Due to extended life expectancy across the world, the incidence of age-related neurodegenerative diseases is on the rise in most industrialized countries.1 Consequently, conditions characterized by mild, moderate, or severe cognitive impairment are becoming a major socioeconomic burden on the society. According to a recent report, the annual cost associated with mental and neurological disorders and diseases in the US exceeds $1.5 trillion.2 Current efforts of the medical research community are aimed at identifying pharmaceutical agents capable of postponing or controlling the symptoms of neurodegenerative diseases, which affect everyday life and functioning of the elderly population.
The central nervous system (CNS) is very sensitive to oxidative stress because it abounds in oxidant substrates and has a relatively low content of antioxidant enzymes.3 Reactive oxygen species (ROS) – induced oxidative damage of neurons has been implicated in the pathogenesis of a wide range of neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease.1,4 Developing therapeutic agents capable of maintaining cerebral redox homeostasis and boosting the antioxidant capacity of brain cells is one of the strategies for battling neurodegenerative diseases.5
On this path, the identification of biological materials and compounds capable of exerting protective effects against ROS-induced neurodegeneration has become of great interest to the food, biotech and pharma companies, as well as the general scientific community. To help in the identification of compounds with neuroprotective effects, scientists have developed a range of in vitro cell-based assays. This article will give an overview of three such assays conducted on human neuroblastoma cells or neuron cells.
Glutathion expression level as a marker of antioxidant status of neurons. Glutathione (GSH) is an endogenous (intracellular) tripeptide, thiol antioxidant that protects important cellular components from ROS-induced oxidative damage by providing reducing equivalents for the reduction of lipid hydroperoxidases. GSH is one of the most efficient and abundant antioxidants; intracellular concentrations of GSH usually range from 1 to 10 mM.6 The concentration of GSH inside the brain cells is typically between 1 and 2 mM.2 The depletion of GSH and a disbalance in the regulation of GSH-related enzymes such as glutathione transferase, has been associated with the onset of both Parkinson’s and Alzheimer’s disease. This assay evaluates the effect of a test material on brain cell protection from buthionine sulfoximine (BSO)/H2O2 – induced oxidative stress by assessing the GSH level as a marker of antioxidant status of neurons.
Glial fibrillary acidic protein (GFAP) expression level as a marker of strength and function of neurons. Glial fibrillary acidic protein is an important intermediate filament protein that belongs to the same family of proteins as vimentin and nestin, which serve largely cyto-architectural functions.7 GFAP is expressed in astrocytes – characteristic, star-shaped glial cells of the CNS, as well as is certain other types of CNS cells such as ependymal cells. Although the mechanism is not fully understood, it has been determined that normal expression of GFAP is required for the structural integrity and mechanical strength of astrocytes, as well as long-term maintenance of myelination.8 In research studies, GFAP is utilized as a marker for immunohistochemical identification of astrocytes, especially reactive astrocytes that are responding to some type of CNS injury.7 This assay monitors the GFAP expression triggered by buthionine sulfoximine (BSO)/H2O2 – induced oxidative stress and evaluates the effect of a test material on brain cell protection. Upon exposing the neurons to oxidative stress, the test material’s neuroprotective effects are quantitatively reflected by the GFAP expression levels.
Assessing protective effects against glutamate toxicity. Glutamate is the primary excitatory neurotransmitter in the CNS; it has a critical role in a range of regulated cognitive functions including learning and memory.9 While an optimal dose of glutamate is needed for normal brain functioning, a disbalance in glutamate levels (excessively low or high concentrations) can trigger neurotoxic or excitotoxic cascades and have deleterious consequences.4 Glutamatergic neurotransmission in the CNS is facilitated by ionotropic and metabotropic glutamate receptors,8 of which the N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors have been specifically recognized for their roles in glutamate signaling. In this assay, human neuroblastoma cells are treated with pathologically high levels of glutamate and the effect of a test material on the extent of DNA damage is monitored via γH2AX – a marker of DNA double strand breaks and genomic instability. The assay allows for quantitative assessment of a test material’s neuroprotective properties against glutamate toxicity.
The pharmaceutical industry utilizes cell-based assays, in the scope of preclinical testing, in the identification of potential candidates for new product development. Cell-based assays have already been proven effective in testing the functionality of biological molecules in vitro, including their immunomodulatory, anticancer, and neuroprotective properties. The food industry utilizes cell-based assays in the identification of biologically active components of food products.
In the wake of increasing socioeconomic burden of neurodegenerative diseases, Brunswick Labs is committed to staying at the forefront of research efforts and has developed and implemented screening assays to assess the neuroprotective properties of therapeutic agents, dietary supplements and functional food components.
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Author Jasenka Piljac Zegarac is a scientist and freelance writer. She holds a PhD in biology and a BS degree in biochemistry, and contributes on a regular basis to several health and science blogs. Her research articles have gathered more than 1200 citations. She may be contacted for assistance with a variety of science and medical writing projects. Find Jasenka on LinkedIn.