Real Time Report: The Oxidative Stress Interactome Project

Blog post by Dr. Tamara Maiuri

We’re going live! Welcome to the first of a series of blog posts aiming to report our findings in real time. This post reports the first steps in the Oxidative Stress Interactome Project, funded by the HDSA Berman/Topper HD Career Advancement Fellowship.

The Oxidative Stress Interactome Project

Oxidative stress is something that happens in our brains as we age, and the inability to deal with it properly has been linked to neurodegenerative diseases including HD as well as Alzheimer’s and Parkinson’s. We know that the huntingtin protein interacts with many other proteins in response to oxidative stress. These proteins come together to repair DNA damaged by oxidation. What if some of those protein-protein interactions have gone wrong when the huntingtin protein is expanded, as it is in HD? That’s what we aim to find out. Then we’ll look for drugs that might fix the problem.

Setting The Scene (aka optimizing experimental set-up)

I will be purifying the huntingtin protein, and any other proteins interacting with it, from skin cells that came from real HD patients. The skin cells are grown in a plastic dish. First, I’ll treat the cells with oxidants such as hydrogen peroxide or potassium bromate, to mimic the oxidation that’s happening in the aging brain. Then I’ll compare the list of “huntingtin interactors” in the presence or absence of oxidation, to see which ones are recruited to the job under conditions of oxidative stress. Down the road, I will compare the normal and expanded huntingtin protein. But one step at a time…

The interacting proteins will be identified by a technique called mass spectrometry. I’ll explain mass spec in a future blog post, because before we get to that step, several things need to be running smoothly. For example, there’s no sense sending your samples for mass spec analysis if not enough huntingtin (and its interactors) was purified from cells in the first place!

To set the scene, I tested two aspects of the experiment:

  • How to break up the cells and get at the protein (fractionation)
  • How to keep the proteins from coming apart during the purification process (crosslinking)

In a fractionation optimization experiment that you can read about on Zenodo, I tested sonication, which basically uses sound energy to break up the cell nucleus and release DNA-bound proteins. It turns out that although sonication released more proteins from the cells, it may have disrupted protein-protein interactions or interfered with the purification of huntingtin, since less huntingtin and its interaction partners were recovered in the end. However, I could see that many more proteins interact with huntingtin upon treatment of the cells with oxidants. Those are the ones we want to identify!

After further fractionation optimization, and comparing notes with other lab members who are purifying huntingtin from cells, I settled on treatment with DNase (a protein that chews up DNA) to release the DNA-bound proteins from the cell.

In a crosslinking optimization experiment (also on Zenodo), I tested 2 different ways to link proteins together. One of the crosslinking chemicals (Lomant’s reagent) was quite finicky and came out of solution. The other (paraformaldehyde, or PFA) actually seemed to improve the cell fractionation process. Both chemicals helped keep the interacting proteins together. Since PFA gave better results, this is the crosslinking agent I’ll move forward with.

The scene is coming together, but there are a few more things to do to make sure we get the best/most informative samples of huntingtin and its interactors. These optimization steps have been done in different types of human cells, because they grow quickly and yield good amounts of protein. Skin cells from patients are not as easy to work with, but it’s time to test the system in those “fibroblasts”. That’s what I’ll be doing next. Stay tuned!

Support the Report

In April of 2017 the Canadian Government released the Fundamental Science Review report from a committee headed by David Naylor. This is the first time in decades any government in Canada has asked for an expert review of how we fund science, as these policies are historically political, and often in contradiction to expert opinion of how to best spend what few tax dollars Canada has ever dedicated to basic science research.

Our review was a highlight of failure, as a country we spend less than 0.5% of our health care spending on the CIHR annual budget, research that ironically impacts lower health care costs on Canada.  Per capita, this is 1/4 what the US spends (1/40th in total dollars), and less than all the G8. Yet despite this, for a tiny country that could fit into most US states, we have a bizarre number of agencies and programs duplicating administrative overhead costs, diverting funds from labs.

Most research grant dollars support people, well-trained people and young people who want to change the world and make life better for the sick. Somehow, priorities of historic Canadian governments have decided that the military is 15X more important for the future of Canadians than health research. As the United States enjoys a robust biotech and Pharmaceutical industry, owning 45% of the world’s pharmaceutical market, Canada’s pharma impact is mostly by outstanding scientist ex-repatriates doing research and development  in the US.

If we ever see the F-35 jets, keeping then parked in hangars every year will cost more than the entire CIHR budget per year.

We encourage The Prime Minster and Minister of Youth to reverse the historic apathy of support for Canadian Science and adopt the recommendations of the Naylor Report.

1% can make all the difference. #SupportTheReport

 

Master of Science

Congratulations to Susie Son on the successful defense of her Master’s thesis. She can now drink from the McMaster Chalice of Knowledge™.

susiedefense1

She’s off in September to Dental School at the University of Toronto.

Susie’s work has progressed to a manuscript in preparation on the interaction of huntingtin protein and HMGB1, a critical factor in DNA repair and autophagy control.

Son defense 2

Tam’s not here, she’s in Chicago at HDSA.

Dr. Tamara Maiuri Wins the Berman-Topper Family HD Career Development Fellowship Award from the Huntington’s Disease Society of America.

Tam topperDr. Maiuri’s research for the next three years will be extending from her 2017 Human Molecular Genetics publication:

Huntingtin is a scaffolding protein in the ATM oxidative DNA damage response complex.

Maiuri T, Mocle AJ, Hung CL, Xia J, van Roon-Mom WM, Truant R. Hum Mol Genet. 2017 Jan 15;26(2):395-406. doi: 10.1093/hmg/ddw395. PMID:28017939

 

This was the first manuscript to define endogenous human huntingtin as a DNA repair protein and to discover that a pathway defective in HD is in common with many genetic ataxias. This work puts forth a new hypothesis for Huntington’s disease: That the age-onset trigger of this disease is elevated Reactive Oxygen Species (ROS) damage to neuronal DNA, which is supposed to be corrected by huntingtin protein, but is actually inhibited by the mutant huntingtin protein in HD.  For these studies, Dr. Maiuri took the approach of avoiding models of disease with synthetic alleles by instead focusing on human cells derived from HD patients with typical clinical HD alleles of CAG 40-45. These new lines are now being shared with the HD research community, and importantly, these lines are not transformed to cancer cells by inhibition of P53. 

P53 is critical to understanding Huntington’s disease, as this critical node for cancer is also critical for DNA damage repair and a known activator of huntingtin protein. P53 regulated proteins, in addition to huntingtin, have been discovered as significant modifiers of HD age of onset from HD genome-wide association studies (GWAS). This 2015 manuscript in CELL:

Identification of Genetic Factors that Modify Clinical Onset of Huntington’s Disease.

Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. Cell. 2015 Jul 30;162(3):516-26. doi: 10.1016/j.cell.2015.07.003.

And this study of the significant HD genes in other CAG expansion diseases:

 

DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases.

Bettencourt C, Hensman-Moss D, Flower M, Wiethoff S, Brice A, Goizet C, Stevanin G, Koutsis G, Karadima G, Panas M, Yescas-Gómez P, García-Velázquez LE, Alonso-Vilatela ME, Lima M, Raposo M, Traynor B, Sweeney M, Wood N, Giunti P; SPATAX Network, Durr A, Holmans P, Houlden H, Tabrizi SJ, Jones L. Ann Neurol. 2016 Jun;79(6):983-90. doi: 10.1002/ana.24656

 

Indicate that when we look at humans with these diseases in an unbiased manner, the pathways that influence these diseases by decades are related to ROS control, and DNA repair, which makes sense in terms of the age-onset aspect of these diseases: as the CAG alleles are expanded, the ability of these patients to repair the everyday damage to their DNA is decreased. This is why the Truant lab is now taking a comparative Cell Biology and Chemical Biology approach to study Spinocerebellar Ataxia and Huntington’s diseases.  The other reason is that these pathways are amenable to drugs.

Consistent with humans with HD, The Truant lab also defined a role of huntingtin as a ROS sensor. A single amino acid, one of 3,144 can allow huntingtin to sense elevated ROS outside of the nucleus, to them travel inside the nucleus to DNA damage caused by ROS.

Huntingtin N17 domain is a reactive oxygen species sensor regulating huntingtin phosphorylation and localization. DiGiovanni LF, Mocle AJ, Xia J, Truant RHum Mol Genet. 2016 Sep 15;25(18):3937-3945. doi: 10.1093/hmg/ddw234.

The future remains in people, not mice. Mouse models of this disease do not get sick at typical alleles seen in the clinic. Mice only live 9 months in the wild, and a few years in the lab. The ROS stress seem in human brains is just not seen in mice.

In the past, the Truant Lab defined the hypo-phosphorylation of huntingtin N17 domain as a defect in HD, which appeared to be corrected by IKK beta kinase inhibitors.

Kinase inhibitors modulate huntingtin cell localization and toxicity.

Atwal RS, Desmond CR, Caron N, Maiuri T, Xia J, Sipione S, Truant R. Nat Chem Biol. 2011 May 29;7(7):453-60. doi: 10.1038/nchembio.582.

Now, we realize that IKK kinase inhibition and inhibition of NFKb signaling can increase ROS damage to DNA, which triggers CK2 kinase activity on huntingtin N17.

 

Dr. Maiuri’s goals will be to define exactly how huntingtin is involved in DNA damage repair, in humans. Already consistent with her work, Dr. Rachel Harding, at the University of Toronto, has been using pure, full huntingtin to discover that this protein can bind DNA.  Our hope is that Dr. Maiuri’s studies will reinforce the first fundamentally new hypothesis in HD research towards a therapeutic in almost 25 years.