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!