The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

William R. Mundy - One of the best experts on this subject based on the ideXlab platform.

  • quantitative assessment of Neurite Outgrowth in human embryonic stem cell derived hn2 cells using automated high content image analysis
    Neurotoxicology, 2010
    Co-Authors: Joshua A Harrill, Theresa M. Freudenrich, Dave W Machacek, Steve L Stice, William R. Mundy
    Abstract:

    Throughout development neurons undergo a number of morphological changes including Neurite Outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary neural cultures and immortalized human and non-human clonal cell lines have been used to investigate the molecular mechanisms controlling Neurite Outgrowth and examine chemical effects on this process. The present study characterizes the molecular phenotype of hN2 human embryonic stem cell (hESC)-derived neural cells and uses automated high-content image analysis to measure Neurite Outgrowth in vitro. At 24h post-plating hN2 cells express a number of protein markers indicative of a neuronal phenotype, including: nestin, beta(III)-tubulin, microtubule-associated protein 2 (MAP2) and phosphorylated neurofilaments. Neurite Outgrowth in hN2 cells proceeded rapidly, with a majority of cells extending one to three Neurites by 48h in culture. In addition, concentration-dependent decreases in Neurite Outgrowth and ATP-content were observed following treatment of hN2 cells with either bisindolylmaleimide I, U0126, lithium chloride, sodium orthovanadate and brefeldin A, all of which have previously been shown to inhibit Neurite Outgrowth in primary rodent neural cultures. Overall, the molecular phenotype, rate of Neurite Outgrowth and sensitivity of hN2 cells to Neurite Outgrowth inhibitors were comparable to other in vitro models previously characterized in the literature. hN2 cells provide a model in which to investigate chemical effects on Neurite Outgrowth in a non-transformed human-derived cells and provide an alternative to the use of primary rodent neural cultures or immortalized clonal cell lines.

  • comparison of pc12 and cerebellar granule cell cultures for evaluating Neurite Outgrowth using high content analysis
    Neurotoxicology and Teratology, 2010
    Co-Authors: Nicholas M. Radio, Theresa M. Freudenrich, Ia L Robinette, Kevi M Crofto, William R. Mundy
    Abstract:

    Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite Outgrowth is one such critical cellular process underlying nervous system development that can be quantified using automated microscopy and image analysis (high content analysis). The present study characterized and compared the PC-12 cell line (NS-1) and primary cultures of cerebellar granular cells (CGC), as models for assessing chemical effects on Neurite Outgrowth. High content analysis of Neurite Outgrowth was performed using the Cellomics ArrayScan V(Ti) automated epifluorescent imaging system to acquire and analyze images of beta-tubulin immunostained cells in 96-well plates. Cell viability was assessed using the CellTiter-Glo assay. Culture of NS-1 or CGC in nerve growth factor or serum respectively, rapidly induced Neurite Outgrowth that increased over four days in vitro. Seven compounds previously shown to affect Neurite Outgrowth in vitro were tested in both models for changes in total Neurite length and cell viability. In NS-1 cells, four chemicals (PKC inhibitor Bis-I, MEK inhibitor U0126, trans-Retinoic acid, methylmercury) inhibited Neurite Outgrowth, while lead, amphetamine and valproic acid had no effect. In CGC, five chemicals inhibited Neurite Outgrowth (Bis-I, U0126, lead, methylmercury, and amphetamine), while trans-Retinoic acid decreased cell viability but not Neurite Outgrowth. Valproic acid was without effect. The sensitivity of the two models was chemical specific: NS-1 cells were more sensitive to Bis-I, methylmercury and trans-Retinoic acid, while CGC were more sensitive to U0126, lead, and amphetamine. For every chemical (except trans-Retinoic acid), Neurite Outgrowth was equal to or more sensitive than cell viability. In comparison, out of seven chemicals without prior evidence for effects on Neurite Outgrowth, only one decreased Neurite Outgrowth (diphenhydramine in CGC). These findings demonstrate that the effects of chemicals on Neurite Outgrowth may be cell type specific.

  • assessment of chemical effects on Neurite Outgrowth in pc12 cells using high content screening
    Toxicological Sciences, 2008
    Co-Authors: Nicholas M. Radio, Joseph M Breier, Timothy J Shafer, William R. Mundy
    Abstract:

    Identification of chemicals that pose a hazard to the developing nervous system is the first step in reducing human exposure and preventing health risks to infants and children. In response to the need for more efficient methods to identify potential developmental neurotoxicants, the present study evaluated the utility of an automated high content screening system to detect chemical effects on Neurite Outgrowth in Neuroscreen-1 cells (NS-1), a subclone of PC12 cells. Plating 2000 NS-1 cells per well with 100 ng/ml nerve growth factor for 96 h produced optimal Neurite growth in a 96-well format. Using this protocol, five chemicals that had been previously shown to inhibit Neurite Outgrowth in PC12 cells were examined. Inhibition of Neurite Outgrowth (assessed as total Neurite length per cell) was observed for all five chemicals. For three of the chemicals, inhibition was associated with decreased cell viability. To demonstrate the utility of this approach for screening, a further set of chemicals (eight known in vivo developmental neurotoxicants and eight chemicals with little evidence of in vivo neurotoxicity) were tested over a wide concentration range (1nM‐100mM). Trans-retinoic acid, dexamethasone, cadmium, and methylmercury inhibited Neurite Outgrowth, although dexamethasone and cadmium only affected Neurite Outgrowth at concentrations that decreased viability. Amphetamine facilitated Neurite Outgrowth, whereas valproic acid, diphenylhydantoin, and lead had no effect. Of the chemicals that were not neurotoxic, there were no effects on cell viability, but two (dimethyl phthalate and omeprazole) increased Neurite Outgrowth at the highest concentration tested. These results demonstrate that a high content screening system can rapidly quantify chemical effects on Neurite Outgrowth in vitro. Concentrationresponse data for both Neurite Outgrowth and cell viability allowed for the determination of the specificity of chemical effects on a neurodevelopmental endpoint. Further studies will examine the utility of other in vitro preparations for cell-based assays of Neurite

  • developmental neurotoxicity testing in vitro models for assessing chemical effects on Neurite Outgrowth
    Neurotoxicology, 2008
    Co-Authors: Nicholas M. Radio, William R. Mundy
    Abstract:

    In vitro models may be useful for the rapid toxicological screening of large numbers of chemicals for their potential to produce toxicity. Such screening could facilitate prioritization of resources needed for in vivo toxicity testing towards those chemicals most likely to result in adverse health effects. Cell cultures derived from nervous system tissue have proven to be powerful tools for elucidating cellular and molecular mechanisms of nervous system development and function, and have been used to understand the mechanism of action of neurotoxic chemicals. Recently, it has been suggested that in vitro models could be used to screen for chemical effects on critical cellular events of neurodevelopment, including differentiation and Neurite growth. This review examines the use of neuronal cell cultures as an in vitro model of Neurite Outgrowth. Examples of the cell culture systems that are commonly used to examine the effects of chemicals on Neurite Outgrowth are provided, along with a description of the methods used to quantify this neurodevelopmental process in vitro. Issues relating to the relevance of the methods and models currently used to assess Neurite Outgrowth are discussed in the context of hazard identification and chemical screening. To demonstrate the utility of in vitro models of Neurite Outgrowth for the evaluation of large numbers of chemicals, efforts should be made to: (1) develop a set of reference chemicals that can be used as positive and negative controls for comparing Neurite Outgrowth between model systems, (2) focus on cell cultures of human origin, with emphasis on the emerging area of neural progenitor cells, and (3) use high-throughput methods to quantify endpoints of Neurite Outgrowth.

Lakshmi P. Kotra - One of the best experts on this subject based on the ideXlab platform.

  • Guaifenesin derivatives promote Neurite Outgrowth and protect diabetic mice from neuropathy
    Journal of Medicinal Chemistry, 2013
    Co-Authors: Mallinath B Hadimani, Meena K. Purohit, Chandrashaker Vanampally, Randy Van Der Ploeg, Dwane Morrow, Katie E. Frizzi, Victor Arballo, Paul Fernyhough, Nigel A Calcutt, Lakshmi P. Kotra
    Abstract:

    In diabetic patients, an early index of peripheral neuropathy is the slowing of conduction velocity in large myelinated neurons and a lack of understanding of the basic pathogenic mechanisms hindered therapeutics development. Racemic (R/S)-guaifenesin (1) was identified as a potent enhancer of Neurite Outgrowth using an in vitro screen. Its R-enantiomer (R)-1 carried the most biological activity, whereas the S-enantiomer (S)-1 was inactive. Focused structural variations to (R/S)-1 was conducted to identify potentially essential groups for the Neurite Outgrowth activity. In vivo therapeutic studies indicated that both (R/S)-1 and (R)-1 partially prevented motor nerve conduction velocity slowing in a mouse model of type 1 diabetes. In vitro microsomal assays suggested that compounds (R)-1 and (S)-1 are not metabolized rapidly, and PAMPA assay indicated moderate permeability through the membrane. Findings revealed here could lead to the development of novel drugs for diabetic neuropathy.

  • Guaifenesin Derivatives Promote Neurite Outgrowth and Protect Diabetic Mice from Neuropathy
    2013
    Co-Authors: Mallinath B Hadimani, Meena K. Purohit, Chandrashaker Vanampally, Randy Van Der Ploeg, Dwane Morrow, Katie E. Frizzi, Victor Arballo, Paul Fernyhough, Nigel A Calcutt, Lakshmi P. Kotra
    Abstract:

    In diabetic patients, an early index of peripheral neuropathy is the slowing of conduction velocity in large myelinated neurons and a lack of understanding of the basic pathogenic mechanisms hindered therapeutics development. Racemic (R/S)-guaifenesin (1) was identified as a potent enhancer of Neurite Outgrowth using an in vitro screen. Its R-enantiomer (R)-1 carried the most biological activity, whereas the S-enantiomer (S)-1 was inactive. Focused structural variations to (R/S)-1 was conducted to identify potentially essential groups for the Neurite Outgrowth activity. In vivo therapeutic studies indicated that both (R/S)-1 and (R)-1 partially prevented motor nerve conduction velocity slowing in a mouse model of type 1 diabetes. In vitro microsomal assays suggested that compounds (R)-1 and (S)-1 are not metabolized rapidly, and PAMPA assay indicated moderate permeability through the membrane. Findings revealed here could lead to the development of novel drugs for diabetic neuropathy

Kenji Hashimoto - One of the best experts on this subject based on the ideXlab platform.

  • potentiation of ngf induced Neurite Outgrowth in pc12 cells by papaverine role played by plc γ ip3 receptors
    Brain Research, 2011
    Co-Authors: Kanako Itoh, Tamaki Ishima, Jan Kehler, Kenji Hashimoto
    Abstract:

    Abstract Papaverine, an inhibitor of phosphodiesterase (PDE) 10A, is gaining attention for its potential in the treatment of neuropsychiatric diseases such as schizophrenia. However, the precise mechanisms underlying the putative neuroprotective/neurotrophic actions of papaverine remain unclear. Thus, we investigated the effects of papaverine on nerve growth factor (NGF)-induced Neurite Outgrowth in PC12 cells. Papaverine potentiated NGF-induced Neurite Outgrowth in PC12 cells in a concentration-dependent manner. In contrast, the selective PDE10A inhibitor MP-10 had no effect on NGF-induced Neurite Outgrowth. The potentiation of NGF-induced Neurite Outgrowth by papaverine was blocked by the PLC-γ inhibitor U73122. Furthermore, papaverine's potentiation of NGF-induced Neurite Outgrowth was also blocked by the co-administration of inositol 1,4,5-trisphosphate (IP 3 ) receptor antagonists (xestospongin C and 2-aminoethoxydiphenyl borate (2-APB)) and by reduced expression of IP 3 receptor gene (i.e., itpr1 and itpr3) by siRNA. Our findings suggest that papaverine could potentiate NGF-induced Neurite Outgrowth, and that activation of PLC-γ and IP 3 receptors might be involved in the mechanism underlying papaverine's potentiation of Neurite Outgrowth in PC12 cells.

  • potentiation of ngf induced Neurite Outgrowth in pc12 cells by papaverine role played by plc γ ip3 receptors
    Brain Research, 2011
    Co-Authors: Kanako Itoh, Tamaki Ishima, Jan Kehler, Kenji Hashimoto
    Abstract:

    Abstract Papaverine, an inhibitor of phosphodiesterase (PDE) 10A, is gaining attention for its potential in the treatment of neuropsychiatric diseases such as schizophrenia. However, the precise mechanisms underlying the putative neuroprotective/neurotrophic actions of papaverine remain unclear. Thus, we investigated the effects of papaverine on nerve growth factor (NGF)-induced Neurite Outgrowth in PC12 cells. Papaverine potentiated NGF-induced Neurite Outgrowth in PC12 cells in a concentration-dependent manner. In contrast, the selective PDE10A inhibitor MP-10 had no effect on NGF-induced Neurite Outgrowth. The potentiation of NGF-induced Neurite Outgrowth by papaverine was blocked by the PLC-γ inhibitor U73122. Furthermore, papaverine's potentiation of NGF-induced Neurite Outgrowth was also blocked by the co-administration of inositol 1,4,5-trisphosphate (IP 3 ) receptor antagonists (xestospongin C and 2-aminoethoxydiphenyl borate (2-APB)) and by reduced expression of IP 3 receptor gene (i.e., itpr1 and itpr3) by siRNA. Our findings suggest that papaverine could potentiate NGF-induced Neurite Outgrowth, and that activation of PLC-γ and IP 3 receptors might be involved in the mechanism underlying papaverine's potentiation of Neurite Outgrowth in PC12 cells.

Yasuyoshi Watanabe - One of the best experts on this subject based on the ideXlab platform.

Paul Fernyhough - One of the best experts on this subject based on the ideXlab platform.

  • Image_3_Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons.JPEG
    2018
    Co-Authors: Mohammad G. Sabbir, Nigel A Calcutt, Paul Fernyhough
    Abstract:

    The muscarinic acetylcholine type 1 receptor (M1R) is a metabotropic G protein-coupled receptor. Knockout of M1R or exposure to selective or specific receptor antagonists elevates Neurite Outgrowth in adult sensory neurons and is therapeutic in diverse models of peripheral neuropathy. We tested the hypothesis that endogenous M1R activation constrained Neurite Outgrowth via a negative impact on the cytoskeleton and subsequent mitochondrial trafficking. We overexpressed M1R in primary cultures of adult rat sensory neurons and cell lines and studied the physiological and molecular consequences related to regulation of cytoskeletal/mitochondrial dynamics and Neurite Outgrowth. In adult primary neurons, overexpression of M1R caused disruption of the tubulin, but not actin, cytoskeleton and significantly reduced Neurite Outgrowth. Over-expression of a M1R-DREADD mutant comparatively increased Neurite Outgrowth suggesting that acetylcholine released from cultured neurons interacts with M1R to suppress Neurite Outgrowth. M1R-dependent constraint on Neurite Outgrowth was removed by selective (pirenzepine) or specific (muscarinic toxin 7) M1R antagonists. M1R-dependent disruption of the cytoskeleton also diminished mitochondrial abundance and trafficking in distal Neurites, a disorder that was also rescued by pirenzepine or muscarinic toxin 7. M1R activation modulated cytoskeletal dynamics through activation of the G protein (Gα13) that inhibited tubulin polymerization and thus reduced Neurite Outgrowth. Our study provides a novel mechanism of M1R control of Gα13 protein-dependent modulation of the tubulin cytoskeleton, mitochondrial trafficking and Neurite Outgrowth in axons of adult sensory neurons. This novel pathway could be harnessed to treat dying-back neuropathies since anti-muscarinic drugs are currently utilized for other clinical conditions.

  • Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons
    Frontiers Media S.A., 2018
    Co-Authors: Mohammad G. Sabbir, Paul Fernyhough, Nigel A Calcutt
    Abstract:

    The muscarinic acetylcholine type 1 receptor (M1R) is a metabotropic G protein-coupled receptor. Knockout of M1R or exposure to selective or specific receptor antagonists elevates Neurite Outgrowth in adult sensory neurons and is therapeutic in diverse models of peripheral neuropathy. We tested the hypothesis that endogenous M1R activation constrained Neurite Outgrowth via a negative impact on the cytoskeleton and subsequent mitochondrial trafficking. We overexpressed M1R in primary cultures of adult rat sensory neurons and cell lines and studied the physiological and molecular consequences related to regulation of cytoskeletal/mitochondrial dynamics and Neurite Outgrowth. In adult primary neurons, overexpression of M1R caused disruption of the tubulin, but not actin, cytoskeleton and significantly reduced Neurite Outgrowth. Over-expression of a M1R-DREADD mutant comparatively increased Neurite Outgrowth suggesting that acetylcholine released from cultured neurons interacts with M1R to suppress Neurite Outgrowth. M1R-dependent constraint on Neurite Outgrowth was removed by selective (pirenzepine) or specific (muscarinic toxin 7) M1R antagonists. M1R-dependent disruption of the cytoskeleton also diminished mitochondrial abundance and trafficking in distal Neurites, a disorder that was also rescued by pirenzepine or muscarinic toxin 7. M1R activation modulated cytoskeletal dynamics through activation of the G protein (Gα13) that inhibited tubulin polymerization and thus reduced Neurite Outgrowth. Our study provides a novel mechanism of M1R control of Gα13 protein-dependent modulation of the tubulin cytoskeleton, mitochondrial trafficking and Neurite Outgrowth in axons of adult sensory neurons. This novel pathway could be harnessed to treat dying-back neuropathies since anti-muscarinic drugs are currently utilized for other clinical conditions

  • Guaifenesin derivatives promote Neurite Outgrowth and protect diabetic mice from neuropathy
    Journal of Medicinal Chemistry, 2013
    Co-Authors: Mallinath B Hadimani, Meena K. Purohit, Chandrashaker Vanampally, Randy Van Der Ploeg, Dwane Morrow, Katie E. Frizzi, Victor Arballo, Paul Fernyhough, Nigel A Calcutt, Lakshmi P. Kotra
    Abstract:

    In diabetic patients, an early index of peripheral neuropathy is the slowing of conduction velocity in large myelinated neurons and a lack of understanding of the basic pathogenic mechanisms hindered therapeutics development. Racemic (R/S)-guaifenesin (1) was identified as a potent enhancer of Neurite Outgrowth using an in vitro screen. Its R-enantiomer (R)-1 carried the most biological activity, whereas the S-enantiomer (S)-1 was inactive. Focused structural variations to (R/S)-1 was conducted to identify potentially essential groups for the Neurite Outgrowth activity. In vivo therapeutic studies indicated that both (R/S)-1 and (R)-1 partially prevented motor nerve conduction velocity slowing in a mouse model of type 1 diabetes. In vitro microsomal assays suggested that compounds (R)-1 and (S)-1 are not metabolized rapidly, and PAMPA assay indicated moderate permeability through the membrane. Findings revealed here could lead to the development of novel drugs for diabetic neuropathy.

  • Guaifenesin Derivatives Promote Neurite Outgrowth and Protect Diabetic Mice from Neuropathy
    2013
    Co-Authors: Mallinath B Hadimani, Meena K. Purohit, Chandrashaker Vanampally, Randy Van Der Ploeg, Dwane Morrow, Katie E. Frizzi, Victor Arballo, Paul Fernyhough, Nigel A Calcutt, Lakshmi P. Kotra
    Abstract:

    In diabetic patients, an early index of peripheral neuropathy is the slowing of conduction velocity in large myelinated neurons and a lack of understanding of the basic pathogenic mechanisms hindered therapeutics development. Racemic (R/S)-guaifenesin (1) was identified as a potent enhancer of Neurite Outgrowth using an in vitro screen. Its R-enantiomer (R)-1 carried the most biological activity, whereas the S-enantiomer (S)-1 was inactive. Focused structural variations to (R/S)-1 was conducted to identify potentially essential groups for the Neurite Outgrowth activity. In vivo therapeutic studies indicated that both (R/S)-1 and (R)-1 partially prevented motor nerve conduction velocity slowing in a mouse model of type 1 diabetes. In vitro microsomal assays suggested that compounds (R)-1 and (S)-1 are not metabolized rapidly, and PAMPA assay indicated moderate permeability through the membrane. Findings revealed here could lead to the development of novel drugs for diabetic neuropathy