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Georgina Harris, MSc
Thesis Defense Seminar
Environmental Health and Engineering
Adviser: Thomas Hartung, MD, PhD
18th May 2018
Lifetime exposures ultimately impact our phenotype and can increase our risk of disease
"Thee-hit hypothesis"
Daskalakis et al., 2013
Substantia Nigra
Dopaminergic
Neurons
Mackenzie et al. The pathology of Parkinson’s disease
In vitro 3D models, using HUMAN cells can overcome some limitations towards faster, less costly testing
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
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
Rotenone
Rotenone mechanism of action
Metabolic (mitochondrial viability)
20% decrease
Cell death (LDH release)
no effect
Smirnova L., Harris G. et al., 2016
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
What happens at the molecular target - complex I inhibition?
Surviving cells
Acute, 24h
Wash-out
Harris G., Eschment M. et al., revised
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
Higher ATP levels despite continued complex I inhibition
Are changes in ATP due to changes in mitochondria?
Harris G., Eschment M. et al., revised
*
Harris G., Eschment M. et al., revised
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
uncorrected p < 0.01
fold change > 1.5
~ 800 genes
Acute
~ 100 genes
Permanent/
Delayed
Harris G., Eschment M. et al., revised
CD200 - Type I membrane glycoprotein
CCK - Cholecystokinin
Aim 2. Conclusions
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?
First Hit
Second Hit
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
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?
- Rotenone 30 nM selected for repeated-dose exposure
Viability (repeated dose)
Harris G., et al., in preparation
Impaired outgrowth despite ATP surplus
Harris G., et al., in preparation
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
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.
Testing drug delivery nanoparticles in 3D LUHMES
Gold NPs - functionalized with sodium citrate or polyethylene glycol
Polylactic acid NPs with green fluorescent tag
Imaging of fluorescent PLA-NP
Gold Nanoparticles - ICP-MS
100 nm
Harris G., et al., Submitted
Cytotoxicity
Control Au-SC Au-PEG PLA-NP
Harris G., et al., Submitted
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
We developed a 3D dopaminergic model which allows for wash-out and repeated-dose experiments
"It's not about how hard you fall, but whether you get up"
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