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ACKNOWLEDGEMENTS: This work was funded by the Fight MND Cure grant, the MND Research Australia’s 2020 PhD top-up scholarship grant, and the Tasmanian Masonic Medical Research Foundation. QUESTIONS? FEEDBACK? WANT TO COLLABORATE? Contact details: Courtney Clark: [email protected] Prof Tracey Dickson: [email protected] Neuropeptide Y reduces neuronal firing of mouse SOD1 G93A cortical neurons Courtney Clark, Marcus Dyer, Rosie Clark, Catherine Blizzard & Tracey Dickson Menzies Institute for Medical Research, University of Tasmania, Australia BACKGROUND/AIMS Aim 1: Investigate the mechanism of which NPY modu- lates excitability using patch clamp electrophysiology. Aim 2: Determine the effect of NPY on the excitability of SOD1 G93A cortical cultures using multi-electrode arrays. Aim 3: Compare the NPY signalling pathways in wild type and SOD1 G93A cortical cultures. Hypothesis: NPY will reduce hyperexcitability in SOD1 G93A cortical neurons Cortical hyperexcitability has been implicated as an early event which contributes to dysfunction in ALS (1, 2). Therefore, modulating neuronal excitability may be a criti- cal measure to slow the progression of dysfunction within the cortex. Neuropeptide Y (NPY) is an inhibitory neuro- modulator which has been documented to prevent neu- ronal hyperexcitability, particularly in epileptic disorders (3-5). However, little is known about NPY in the context of ALS. 1) NEUROPEPTIDE Y MODULATES THE INTRINSIC EXCITABILITY OF PYRAMIDAL AND INHIBITORY NEURONS References: 1. Menon et al. 2015; Clin Neurophysiol 216(4):803-9. 2. Vucic et al. 2008; Brain 131(2):1540-50. 3. El Bahh et al. 2005; Eur J Neurosci 22(6):1417-30. 4. Colmers et al. 2003; Epilepsy Curr 3(2):53-8. 5. Wickham et al. 2019; Sci Rep 9(1):19393. 6. Marcuzzo et al. 2019; Neuroscience 416:88-99. Results Puffing NPY onto inhibitory (n = 11) and pyramidal neurons (n = 7) significantly altered the rheobase but not the maximum firing frequency. The action potential threshold is significantly reduced in inhibitory neurons after exposure to NPY and significantly increased in pyramidal cells. 2) NEUROPEPTIDE Y REDUCES THE ACTIVITY OF SOD1 G93A CORTICAL NEURONS. Results Neuronal spikes/min and mean spike rate were significantly increased in SOD1 G93A cortical neu- rons (n = 10 embryos) compared to wild type (n = 18). Both 100nM and 1uM of NPY significantly reduced the neuronal spikes/min and mean spike rate in SOD1 G93A cortical cutlures but not wild type. 3) NEUROPEPTIDE Y RECEPTOR PATHWAY ALTERED IN SOD1 G93A MODEL OF ALS CONCLUSIONS Results from this study are in line with previous work (6) documenting cortical hyperexcitability early in the SOD1 G93A mouse. NPY significantly reduced the spiking activity of SOD1 G93A cortical cultures, implicating NPY as a potential therapeutic against preclinical hyperexcitability in this model. Loss of cortical inhibition occurs early on in ALS patients. There- fore, NPY could act as a therapeutic intervention to increase the intrinsic excitability of inhibitory neurons. While a statistically significant increase in the expression of Y1 receptors was observed, the downstream signal- ling pathways for NPY appeared unchanged in the SOD1 G93A model at this time point. Future studies will focus on determining the effect of NPY on the excitability of neurons from ALS patient induced pluripotent stem cell lines. To indicate whether NPY can prevent hyperexcitability in human models of the disease. These investigations will also confirm whether changes to the Y receptors are present in human models of ALS. Results NPY Y1 receptors situated on the soma of excitatory cortical neurons were significantly increased in SOD1 G93A cultures (n = 3) compared to wild type (n = 7). A single dose of 100nM NPY had no long term effect on Y1 receptor expression. There is no observed difference in phosphorylated or total Akt and ERK1/2 protein expression between SOD1 G93A (n = 7) and wild type cultures (n = 13). NPY had no statistically significant effect on phosphorylated or total Akt and ERK1/2 protein expression. METHODS Whole cell patch clamp electrophysiology The effect of NPY on cortical pyramidal and inhibitory neurons was investigated in P60 C57BL/6 cortical slices. NPY (1uM) was puffed onto patched cells and recordings were taken 1 minute prior to NPY application. Statistical analysis: Paired t-tests. Effect of NPY on SOD1 G93A neuron excitability and receptor pathway investigations Cortical neurons of Thy1YFP x SOD1 G93A mouse embryos and their wild type littermates were cultured at E15.5 and grown until 14 days in vitro. MEA: Neuronal cultures treated with either 100nM or 1uM of NPY were compared to vehicle controls using multi-electrode array recordings (10 mins). Immunocytochemistry: Y1 receptor expression was compared in wild type and SOD1 G93A cortical neurons normalised to wild type naive controls. 100nM NPY was added to neurons for 24 hours to investigate treatment effect. Western blots: effect of 100nM of NPY on Akt and ERK1/2 phosphorylation was compared at 10mins and 24hrs post treatment. Statistical analysis:Two-Way ANOVA and Bonferroni’s post hoc test. * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001. Naive 10 min NPY 24 hr NPY Naive 10 min NPY 24 hr NPY 0.0 0.5 1.0 1.5 2.0 phos-Akt / t-Akt Immunoreactivity Naive 10 min NPY 24 hr NPY Naive 10 min NPY 24 hr NPY 0.0 0.5 1.0 1.5 phos-ERK1/2 / t-ERK1/2 Immunoreactivity Naive 10 min NPY 24 hr NPY Naive 10 min NPY 24 hr NPY 0 2 4 6 8 Protein fold change (Akt pan) relative to GAPDH Naive 10 min NPY 24 hr NPY Naive 10 min NPY 24 hr NPY 0 5 10 15 Protein fold change (total ERK1/2) relative to GAPDH Naive NPY treatment Naive NPY treatment 0 1 2 3 YFP +ve neuron Y1R expression (average CTCF) Wild type SOD1G93A !
