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Marc Freeman, PhD |
Scientists at UMass Medical School have developed a new fruit fly model to investigate pathological changes in neurons with neurodegenerative mutations as they age with unprecedented resolution. This powerful new tool will allow investigators to probe the underlying causes of neurodegenerative diseases and has already been used to identify two new genes—hat-trick and xmas-2—that play an important role in motor neuron death in amyotrophic lateral sclerosis (ALS). Details of the study were published in Current Biology.
“With this fly model, we can now perform powerful, genome-wide forward genetic screens that aren’t possible in other animal models to search for degenerative events in individual neurons as they age,” said Marc Freeman, PhD, Howard Hughes Medical Institute Investigator and vice chair and professor of neurobiology. “This will help us pick apart the disease pathology so we can understand how and why diseases are triggered and how they progress—a key to developing therapeutic interventions.”
ALS is a progressive, neurodegenerative disorder affecting the motor neurons in the central nervous system. As motor neurons die, the brain’s ability to send signals to the body’s muscles is compromised. People who develop the disease begin to experience symptoms as they get older, usually between the ages of 40 and 70. Survival after the onset of symptoms is typically only three to five years. Huge strides have been made in identifying genetic mutations that cause ALS, but how these mutations affect the neurons and cause toxicity is still not fully understood.
The animal model Drosophila, more commonly known as the fruit fly, has been a powerful tool for studying nearly all aspects of basic animal biology, but also the brain and neurodegenerative diseases, such as ALS. Though they may appear quite different from us on the outside, the genes that drive development and physiological functions are remarkably similar in Drosophila and humans. Additionally, because of its short maturation period and its ability to generate large numbers of offspring quickly, the fruit fly is a great model for performing powerful genetic screens to identify the mechanisms that underlie disease biology.
The majority of current fruit fly models for studying ALS require that all neurons in the animal carry the disease mutation. This shortens the life span of the animal from the typical 30 days to only three or four. These and other models also rely on relatively crude measurements of neuron health to track disease progression, such as animal death or whole tissues destruction. This makes it difficult for scientists to understand exactly what is going wrong in sick neurons and compare it to humans, or to study how the disease progresses in individual neurons as they age in vivo.
“We wanted to find a way to better model ALS in Drosophila,” said Dr. Freeman, “a model that would allow us to visualize with high resolution subsets of neurons—and the key axonal and synaptic compartments of the neuron—so we could tease apart the cellular changes that occur as the neurons began to fail.”
The key to developing this new fly model for Freeman and colleagues was to increase the efficiency of a currently available technology known as “MARCM” or “mosaic analysis with a repressible cell marker.” MARCM is an extremely useful genetic technique for labeling individual cells in Drosophila. In particular, MARCM allows for the production of small numbers of homozygous mutant cells, which are also uniquely labeled with a green fluorescent protein (GFP). This allows for the examination of the consequences of genetic mutations on single cells in an otherwise wild type animal.
The Freeman laboratory developed new methods for generating these fly clones with high efficiency in motor neurons (the neurons that degenerate in ALS) that can be visualized directly through the fly cuticle. That step also facilitated their performing rapid screens to identify genes that modify disease, and opened the door to examining how motor neurons changed over time as the fly aged.
Using the new model, Freeman and colleagues were able to demonstrate that mutations in the RNA-processing protein TDP-43, a gene known to cause ALS, leads to age-dependent dying of motor neurons in the Drosophila as the flies aged. They next searched for genes that were required for the human mutant TDP-43 to exert these pro-degenerative effects and found three genes, hat-trick, xmas-2 and GSK3, a previously known contributor to ALS progression, that when eliminated, suppressed the toxic effects of TDP-43. This suggests that blocking the activity of these newly identified genes in patients may be used to potentially stall onset or progression of ALS.
“The model we’ve created offers far greater cellular precision and can be viewed at a higher resolution that what is currently available,” said Freeman. “In addition to TDP-43, the system can be used to model any motor neuron disease in live motor neurons, and we can then quickly screen for new genes required for toxicity.
“We are excited that this model has already yielded two new molecules required for TDP-43-mediated toxicity that can potentially be targeted for therapeutic benefit,” Freeman added.
The next step for Freeman and colleagues is to see if their newly identified genes are also required for TDP-43 toxicity in mouse and human models of disease. In collaboration with Robert H. Brown Jr., DPhil, MD, the Leo P. and Theresa M. LaChance Chair in Medical Research, chair and professor of neurology at UMMS, Freeman will continue to use the model to further explore the cellular effects other ALS mutations have on motor neurons.
