neuroprotective agents to reduce neuronal loss following sci

Annabel Ola15.9.2011

Spinal Cord Injury (SCI)

Traumatic insult to the spinal cord causes mechanical damage and tissue degeneration

Primary injury: Mechanical trauma Spinal cord swells, filling spinal canal Necrosis

Secondary injury: Ischaemia and apoptosis The site of injury is expanded Therapeutic benefit would be stopping this process

Background to this project

Neuroprotective action of fibronectin peptides (2010 King et al.)

The peptide PRARIY decreased neuronal death in vivo.

Proline

Arginine

Alanine

Arginine

Isoleucine

Tyrosine

Aims and objectives

Aim: To investigate the activity of PRARIY in an in vitro model of spinal cord injury

Objectives:1) Set up a culture system to investigate neuronal cell death.

2) Characterise the uptake and localisation of fluorescently labelled peptide PRARIY in neuronal cells.

a) Is this peptide protective?

b) Where is the peptide localised?

3) Compare neuroprotection seen with PRARIY to a scrambled control peptide YRPIRA, to investigate whether it is this particular sequence of amino acids in the peptide which confers the neuroprotective characteristics.

Approaches to creating a model of cell death in vitro 3D cell culture of 2 neuronal cell lines

Pure neuronal cultures More reproducible than primary cultures Traps the cells in collagen matrix/scaffold Viability was assessed before setting in gels

10 mM Glutamate & 50 µM Glycine Act on NMDA receptors and induce excitotoxicity Glycine is a co-agonist of the NMDA receptor

Approaches to creating a model of cell death in vitro (2) Other stimuli – induce cell death in the CNS

1 μg/mL TNFα – inflammatory stimulus 100 mM NMDA – excitotoxic stimulus

Live/dead staining 200 μg/mL Propidium iodide

Red stain for dead cells 1 μg/mL Hoechst

Blue stain for all cells regardless of viability

Approaches to creating a model of cell death in vitro

BA

Figure 1. Examples of staining with 200 μg/mL Propidium iodide (A) and 1 μg/mL Hoechst (B) in live/dead staining of collagen gels.

Optimising treatment protocol

Overview of experiments

Time course death experiment Excitotoxic/inflammatory stimuli comparison PRARIY localisation PRARIY rescue from death by glutamate Dose-dependent effect of PRARIY Comparison of PRARIY and scrambled peptide

YRPIRA

Time course death of B104 cells

Differences in death were seen between control and treated gels, shown in figure 2 with the optimised protocol.

At 24 and 48 hours, there were significant differences between control and treated.

***

***

Similar amounts of death were elicited by each of the stimuli.

Cell death was seen with and without the presence of FCS. More death was seen without, so this was chosen as the treatment vehicle.

Excitotoxic and inflammatory stimuli comparison – B104

PRARIY localisation in B104 2D culture

Figure 4. B104 24hr Fluorescent peptide uptake.

Hoechst with FITC-PRARIY (A), FITC-PRARIY only (B), an overlay of 3 images (C) and FITC-PRARIY with CM DiI (D).

A B

C D

FITC-PRARIY entered the B104 cells.

The peptide is largely cytoplasmic (figure 4 A and C), creating an orange colour with the co-localisation of the CM DiI.

D

PRARIY rescue of B104 cells

PRARIY with glutamate had a significant effect on the amount of cell death by glutamate at 24 hours, but the level of death in the control was very high.

***

***

Dose response of B104 cells to PRARIY

There was no dose-dependent effect seen with B104 cells but there were significant differences between glutamate treated gels and the different doses of the peptide.

There could however be a dose-dependent effect with lower doses and further investigation is needed.

***

**

Dose response of NG108 cells to PRARIY

Again there was no dose-dependent effect seen in NG108 cells, but PRARIY brought the levels of death to that seen in the control.

This is the only NG108 experiment where the control levels of death has been significantly lower than that seen with glutamate.

**

******

*

Initial comparison of PRARIY and YRPIRA

No difference between the two peptides but both reduced glutamate levels of death however glutamate levels of death were not high.

More investigation is needed but there was limited time to repeat these experiments etc.

******

Scrambled peptide YRPIRA elicits protection in NG108 cells

There was a significant difference between the two peptides (n=4, P<0.05).

Also PRARIY and YRPIRA significantly reduced cell death. And again control levels of death were lower than previously seen.

*****

*

Conclusions

There was variability between experiments, especially with the NG108 cells.

I could not properly investigate what I had set out to and results were inconclusive.

PRARIY enters B104 and NG108 cells.

PRARIY appears to reduce levels of death elicited with glutamate.

Future experiments

Improve the cell death model to isolate cells so there is less variability between experiments.

Establish a full time course death experiment.

Peptide rescue in cell death caused by other stimuli.

Investigate the localisation of PRARIY at different time points

Acknowledgements

The Open University - Dr James Phillips, Dr John Golding, Dr Jane Loughlin and Melanie Georgiou

The School of Pharmacy - Dr Rosemary Smyth and Dr Mike Munday

Thank you for listening

Questions?