1. Investigation of the Effects
of Innate Immune
Activation on Cell Death on
a Neuronal Cell Line
Kelly Moore
4th Year Project
Supervisor: Dr. Declan McKernan
Lab Mentor: Laura Olsen

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

9. Aim
To determine if viral infection in a cell culture
system exacerbates the cell death induced by
Parkinson’s disease.
Hypothesis
Viral infection mimicked by Poly (I:C) in SH-SY5Y
neuronal cells will exacerbate the cell death
induced by a neurotoxin MPTP.

10. SHSY-5Y Cells
• Neuroblastoma cells
• Bone marrow from 4
year old female
• Characteristics of
dopaminergic neurons
Experimental Design

11. 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

12. Methods
• MTT assay
• Western blotting

13. MTT Assay

14. Western Blotting
Gel electrophoresis Transfer Antibody

15. Results

16. 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 kDa
87 kDa
Figure 2: Zoomed image of the
cPARP band.
Showing the presence of two bands
of different molecular weight.

17. Figure 2: Ratio of 87kDa cPARP over β-actin
for each treatment group. n=3
Mean +/- 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.

18. 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.

19. 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.

20. 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.

21. 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.

22. 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.

23. 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.

24. Thank You!