronal damage and synaptic dysfunction. Importantly, sesamol could balance the cholinergic system by suppressing the AChE activity and increasing the ChAT activity and M1 mAChR expression. Sesamol treatment also inhibited the expression of inflammatory factors and overactivation of microglia in SCOP-treated mice. Meanwhile, sesamol improved the antioxidant enzyme activity and suppressed oxidative stress in SCOP-treated mice and ameliorated the oxidized cellular status and mitochondrial dysfunction in SCOP-treated SH-SY5Y cells. In conclusion, these results indicated that sesamol attenuated SCOP-induced cognitive dysfunction via balancing the cholinergic system and reducing neuroinflammation and oxidative stress.

Pleiotropic effects of proopiomelanocortin and VGF nerve growth factor inducible neuropeptides for the long-term regulation of energy balance.

Seasonal rhythms in energy balance are well documented across temperate and equatorial zones animals. The long-term regulated changes in seasonal physiology consists of a rheostatic system that is essential to successful time annual cycles in reproduction, hibernation, torpor, and migration. Most animals use the annual change in photoperiod as a reliable and robust environmental cue to entrain endogenous (i.e. circannual) rhythms. Research over the past few decades has predominantly examined the role of first order neuroendocrine peptides for the rheostatic changes in energy balance. These anorexigenic and orexigenic neuropeptides in the arcuate nucleus include neuropeptide y (Npy), agouti-related peptide (Agrp), cocaine and amphetamine related transcript (Cart) and pro-opiomelanocortin (Pomc). Recent studies also indicate that VGF nerve growth factor inducible (Vgf) in the arcuate nucleus is involved in the seasonal regulation of energy balance. In situ hybridization, qPCR and RNA-sequencing studies have identified that Pomc expression across fish, avian and mammalian species, is a neuroendocrine marker that reflects seasonal energetic states. Here we highlight that long-term changes in arcuate Pomc and Vgf expression is conserved across species and may provide rheostatic regulation of seasonal energy balance.

Maternal Deprivation and Sex Alter Central Levels of Neurotrophins and Inflammatory Cytokines in Rats Exposed to Palatable Food in Adolescence.

Maternal deprivation (MD) in rodents is used to simulate human-infant early life stress, which leads to neural, hormonal, and behavioral alterations. Palatable food (PF) can reduce the stress response, and individuals use it as a self-applied stress relief method. Thus, the present study aimed to evaluate the effect of the association between MD in the early life (P1-P10) and PF consumption (condensed milk, P21-P44) in the central neuroplasticity (BDNF/NGF levels) and central neuroinflammatory parameters (TNF-α, IL-6, and IL-10 levels) in male and female Wistar rats in the adolescence. In addition, weight-related parameters (weight gain, Lee Index, and relative adipose tissue weight) were evaluated. PF exposure increased relative adipose tissue weight; however, it did not lead to a change in animals' body weight. MD reduced hypothalamic BDNF and NGF levels, and hippocampal TNF-α levels in male and female rats. Animals of both sexes that received PF, exhibited reduced hypothalamic NGF levels. Neuroinflammatory marker evaluations showed that male rats were more susceptible to the interventions than female rats, since MD reduced their cortical IL-10 levels and PF increased their IL-6 levels. Differences in the Lee index, central BDNF, TNF-α, and IL-6levels were observed between sexes. Male animals per se presented greater Lee index. Female rats had higher BDNF and IL-6 levels in the hippocampus and hypothalamus and higher hypothalamic TNF-α levels than those observed in males. In conclusion, there were more noticeable effects of MD than PF on the variables measured in this study. Sex effect was identified as an important factor and influenced most of the neurochemical measures in this study. In this way, we suggest including both female and male animals in researches to improve the quality of translational studies.

BDNF and NT3 Reprogram Human Ectomesenchymal Dental Pulp Stem Cells to Neurogenic and Gliogenic Neural Crest Progenitors Cultured in Serum-Free Medium.

BACKGROUND/AIMS: Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising types of cells to regenerate nerve tissues. Standard DMEM+10% fetal bovine serum (FBS) culture medium allows a fast expansion of hDPSC as a surface-adherent cell monolayer. However, the use of FBS also compromises the clinical use of these protocols, and its longterm presence favors hDPSCs differentiation toward mesenchymal cell-derived lineages, at the expense of a reduced capability to generate neural cells. The objective of this work was to characterize the role of neurotrophin signaling on hDPSCs using a serum-free culture protocol, and to assess the neurogenic and gliogenic capacity of hDPSCs for future nerve tissue bioengineering and regeneration. METHODS: We compared the different expression of neurotrophin receptors by RT-PCR, Q-PCR, and IF of hDPSCs cultured with different growth media in the presence or absence of serum. Moreover, we assessed the response of hDPSCs to stimulation of neurotransmitter receptors by live cell calcium imaging under these different media. Finally, we compared the osteogenic potential of hDPSCs by Alizarin red staining, and the differentiation to gliogenic/neurogenic fates by immunostaining for Schwann lineage and neuronal lineage markers. We tested a commercial serum-free medium designed for the growth of mesenchymal stem cells: StemPro MSCTM (STP). RESULTS: hDPSCs cultured in STP generated small non-adherent floating dentospheres that showed very low proliferation rates, in contrast to standard FBS-containing medium. We found that hDPSCs grown in STP conditions overexpressed neurotrophin receptor genes NTRK2 (TrkB) and NTRK3 (TrkC). Interestingly, the stimulation of these receptors by adding their respective ligands BDNF and NT-3 to STP medium enhanced the neural crest (NC) progenitor features of cultured hDPSCs. We observed a 10 to 100-fold increase of migratory NC cell markers HNK1 and P75NTR, and a significant overexpression of pluripotency core factors SOX2, OCT4 and NANOG. Moreover, hDPSCs cultured in BDNF/NT-3 supplemented STP showed a largely increased potential to differentiate towards neuronal and Schwann glial lineage cells, assessed by positive immunostaining for DCX, NeuN and S100ß, p75NTR markers, respectively. CONCLUSION: Our results demonstrate that the use of BDNF and NT-3 combined with STP induced the partial reprogramming of ectomesenchymal hDPSCs to generate early NC progenitor cells, which are far more competent for neuronal and glial differentiation than hDPSCs grown in the presence of FBS.

Pigment epithelium-derived factor (PEDF) reduced expression and synthesis of SOST/sclerostin in bone explant cultures: implication of PEDF-osteocyte gene regulation in vivo.

Mutations in Serpinf1 gene which encodes pigment epithelium-derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective matrix mineralization. We reported previously that PEDF reduced expression and synthesis of Sost/Sclerostin as well as other osteocytes genes encoding proteins that regulate matrix mineralization [1]. To determine whether PEDF had an effect on osteocyte gene expression in bone, we used bone explant cultures. First, osteocytes were isolated from surgical waste of bone fragments obtained from patients undergoing elective foot surgeries under approved IRB protocol by Penn State College of Medicine IRB committee. Primary osteocytes treated with PEDF reduced expression and synthesis of Sost/Sclerostin and matrix phosphoglycoprotein (MEPE) as well as dentin matrix protein (DMP-1). On the whole, PEDF reduced osteocyte protein synthesis by 50% and by 75% on mRNA levels. For bone explants, following collagenase digestion, bone fragments were incubated in alpha-MEM supplemented with 250 ng/ml of PEDF or BSA. After 7 days of incubation in a medium supplemented with PEDF, analysis of mRNA by PCR and protein by western blotting of encoded osteocyte proteins showed reduced Sclerostin synthesis by 39% and MEPE by 27% when compared to fragments incubated in medium supplemented with BSA. mRNA expression levels of osteocytes in bone fragments treated with PEDF were reduced by 50% for both SOST and MEPE when compared to BSA-treated bone fragments. Taken together, the data indicate that PEDF has an effect on osteocyte gene expression in bone and encourage further studies to examine effect of PEDF on bone formation indices in animal models and its effect on osteocyte gene expression in vivo following PEDF administration.

Medicinal Leech CNS as a Model for Exosome Studies in the Crosstalk between Microglia and Neurons.

In healthy or pathological brains, the neuroinflammatory state is supported by a strong communication involving microglia and neurons. Recent studies indicate that extracellular vesicles (EVs), including exosomes and microvesicles, play a key role in the physiological interactions between cells allowing central nervous system (CNS) development and/or integrity. The present report used medicinal leech CNS to investigate microglia/neuron crosstalk from ex vivo approaches as well as primary cultures. The results demonstrated a large production of exosomes from microglia. Their incubation to primary neuronal cultures showed a strong interaction with neurites. In addition, neurite outgrowth assays demonstrated microglia exosomes to exhibit significant neurotrophic activities using at least a Transforming Growth Factor beta (TGF-ß) family member, called nGDF (nervous Growth/Differentiation Factor). Of interest, the results also showed an EV-mediated dialog between leech microglia and rat cells highlighting this communication to be more a matter of molecules than of species. Taken together, the present report brings a new insight into the microglia/neuron crosstalk in CNS and would help deciphering the molecular evolution of such a cell communication in brain.

Pigment Epithelium-derived Factor Protects Retinal Pigment Epithelial Cells Against Cytotoxicity "In Vitro".

Oxidative stress has been implicated in neurodegenerative diseases, such as age-related macular degeneration. Hydrogen peroxide and sodium iodate can mediate oxidative injury. Sodium iodate induces a selective retinal degeneration targeting the RPE. We describe a method of chronic sodium iodate-mediated injury on RPE cells that may serve to evaluate protective factors against oxidative stress. Cytotoxicity and cell viability curves of ARPE-19 cells with sodium iodate were generated. The antioxidant pigment epithelium-derived factor decreased sodium iodate-mediated cytotoxicity without affecting ARPE-19 cell viability. A cell culture system to evaluate protection against oxidative stress injury with PEDF is discussed.

Neurotrophic Factors in Parkinson's Disease: What Have we Learned from Pre-Clinical and Clinical Studies?

BACKGROUND: Parkinson´s Disease (PD) is a chronic, progressive condition, being the second most common neurodegenerative disorder worldwide. The classical features include: bradykinesia, resting tremor, rigidity and festination. These neurological alterations are probably due to the death of dopaminergic neurons in the Substantia Nigra pars compacta and consequent reduction of dopamine input into the striatum. The decrease of dopamine levels may also be involved in the emergence of non-motor symptoms, including cognitive impairment, anxiety and depression symptoms. Neurotrophic Factors (NF) are proteins that modulate neuronal function, development, and survival. It has been reported that NF might exert a protective role in PD. OBJECTIVE: We aim to discuss the emerging evidence from pre-clinical and clinical studies regarding the role of NF in PD as well as their potential as promising therapeutic strategies. METHODS: We carried out an extensive literature search in PubMed central. RESULTS: Pre-clinical studies using NF to treat PD are divergent probably due to several methodological differences, thus precluding any conclusion. Clinical studies findings obtained with the administration of NF in patients with PD were even more disappointed. On the other hand, pre-clinical and clinical studies generally support that physical activity is a low-cost, non-pharmacologic strategy with good results to treat PD. CONCLUSION: The use of NF as a treatment for PD is still a promise not incorporated in clinical practice. Methods to deliver NFs, doses and compounds administered, side effects, population characteristics and duration of disease may probably contribute to the unsuccessful results.

Galanin Administration Partially Restores Erectile Function After Cavernous Nerve Injury and Mediates Endogenous Nitrergic Nerve Outgrowth In Vitro.

BACKGROUND: Previously, we found that the neuropeptide galanin was strongly upregulated soon after bilateral cavernous nerve injury (BCNI) and that galanin and its receptors were expressed in nitrergic erectile innervation. Galanin has been observed to exert neuroregenerative effects in dorsal root ganglion neurons, but evidence for these effects in the major pelvic ganglion (MPG) after BCNI is lacking. AIM: To evaluate the neurotropic effects of galanin receptor agonists and antagonists in vitro in nitrergic neurons and MPG and in vivo in rats after BCNI. METHODS: Male Sprague-Dawley rats underwent BCNI and sham surgery. Organ culture and single-cell neuron culture of the MPG were performed. Osmotic pump treatment with the galanin agonist in vivo and measurement of erectile response to electrostimulation after BCNI, immunohistochemical localization of galanin and receptors in the human neurovascular bundle, and myographic analysis of rat corpus cavernosum smooth muscle relaxation to galanin receptor agonists were investigated. OUTCOMES: Neurite outgrowth in vitro and erectile response to electrostimulation after BCNI in vivo, immunohistochemical localization of galanin and receptors, and penile muscle relaxation in vitro. RESULTS: Galanin showed neurotrophic action in vitro and inhibition of endogenous galanin significantly impaired neurite outgrowth in nitrergic but not in sympathetic MPG neurons. In vivo administration of a selective galanin receptor-2 agonist, M1145, resulted in partial recovery of erectile function (EF) after BCNI. Galanin did not act as a direct vasodilator on corpus cavernosum muscle strips. CLINICAL TRANSLATION: Endogenous neurotrophins such as galanin could be used as a strategy to improve EF for patients after BCNI from radical prostatectomy. STRENGTHS AND LIMITATIONS: We evaluated the effect of galanin on nerve regeneration and EF recovery in vivo and in vitro. Limitations include the lack of washout period for the in vivo experiment and absence of differences in the expression of neuronal markers between treatment groups. CONCLUSIONS: We identified galanin as a potential endogenous mechanism for nerve regeneration after BCNI, which could play a physiologic role in EF recovery after radical prostatectomy. In vivo treatment with exogenous galanin was beneficial in enhancing EF recovery after BCNI, but further research is necessary to understand the underlying mechanisms. Weyne E, Hannan JL, Gevaert T, et al. Galanin Administration Partially Restores Erectile Function After Cavernous Nerve Injury and Mediates Endogenous Nitrergic Nerve Outgrowth In Vitro. J Sex Med 2018;15:480-491.

Neuroprotective strategies for retinal disease.

Diseases that affect the eye, including photoreceptor degeneration, diabetic retinopathy, and glaucoma, affect 11.8 million people in the US, resulting in vision loss and blindness. Loss of sight affects patient quality of life and puts an economic burden both on individuals and the greater healthcare system. Despite the urgent need for treatments, few effective options currently exist in the clinic. Here, we review research on promising neuroprotective strategies that promote neuronal survival with the potential to protect against vision loss and retinal cell death. Due to the large number of neuroprotective strategies, we restricted our review to approaches that we had direct experience with in the laboratory. We focus on drugs that target survival pathways, including bile acids like UDCA and TUDCA, steroid hormones like progesterone, therapies that target retinal dopamine, and neurotrophic factors. In addition, we review rehabilitative methods that increase endogenous repair mechanisms, including exercise and electrical stimulation therapies. For each approach, we provide background on the neuroprotective strategy, including history of use in other diseases; describe potential mechanisms of action; review the body of research performed in the retina thus far, both in animals and in humans; and discuss considerations when translating each treatment to the clinic and to the retina, including which therapies show the most promise for each retinal disease. Despite the high incidence of retinal diseases and the complexity of mechanisms involved, several promising neuroprotective treatments provide hope to prevent blindness. We discuss attractive candidates here with the goal of furthering retinal research in critical areas to rapidly translate neuroprotective strategies into the clinic.

The role of chalcones: helichrysetin, xanthohumol, and flavokawin-C in promoting neurite outgrowth in PC12 Adh cells.

Chalcones are a group of compounds widely distributed in plant kingdom. The aim of this study was to assess the neurite outgrowth stimulatory activity of selected chalcones, namely helichrysetin, xanthohumol and flavokawin-C. Using adherent rat pheochromocytoma (PC12 Adh) cells, the chalcones were subjected to neurite outgrowth assay and the extracellular nerve growth factor (NGF) levels were determined. Xanthohumol (10 µg/mL) displayed the highest (p < 0.05) percentage of neurite-bearing PC12 Adh cells and the highest (p < 0.05) NGF level in the culture medium of xanthohumol-treated cells. While, helichrysetin induced a moderately high numbers of neurite-bearing cells, flavokawin-C did not stimulate neurite outgrowth. This work supports the potential use of xanthohumol as a potential neuroactive compound to stimulate neurite outgrowth.

Effects of neuroactive agents on axonal growth and pathfinding of retinal ganglion cells generated from human stem cells.

We recently established a novel method for generating functional human retinal ganglion cells (RGCs) from human induced pluripotent cells (hiPSCs). Here, we confirmed that RGCs can also be generated from human embryonic stem cells (hESCs). We investigated the usefulness of human RGCs with long axons for assessing the effects of chemical agents, such as the neurotrophic factor, nerve growth factor (NGF), and the chemorepellent factors, semaphorin 3 A (SEMA3A) and SLIT1. The effects of direct and local administration of each agent on axonal projection were evaluated by immunohistochemistry, real-time polymerase chain reaction (PCR), and real-time imaging, in which the filopodia of the growth cone served as an excellent marker. A locally sustained agent system showed that the axons elongate towards NGF, but were repelled by SEMA3A and SLIT1. Focally transplanted beads that released SLIT1 bent the pathfinding of axons, imitating normal retinal development. Our innovative system for assessing the effects of chemical compounds using human RGCs may facilitate development of novel drugs for the examination, prophylaxis, and treatment of diseases. It may also be useful for observing the physiology of the optic nerve in vitro, which might lead to significant progress in the science of human RGCs.

Pigment Epithelium-Derived Factor (PEDF) Prevents Hepatic Fat Storage, Inflammation, and Fibrosis in Dietary Steatohepatitis of Mice.

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of inflammation through its anti-oxidative property. Since oxidative response is considered to play the pivotal role of the development and progression of nonalcoholic steatohepatitis (NASH), it is conceivable that PEDF may play a protective role against NASH. In this study, we examined whether administration of PEDF slowed the progression of NASH in mice models. METHODS: Mice were fed methionine- and choline-deficient (MCD) diet with or without intramuscular administration of adenovirus-expressing PEDF (Ad-PEDF). Effects of PEDF administration on NASH were histologically and biochemically evaluated. RESULTS: Administration of Ad-PEDF significantly decreased hepatic fat storage as well as serum levels of ALT in MCD diet-fed mice. Dihydroethidium staining showed that MCD diet-triggered oxidative stress was reduced in the liver of Ad-PEDF-administered mice compared to that of PBS- or Ad-LacZ-administered mice. Activation of Kupffer cells and hepatic fibrosis was also inhibited by Ad-PEDF administration. Quantitative real-time RT-PCR revealed that MCD diet up-regulated expressions of TNF-α, IL-1ß, IL-6, TGF-ß, collagen-1, and collagen-3 mRNA, which were also attenuated with Ad-PEDF administration, whereas MCD diet-induced down-regulation of expressions of PPAR-γ mRNA was restored with Ad-PEDF administration. Furthermore, immunoblotting analysis showed that MCD diet-induced up-regulation of NADPH oxidase components was significantly decreased in Ad-PEDF-administered mice. CONCLUSIONS: The present results demonstrated for the first time that PEDF could slow the development and progression of steatohepatitis through the suppression of steatosis and inflammatory response in MCD diet-fed mice. Our study suggests that PEDF supplementation may be a novel therapeutic strategy for the treatment of NASH.

A Combined In Vitro Assay for Evaluation of Neurotrophic Activity and Cytotoxicity.

Neurotrophic assays are phenotypic methods to identify molecules that stimulate differentiation of neuronal cells. Bioactive small molecules with neurotrophic actions hold great promise as therapeutic agents for the treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth. A combined in vitro method, which measures neurotrophic activity and cytotoxicity in a single assay, has been described. This assay, performed in 96-well microplates with PC12 and Neuroscreen-1 (NS-1; a subclone of PC12) cells, is a simple tool for identification of new neurotrophic agents. Stimulation of neurite outgrowth was measured with NIS software by analysis of digital cell images as multiple parameters, namely, mean neurite length, neurite length/cell, nodes/cell, and number of neurites/cell. The assay has been standardized and validated with dose-response analysis for nerve growth factor (NGF) and mechanism-based inhibitors of NGF-induced neurite outgrowth, namely, SU6656 (an Src family kinase inhibitor) and PD98059 (a MEK inhibitor). The assay has been successfully applied for screening natural and synthetic compound libraries for cytotoxicity and neurotrophic activity. Screening of a set of harmala alkaloids identified harmine as a potential neurotrophic molecule that significantly stimulated NGF-induced neurite outgrowth in the NS-1 cells. Important advantages of this method are its simplicity and determination of cytotoxicity and neurotrophic activity in a single assay. This assay may be suitable for primary and cultured neuronal cells.

Dose-Dependent Differential Effect of Neurotrophic Factors on In Vitro and In Vivo Regeneration of Motor and Sensory Neurons.

Although peripheral axons can regenerate after nerve transection and repair, functional recovery is usually poor due to inaccurate reinnervation. Neurotrophic factors promote directional guidance to regenerating axons and their selective application may help to improve functional recovery. Hence, we have characterized in organotypic cultures of spinal cord and dorsal root ganglia the effect of GDNF, FGF-2, NGF, NT-3, and BDNF at different concentrations on motor and sensory neurite outgrowth. In vitro results show that GDNF and FGF-2 enhanced both motor and sensory neurite outgrowth, NGF and NT-3 were the most selective to enhance sensory neurite outgrowth, and high doses of BDNF selectively enhanced motor neurite outgrowth. Then, NGF, NT-3, and BDNF (as the most selective factors) were delivered in a collagen matrix within a silicone tube to repair the severed sciatic nerve of rats. Quantification of Fluorogold retrolabeled neurons showed that NGF and NT-3 did not show preferential effect on sensory regeneration whereas BDNF preferentially promoted motor axons regeneration. Therefore, the selective effects of NGF and NT-3 shown in vitro are lost when they are applied in vivo, but a high dose of BDNF is able to selectively enhance motor neuron regeneration both in vitro and in vivo.

Pigment epithelium derived factor upregulates expression of vascular endothelial growth factor by human mesenchymal stem cells: Possible role in PEDF regulated matrix mineralization.

Pigment epithelium-derived factor (PEDF) encoded by serpinf1 is a potent antiangiogenic factor found in a wide variety of fetal and adult tissues. Several reports have shown that lack of PEDF leads to osteogenesis imperfecta (OI) type VI whose hallmark is a defect in mineralization that leads to excessive osteoid build up that fails to mineralize. Because PEDF is antiangiogenic factor it would pose serious consequences on bone development and healing of fractures. To understand possible mechanisms by which PEDF plays a role in bone development and regulation of matrix mineralization, we determined the effects of exogenous PEDF on vascular endothelial growth factor (VEGF) expression by human mesenchymal stem cells (hMSCs) and mechanisms of its regulation by PEDF. Human MSCs incubated in normal medium supplemented with exogenous PEDF increased VEGF expression; this increase was also seen when PEDF was added to hMSCs undergoing osteogenic differentiation. MSCs maintained in osteogenic medium increased synthesis of both VEGF and PEDF but both factors were maintained relatively in balance during differentiation. To understand mechanisms by which exogenous PEDF regulated VEGF expression, hMSCs exposed to PEDF activated Erk signaling pathway in MSCs; inhibition of Erk signaling reduced VEGF mRNA expression as well as protein production suggesting that PEDF regulates VEGF expression in MSCs via Erk signaling pathway. In conclusion, PEDF increases VEGF expression by MSCs suggesting that regulation of VEGF by PEDF may be part of the mechanisms by which PEDF regulates osteoblastic mineralization.

Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.

Antimanic-like activity of candesartan in mice: Possible involvement of antioxidant, anti-inflammatory and neurotrophic mechanisms.

Activation of the brain angiotensin II type 1 receptor (AT1R) triggers pro-oxidant and pro-inflammatory mechanisms which are involved in the neurobiology of bipolar disorder (BD). Candesartan (CDS) is an AT1 receptor antagonist with potential neuroprotective properties. Herein we investigated CDS effects against oxidative, neurotrophic inflammatory and cognitive effects of amphetamine (AMPH)-induced mania. In the reversal protocol adult mice were given AMPH 2 mg/kg i.p. or saline and between days 8 and 14 received CDS 0.1, 0.3 or 1 mg/kg orally, lithium (Li) 47.5 mg/kg i.p., or saline. In the prevention treatment, mice were pretreated with CDS, Li or saline prior to AMPH. Locomotor activity and working memory performance were assessed. Glutathione (GSH), thiobarbituric acid-reactive substance (TBARS) and TNF-α levels were evaluated in the hippocampus (HC) and cerebellar vermis (CV). Brain-derived neurotrophic factor (BDNF) and glycogen synthase kinase 3-beta (GSK-3beta) levels were measured in the HC. CDS and Li prevented and reversed the AMPH-induced increases in locomotor activity. Only CDS prevented and reversed AMPH-induced working memory deficits. CDS prevented AMPH-induced alterations in GSH (HC and CV), TBARS (HC and CV), TNF-α (HC and CV) and BDNF (HC) levels. Li prevented alterations in BDNF and phospho-Ser9-GSK3beta. CDS reversed AMPH-induced alterations in GSH (HC and CV), TBARS (HC), TNF-α (CV) and BDNF levels. Li reversed AMPH-induced alterations in TNF-α (HC and CV) and BDNF (HC) levels. CDS is effective in reversing and preventing AMPH-induced behavioral and biochemical alterations, providing a rationale for the design of clinical trials investigating CDS׳s possible therapeutic effects.