The use of cell models in determining neuronal responses to EASs - - PowerPoint PPT Presentation
The use of cell models in determining neuronal responses to EASs Professor Robert A. Smith School of Life Sciences University of Glasgow Scotland (UK) Nervous System Complexity CNS and PNS Cellular
Synaptic contacts/neural networks
Regional specialisation Blood Brain Barrier Motor/sensory pathways Cognitive
Developmental stages Maturation after birth Adult
(especially NMDA receptors)
■ adrenergic, cholinergic, dopaminergic ■ -TH, estrogen and androgen
Sex steroids and hormones crucial in developing brain
Control of Hypothalamic/neuroendocrine axis Spatial cognitive functions Memory Effects of: Insecticides - DDT (estrogen agonist) Polychorinated biphenyls (TH receptors) Phthalates (androgen antagonists)
Early
alternative approaches to animal testing (1986)
– Israel
Shahar & Goldberg, 1987
Atterwill et al., 1994
Harry et al., 1998
Recent 3rd Intl Conference on Alternatives for DNT (2011) – Italy Bal-Price et al., 2012 Xi’an International Neurotoxicology conference (2011) – China
Llorens et al., 2012
– Japanese Teratology Society
Crofton et al., 2012
10th Intl Early Toxicity Screening conference (2012)
– USA
Neurotoxicity (opening session)
Undifferentiated - proliferation Differentiated
(Seki et al., 2011)
and caspase-3 activity increased
(Hendriks et al., 2012)
(Axelrad et al., 2003)
mouse cerebellum derivation
(Lunqvist et al., 2012)
Silva et al. (2006) Suňol et al. (2008) Briz et al. (2011)
Matsunaga et al. (2010)
Mundy et al. (2006) Suňol et al. (2008) Smith (2009) Briz et al. (2011)
Xiong et al. (2012)
Iwakura et al. (2010)
(Mundy et al., 2008; Bal-Price et al., 2010; Briz et al., 2011)
Paquet-Durand et al. (2003)
Cell membrane potential assay - AcuteTox project chemicals
Gustafsson et al. (2010)
Undifferentiated Differentiated Sanfeliu et al. (1999) Cheung et al. (2009) Tuj-1
Phase 40X Phase 40X
Neurite outgrowth and network formation assays
Frimat et al. (2010); van Thriel et al. (2012)
LUHMES cells (NPCs from female fetal brain) Neurite quantification in live cells
(Stiegler et al., 2011)
(Buzanska et al., 2005)
(Generated from embryonic line)
(Zimmer et al., 2012)
(Conversion of fetal and postnatal fibroblasts)
(Pang et al., 2010)
hNSC
(De Filippis et al., 2007)
ReNcell CX
(Breier et al, 2008)
PCBs disrupt TH-dependent
(Fritsche et al., 2005;
neural & glial differentiation
Schreiber et al., 2010)
Human (or Rodent) Fetal NPCs Proliferation -
* Quantify by several methods
Migration –
* NPCs leave sphere following growth factor withdrawal
Differentiation –
* Neuronal & glial markers
Breier et al. (2010): Neurotox. Teratol. 32: 4-15
Schreiber et al. (2010): Environ Health Perspect 118, 572-578
Control 10 µM PBDE- 47 10 µM PBDE- 99 Tuj-1 O4
Migration and differentiation of neurons and oligodendrocytes reduced following exposure to PBDEs
Zychowicz et al. (2011): Acta Neurobiol. Exp. 71, 12-23
ECM bioengineered printed arrays
+ 2% Serum Serum free PLL FN Tuji-1: green Ki-67 (proliferation): red Hoechst: nuclei
Culture environment effect - PLL – undifferentiated FN – differentiated Incorporate electrodes Potential in neurotoxicity screens
proliferation differentiation migration neurite outgrowth electrophysiology
Used to assay migration impairment
following exposure to chemicals
Zimmer et al. (2010): Environ. Health Perspect. 120, 1116-1122
Untreated Treated
Tuj-1 Peripherin DNA Tuj-1 Brn3a Tuj-1 NeuN
Peripheral markers expressed in neural cells from hESC
Gene expression changes in murine NSCs following chemical exposure Need define thresholds between adaptive v. adverse responses
(Pennings et al., 2012; Theunissen et al., 2012)
Transgene activation and transcription factor induction
(Pang et al., 2011; Kumar et al., 2012)
ToxCast Programme (EPA)
– stem and NPCs of particular merit
– proliferation – migration – neurite outgrowth – functional activity