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)”.
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”.