SPECTROstar Nano
Absorbance plate reader with cuvette port
Biological assays, including aggregation, cytotoxicity, and signaling assays, are crucial to help researchers understand the underlying mechanisms of these diseases, as well as identify promising therapeutics in the early stage of drug development. This article details the importance of these assays and the need for sensitive and fast instruments that can analyze assays using a range of detection methods.
What are neurodegenerative diseases?
The term refers to diseases that are typically identified by symptoms such as lack of motor control, mood disorders, and cognitive deficits. As the name indicates, the disorders are characterized by degeneration of the neurons. Neurons are nerve cells in the spinal cord that do not replicate. If the neurons are damaged, they do not recover leading to brain dysfunction and incurable diseases. Although similarities between neurodegeneration can be found in the related diseases, the root causes vary: Huntington’s disease is caused by a gene mutation, Creutzfeldt Jakob prion disease is induced by incorrectly folded protein (prion), and Parkinson's and Alzheimer's are suspected to be caused by a combination of genetic and environmental factors and typically occur in people over 60. The disruption of mitochondrial function is also known to be linked to neurodegenerative diseases.
A similarity shared among these neurodegenerative diseases is the way how damage is induced in the neurons and consequently brains. Misfolded proteins occur in the course of all the mentioned disorders. They form amyloid fibrils, protein aggregates that build plaques and potentially harm neuronal cells and brain tissue. Understanding and interfering with this alteration is an important approach for treating neurodegeneration as it may benefit patients with any of the diseases.
Why is it important to research neurodegenerative diseases?
Neurodegenerative diseases follow only cardiovascular diseases and cancer as primary causes of death and unlike cardiovascular diseases and cancer, neurodegenerative diseases cannot be cured or slowed significantly.
Symptoms of the nerve disorders include impaired movement, memory loss, mood changes, impaired talking, and many more. Initially, these dysfunctions do not significantly impair patients and they are able to continue to lead an independent life. However, as these diseases progress, a patient’s quality of life drastically diminishes until they require full-time care. Consequently, the social and financial burden also increases during disease progression.
In Europe, it currently costs approximately €130 billion per year to care for people with dementia, a consequence of neurodegenerative diseases. With the aging population on the rise, the number of affected people rises. In 2015, Alzheimer’s Disease affected 40 million people worldwide, and this is predicted to grow to 130 million by 2030, with one-third to one-half of people above 85 years of age developing this disease. Alzheimer’s Disease has an average duration of 2 to 10 years, which no doubt has an impact on the predicted economic toll in the USA of $1 trillion per annum of this disease by 2050.
To date, there are a limited number of therapies that treat neurodegenerative diseases, and most of these only treat symptoms. In fact, no new drugs for Alzheimer’s Disease have been approved in Europe in the last five years. Therefore, effective treatments to delay or reduce the symptoms of these debilitating diseases are essential to limit the devastating impact on individuals, families, societies, and economies. A delay in the onset of Alzheimer’s Disease by just five years would reduce the financial burden in the USA by 50% and demonstrates even a limited delay would be beneficial, yielding improved autonomy of the patient and relief to the commitment of the family and the public health bill.
Identifying drug targets for neurodegenerative diseases
In order to identify potential new drugs for the treatment of neurodegenerative disorders, it is sensible to understand the disease itself. Due to their wide and varying symptoms and the typical slow onset, diagnosis is often only made when the disease is well underway. As such, there are still gaps in the understanding of these disorders, in particular the triggers and early stages of the diseases. Therefore, basic research is ongoing to understand these diseases in more depth and help identify new drugs.
The development of novel assays in the last 10 years made it possible to study neurodegeneration processes in vitro and in high throughput. These tools offer the possibility to test multiple experimental conditions or multiple possible drugs in very short times. Several key assays are valuable tools for analyzing the disease pathway and evaluating the effects of potential drugs. Following, we will explain assays testing aggregation, cytotoxicity, signaling, and protein quantity and we show how they help to understand neurodegenerative diseases.
Aggregation assays in neurodegenerative diseases research
A key feature of neurodegenerative diseases is the formation of soluble, functional proteins into insoluble, highly ordered protein aggregates termed amyloid fibrils or plaques. This transition begins with the formation of prefibrillar species (dimers, tetramers, hexamers, etc.) before developing into large protein aggregate complexes. In the case of prion research, whole animal models have traditionally been used to monitor these protein aggregates through lengthy bioassays that could take up to 6 months. In 2012, a faster, higher throughput aggregation assay for prion seeding monitoring was developed by researchers at Rocky Mountain Laboratories in Montana called real-time quaking-induced conversion assay (RT-QuIC).
RT-QuIC uses fluorescence intensity to measure the aggregation of prion proteins. The fluorescent dye thioflavin T (ThT) is added to recombinant proteins. The molecule binds to beta-sheets formed during fibril formation, which induces a fluorescence increase. The method is performed in microplates and uses recombinant prion protein, tau protein, or alpha-synuclein. The plate is intermittently shaken to permit break-up of fibrils during shaking and new fibril formation during quiescence. The process takes up to 168 h, but fibrils formation is accelerated at higher temperatures. Therefore, the protein aggregation assay is often performed at 37 °C or higher. The combination of high temperature, intermittent shaking, and long-run times places high demands on the measuring instrument. The Omega microplate reader series has proven robust and reliable to perform the tedious work as outlined in the application note “Real-time quaking-induced conversion assay for prion seeding”. Features such as high-temperature incubation (up to 65 °C), an improved plate carrier, and a more robust transport system have made the Omegas the readers of choice for aggregation assays. This scientific talk shows tips and tricks on how to optimize your RT-QuiC measurements.
A study published in 2016 found the RT-QuiC performed with cerebrospinal fluid to be a reliable test for sporadic Creutzfeldt Jakob disease. Eleven centers based in Europe, Asia, and Australia analyzed the human CSF samples using RT-QuiC and BMG LABTECH plate readers (with one exception). The centers diagnosed Creutzfeldt Jakob disease with 100 % accordance (McGuire et al. 2016).
A method suited to monitor the onset of aggregation employs a novel fluorophore: bis(triphenyl phosphonium) tetraphenylethene (TPE-TPP). The dye has superior characteristics to the ThT dye as it binds more efficiently. Furthermore, the monitoring of its fluorescence polarization (FP) reports on early stages (dimers, tetramers…) of aggregation. FP measures the rotation of molecules in solution. Small molecules move fast and depolarize emission whereas large molecules move more slowly and retain emission polarization. The binding of TPE-TPP to (pre-)amyloid structures slows down its rotation leading to measurable changes in FP. The assay was developed by a research group in Australia that used a CLARIOstar microplate reader to perform the FP measurement. A detailed assay description can be found in the Application Note “Novel aggregation-specific fluorogen monitors prefibrillar protein aggregation by fluorescence polarisation (FP)”.
Viability assays and toxicity assays assist in understanding and modifying neuronal death
Besides protein aggregation, neuronal cell death is a hallmark of neurodegeneration. While neurons are restricted in cell death in healthy adults, they die during neurodegenerative diseases leading to loss of brain function. The neurons die via mechanisms of programmed cell death which can be induced by oxidative damage of mitochondria or DNA, membrane damage by protein aggregates, and others. Programmed cell death mechanism reported in neuronal cells is apoptosis, necrosis, and autophagy. Understanding the causes of neuronal cell death, its mechanisms, and how to interfere with it is an approach to finding medication for neurodegenerative diseases. Many assays study cell death and help to decipher and modulate neuronal cell death. An overview of general cytotoxicity assays gives our blog post “Cytotoxicity – These assays tell you what your cells don’t like”.
The most commonly used cytotoxicity assays are based on tetrazolium salts. Their reduction by viable cells leads to an absorbance shift that is read by microplate readers and indicates metabolic activity. Researchers from the National Medical Research Center, Moscow, Russia used the assay to prove that insulin limits excitotoxicity in cortical neurons, an effect inducing cell death in neurons that are linked to neurodegenerative diseases (Krasil’nikova et al. 2019).
ATP-dependent luciferase viability assays are popular because of their simplicity and sensitivity. They measure the ATP content of lysed cells by an ATP-dependent luciferase. The light output measured by a microplate reader is directly related to cell number. The assay helped Singapore researchers to reveal a cytotoxic effect of mononamine oxidase (MAO) activity in a huntingtin cell model (Ooi et al. 2015).
Recently developed cell death assays not only report on toxicity at one-time point but monitor cytotoxic effects over time. The RealTime-Glo™ viability assay produces light in presence of viable cells. If cells are kept in a culturing environment (37 °C, 5 % CO2) by an atmospheric control unit regulating gases inside the reader, it can be used for real-time viability monitoring over days: The Atmospheric Control Unit (ACU) for the CLARIOstar and VANTAstar provides versatility in long-term cell-based assays. Using the assay on a CLARIOstar microplate reader, a protective effect of a specific fusion protein against neurotoxins was studied (Paliga et al. 2019).
Signaling assays shed light on neuron function
An early event of neurodegeneration is impaired neuron signaling. Altered signaling processes include Ca2+ signaling regulating synaptic transmission, energy metabolism, and cell survival or signal transmission via receptors.
Ca2+ signaling in neurons is vital for their cellular function as information transmitters. Intracellular Ca2+ changes can be monitored by fluorescent dyes such as Fura-2, Fluo-8, or Cal- 520. The application note “Monitoring intracellular calcium using fluorescent dyes in a mid-throughput assay” compares the different assays monitoring Ca2+. An example of Ca2+ assays helping neurodegenerative diseases research is the use of Fura-2 to develop a model of differentiated neurons that better reflect neurons than the human neuroblastoma cells they are derived from (Ferguson et al. 2016). Fura-2 intracellular Ca2+ measurements showed the stimulation of differentiated cells with KCl, while non-differentiated cells did not increase intracellular Ca2+ levels upon KCl.
Neurodegeneration biomarker quantification
Several proteins have been found to show altered expression during the different neurodegenerative diseases. Tau protein is implicated in Alzheimer's disease, the TREM2 receptor levels correlate with the risk to develop Alzheimer’s, and the BDNF growth factor is decreased in Alzheimer’s. For Parkinson’s increased levels of neuroinflammation such as IL6, IL β1, and TNF α are detected.
A popular method to quantify specific proteins in solution is ELISA assays. Our application note “Fast and accurate detection of Alzheimer’s Disease targets with SimpleStep ELISA® kits and SPECTROstar® Nano” explains the ELISA principle and shows how it was used to quantify proteins related to neurodegeneration. A research group based in Sydney/Australia used a TNF-alpha ELISA to study the effect of chronic microglial activation on the inflammatory marker. They found pro-inflammatory TNF-alpha to be elevated at all ages of a mouse model with chronic microglial activation, a feature of neurodegenerative diseases (Gyengesi et al. 2019). Recently, phospho-tau sites Thr181 and Thr217 have shown potential as biomarkers of disease. When comparing site specific phospho-tau in normal and Alzheimer’s disease brains, these biomarkers were confirmed to functionally distinguish between healthy and diseased tissue.
Instrumentation for neurodegeneration research
These studies demonstrate high throughput methods used in the research of neurodegenerative diseases. As they require various detection modes, the use of multimode plate readers is recommended for neurodegenerative disease research.
For the performance of aggregation assays, it is essential that the analysis instrumentation can shake and incubate microplates over long periods of time, up to 7 days. BMG Labtech is a technology leader in the microplate field. The multimodal plate readers include the CLARIOstar® Plus and Omega series, which have a dedicated plate carrier and provide robustness in long-term shaking aggregation measurements.
For further information on how BMG LABTECH plate readers help neurodegeneration diseases research have a look at our neuroscience research area.
References
- Lassonade M. et al. (2017). The Challenge of Neurodegenerative Diseases in an Aging Population. https://royalsociety.org/-/media/about-us/international/g-science-statements/2017-may-aging-population.pdf?la=en-GB&hash=C665B04DAB77DE2C053D8F51E61E4379
- EU Joint Programme – Neurodegenerative Disease Research. (2019). Why Choose Neurodegenerative Diseases? https://www.neurodegenerationresearch.eu/why/
- Sullivan T. (2019). A Tough Road: Cost to Develop One New Drug is $2.6 Billion; Approval Rate for Drugs Entering Clinical Development is less than 12%. https://www.policymed.com/2014/12/a-tough-road-cost-to-develop-one-new-drug-is-26-billion-approval-rate-for-drugs-entering-clinical-de.html
- Nakamura M. (2012). Real-Time Quaking Induced Conversion Assay for Prion Seeding. BMG LABTECH App Note 232.
- McGuire et al. (2016) Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic creutzfeldt-jakob disease: An international study. Ann Neurol. 2016 Jul;80(1):160-5. doi: 10.1002/ana.24679. Epub 2016 Jun 1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982084/
- Kumar M et al. (2017) Monitoring Early-Stage Protein Aggregation by an Aggregation-Induced Emission Fluorogen. Anal. Chem. 2017, 89, 17, 9322-9329. https://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b02090
- Krasil’nikova et al. 2019. Insulin Protects Cortical Neurons Against Glutamate Excitotoxicity Front. Neurosci., 24 September 2019. https://doi.org/10.3389/fnins.2019.01027
- Ooi J et al. 2015. Inhibition of Excessive Monoamine Oxidase A/B Activity Protects Against Stress-induced Neuronal Death in Huntington Disease. Mol Neurobiol. 2015; 52(3): 1850–1861. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586002/
- Paliga D et al. (2019). Lethal Factor Domain-Mediated Delivery of Nurr1 Transcription Factor Enhances Tyrosine Hydroxylase Activity and Protects from Neurotoxin-Induced Degeneration of Dopaminergic Cells Mol Neurobiol. 2019; 56(5): 3393–3403. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476859/
- Ferguson R et al. (2016) PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes. PLoS One. 2016; 11(7): e0159051. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938384/
- Gyengesi et al. (2019) Chronic Microglial Activation in the GFAP-IL6 Mouse Contributes to Age-Dependent Cerebellar Volume Loss and Impairment in Motor Function. Front. Neurosci., 03 April 2019. https://www.frontiersin.org/articles/10.3389/fnins.2019.00303/full
