The use of cell models in determining neuronal
responses to EASs
Professor Robert A. SmithSchool of Life SciencesUniversity of GlasgowScotland (UK)
Nervous System Complexity
���� CNS and PNS ���� Cellular heterogeneity
Neuronal and glial cellsSynaptic contacts/neural networks
���� Functional diversityRegional specialisationBlood Brain BarrierMotor/sensory pathwaysCognitive
���� Age Related susceptibility Developmental stages Maturation after birthAdult
���� Acute/Chronic/Delayed Responses
Brain Vulnerability…
���� Potentially more susceptible to damage by hazardous chemicals (including EASs)
- high metabolic rate - consumes more oxygen than other tissues.
���� Reactive Oxygen Species (ROS) generated
- mitochondrial dysfunction
- over activation of glutamate receptors (especially NMDA receptors)
- leads to Ca 2+ influx
- cell death pathways triggered
The Developing Brain: Additional challenges…
���� Blood Brain Barrier incomplete
���� Cell proliferation/ neurogenesis
���� Cell migration
� Neuronal cell differentiation - axon- dendrites- receptors
■ adrenergic, cholinergic, dopaminergic■ -TH, estrogen and androgen
- synaptogenesis
���� Glial maturation
Neural targets of Endocrine Disruptors in vivoSex steroids and hormones crucial in developing bra in
- Hypothalamus
- Pituitary
- Hippocampus
- Cerebral Cortex
- Cerebellum
Control of Hypothalamic/neuroendocrine axis
Spatial cognitive functions
Memory
Effects of:
Insecticides - DDT (estrogen agonist)
Polychorinated biphenyls (TH receptors)
Phthalates (androgen antagonists)
Neurotoxicity Testing Alternatives - Initiatives
Early
���� 31st OHOLO Conference: Model systems in neurotoxicology: alternative approaches to animal testing (1986) – Israel
Shahar & Goldberg, 1987
����ECVAM Workshop: In vitro neurotoxicity testing – Italy Atterwill et al., 1994
����WHO/IPCS: In vitro techniques for assessing neurotoxicity (1997) – USAHarry et al., 1998
Recent
�3rd Intl Conference on Alternatives for DNT (2011) – Italy Bal-Price et al., 2012
�Xi’an International Neurotoxicology conference (2011) – ChinaLlorens et al., 2012
���� Developmental neurotoxicity testing (2011) – Japanese Teratology SocietyCrofton et al., 2012
�10th Intl Early Toxicity Screening conference (2012) – USANeurotoxicity (opening session)
In Vitro endpoints for predicting and assessing neurotoxicity
Cell proliferation
Cell death
Migration assays
Neurite outgrowth/ network formation
Protein marker expressions
- Axonal
- Dendrtic
- Synaptic
- Glial cell
Receptor expression
In vitro systems available for determining neural responses
� Continuous and immortalised cells lines
- rodent/human- undifferentiated/differentiated
� Primary neurons
- mainly rodent
� Brain slices - e.g. hippocampus
� Stem and Progenitor cells
- rodent/human- embryonic/induced pleuripotent
Adherent cultures or 3D floating neurosphere masses
Cell Lines - RodentPC12 – rat pheochromocytoma (dopaminergic)
Undifferentiated - proliferationDifferentiated - neurite outgrowth
���� Bisphenol-A (BPA) – neurites suppressed (Radio & Mundy, 2008)
���� BPA inhibited MAPK phosphorylation (Seki et al., 2011)
B35 – rat neuroblastoma (cholinergic)
���� exposure to tetrabromo-BPA – ROS production, [ Ca2+]iand caspase-3 activity increased (Hendriks et al., 2012)
NB2a – mouse neuroblastoma (cholinergic)
���� neurite outgrowth (Axelrad et al., 2003)
GH3 – rat pituitary
���� BPA induced Growth Hormone release (Dang et al., 2007; 2009)
C17.2 – mouse cerebellum derivation (Lunqvist et al., 2012)
However,
majority established from tumours…
Limitation of Continuous Cell Lines
Primary cultured neurons
Cerebral cortex Silva et al. (2006)
Suňol et al. (2008) Briz et al. (2011)
Hippocampus Matsunaga et al. (2010)
Cerebellum- Granule cells Mundy et al. (2006)
Suňol et al. (2008)Smith (2009)Briz et al. (2011)
- Purkinje cells Xiong et al. (2012)
Hypothalamus Iwakura et al. (2010)
Primary cultured rodent cerebellar granule cellsHomogeneous neuronal cultures
Prepared from post-natal pups
Functional glutamate receptors by 6-8 div
Functional estrogen receptors
Extensive neurite production
- quantitative analysis of changes
Expression of neurotypic proteins
- cytoskeleton
- growth cones
- synapses
Basis of many neurotoxicity studies (Mundy et al., 2008; Bal-Price et al., 2010; Briz et al., 2011)
Still in search of the Human Dimension…
Advantages of primary neurons over using cell lines established from tumours therefore -
���� More normal functional phenotype
BUT…
Culture preparation with potential variability
Relatively low -medium throughput screening
Majority not of human origin
Human Cell Lines
NTera-2 – teratocarcinoma (cholinergic) Paquet-Durand et al. (2003)
���� Differentiated cells express neuronal polarity mark ers
Human origin but
- lack advantage of 1˚ cultures
- derived from tumours
� Cell membrane potential assay - AcuteTox project che micals Gustafsson et al. (2010)
SH-SY5Y – neuroblastoma (dopaminergic) cells
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)
HUB-NSC (Human umbilical cord derived) (Buzanska et al., 2005)
Human Neural Crest Cells(Generated from embryonic line) (Zimmer et al., 2012)
Induced Neuronal Cells(Conversion of fetal and postnatal fibroblasts) (Pang et al., 2010)
The way forward…. Human Neural Stem Cells
Immortalised stem cells (fetal brain)
hNSC (De Filippis et al., 2007)
ReNcell CX (Breier et al, 2008)
Neuronal Precursor Cells
PCBs disrupt TH-dependent (Fritsche et al., 2005;
neural & glial differentiation Schreiber et al., 2010)
The Neurosphere Assay for Developmental Neurotoxicity Testing
Human (or Rodent) Fetal NPCs
Proliferation -
* Quantify by several methods
Migration –
* NPCs leave sphere followinggrowth factor withdrawal
Differentiation –
* Neuronal & glial markers- Tuj-1 (green) - O4 (red)
Breier et al. (2010): Neurotox. Teratol. 32: 4-15
Polybrominated Diphenyl Ethers inhibit
differentiation of hNPCs
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 oligod endrocytes reduced following exposure to PBDEs
HUCB-NSC – Buzanska Lab
Zychowicz et al. (2011): Acta Neurobiol. Exp. 71, 12-23
ECM bioengineered printed arrays
+ 2% Serum Serum free
PLL
FN
Tuji-1: greenKi-67 (proliferation ): redHoechst: nuclei
Culture environment effect -
PLL – undifferentiated
FN – differentiated
Incorporate electrodes
Potential in neurotoxicity screens
proliferation
differentiation
migration
neurite outgrowth
electrophysiology
hESC-derived NC cells used in MINC assay
Used to assay migration impairment of NC cells (MINC) 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
Other promising approaches
���� Genomic and metabolomic analyses
Gene expression changes in murine NSCs following ch emical exposure
Need define thresholds between adaptive v. adverse responses
(Pennings et al., 2012; Theunissen et al., 2012)
���� Neuronal cells from fetal & postnatal fibroblasts
Transgene activation and transcription factor induc tion
(Pang et al., 2011; Kumar et al., 2012)
�Mathematical & computational initiatives
ToxCast Programme (EPA)
Unresolved Issues…
In vitro methods unable to mimic complexity of brain
Yet to achieve brain-region specific human neural c ells
Cognitive and behavioural aspects
Selection of appropriate battery of neurotoxicity t ests
Validation
Routine High Throughput Screening
Current State of the Available Art…
Evolution of neural cell models for neurotoxicity
Exciting advances in human cell technologies
– stem and NPCs of particular merit
Reliable endpoints for testing neurotoxicity (incl. DNT)
– proliferation– migration– neurite outgrowth
– functional activity
Data on mechanisms of action
Relevance to investigation of responses to EASs