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Turning Off Mutant TDP-43 Stops ALS in an Animal Model

Talan, Jamie

doi: 10.1097/01.NT.0000413077.17444.76
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Investigators reported that in a transgenic rat model of ALS, mutant TDP-43 expressed in the motor neurons alone was sufficient to cause paralysis. When the investigators halted the expression of the abnormal protein, the rats with limited loss of motor neurons damage showed “a dramatic recovery of motor function.”

Sometimes a rat is mightier than a mouse. At least that's the way some neuroscientists see it. A team of investigators at Thomas Jefferson University developed many different lines of transgenic rats expressing the human mutant form of TAR DNA binding protein 43 (TDP-43) that is linked to amyotrophic lateral sclerosis (ALS). They showed that turning off expression of the mutant early enough stops disease progression and partially restores movement.

The finding, reported in the January Journal of Clinical Investigation, proves for the first time that the mutant TDP-43 expressed in the motor neurons alone was sufficient to cause paralysis. What's more, when the investigators halted the expression of the abnormal protein, the rats with limited loss of motor neurons damage showed “a dramatic recovery of motor function,” said Xu-Gang Xia, MD, PhD, associate professor of pathology and lead investigator of the study. The disease stopped progressing after the mutant gene was turned off even in the rats with profound loss of motor neurons.

Experts say that it is a great leap to even think about such a strategy in ALS patients. For now, it is not possible to turn genes on and off. But it is a proof of concept that it may be possible to develop drugs that turn off the expression of the mutant protein and deliver the drug early enough to prevent paralysis and stop the degeneration of motor neurons.

“We do not have targets for TDP-43 but we are moving forward to look for substances that could block this mutant protein in humans,” said Dr. Xia.

He said that he has always felt that rats are better research tools, which is why he set out to create transgenic ALS rat models. “Rats have bigger brains, more genes and they are easier to use in behavioral studies,” he said. He added that the mouse models do not recapitulate the motor neuron damage that they have created in their new rat model.

“In humans, we never know if it is one gene or one cell type that leads to ALS,” he explained. “But our study showed that the expression of the human mutant form of TDP-43 in the motor neurons was enough to cause paralysis and when we turned off the gene we stopped the disease progression. That means if we can find a therapy that targets TDP-43 and give it early enough we may be able to prevent ALS from progressing.”

The animals that were partially rescued from this disease did not die from this disease, he added.

The researchers used transgenic techniques to overexpress TDP-43 in motor neurons, all neurons and skeletal muscles. The overexpression in all types of neurons and in skeletal muscle led to severe damage in motor axons and skeletal muscle but there was limited motor neuron loss. When they restricted transgene overexpression to the motor neurons alone, there was a marked loss of motor neurons at the end stages of the disease. More specifically, they turned on the mutant TDP-43 at 60 days and within two weeks the animals began losing grip strength and mobility. A week later, they were losing their ability to retract their hind legs.



To switch on and off the transgene, they used a tetracycline regulatory system, a well-established technique that is widely used in transgenic animal models. To shut off expression of mutant TDP-43, they added doxycycline (Dox) to the rat's diets. It works like a genetic switch to shut off the mutant gene. With the mutant gene shut down, there was no more damage and death to motor neurons.



When the animals died, the investigators were able to count the number of motor neurons in a long segment of the lumbar cord and compare all of the rat lines they created. They found a remodeling of motor units in those Dox-treated transgenic rats.

The experiments offer hope that a similar technique could one day be used in patients in the earliest stages of the disease when motor neuron damage is limited.

At first, it was thought that TDP-43 was a DNA-binding protein but newer research suggests that TDP-43 binds RNA and regulates splicing and stabilization of RNAs. How it exactly works is not yet known. There have been more than a dozen different TDP-43 mutations in ALS patients identified in the last several years. Having one bad copy is enough to cause the disease.

Dr. Xia and his colleagues are now screening libraries of compounds to see if any medicines stop the gene from turning on the mutant TDP-43 protein. Abnormal clumps of the protein have been identified in some patients with ALS or frontotemporal dementia, a discovery first reported in 2006 in Science by University of Pennsylvania investigators John Trojanowski, MD, PhD, and Virginia Lee, PhD.

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Edward B. Lee, MD, PhD, assistant professor in the department of pathology and laboratory medicine in the Division of Neuropathology in the Perelman School of Medicine at the University of Pennsylvania, studied under Dr. Lee and Dr. Trojanowski, and worked with them in developing the TDP-43 mouse model.

Dr. Lee heads the Translational Neuropathology Research Laboratory where he is studying ALS and Alzheimer disease and how certain metabolic activities affect disease states.

“This is a beautiful paper,” he said of the JCI study. “The study validates the importance of TDP-43. This group showed that they could stop the disease from progressing. If we can figure out a way to target the underlying mechanisms of disease early enough in patients we may be able to halt disease progression.”

In the transgenic mouse developed at the University of Pennsylvania, they turned on the mutated TDP-43 gene to provoke disease but they did not turn it off. “This is the first time that scientists turned off the gene and showed that it can halt the disease process.”

But, he added, “we don't know how to turn genes on and off in humans.”

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• Huang C, Tong J, Xia Z, et al. Mutant TDP-43 in motor neurons promotes the onset and progression of ALS in rats. J Clin Invest 2012:122:1;107-118.
    • Lee EB, Lee VM, Trojanowski JQ. Gains or losses: molecular mechanisms of TDP-43-mediated neurodegeneration. Nature Rev Neurosci 2011; 13(1):38-50.
      • Igaz LM, Kwong L,Lee, EB, et al. Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. J Clin Invest 2011:121(2);726-738
        ©2012 American Academy of Neurology