2016 NETP Investigator Vignette

Title: Proteomic Methods for Analyzing Protein in Dopaminergic Neurons

Investigator: Angus Nairn, Ph.D., Yale University School of Medicine

The goal of the research supported by the DoD grant was to try to develop methods that would enable us to understand really the very initial steps that go wrong in Parkinson's disease. And we hypothesized that those might be dysfunction in mitochondria, the powerhouse of the cell that may be occurring in very, very specific parts of the cells that become dysfunctional. And so we used a culmination of genetic and proteomic methods to extract information about the mitochondria and the proteins in the mitochondria and discreet parts of the brain.

The simple model in Parkinson's disease is that the dopamine neurons, which project to the striatum that’s involved in motor control, slowly over time degenerate. So they send these axons from their cell bodies in one part of the brain and the axons go to another part of the brain. And there at those axon terminals is where they communicate with other cells that they contact. And in Parkinson's disease those terminals actually start to become less efficient and stop working generally, until actually, eventually, they’re lost completely, physically. There’s a physical loss of those connections.

One of the very critical parts of Parkinson's disease research over the last two decades has been the identification of familial genetic relationships that cause the disease. And so now we know I think there are 10 or more Park genes, and a number of them play a critical role in the function of mitochondria. And so in part because we were interested in the functionality of these nerve terminals, and in part because of the genetics that pointed toward mitochondria, we thought that mitochondria would be a good target to study because one of the very first stages in the dysfunction of the release in these dopamine neurons would be very subtle changes in the way the mitochondria works, perhaps that would give us a clue as to some of the early stages of the disease.

And so the essence is the challenge of using what we call proteomic approaches to isolate a very small--it’s like a needle in a haystack challenge, you know. How do you get the needle out of the haystack quickly without it changing very much? Well, maybe if I use a really powerful magnet, that needle will stick to the magnet and I can pull it out really quickly. And so that’s essentially what we did. We used an approach which was based on manipulating genetically the mouse model that we had only to express a particular protein called a green fluorescent protein on the outside of the mitochondria, and only mitochondria in the dopamine neurons.

And so we then took an antibody to the green fluorescent protein and we actually fixed it on magnetic beads, little iron balls, and we mixed those beads with the extract of the part of the brain that we’re interested in pulling the needle out of, and then we actually put it down a big magnet, a really powerful magnet, and so we can do that really quickly.

The other organelles that we’ve felt that we had a possibility to study were the nucleus of the cell, which contains the DNA. And then synaptic vesicles, which are organelles, much smaller organelles, that contain the neurotransmitters that are released from one nerve cell and then modulate the activity of the next nerve cell. And so in parallel, in the work that was funded, we developed tools to approach each of those three organelles. And we feel that such an approach would enable people to put protein analysis or so-called proteomics on the same numerical scale as other groups who are studying DNA and RNA molecules within the same discreet nerve cells that we’re interested in.

So the DoD program was fundamental for the whole project; the project wouldn’t have happened if it hadn't been for the support. The support was excellent. I don’t think it would really have been feasible through other normal NIH-type mechanisms at that time. So the DoD program was really a nice opportunity to fund something which was new and different for my lab, and also was new for the general field of proteomics in the nervous system.