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In Vitro Resilience and Nanotoxicity in 3D Brain models

Georgina Harris, MSc

Thesis Defense Seminar

Environmental Health and Engineering

Adviser: Thomas Hartung, MD, PhD

18th May 2018

BACKGROUND

"It's not about how hard you fall,

but whether you get up"

Adapted from Vince Lombardi

RESILIENCE

We are exposed to thousands of compounds daily

Exposures and risk of neurodegenerative disease

  • The central nervous system (CNS) can be especially vulnerable to toxicity due to limited regeneration and high energy demands.
  • With an aging population, there in an increase in neurodegenerative diseases.
  • Only 85 substances have been tested for neurotoxicity (EPA).

  • Over 1500 chemicals identified as potential neurotoxicants.

Lifetime exposures ultimately impact our phenotype and can increase our risk of disease

"Thee-hit hypothesis"

Daskalakis et al., 2013

Parkinson's Disease

  • Genetics are known to play a role, 90% of cases are sporadic

  • Epidemiological studies have shown that exposure to pesticides can increase the risk of developing Parkinson's

Substantia Nigra

Dopaminergic

Neurons

Mackenzie et al. The pathology of Parkinson’s disease

  • Current therapies treat symptoms and diagnosis is too late for prevention

Current in vivo and in vitro models

  • For toxicity testing animal models are the gold standard.

  • Inbreeding leads to a lack of genetic diversity which plays a major role in chronic disease

  • Animals serve poorly for chronic neurodegenerative diseases such as Parkinson's and Alzheimer's

In vitro 3D models, using HUMAN cells can overcome some limitations towards faster, less costly testing

  • In vitro models test high concentrations and current models are usually cancer cell lines or animal cells

  • 2D culture.... " Cells are bored to death" T. Hartung

Aim 1. To develop and characterize a human 3D in vitro dopaminergic cell model

The LUHMES Cell Line

  • Human immortalized cell line (non-cancer)
  • Dopaminergic neurons - affected in Parkinson's disease

3D

LUHMES

model

  • Cultured in monolayer (2D)
  • Can be kept in culture for up to 9 days

LUHMES differentiation in 3D

Compound diffusion

Differentiation marker expression

Aggregate Size

Continuous gyratory shaking to generate homogeneous spheres

Differentiation marker expression in 3D vs 2D

Aggregates

Smirnova L., Harris G. et al., 2016

3D LUHMES in vitro model

  • LUHMES differentiate in 3D and can be kept in culture for > 21 days

  • Close cell interactions allow for better signaling between cells
  • Cultured in suspension - allowing compound wash-out

Current in vitro models

Current in vitro models study acute effects after short-term exposures.

Most assays are developed to study toxicity pathways and do not measure whether cells adapt or recover.

Chronic Diseases manifest long after exposure is removed

To mimic this in vitro, compounds need to be washed-out

Cellular recovery and resilience

Most in vitro work performed to date!

Smirnova L., Harris G. et al., ALTEX, 2015

Aim 2. To study acute effects and recovery after rotenone exposure in 3D LUHMES

Rotenone

  • Non-selective pesticide - Natural origin (labelled organic)

  • Currently still used in developing countries and for organic farming

  • Highly lipophillic and can cross the blood brain barrier

  • Occupational exposure is associated with increased risk of Parkinson's disease

Rotenone mechanism of action

100 nM Rotenone selected to study recovery

Metabolic (mitochondrial viability)

20% decrease

  • 100 nM rotenone, 24 h

Cell death (LDH release)

no effect

Smirnova L., Harris G. et al., 2016

Rotenone exposure and wash-out

1. Changing media

2. Transferring aggregates to a new plate

7 day recovery -->

"Free" rotenone quantification in media by mass spectroscopy

D8, 24 h

D15, Wash-out

D8

D15

D15

-- + R100 -Agg +Agg

- Agg +Agg

Harris G., Eschment M. et al., revised

Progressive loss in viability after wash-out

What happens at the molecular target - complex I inhibition?

Surviving cells

Acute, 24h

Wash-out

Harris G., Eschment M. et al., revised

Complex I activity is permanently inhibited after wash out

low-levels of rotenone remain bound to aggregates after wash-out

Does complex I inhibition alter ATP levels after wash-out?

Harris G., Eschment M. et al., revised

ATP levels decrease acutely but are restored after wash-out

Higher ATP levels despite continued complex I inhibition

Are changes in ATP due to changes in mitochondria?

Harris G., Eschment M. et al., revised

3D

differentiation

Effects on mitochondrial number and morphology

*

Harris G., Eschment M. et al., revised

Acute effects on neurite outgrowth are reversible

LUHMES-RFP

3D LUHMES are electrically active on Day 15

3D Aggregates functionally recover after 100 nM rotenone wash-out

Do permanent molecular changes take place?

Are they electrically active??

Harris G., Eschment M. et al., revised

Changes in gene expression

uncorrected p < 0.01

fold change > 1.5

~ 800 genes

Acute

~ 100 genes

Permanent/

Delayed

Harris G., Eschment M. et al., revised

CD200 and CCK

CD200 - Type I membrane glycoprotein

  • Immune signalling
  • Downregulated in the brain of aging rats (Wang et al., 2011)

CCK - Cholecystokinin

  • Enriched in the midbrain, regulates dopamine release
  • Polymorphisms associated with PD symptoms (Lenka et al., 2016)

Aim 2. Conclusions

  • Our 3D model shows that LUHMES functionally recover from acute 100 nM rotenone exposure in vitro.

  • Wash-out experiments provide a new strategy to study acute, delayed or permanent effects.

Cellular Resilience

Smirnova L., Harris G. et al., ALTEX 2015

Aim 3. Do pre-exposed (recovered) cells retain 'memory' from past exposures?

How do they respond to a second exposure?

Exposure timeline to study resilience/susceptibility

First Hit

Second Hit

Pre-exposures lead to 'memory' .....and RESILIENCE

100 nM

50 nM

25 nM

Harris G., Eschment M. et al., revised

Higher viability is not due to increase in number of cells or proliferation after recovery

The response to a second exposure is different to the first

  • Selected genes known to be altered by rotenone

Control First hit Second hit

Stronger response after second hit

Weaker response after second hit

Same response after second hit

"threshold/memory"

What could underlie resilience?

  • High glycolytic capacity (ATP generation)
  • Permanent activation of survival pathways

- Increased metabolism

- Changes in transporter levels

- Autophagy mechanisms which can remove damaged proteins and organelles

  • Epigenetic mechanisms

If not given the time to recover...

How do cells respond to repeated-dose exposures?

Exposure timeline to study repeated low-dose effects

- Rotenone 30 nM selected for repeated-dose exposure

ATP increases with repeated low-dose exposure

Viability (repeated dose)

Harris G., et al., in preparation

Repeated-dose effects on neurite outgrowth

Impaired outgrowth despite ATP surplus

Harris G., et al., in preparation

PD-related genes are up-regulated with repeated low-dose exposure

DOPAMINE SYNTHESIS

DAMAGED PROTEIN REMOVAL

Wash-out, 100 nM

RD30

Low-dose repeated exposures could mimic early molecular events leading to long-term degeneration

NEURO-PROTECTION

CARBON METABOLISM

Harris G., et al., in preparation

Wash-out and repeated-dose experiments can help identify recovery vs disease pathways

- development of new preventive therapies

- better in vitro to in vivo extrapolation than acute studies

Limitations and further research

Single Cell type

Single concentration for most endpoints

Limited throughput for certain endpoints

'Healthy' cells, normal response

Only tested female cells

Identifying whether the observed effects are compound or cell specific

Determining how cells compensate for the acute effects and how gene variants may interfere with recovery using genome editing

Testing nanoparticles, for example those designed for drug delivery

Nanoparticles as Drug Delivery Systems

Nanoparticles (NP) are between 1-100 nm in size

> 800 NP types found across industries

Their phisicochemical properties can be modified to cross biological barriers such as the blood brain barrier and target specific cells for drug delivery.

Nanotoxicity

and Drug Delivery

Testing drug delivery nanoparticles in 3D LUHMES

Gold NPs - functionalized with sodium citrate or polyethylene glycol

Polylactic acid NPs with green fluorescent tag

Nanoparticle size and internalization

Imaging of fluorescent PLA-NP

Gold Nanoparticles - ICP-MS

100 nm

Harris G., et al., Submitted

Effects on mitochondrial membrane potential and cytotoxicity

Cytotoxicity

Control Au-SC Au-PEG PLA-NP

Harris G., et al., Submitted

Effects on mitochondrial membrane potential and viability

The studied NPs showed dose-dependent effects on mitochondria and viability therefore further studies are needed to determine their applicability to deliver drugs targeted at dopaminergic cells.

Harris G., et al., Submitted

Final Thoughts...

We developed a 3D dopaminergic model which allows for wash-out and repeated-dose experiments

  • Cells may retain short-term memory from past exposures

"It's not about how hard you fall, but whether you get up"

Final Thoughts...

  • When not given time to recover, our model shows repeated-doses lead to expression of disease-related genes

  • 3D LUHMES can be applied to study toxicity of NPs which target dopaminergic cells for drug delivery

Acknowledgements

Advisors

Prof. Thomas Hartung

Dr. Lena Smirnova

Collaborations

Prof. Michael Mccaffery

Sebastian J Perez

Erin Pryce

Dr. Daniel Severin

Dr. Paulo Emílio Corrêa Leite

Dr. Mariana Rodrigues

Prof. Marcel Leist

Richard Maclennan

Dana Freeman

CAAT Lab

Melanie Echsment

Johannes Delp

Dr. Helena Hogberg

Dr. David Pamies

Shelly Odwin

Megan Chesnut

Dr. Alex Maertens

Dr. Andre Kleensang

Prof. Alan Goldberg

Ruth Brady

Michelle Downes

Thesis Committee

Prof. Marsha Wills-Karp Prof. Daniele Fallin

Dr. Anne Hamacher-Brady

Dr. Sin-ichi Kano

Dr. Wan-Yee Tang

Dr. Jiou Wang

Dr. Zhibin Wang

Funding Sources

Center for Alternatives to Animal Testing

International Foundation for Ethical Research

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