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