investigation of the effects of innate immune activation on cell death on a neuronal cell line
TRANSCRIPT
Investigation of the Effects of Innate
Immune Activation on Cell Death on a
Neuronal Cell Line
Kelly Moore 4th Year ProjectSupervisor: Dr. Declan McKernan Lab Mentor: Laura Olsen
Innate Immune System• Activated immediately following infection or injury• Body’s first line of defence • Invading pathogen is detected by Dendritic cells and
macrophages• Network of pattern recognition receptors (PRRs) e.g.
Toll-like receptors• Recognise pathogen associated molecular patterns
(PAMPs)• Production of pro-inflammatory cytokines &
chemokines• Causing either inflammation or phagocytosis
Toll-Like Receptors
• Transmembrane signalling proteins expressed on immune cells
• 10 TLRs in humans• Located on either plasma membrane or internal
membrane• Expressed in tissues involved in immune function,
such as spleen leukocytes, lung , GIT and Brain. • Activation of TLRs leads to production of Cytokines,
chemokines or IFNs• Causes immune response
Toll-like Receptor 3
• Expressed on endosomes • Recognize dsRNA produced
during viral replication• Mediates signals via TRIF • Activates the transcription
factors IRF-3, NF-κB,AP-1• In turn, induces the production
of type 1 interferons• Anti-viral host defence
TLR3 in the brain
TLR3 causes inflammatory responses and breaks down the BBB
The TLR3 triggers IFN expression and restricts viral replication and spread of virus beyond the site of entry
TLR3 can cause the formation of Lewy bodies a hallmark of Parkinson’s disease
Parkinson’s Disease
Neuroinflammation triggered by pathogens can be associated with PD
Progressive neurodegenerative disorder Results from a pathophysiologic loss or degeneration of
dopaminergic neurons in the substantia nigra and the presence of α-synuclein aggregates , Lewy Bodies
Models of PD – Rotenone, 6-OHDA or MPTP
Cell death
Can be attributed to both the Immune activation of TLR3 and PD
Cells receive apoptotic stimuli Mitochondria releases cytochrome c Recruits caspase-9 Results in a caspase cascade
pyr
AimTo determine if viral infection in a cell culture system exacerbates the cell death induced by Parkinson’s disease.
HypothesisViral infection mimicked by Poly (I:C) in SH-SY5Y neuronal cells will exacerbate the cell death induced by a neurotoxin MPTP.
SHSY-5Y Cells
• Neuroblastoma cells
• Bone marrow from 4 year old female
• Characteristics of dopaminergic neurons
Experimental Design
Group 1: Control
• No drug treatment
Group 2: Poly IC
• Poly IC• Medium
Group 3: MPP+
• Medium• MPP+
Group 4: Experimental
• Poly I:C• MPP+
Treatments
MPP+: neurotoxin, mimics PD,• 20ug/ml for 24 hours
Poly I:C: synthetic analogue of dsRNA• 20ug/ml for 24 hours
Methods
• MTT assay • Western blotting
MTT Assay
Western Blotting
Gel electrophoresis Transfer Antibody
Results
Cleaved PARP Western Blot
cPARP 89 kDa
Β-Actin 42 kDA
Figure 1: Western blot with cPARP compared to β-Actin, showing all treatment groups
89 kDa87 kDa
Figure 2: Zoomed image of the cPARP band.Showing the presence of two bands of different molecular weight.
Figure 2: Ratio of 87kDa cPARP over β-actin for each treatment group. n=3Mean +/- SEM, Significant if P<0.05.
Figure 3: Ratio of 89 kDa cPARP over β-actin for each treatment group. n=3 Mean +/- SEM, Significant if P<0.05.
Figure 4: Bar chart representing the % of viable cells for each treatment group. n =6.
Presented as Mean +/- SEM, *** p< 0.001, significant difference compared to both control and Poly (I:C) groups.
Discussion Caspase 3 cleaves many different substrates including PARP. cPARP can be used as a marker of cleaved Caspase 3 activity,
indirectly measuring apoptosis. cCaspase-3 can stimulate apoptosis.
Discussion From the western blot analysis we can see a trend, showing an
increase in cPARP in each treatment group, especially in the Poly I:C group. Indicates there is caspase 3 activity.
We see a significant decrease in the amount of viable cells, when treated with MPP+ and the combination of Poly (I:C) and MPP+.
However, MTT assay can only tell us that cell death occurred, and can’t specify what type of cell death.
MPP+ could be causing other forms of cell death apart form apoptosis, this is why the western blot doesn’t correlate with the MTT assay, as cCaspase-3 can only indicate apoptosis.
Conclusion
The neurotoxin MPP+ mimicking PD, significantly decreases cell viability, however we do not know by what means.
Poly I:C, the viral mimetic causes an increase cPARP expression indicating cCaspase-3 activity, indicting apoptosis may be occurring.
Further Studies Further studies should be carried out to determine what types of cell
death occurs. Perhaps doing more western blots probing for proteins involved in other forms of cell death, such as necrosis or pyroptosis.
Different time points should be carried out as the dose of Poly I:C used is quiet small and doesn’t cause cell death immediately, it usually causes inflammation first.
Future experiments, should increase the n number, to get a better picture of the effects of both drugs in combination.
Acknowledgements I would like to thank Dr. Declan McKernan
and Ms Laura Olsen. I would also like to thank all the staff in
the department of Pharmacology, especially the staff in the CNS lab.
Finally I would like to thank the girls in my group for all their help and support.
Thank You!