Author: raytruant

Blog post by Dr. Tamara Maiuri

In a previous post, I described two new hypotheses generated by the findings of the HDSA-funded project looking for DNA repair-relevant huntingtin interacting proteins: that huntingtin binds poly ADP ribose (PAR), and that PAR signaling is dysregulated in HD. Last time, I reported our preliminary results supporting the first hypothesis. In the current post, I’ll report our first solid clue that PAR signaling may be dysregulated in HD: cells from HD patients have higher levels of PAR.

Let’s take a step back and look at the bigger picture for a moment. We know that suboptimal DNA repair plays a role in many neurodegenerative diseases [1–3], and DNA repair genes are implicated in HD symptom onset [4–6]. PAR synthesis is one of the first steps of DNA repair: PARP proteins recognize breaks in DNA and string together chains of PAR to help recruit the DNA repair machinery. If all goes well, the damage is repaired, the PAR is broken down for recycling, and the cell can go about its business as usual.

If the DNA is not repaired properly, then PARPs keep trying to bring in the recruits–they keep generating PAR. This wouldn’t be a huge problem, except that it seriously messes with the cell’s energy factories, aka mitochondria, in a number of ways. First off, the raw material that PARPs use to make PAR (called NAD+) is also needed by mitochondria to convert food into energy. If PARPs use up all the NAD+ stores, then cells undergo “energetic collapse” and die [7]. Bad news for high-energy-burning neurons. To make matters worse, the PAR produced in the nucleus also travels out to mitochondria and signals them to carry out a form of programmed cell death called Parthanatos [8]. Additional “nucleus-to-mitochondria death signaling” is thought to contribute to neuronal death in a number of neurodegenerative diseases [9,10]. In fact, many of the phenotypes we see in HD models and tissues could be explained by energy-draining hyper-PARylation, including protein aggregation [11–13], ATP depletion [14], and mitochondrial dysfunction [15].

We know that the expanded huntingtin protein is the cause of HD. We also know that huntingtin physically locates to sites of DNA damage and scaffolds DNA repair proteins [16], and that neurons are exposed to more and more DNA-damaging reactive oxygen species (ROS) as we age [17]. So, it makes sense to hypothesize that

Suboptimal mutant huntingtin function in the repair of nuclear DNA leads to hyper-PARylation in the high-ROS-load neurons of the striatum.

Suboptimal DNA repair by expanded huntingtin would have some observable consequences. For one, there would be more damaged DNA accumulating in HD tissues. This is been seen in patient cells by us [16] and others [18]. Secondly, we would expect hyper-PARylation if PARPs are constantly recognizing DNA breaks and generating PAR. That’s what Truant lab member Carlos Barba Bazan tested in these experiments deposited to Zenodo. Carlos found that PAR levels are elevated in two different HD patient cell lines compared to control.

We’re pretty excited about this finding and what it might mean. Is hyper-PARylation the culprit behind neuronal death in HD brains? We have more experiments to do before we know the answer to that. But one important clue is that a PARP inhibitor was beneficial in an HD mouse model [19,20]. PARP inhibitors are commonly used drugs in the cancer field, which means there’s a tonne of information about them, and many have already been tested for safety. We have a ways to go before we know whether any of these drugs might be suitable to treat HD. We will share all of our results about how important PAR signaling might be to HD as soon as we get them!

1. Maynard S, Fang EF, Scheibye-Knudsen M, Croteau DL, Bohr VA. DNA Damage, DNA Repair, Aging, and Neurodegeneration. Cold Spring Harb Perspect Med. 2015;5. doi:10.1101/cshperspect.a025130
2. Leandro GS, Sykora P, Bohr VA. The impact of base excision DNA repair in age-related neurodegenerative diseases. Mutat Res. 2015;776: 31–39.
3. Jeppesen DK, Bohr VA, Stevnsner T. DNA repair deficiency in neurodegeneration. Prog Neurobiol. 2011;94: 166–200.
4. Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. Identification of Genetic Factors that Modify Clinical Onset of Huntington’s Disease. Cell. 2015;162: 516–526.
5. Bettencourt C, Hensman-Moss D, Flower M, Wiethoff S, Brice A, Goizet C, et al. DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases. Ann Neurol. 2016;79: 983–990.
6. Moss DJH, Pardiñas AF, Langbehn D, Lo K, Leavitt BR, Roos R, et al. Identification of genetic variants associated with Huntington’s disease progression: a genome-wide association study. Lancet Neurol. 2017;16: 701–711.
7. Cantó C, Menzies KJ, Auwerx J. NAD(+) Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus. Cell Metab. 2015;22: 31–53.
8. David KK, Andrabi SA, Dawson TM, Dawson VL. Parthanatos, a messenger of death. Front Biosci . 2009;14: 1116–1128.
9. Narne P, Pandey V, Simhadri PK, Phanithi PB. Poly(ADP-ribose)polymerase-1 hyperactivation in neurodegenerative diseases: The death knell tolls for neurons. Semin Cell Dev Biol. 2017;63: 154–166.
10. Fang EF, Scheibye-Knudsen M, Chua KF, Mattson MP, Croteau DL, Bohr VA. Nuclear DNA damage signalling to mitochondria in ageing. Nat Rev Mol Cell Biol. 2016;17: 308–321.
11. Dahl J-U, Gray MJ, Jakob U. Protein quality control under oxidative stress conditions. J Mol Biol. 2015;427: 1549–1563.
12. Squier TC. Oxidative stress and protein aggregation during biological aging. Exp Gerontol. 2001;36: 1539–1550.
13. Weids AJ, Ibstedt S, Tamás MJ, Grant CM. Distinct stress conditions result in aggregation of proteins with similar properties. Sci Rep. 2016;6: 24554.
14. Mochel F, Haller RG. Energy deficit in Huntington disease: why it matters. J Clin Invest. 2011;121: 493–499.
15. Carmo C, Naia L, Lopes C, Rego AC. Mitochondrial Dysfunction in Huntington’s Disease. Adv Exp Med Biol. 2018;1049: 59–83.
16. Maiuri T, Mocle AJ, Hung CL, Xia J, van Roon-Mom WMC, Truant R. Huntingtin is a scaffolding protein in the ATM oxidative DNA damage response complex. Hum Mol Genet. 2017;26: 395–406.
17. Mattson MP, Magnus T. Ageing and neuronal vulnerability. Nat Rev Neurosci. 2006;7: 278–294.
18. Askeland G, Dosoudilova Z, Rodinova M, Klempir J, Liskova I, Kuśnierczyk A, et al. Increased nuclear DNA damage precedes mitochondrial dysfunction in peripheral blood mononuclear cells from Huntington’s disease patients. Sci Rep. 2018;8: 9817.
19. Cardinale A, Paldino E, Giampà C, Bernardi G, Fusco FR. PARP-1 Inhibition Is Neuroprotective in the R6/2 Mouse Model of Huntington’s Disease. PLoS One. 2015;10: e0134482.
20. Paldino E, Cardinale A, D’Angelo V, Sauve I, Giampà C, Fusco FR. Selective Sparing of Striatal Interneurons after Poly (ADP-Ribose) Polymerase 1 Inhibition in the R6/2 Mouse Model of Huntington’s Disease. Front Neuroanat. 2017;11: 61.

Blog post by Dr. Tamara Maiuri

Previously I explained our rationale for hypothesizing that huntingtin may bind poly ADP-ribose (PAR). If so, this could be the way it gets to damaged DNA, and this might be dysregulated in HD. Since we know DNA repair genes impact whether HD patients get sick early or late in life, this is a good place to look for problems—then we can look for drugs that fix the problems.

But first things first: does huntingtin bind PAR? There are many “domains” or regions of proteins that are capable of binding PAR: PAR-binding motifs, macrodomains, and WWE domains to name a few. A quick scan of the huntingtin sequence revealed four potential PAR-binding motifs. We can look at the recently solved huntingtin structure to get some more clues:

This is pure speculation at this point—many regions of the protein were left out of this structure so it’s too early to know for sure—but it’s fun to guess: the barrel of the huntingtin solenoid is the right size to accommodate DNA, as it is the same size as the DNA binding regions of other DNA repair proteins such as MSH2, MSH6, and PCNA (pictured above). Similar to MSH2, three of the potential PAR-binding motifs within huntingtin are exposed to the outer surface of the DNA binding region, suggesting a mechanism by which huntingtin is recruited by PAR, followed by direct binding to DNA. (Direct binding of purified full-length huntingtin to DNA has been shown by Dr. Rachel Harding and deposited to Zenodo: https://zenodo.org/record/801606#.Wr4eG5PwZ-V).

One way to get a clue about this is to immobilize huntingtin protein on a membrane, then overlay it with purified PAR polymer. If huntingtin binds PAR, it too will be stuck to the membrane. After washing away unbound PAR, you can detect whatever is left with an anti-PAR antibody. This is called a PAR overlay assay.

The first few experiments (deposited to Zenodo) look promising. Purified full length huntingtin (produced by Dr. Harding) reproducibly bound PAR in several experiments, as did a fragment made up of amino acids 78-426 (which conveniently contains one of the potential PAR binding motifs mentioned above). I also tried two preparations of expanded huntingtin (Q46 and Q54) in one experiment, with confusing results: huntingtin Q54 bound PAR while huntingtin Q46 did not. This could be because the Q46 prep was from an older stock—I’m currently repeating the experiment to find out what’s going on.

These are very early results, and I need to make mutations in the potential PAR-binding motifs to see whether they are specifically mediating the PAR interaction, or whether this is nonspecific binding. But we may be on to something here… in the next blog post I’ll share more preliminary data implicating PAR in huntingtin chromatin recruitment in cells.

Blog post by Dr. Tamara Maiuri

Forgive me readers for I have been busy, it has been 3 months since my last blog post. During this time, we published a very exciting story identifying a lead compound and how we think it works in HD. Also during this time, I have gone through the list of huntingtin interactors identified in my HDSA-funded project to find drug targets that are relevant to the process of DNA repair. We now have some interesting results!

As explained in my last post, the original aim to identify huntingtin interactors from human cells was not technically feasible and mouse cells were used instead. Several hundred protein-protein interactions were detected, then the list was further refined by only considering high and medium confidence hits. We now have a list of 314 proteins that interact with huntingtin reproducibly across biological replicates. See the end of this post for the lists of ROS-dependent interactors (Tables 1-3), proteins that interacted with huntingtin only in untreated cells (Table 4), and proteins found to be modified by poly ADP-ribose (PARylated proteins; Table 5). These tables have also been deposited to Zenodo.

Indeed, the most notable finding of the ROS-dependent interactome analysis was the high degree of overlap with datasets of “PARylated” proteins. PAR is of interest to us because it is generated in response to DNA damage, and acts to recruit DNA repair proteins to damage sites.

This result led us to pursue two hypotheses with the aim of identifying drug targets relevant to DNA repair:

Huntingtin is a PAR-binding protein: Huntingtin may use PAR binding to interact with PARylated proteins. If so, this is likely to be the mechanism by which huntingtin interacts with chromatin and assembles DNA repair factors in its role as a scaffold, and this may be dysregulated in HD.

PAR signaling is dysregulated in HD: Regardless of whether huntingtin physically binds PAR, it is still possible that we have uncovered a previously unexplored aspect of HD pathology: there is strong genetic evidence that DNA repair genes influence disease [1-3], huntingtin participates in the process of DNA repair [4], and elevated levels of damaged DNA are common to HD tissues and models [4-8].

In response to DNA damage, PAR chains are the first scaffold generated for DNA repair factor assembly: a sticky web bound by proteins involved in DNA repair. So it’s possible that in HD, excess DNA damage accumulates due to sub-optimal huntingtin function in the repair process, leading to Poly ADP-Ribose Polymerase (PARP) hyperactivation and elevated PAR levels.

High PAR levels could not only interfere with huntingtin function if it binds PAR, but also cause an “energy crisis” in high-energy-consuming neurons [9-11]. In fact, energy deficits have been frequently observed across HD models and in HD patients [12]. Similarly, PARP hyperactivation is linked to cerebellar ataxia [13], and PARP inhibition has been reported to improve phenotypes in a HD mouse model, although by a different mechanism [14,15].

In the coming blog posts, I will share preliminary data that suggests huntingtin can bind PAR in a test tube, and that PAR is at least partially responsible for huntingtin recruitment to chromatin. We have also detected increased amounts of PAR and of chromatin-bound huntingtin in cells from an HD patient. Stay tuned!

References:

1. Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. Identification of Genetic Factors that Modify Clinical Onset of Huntington’s Disease. Cell. 2015;162: 516–526.
2. Bettencourt C, Hensman-Moss D, Flower M, Wiethoff S, Brice A, Goizet C, et al. DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases. Ann Neurol. 2016;79: 983–990.
3. Moss DJH, Pardiñas AF, Langbehn D, Lo K, Leavitt BR, Roos R, et al. Identification of genetic variants associated with Huntington’s disease progression: a genome-wide association study. Lancet Neurol. 2017;16: 701–711.
4. Maiuri T, Mocle AJ, Hung CL, Xia J, van Roon-Mom WMC, Truant R. Huntingtin is a scaffolding protein in the ATM oxidative DNA damage response complex. Hum Mol Genet. 2017;26: 395–406.
5. Bogdanov MB, Andreassen OA, Dedeoglu A, Ferrante RJ, Beal MF. Increased oxidative damage to DNA in a transgenic mouse model of Huntington’s disease. J Neurochem. 2001;79: 1246–1249.
6. Kovtun IV, Liu Y, Bjoras M, Klungland A, Wilson SH, McMurray CT. OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells. Nature. 2007;447: 447–452.
7. Enokido Y, Tamura T, Ito H, Arumughan A, Komuro A, Shiwaku H, et al. Mutant huntingtin impairs Ku70-mediated DNA repair. J Cell Biol. 2010;189: 425–443.
8. Askeland G, Dosoudilova Z, Rodinova M, Klempir J, Liskova I, Kuśnierczyk A, et al. Increased nuclear DNA damage precedes mitochondrial dysfunction in peripheral blood mononuclear cells from Huntington’s disease patients. Sci Rep. 2018;8: 9817.
9. Morales J, Li L, Fattah FJ, Dong Y, Bey EA, Patel M, et al. Review of poly (ADP-ribose) polymerase (PARP) mechanisms of action and rationale for targeting in cancer and other diseases. Crit Rev Eukaryot Gene Expr. 2014;24: 15–28.
10. Andrabi SA, Umanah GKE, Chang C, Stevens DA, Karuppagounder SS, Gagné J-P, et al. Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis. Proc Natl Acad Sci U S A. 2014;111: 10209–10214.
11. Fouquerel E, Goellner EM, Yu Z, Gagné J-P, Barbi de Moura M, Feinstein T, et al. ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion. Cell Rep. 2014;8: 1819–1831.
12. Dickey AS, La Spada AR. Therapy development in Huntington disease: From current strategies to emerging opportunities. Am J Med Genet A. 2017; doi:10.1002/ajmg.a.38494
13. Hoch NC, Hanzlikova H, Rulten SL, Tétreault M, Komulainen E, Ju L, et al. XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia. Nature. 2017;541: 87–91.
14. Cardinale A, Paldino E, Giampà C, Bernardi G, Fusco FR. PARP-1 Inhibition Is Neuroprotective in the R6/2 Mouse Model of Huntington’s Disease. PLoS One. 2015;10: e0134482.
15. Paldino E, Cardinale A, D’Angelo V, Sauve I, Giampà C, Fusco FR. Selective Sparing of Striatal Interneurons after Poly (ADP-Ribose) Polymerase 1 Inhibition in the R6/2 Mouse Model of Huntington’s Disease. Front Neuroanat. 2017;11: 61.

Blog post by Dr. Tamara Maiuri

In my last real-time report of the HDSA-funded project to identify of oxidation-related huntingtin protein-protein interactions, I was happy to report the successful purification of huntingtin and its interacting proteins from mouse cells. I was quite optimistic that the experiment would work using cells from an HD patient. This turned out not to be the case. Despite growing large amounts of cells, there was simply not enough starting material. Although we want to answer our questions about HD using human sources of information, it is just not technically possible with patient fibroblasts.

The good news is that I was able to generate two more replicates of the experiment in mouse cells. The total list of proteins identified by mass spectrometry can be found on Zenodo, and further refinement of the data was done by quantifying the intensity of each peptide (bit of protein) to give us a better sense of the most abundant hits. This has also been deposited on Zenodo.

Sifting through the data is taking some time—being a scaffold, huntingtin interacts with several hundred proteins. We are also in the final revision stages of a few manuscripts for which experiments have been prioritized (one manuscript describes how we turned HD patient skin cells into a tool for the HD research community—a pre-print can be read on Bioarchive). I will post a more detailed analysis in the coming weeks, but here are some general conclusions from the most reproducible results:

Stable interactions:

The proteins that interact with huntingtin in cells treated with DNA damaging agents also interact with huntingtin in untreated cells. This could be because

• The treatment didn’t work, or the untreated cells are under an unintended form of stress
• Huntingtin transiently “samples” interactions with many proteins in unstressed conditions, which it binds more tightly upon stress. In this case, the cross-linking step may cause us to capture weak interactions
• Some of the interactions may be non-specific artifacts of the experimental set-up

These possibilities will be tested by following up on interesting hits in our human fibroblast system.

A connection to poly ADP ribose:

Many of the proteins that interact with huntingtin are also found in data sets of “PARylated” and “PAR-binding” proteins (see references below). Poly ADP ribose, or PAR, is a small biomolecule that plays a role in the process of DNA repair (among many other cellular processes). When the DNA repair protein “PARP1” notices some damaged DNA, it starts to attach chains of PAR to nearby proteins. This forms a sort of net to recruit other DNA repair factors. The overlap between our list of huntingtin interacting proteins and PARylated/PAR-binding proteins suggests that huntingtin may also bind PAR, just like many other DNA repair proteins. In fact, I have preliminary results suggesting it does just that. I will post them soon!

Data sets of PARylated and PAR-binding proteins:

Gagné J-P, Isabelle M, Lo KS, Bourassa S, Hendzel MJ, Dawson VL, et al. Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes. Nucleic Acids Res. 2008;36: 6959–6976.

Jungmichel S, Rosenthal F, Altmeyer M, Lukas J, Hottiger MO, Nielsen ML. Proteome-wide identification of poly(ADP-Ribosyl)ation targets in different genotoxic stress responses. Mol Cell. 2013;52: 272–285.

Zhang Y, Wang J, Ding M, Yu Y. Site-specific characterization of the Asp- and Glu-ADP-ribosylated proteome. Nat Methods. 2013;10: 981–984.

Blog post by Dr. Tamara Maiuri

Well it’s been a long haul, but I’m happy to say I finally have a list of proteins that interact with the huntingtin protein (expanded versus normal) under conditions of reactive oxygen species (ROS) stress. This is the very first step to achieving the goal of the project: to identify drug targets that are relevant to the process of DNA repair, which, through powerful genetic studies, has been repeatedly implicated in the progression of HD.

This first step was not without its obstacles. The goal at the outset was to identify proteins out of real HD patient cells, a more relevant system than cells from an HD mouse model. Unfortunately, it’s nearly impossible to grow up enough cells to yield the protein needed for mass spectrometry. My solution to this problem was to treat cells in batches, snap freeze them, and store them for processing once I had enough.

After working out the conditions for cross-linking and fractionation, inducing oxidative stress, and pulling huntingtin-associated proteins out of HD patient cells, I started growing up batches of cells. On the day I harvested the largest batch yet, the ROS inducer, 3NP, didn’t show the tell-tale signs of working (floating cells, larger cell pellet). When I tested a sample for interacting DNA repair proteins, I found almost no interaction. That was a bad day. This batch cannot be used–it amounted to a waste of time and resources. I spent a few weeks trying to figure out what went wrong with the 3NP, but no dice.

At this point, it was time to consider options and cut losses: we need to move on with this project. So, I considered switching to mouse striatal cells. They may not be as accurate a model as human fibroblasts, but we can get a list of huntingtin-interacting proteins from mouse cells and verify them in human cells. I revisited the other ROS sources tested in the past, and decided on H2O2 (see the optimization experiment on Zenodo).

It was much faster growing up enough striatal cells for mass spec analysis. The experiment wasn’t perfect–the untreated HD cells showed undue signs of stress, and so this will have to be repeated to be sure of our results. But we now have a list! Here are the preliminary results, in a nut shell:

After eliminating likely false positives (ribosomal proteins, chaperones, cytoskelton), there are:

• 92 proteins that interact with huntingtin under basal conditions and are released upon ROS stress
  • 36 of these are inappropriately maintained by expanded huntingtin
• 38 new interactions formed upon ROS stress
  • 29 of which do not happen with expanded huntingtin
• 52 proteins that interact with expanded, but not normal, huntingtin upon ROS stress

Of note, HMGB1 was identified in the immunoprecipitates from H2O2-treated cells (both Q7 and Q111), which we have previously identified as a huntingtin interacting protein. Further, the list of proteins from H2O2-treated Q111 cells includes FEN1, PCNA, Formin1 (the actin binding protein required for DNA repair), CENPJ, PARP8, and many other DNA repair proteins. The full list and experimental conditions are available on Zenodo.

The good news is that we now know these conditions worked very well in the mass spec analysis, and it may be feasible to grow up enough human cells after all. Since our TruHD-Q43Q17 cells (from a patient with 43 CAG repeats) grow the fastest, I started with those. Last week, I sent samples from the TruHD-Q43Q17 cells treated with a DNA damaging agent called MMS for mass spec analysis. It will take a few more weeks to get enough TruHD-Q21Q18 cells (from a spousal control). Stay tuned for the results!

This project is funded by the HDSA Berman/Topper HD Career Advancement Fellowship. 

Blog post by Dr. Tamara Maiuri

Image credit: Justinas

Previously I described a method to measure DNA repair capacity in cells: the GFP reactivation assay. This worked nicely in mouse striatal cells, with HD cells consistently showing about half the repair capacity of wild type cells. I have since tried it in cells from HD patients, using a different method to measure the GFP signal (microscopy instead of flow cytometry). The results were similar: a lower repair capacity was seen in HD cells (see experiment on Zenodo). The difference wasn’t as big as with mouse striatal cells, which is to be expected from clinically relevant CAG lengths compared to a model system that exaggerates the effect of expanded huntingtin. But the experiment was done twice with very similar results each time. In the coming weeks I will test whether this small but consistent difference is exacerbated by treating cells with DNA damaging agents. I will also make sure we’re measuring DNA damage pathways, and not some other phenomenon, by knocking down or inhibiting PARP. Stay tuned…

I also previously reported a way to visualize huntingtin protein at sites of DNA damage: stable cell lines expressing an inducible, huntingtin-specific YFP-tagged intrabody. I’m happy to say that the stable cell lines are growing, albeit slowly. If the growth rates recover, we will have available TruHD-Q21Q18, TruHD-Q41Q17, TruHD-Q43Q17, and TruHD-Q50Q40 cell lines in which huntingtin protein can be visualized in real time by addition of doxycycline to the media. The slow growth may be because of the combined toxicity of nucleofection and G418 selection, or due to leaky expression of the intrabody, which interferes with cell division. I’m currently testing the first idea by lowering the G418 concentration. If this doesn’t work, I may have to use alternate methods of detecting endogenous huntingtin. Fingers crossed!

Blog post by Dr. Tamara Maiuri

I am still busily collecting cells to be sent for mass spec for our goal of obtaining a list of proteins that interact with huntingtin upon oxidative DNA damage. Unfortunately I’ve run into a few road blocks, which I will blog about in the coming weeks (hopefully with a resolution!).

Meanwhile, I’ve been working on methods to assess the hit proteins for their physiological relevance as potential drug targets. Last time I described one such approach: the GFP reactivation assay. Since then, data from 3 experiments have been combined and look promising. While repair efficiency varies from experiment to experiment, mouse HD cells consistently show approximately half the repair efficiency of normal cells (an average of 44.8% over 3 experiments). This is a readout we can use to test the effects of manipulating our hit proteins.

Another approach involves measuring how long huntingtin hangs around at sites of DNA damage, and whether expanded huntingtin lingers too long. We know expanded huntingtin has no trouble reaching damaged DNA, so maybe the problem is that it can’t get off, inappropriately gluing down all the proteins it is scaffolding.

To test this hypothesis, we first need a way to visualize huntingtin protein at sites of DNA damage. While most researchers use overexpression (getting cells to generate protein from externally supplied DNA) to visualize their protein of interest, this is very difficult with huntingtin because of its huge size. To get around this, many HD researchers express small fragments of the huntingtin protein. Overexpression of any protein can have pitfalls because it’s impossible to know if the overexpressed protein is behaving the way it would under normal expression levels in the cell. This is especially true if you’re only using a fragment of the protein—what if the fragment doesn’t fold into the same shape that it would as a whole? What if the missing parts of the protein interact with other important proteins?

Exciting new technologies now allow us to track the behaviour of endogenous huntingtin protein (the huntingtin existing naturally in the cell). We put to use two different intracellular antibodies, or “intrabodies” that recognize and bind the huntingtin protein. We tagged these intrabodies with yellow fluorescent protein (YFP) to generate “chromobodies”. This allowed us to follow their interaction with endogenous huntingtin in live cells. Indeed, we could watch the endogenous huntingtin protein being recruited to sites of DNA damage.

This tool is not without its drawbacks. While it doesn’t seem to interfere with huntingtin’s recruitment to damaged DNA, it must interfere with its role in cell proliferation. We know this because we can’t stably express the chromobody in cells over time. When we watch cells expressing the chromobody try to divide, they just die (unlike the cells expressing only YFP, which happily multiply).

This roadblock can be hurdled using an inducible system: the cells carry the DNA expressing the chromobody, but it isn’t turned on to generate protein until you add a drug called doxycycline. So I first cloned the chromobody into an inducible vector (cloning experiment deposited to Zenodo). When co-transfected with the doxycycline-responsive Tet3G transcriptional activator, it showed beautiful induction by doxycycline in mouse striatal cells (induction experiment deposited to Zenodo).

But we want to work with cells from HD patients. It’s harder to get DNA into these cells, but we can do it with electroporation. To avoid this labour-intensive process every time I want to do an experiment, I’m making HD patient cell lines that stably express the inducible chromobody and doxycycline-responsive Tet3G activator. The Tet3G vector carries a drug resistance gene, so I can select the cells with the drug G418. A simple experiment (deposited to Zenodo) showed that the optimal concentration for G418 selection in fibroblasts is 50 ug/mL.

At this point, my luck ran out. The beautiful induction I saw in mouse striatal cells did not happen in HD patient fibroblasts. From the first few failed attempts, I learned the following:

• If cells become too sparse during the G418 selection process, they die. Need to transfect a larger number of cells so that they can be downsized during the selection process and still maintain confluency >50% for cell health.
• Transfection of fibroblasts is an issue. Need to use electroporation, and co-transfect H2B-mCherry to identify transfected cells
• Transfection of pTRE-nucHCB2 (inducible chromobody), pEF1a-Tet3G (doxycycline-responsive transcriptional activator), and H2B-mCherry is far more toxic than the equivalent microgram quantity of sonicated salmon sperm DNA. Need to use pTRE-nucHCB2, sssDNA, and H2B-mCherry as the untransfected control (that is, -Tet3G) in order to compare rates of selection by G418 in untransfected versus transfected cells
• In contrast to striatal cells, fibroblasts don’t seem to be inducing expression of nucHCB2 with doxycycline

After ruling out protein turnover, FBS concentration in the media, and different preps of DNA, the only difference between the nice result in mouse striatal cells and the confusing result in human fibroblasts is the method of transfection (the easier, polymeric method for striatal cells versus the more tedious—and expensive—electroporation method for fibroblasts). But this couldn’t possibly be the problem… could it? Only one way to find out: I set up a direct comparison experiment. To my great surprise, the striatal cells induced when transfected by the polymeric method, but not by electroporation! The experiment is posted on Zenodo.

At this point I recalled a suggestion made a few weeks prior, by Claudia Hung, a student in the lab: she asked whether the size of the plasmids could explain the results. I really didn’t think so at the time, but now that idea might make sense! The Tet3G vector is pretty large (7.9 kb), and sure enough, difficulty transfecting large vectors by electroporation is well documented (once you look for it!). This study by Lesueur et al explains that simply giving the cells a chance to recover from the electroporation before plating them can greatly enhance cell viability and transfection efficiency. This was my next move. There was a glimmer of hope in the results: the longer recovery time resulted in induction in a few cells. After taking a closer look at the Lesueur et al study, in which they used much larger amounts of DNA, I tried increasing the amount of DNA.

Eureka! Finally, after months of trouble shooting, I found conditions in which we can induce expression of a huntingtin-specific chromobody in cells from an HD patient (see the results on Zenodo). Next week I will be electroporating cells from HD patients who have different CAG lengths in their huntingtin genes, and selecting them in G418 to get stable cell lines. The result will be a panel of cell lines with different-sized huntingtin expansions, in which we can visualize the natural huntingtin protein by dropping in doxycycline—a great tool for our lab and HD researchers around the world.

If you’ve made it this far through this tedious blog post, thanks for reading. You now have a sense of the tiny incremental steps it takes to move a project forward. This is only one facet of a much larger goal, and each facet has its own set of obstacles. But with careful, calculated perseverance we can get through each road block and move our understanding of HD forward. This work is funded by the HDSA Berman/Topper HD Career Development Fellowship.