Utilizing Simple Western to Understand Neuronal Markers in Schizophrenia
"It can be difficult to optimize new antibodies, particularly for brain markers where you have a lot of background noise and low levels of your target. The
appropriate control and low variability between plates using Simple Western™ means we can complete our studies in weeks rather than months."
- Michael Harte, PhD Senior Lecturer in Drug Action, Neuroscience, University of Manchester
New approaches in CNS Drug Discovery for Neurodegenerative Diseases
Neuropsychiatric and neurodegenerative diseases are major contributors to the global burden of disease. The nervous system, especially the brain, is one of the most complex tissues, presenting unique challenges to traditional gene expression analysis. Optimizing techniques to investigate the neural mechanisms underlying cognitive deficits seen in neurodegenerative and neuropsychiatric diseases is key in determining their unique and complex pathophysiology, and aid in the identification of novel targets to improve treatments for patients.
Dr Michael Harte, a Senior Lecturer in Drug Action at the University of Manchester, specializes in the development of preclinical models for neurological disorders, combining behavioural, electrophysiological and post-mortem studies for the testing of novel compounds for progression to the clinic. In Michael’s most recent research, he is investigating a range of synaptic and neuronal markers related to Schizophrenia
Cognitive impairments associated with schizophrenia (CIAS) include deficits in executive function and working memory. These severely debilitating, constant and enduring features of the illness occur prior to the onset of psychosis and are resistant to current treatments, greatly reducing patients’ quality of life. To enable the discovery of new therapeutic approaches, carefully validated animal models and optimized protein analysis techniques are required.
Challenges when Detecting Neuronal Markers-Background Noise and Low Target Levels
To better understand cognitive deficit associated biomarker changes within CNS diseases, expression changes for proteins such as Parvalbumin (PV), present in GABAergic interneurons and playing a key role in regulation learning and memory, need to be accurately measured. These PV interneurons are commonly reduced in several preclinical models and associated human post-mortem studies. On top of this, PV is a low molecular weight protein, typically 9-11 kDa, and is notoriously difficult to detect using standard western blotting. Small sample volume is also another challenge that must be taken into account. With Michael’s treatment studies typically consisting of a range of groups with over 60 brains and multiple regions for analysis, traditional, more time-consuming approaches such as immunohistochemistry aren’t always viable.
Less hands-on and more accurate results for Neuroscience Research
That’s where Simple Western™ comes in; offering gel-free, blot-free, and hands-free capillary-based immunoassay platforms that integrate and automate the entire protein separation and detection process. “Simple Western allowed us to switch to a high throughput system with a greater control over validating antibodies, parvalbumin being the perfect example (FIGURE 1). It works perfectly to shorten study time to generate post-mortem results. With colleagues and I having had limited success in the past using traditional western approaches, introducing Simple Western’s advanced western blotting solutions for Neuroscience has allowed for greater consistency and ease of use for all users, achieving excellent reproducibility” says Michael.
Figure 1. Simple Western assay analysis showing (a)–(c) Protein expression levels for parvalbumin (PV) and (d)–(f) postsynaptic density 95 (PSD95) in frontal cortex, dorsal and ventral hippocampus of vehicle and subchronic phencyclidine (scPCP)-treated mice. Data were normalized to levels of glyceraldehyde 3-phosphate dehydrogenase (PV) or b-actin (PSD95) and expressed as percentage of control. scPCP mice showed a significant decrease in all regions for both markers apart from PSD95 in dorsal hippocampus. Data are mean +/–SEM, n = 7–8 per group
Michael and his team continue to use the system in their most recent research investigating inflammasome pathways in Alzheimer’s disease with the team’s ultimate goal to elucidate the neural mechanisms underlying cognitive deficits seen in a range of CNS diseases.
Publication Spotlight
1. Gigg J, McEwan F, Smausz R, Neill J, Harte MK. Synaptic biomarker reduction and impaired cognition in the sub-chronic PCP mouse model for schizophrenia. Journal of Psychopharmacology. 2020;34(1):115-124. doi:10.1177/0269881119874446
2. Tang, A & Harte, M 2021, 'Investigating Markers of the NLRP3 Inflammasome Pathway in Alzheimer’s Disease: A Human Post-Mortem Study', Genes, vol. 12, no. 11, 1753. https://doi.org/10.3390/genes12111753