NAME: Francesca Cicchetti, PhD
POSITION: Professor and Researcher, Neuroscience Department, Centre de Recherche du CHU de Québec, Faculty of Medicine, Laval University
EDUCATION: BS, McGill University, Montréal, QC; MS and PhD in Neuroscience, Université de Laval, Québec, QC; post-doctoral work with Ole Isacson, MD, Harvard University, Cambridge, MA
HOBBIES: Travel; tango and salsa
Dr. Francesca Cicchetti is a neuroscientist at the University of Laval in Québec, Canada. Her 2014 paper “Mutant huntingtin is present in neuronal grafts in Huntington disease patients,” published in Annals of Neurology, 1 was selected by the HD Insights Editorial Board as one of the most influential papers of the year. Her findings suggest that mutant huntingtin (mHTT) can be transmitted through non-cell-autonomous mechanisms, and potentially implicate the immune system in HD pathology. Her discoveries are already leading to new understanding of HD pathogenesis and have the potential to identify novel targets for early HD therapies. Dr. Cicchetti recently spoke with HD Insights about her past and current research. The following is an edited transcript of the conversation.
HD INSIGHTS: Can you tell us how you first became interested in HD?
CICCHETTI: I was doing a PhD thesis under the supervision of Dr. André Parent, a renowned neuroanatomist based here at Laval University in Québec, and had been assigned a project on tract tracing studies in squirrel monkeys. I became very attached to the animals. I had names for each one of them and fed them bananas in the morning. When it was time to sacrifice them, I simply could not do it, I was just too emotional. I told Dr. Parent that I could not continue the project. At that time, he was also keen to initiate a human brain bank, and asked me if I would be interested in taking on this project instead. So, I began to contact pathologists, and I collected close to 500 human brains. The brain bank is still active today in distributing tissue to various research groups. Through this endeavor, I began studying the human brain: the basal ganglia in normal conditions, and then became interested in pathologies related to striatal dysfunction, such as HD.
HD INSIGHTS: What were your first research experiences with HD?
CICCHETTI: Our initial work focused on the characterization of striatal cell populations, and trying to understand their selective vulnerability. We were particularly interested in subpopulations of interneurons that express calcium-binding proteins. These were the very first papers that we published in the field.
HD INSIGHTS: Your paper in Annals of Neurology suggested for the first time that mHTT might be transmissible from nerve cell to nerve cell. Can you discuss?
CICCHETTI: I believe that this paper is our most important contribution to HD research. We were extremely fortunate, of course, to have access to this unique material, the brains of HD patients who had received fetal striatal transplants as an experimental therapy as an attempt to slow the progression of their disease – a trial that was initiated by Thomas Freeman at the University of South Florida. Our paper demonstrated the presence of the mHTT protein within the grafted tissue a decade post-transplantation. We did not see this in early time points following transplantation. The mutant protein was found within the extracellular matrix of the grafted tissue, not specifically within grafted cells. However, we also saw mHTT aggregates in cells associated with blood vessels as well as in perivascular macrophages within the cerebral tissue of the transplanted HD patients. When we first submitted the paper reporting this data, it was clear that the reviewers were interested in this finding, but did not quite believe it. We used every technique we could possibly use on human postmortem tissue—immunohistochemistry, immunofluorescence, confocal and electron microscopy, Western immunoblotting, and infrared spectroscopy, to show that mHTT inclusions were indeed present in the transplanted tissue. This observation, which we were very excited about, raised a whole new set of questions. How did the mutant protein get to the transplanted tissue, which is genetically unrelated to the patient? In the paper, we proposed a series of non-cell-autonomous mechanisms to explain the propagation of mHTT.
HD INSIGHTS: We know that mHTT will be produced within the neurons of individuals with HD. What role would transmission play in the disease and why is that important?
CICCHETTI: This is a very important point. Because of the genetic nature of HD, the mutant protein is expressed ubiquitously, in every cell of the body. The extent to which non-cell-autonomous mechanisms of pathological protein spread may contribute to disease onset and development is unknown, but this observation alone may fundamentally change our understanding of the pathogenesis of HD, and of other neurodegenerative disorders. If non-cell-autonomous mechanisms do indeed play a significant role in disease pathophysiology, developing therapies that are designed to halt the propagation of mHTT will be of significant value.
HD INSIGHTS: I’ll return to the targets in a moment, but I wanted to touch on your mention of other diseases. Dr. Virginia Lee and her colleagues have described trans-synaptic transmission of α-synuclein in PD models.2 Is there a common theme?
CICCHETTI: Certainly. But besides the transsynaptic transmission of α-synuclein demonstrated in the original publication by Luk et al.,2 Lee’s group also proposed the potential prion-like behavior of α-synuclein.3 In HD, I do not personally believe that mHTT propagates in a prion-like fashion, that mHTT seeds pathology and changes the conformation of the protein in neighboring cells. However, I do believe in the trans-synaptic propagation of mHTT, something that we are currently working on. But what we are especially keen on is to explore the involvement of the immune system—blood-borne cells in particular—as a vehicle to transport and spread mHTT.
HD INSIGHTS: So you think immune cells may be transporting mHTT?
CICCHETTI: Other groups have already shown that monocytes, for example, express mHTT very early in the disease course.4 As you pointed out, in HD patients, every cell of the body expresses mHTT, but certain cell types seem to be much more vulnerable to cell death, although the vulnerability of these cell populations is not well understood. So we know that peripheral cells express the mutant protein, and we now have evidence that there is leakage of the blood-brain barrier in HD patients, data that we will shortly submit for publication. Based on this, we believe that the transmigration of peripheral cells to the brain may be facilitated. I do think that that there is a strong possibility that mHTT spreads to the brain via blood-borne cells.
HD INSIGHTS: Which immune cells do you think are involved?
CICCHETTI: Based on current literature, 5 mHTT is detectable in monocytes and T-cells, which specifically correlates with burden of disease scores, further suggesting that these populations could be used as biomarkers. Results from PET studies in HD patients also support the idea that microglial activation is an early event in HD pathogenesis.6 There is strong evidence in favor of an inflammatory or immune-driven response that precedes neurodegeneration in HD.
HD INSIGHTS: Do you think that these immune cells, which are facilitating the propagation of mHTT, are responsible for the spread of mHTT within the brain?
CICCHETTI: I tend to believe so, but I’m not ruling out the possibility that there is a very high expression of mHTT first in the brain, and then we find residues of the protein in the periphery. However, my guess is that it is primarily the other way around, based on the fact that there are early signs of immune dysregulation, including elevated levels of cytokines in HD patients, long before any of the neurological features that are used for diagnosis.
Another avenue of propagation that we are currently exploring is that of exosomes and microvesicles, which can be released from peripheral cells. Their small size would allow them to enter the brain even in the absence of leakage of the blood-brain barrier. What is interesting about these small vesicles is that some of them contain mitochondria, which allows them to travel long distances because they have their own powerhouse. Despite their small size, they are big enough to carry various proteins, and could serve as miniature mHTT cargoes circulating from peripheral blood into the brain.
HD INSIGHTS: Does this also suggest that peripheral markers could serve as biomarkers of HD?
CICCHETTI: Absolutely, and a number of research groups have suggested this. Not only could they serve as biomarkers of HD, but also as markers of treatment efficacy.
HD INSIGHTS: Have we seen any treatments that change these peripheral markers in HD?
CICCHETTI: I do not know of any studies that have specifically monitored the changes in these markers following treatment. However, one study has demonstrated normalization of cytokine levels and moderate motor benefits in animal models of the disease using a bone marrow transplant paradigm7 and a recent publication in Brain has reported that glucan-encapsulated siRNA can significantly reduce HTT levels in monocytes and macrophages derived from pre-manifest HD patients, which in turn decreases the levels of pro-inflammatory cytokines released by these cells.8 There were also a number of studies reporting benefits of the anti-inflammatory drug minocycline in animal models of HD, but the treatment did not translate into meaningful improvements in HD patients.9
HD INSIGHTS: So we could be looking at mHTT and peripheral monocytes to determine whether new therapies are efficacious in HD?
CICCHETTI: If we establish that inflammation or immunity indeed plays a critical role in HD, we could certainly use some of these cell populations to monitor treatment efficacy. But even though inflammation/immunity may be important in driving disease pathology, I suspect the problem is much more complex, and we are likely to need to treat the disease with a combination of approaches. Gene-silencing therapies using antisense oligonucleotides (ASOs) 10 or RNA interference targeting specific SNPs to lower mHTT11, 12 seem very promising in animal models of the disease. These methodologies would not target propagation per se but attack the problem upstream of propagation, aiming at the mHTT mRNA before the gene is translated. However, in anticipated clinical trials, ASOs will be delivered through the cerebrospinal fluid and siRNA directly into the brain to target cerebral mHTT, not the expression of mHTT in the periphery. I assume the delivery of ASOs could eventually be considered via the peripheral system to target peripheral cells.
HD INSIGHTS: And you think these peripheral monocytes could be used as an assay of the efficacy of these interventions?
CICCHETTI: They could be, or any other blood-borne cells. The monocytes are very few compared to other types of blood cells. We cannot discard other populations as potential biomarkers, nor the microvesicles and exosomes that they can release.
HD INSIGHTS: You mentioned that your research has implications for targets for therapy. Could you elaborate?
CICCHETTI: Of the mechanisms that we proposed or suspect underlie mHTT propagation, there are two that we are most eager to investigate. The first is trans-synaptic spread through the cortico-striatal pathway, for which there is accumulating evidence both in vitro and in vivo, especially with the latest paper published by Pecho-Vrieseling et al. in Nature Neuroscience.13 The work of Dr. William Yang showing that modulating the expression of mHTT through the cortico-striatal pathway can actually change some characteristics of the pathology, also provides evidence for the importance of this pathway in non – cell-autonomous mechanisms of protein spread.14 Gene silencing methodologies, either ASOs or siRNAs, applied directly into the brain could block the propagation of the mutant protein. The second possibility for therapeutic development would be to target circulating immune cells, which we hypothesize carry the mutant protein and can transmigrate into the brain via leaky blood vessels. Of course, there may be other explanations for the presence of mHTT within genetically unrelated grafted tissue. Explanations could be oxidative stress, inflammation, or poor trophic support. But again, I think that mHTT propagations via the cortico-striatal pathway and/or the immune system are the most likely scenarios and therefore the best therapeutic targets.
HD INSIGHTS: Do you see any therapies that are currently under development that could potentially address either one of these possibilities?
CICCHETTI: To target the immune system, we could consider vaccines, but this approach has not yielded any benefits in other diseases such as Alzheimer disease, and in fact generates a number of complications. Intrabodies, which are recombinant single chain antibody fragments, have shown significant effects in reducing the striatal mHTT protein load.15 ASOs are also showing great promise in animal studies, and could be used to target both cerebral and peripheral mHTT.10 No matter what the approach, it will be critical to select specific populations of HD patients, before excessive neuropathology has set in. Selecting late-stage patients will prove more difficult because there will be much more aggregated forms of mHTT in such patients, whom will not respond as well to ASOs. We need to silence the gene early, given that the soluble protein is likely to be the toxic form, not the aggregate itself.
HD INSIGHTS: Your paper has created quite a buzz, and was recently recognized as one of the papers of the year by the Editorial Board of HD Insights. Can you tell us who funded the study?
CICCHETTI: No one. But since the publication of the manuscript, we have received all the funding we have applied for. Persistence paid off and I am counting my blessings!
HD INSIGHTS: I assume given its impact that this paper was readily accepted by journals?
CICCHETTI: I can now laugh about it, but publishing this paper was quite a battle. It took close to two-and-a-half years. We first made this observation more than 3 years ago. I went to the microscope and saw the inclusions in the transplant. I remember calling a colleague in Europe and telling him about the observation, and he immediately replied that I needed to publish this paper as soon as possible. But we really, really struggled to get it published. The reviewers were intrigued and interested by the finding, or at least the majority of them, but did not quite believe it, and I think that they were not ready to accept the idea of non-cell-autonomous propagation of pathological proteins in genetic disorders. I have to admit that the first versions of the paper were bold and somewhat speculative, but now the published end product is something of a lobotomized version of what we really wanted to say. What I was most pleased about when HD Insights contacted me to present this at HSG 2014 was the fact that we finally had a platform to present this work, where people wanted to hear what we had to say. And now that I see the paper of Pecho-Vrieseling et al.—which came out just a couple of months after ours—providing in vitro and in vivo evidence of non-cell autonomous mHTT spread, it is clear that we are now open to discussing these ideas and thinking outside the box. This has been so rewarding and it has given me so much energy. I was a workaholic before, but now I am unstoppable. I really want to solve this thing. One of the things I have most enjoyed in the last couple of years is that the landscape of research has completely changed. Now we conduct multidisciplinary and multi-centric research. Through this project we have teamed up with colleagues who are pure immunologists working on arthritis, for example. And together we are attacking this problem from a completely different perspective. It is a very exciting time.
HD INSIGHTS: What is the most exciting aspect for you?
CICCHETTI: It’s being able to rally colleagues from all disciplines to study this. It’s the collaboration with researchers who are completely outside this field and investigating or trying to apply concepts that they have discovered in their own research—in other words, thinking of the problem differently, in ways we had not thought of before. I am also a big fan of recycling medicine that is already out there. It takes so long and it is so expensive to do research and development; why not screen compounds that are already available and have a good safety profile, and see if they can have applications for other disorders? I have been blessed to work with wonderful students and collaborators on the Annals of Neurology paper, and to now have them on board, with new collaborators, to pursue this work.
HD INSIGHTS: So when you are not identifying propagation of mHTT, how do you spend your time?
CICCHETTI: I spend a lot of time in the lab. I’m a bit of a nerd and I must say that my staff is completely fed up with me, because even at Christmas parties, I talk about mHTT propagation! But I also love to travel, spend time with my family and practice tango and salsa. I’m going to see my parents in Florida over the holidays.
HD INSIGHTS: Will you take your microscope?
CICCHETTI: No, but I am definitely taking my computer, and working on our next paper reporting blood-brain barrier leakage in HD.
HD INSIGHTS: Dr. Cicchetti, thank you very much for all your time and for your great insights into a new area of HD research that has clearly captured many people’s attention.
1 Cicchetti F, Lacroix S, Cisbani G, et al. Mutant huntingtin is present in neuronal grafts in Huntington disease patients. Ann Neurol. 2014; 76(1):31-42.
2 Luk KC, Kehm V, Carroll J, et al. Pathological α-Synuclein Transmission Initiates Parkinson-like Neurodegeneration in Nontransgenic Mice. Science (New York, N.Y.). 2012; 338(6109): 949-953.
3 Luk KC, Lee VMY. Modeling Lewy pathology propagation in Parkinson’s disease. Parkinsonism Rel D. 2014;20(Suppl 1):S85- S87.
4 Björkqvist M, Wild EJ, Thiele J, et al. A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington’s disease. J Exp Med. 2008; 205(8):1869-1877.
5 Weiss A, Tr, xE, et al. Mutant huntingtin fragmentation in immune cells tracks Huntington’s disease progression. J Clin Invest. 2012; 122(10):3731-3736.
6 Pavese N, Gerhard A, Tai YF, et al. Microglial activation correlates with severity in Huntington disease: a clinical and PET study. Neurology. 2006; 66(11):1638-1643.
7 Kwan W, Magnusson A, Chou A, et al. Bone marrow transplantation confers modest benefits in mouse models of Huntington’s disease. J Neurosci. 2012; 32(1):133-142.
8 Trager U, Andre R, Lahiri N, et al. HTT-lowering reverses Huntington’s disease immune dysfunction caused by NF?B pathway dysregulation. Brain. 2014; 137 (Pt 3):819-833.
9 Soulet D, Cicchetti F. The role of immunity in Huntington’s disease. Mol Psychiat. 2011; 16(9):889-902.
10 Kordasiewicz HB, Stanek LM, Wancewicz EV, et al. Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis. Neuron. 2012; 74(6): 1031-1044.
11 Pfister EL, Kennington L, Straubhaar J, et al. Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington’s disease patients. Curr Biol. 2009; 19(9):774-778.
12 Drouet V, Ruiz M, Zala D, et al. Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells. PloS One. 2014; 9(6):e99341.
13 Pecho-Vrieseling E, Rieker C, Fuchs S, et al. Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons. Nat Neurosci. 2014; 17(8): 1064-1072.
14 Wang N, Gray M, Lu X-H, et al. Neuronal targets for reducing mutant huntingtin expression to ameliorate disease in a mouse model of Huntington’s disease. Nat Med. 2014;20(5):536-541.
15 Southwell AL, Ko J, Patterson PH. Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington’s disease. J Neurosci. 2009; 29(43):13589-13602.