In this guest post, organic chemistry graduate student Tien Nguyen discusses the findings of a recent study that will hopefully bring us closer to understanding Parkinson's Disease.

Current medical therapies for Parkinson's Disease--which affects about 1 million Americans--only treat the symptoms, with none proven to slow or reverse the disease. Now, scientists may have identified the biological process that causes it.

Scientists know that in humans, inactivation of the parkin gene leads to Parkinson's disease. But they don't know which steps occur in between. Now, researchers at Johns Hopkins University have proposed a possible chain of events.

In a study published in Nature Neuroscience, the researchers showed blocking an enzyme, called poly(ADP-ribose) polymerase-1, or PARP1, stops the development of Parkinson's disease in mice.

"We were excited and surprised by the almost complete protection against dopaminergic cell death by the PARP1 inhibitor," said Yunjong Lee, lead author of the study and post-doctoral researcher in a joint lab run by Professors Valina and Ted Dawson.

Lee and co-workers were able to protect specific brain cells called dopaminergic neurons. These neurons make dopamine, a chemical critical to motor control, arousal, and motivation. Loss over time of dopaminergic neurons is the defining feature of Parkinson's disease.

"We need to start thinking about PARP1 inhibitors," said Dr. Daniel Roque, a neurologist at the University of North Carolina at Chapel Hill School of Medicine who regularly treats patients with Parkinson's disease. About half a dozen pharmaceutical companies are testing drug candidates designed to inhibit PARP1 in human clinical trials against breast, ovarian and lung cancer. Some of these drugs have already passed phase I trials which show they aren't toxic to humans.

PARP1 inhibitors stopped disease progression in mice, but what led to the accumulation of PARP1? The researchers proposed to trace this protein accumulation back to a mutation in the parkin gene.

One of the parkin protein's functions is to maintain low levels of the AIMP2 protein. AIMP2 has an essential role in translating RNA into proteins. Parkin uses small proteins-called ubiquitins-to tag AIMP2. Tagging the AIMP2 protein with a lot of ubiquitins lets the cell know to destroy it.

When the parkin protein is not doing its job, there is a build-up of AIMP2. Brain autopsies of deceased Parkinson's patients tend to show higher levels of AIMP2.

To mimic Parkinson's disease in mice, Lee and his colleagues genetically modified a line of mice to overexpress the AIMP2 protein. They successfully observed the mice gradually producing less dopamine over time. They also saw a decrease in motor skills using the rotarod test-a standard test in which researchers record how long a mouse can walk on an accelerating, rotating rod before it falls off.

The mice showed up to a 60 percent loss of the dopamine producing brain cells, an impressive result, Lee said, given the difficulty of simulating neurodegenerative diseases in animals. This specialized mouse model gave researchers the opportunity to look for possible pathways leading to brain cell death.

The team noticed elevated levels of PAR proteins, which is an indication of PARP1 activation. They proposed that an increased amount of AIMP2 stimulates PARP1 activity. High levels of PAR are known to kill cells through a process called parthanatos-named by Professors Valina and Ted Dawson a few years earlier, after PAR and Thanatos, the Greek god of death, Lee said.

The main finding of the study, the activation of PARP1 by AIMP2, challenges conventional thinking in neurobiology because of the proteins' locations in the cell.

PARP1 resides in the cell's control center, the nucleus, while AIMP2 floats around inside a different compartment, called the cytosol. To enter the nucleus you need a special access code, called a nuclear localization sequence or NLS. Because AIMP2 does not have this code, it shouldn't be able to cross the nuclear membrane.

"We get asked this question a lot," Lee said. He said cells experiencing oxidative stress or DNA damage could allow AIMP2 into cross into the nucleus. They tested this theory by adding a chemical oxidant to the cells. This chemical can cause DNA damage and oxidize compounds comprising the cell membrane, weakening it. They observed AIMP2 in the nucleus, after it apparently crossed the nuclear membrane.

Finding AIMP2 in the nucleus doesn't prove anything by itself. The researchers needed to determine if it interacted with PARP1 once it got inside. The researchers probed this interaction using a technique called co-immunoprecipitation. Here's how they did it.

First, AIMP2 is marked with a small sequence of proteins making it easy to detect. Second, a substance that binds PARP1-called an antibody conjugate-is attached to a large bead.

The loaded bead is added to a test tube containing a cell nucleus. The cell nucleus has the specially marked AIMP2 inside. Placing the test tube inside a centrifuge-essentially a quickly rotating carousel-pushes the heaviest parts to the bottom of the tube. The rest of the cell goo not bound to the bead is washed off.

What you have left is the bead, which binds to PARP1, and anything in the cell that was bound to PARP1. In this mixture the researchers found the specially marked AIMP2 proteins.

"The authors have used the accepted techniques to demonstrate an interaction between AIMP2 and PARP. But the bigger question is whether this interaction has any biological relevance," Alexandow Gow, a neurology professor at Wayne State University, wrote to me in an email. Lots of proteins, when produced at high levels, can have new functions that they wouldn't necessarily have naturally.

"We need solid data that parthanatos is involved in Parkinson's disease," Lee said, and his research lab is looking for that connection. They want to develop mouse models for different proteins that are regulated by parkin or any of the seven genes that are associated with Parkinson's disease.

When asked if the study's results warrant testing of PARP1 inhibitors against Parkinson's disease in human clinical trials, Roque said it can be difficult to anticipate how well mouse models will translate into humans. He said more research was needed before human trials should begin.

"We're all hoping to find anything that will slow down the disease and maybe this is where the answer lies," he said.

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Tien Nguyen is an organic chemistry graduate student with an interest in science journalism at UNC-Chapel Hill.

Image Credit:

Protein PARP1 by Emw