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Iñigo Ruiz-Barrio, first author of the article, and Dr. Jaime Kulisevsky from the movement disorders unit of the Hospital de Sant Pau in Barcelona. Welcome to the podcast and thank you very much for your time.
[00:00:53] Dr. Jaime Kulisevsky: Thank you for inviting us to this podcast. Thank you.
[00:00:57] Dr. Eduardo de Pablo-Fernandez: To start with your [00:01:00] biomarker focuses on protein Tau which may be a bit surprising for people not familiar with the pathophysiology of Huntington's disease. We're more familiarized with the role of huntingtin protein in the pathophysiology of Huntington's disease.
But I would like you to discuss a bit more about the role of tau in the pathogenesis of Huntington's disease and what prompted you to do the study.
[00:01:27] Dr. Jaime Kulisevsky: Yeah, then it is well known that Huntington results from a genetic mutation causing the piled up of mutant huntingtin protein due to this abnormal expansion of the CAG repeats in the HTT gene. But despite this recognized relationship between, for instance, the size of CAG expansion and age of onset the clinical phenotype and disease severity there is a great variability [00:02:00] among patients who share more or less the same CAG expansion, age, or disease burden.
Then this suggests that beyond the effects of directly mediated by the mutant huntingtin team. Other neuropathological mechanisms could variably affect patients and contribute to this clinical variability that also implies a focus of our attention that is cognitive detergent. Then we base it on the work of other people that from some years it's known that there is a potential role of tau protein in HD.
And for instance, in tau, Braak stages, one to three in patients with Huntington in the. pathologic anatomy. In 60 percent of the cases, there are tau pathology. And for instance, in [00:03:00] 80 patients with dementia they exhibited a severe tau pathology consistent with Braak stages 5 and 6. And then also more recent studies have revealed elevated levels of Tau protein in the cerebrospinal fluid of HD patients. And then it is also important that additional works have shown that the mutant huntingtin promotes neurodegeneration through direct interaction with Tau protein, which induces changes in protein structure, Tau for correlation, and four, three, four repeats and three repeats tau deposition.
Then, all these findings have led to the proposal of incorporating HD into the group of secondary tauopathies. Then also in a recent study from our group was focused on cognitive [00:04:00] heterogeneity in HD. We not only confirmed that more severe forms of cognitive deterioration in HD extend beyond the typical executive dysfunction that is considered the typical cognitive dysfunction of HD, and also affect processes with clear posterior cortical dependence such as language, visuospatial impairment.
And this phenotype was, in our work, associated with higher CSF levels of T Tau and P Tau 231, and to a more pronounced pattern of posterior cortical atrophy in specific brain regions closely linked to the cognitive processes affected by Tau. But then this mechanism of Tau pathology and Tau in the CSF correlated with the phenotype moved us to make a [00:05:00] study considering whether tau can be also found in other tissues, for instance, in peripheral tissues as happens in other neurodegenerative diseases such as alpha synuclein in Parkinson's.
And, t aking advantage of pivotal study demonstrating that tau quantification in skin biopsies is present in corticobasal degeneration and in progressive supranuclear palsy. Then we aim to investigate both the feasibility of skin tau quantification and whether it can be a biomarker for enhancing group classification and assessing disease severity across the HD spectrum.
[00:05:48] Dr. Eduardo de Pablo-Fernandez: Okay, so if I got this right, huntingtin is not the only one to blame in Huntington's and there is potential contributing factors and there is evidence that Tau is likely to be one of them from [00:06:00] pathological studies. And now we are trying to develop biomarkers or more accessible tissue biopsies to evaluate that. In your study, you Recruited 23 people with Huntington's disease from the hospital clinic and eight controls and you evaluated and quantified the skin tau in skin samples Can you tell us more about the methods in particular about the processing of the skin biopsy and quantification of the tau protein
[00:06:31] Dr. Ingio Ruiz-Barrio: We designed a study in which we consecutively recruited patients from our Huntington disease cohort, along with healthy control. And in addition to clinical, neuropsychological, and brain MRI assessments, we took a skin biopsy from the ankle area in all patients.
The biopsy process is quite simple and safe. It involves a local anesthetic followed by a three millimeter punch biopsy taken under sterile conditions about 10 centimeters above the ankle. After collection, the sample is transported on [00:07:00] dry ice and stored at minus 80 degrees. And the procedure doesn't even need steeping.
Uh, uh, um, Um, uh, Um, uh, Um, uh, um, uh, um, um, um.
Solid tissue. First we need to homogenize the sample, but a critical step here is removing any blood remnants which could contaminate our trial measurements, from peripheral nerves with peripheral blood taps. To prepare the tissue for ELISA, we start by carefully removing any visible blood vessels with a scalpel, and next we wash the tissue with a buffer, which helps get rid of any leftover blood or debris,
then we incubate the tissue with lysis buffer that has a protease inhibitor in it, and this step breaks down the cell membrane and keeps the protein from degrading. After that, we uh, centrifuge it, and This allows us to collect the supernatant, and this way, we are left with a clear sample, free of any insoluble [00:08:00] material or blood contaminants, making it suitable for ELISA analysis.
And from there, we proceed with the usual ELISA steps, one important check in this process is ensuring the antibody specifically targets peripheral tau. So we used a previously validated antibody in order to study and also run parallel immunofluorescence experiments for visual confirmation.
And to interpret the tau concentration from ELISA, we generate a calibration tool using recombinant tau proteins so that we can get at the end a kinematic value for example, tau concentration in this sample. However, since the skin protein content can vary between individuals, we adjust this by measuring total protein in each sample and reporting the tau level as relative value.
This standardization allows us compare tau levels accurately across different patients, making the result more meaningful and reliable.
[00:08:53] Dr. Eduardo de Pablo-Fernandez: What did the results show? There is a great correlation between all clinical parameters that you evaluated [00:09:00] with tau quantification. Was that surprising to you or what did you expect?
[00:09:06] Dr. Jaime Kulisevsky: It was relatively surprising to us because we didn't expect that most individuals had Tau pathology and it was easy to also quantify because there was an important amount of Tau. Main findings show with significantly elevated skin Tau levels in individuals with manifest HD compared to pre manifest carriers and with healthy controls.
And these elevated tau levels correlated with several key clinical metrics. Specifically, there was a significant association with the CAG repeat length and the CAG age product score that is used in Huntington and both genetic indicators of disease burden. Also [00:10:00] clinically higher Tau levels corresponded to poorer scores on the unified Huntington disease rating scale and total motor score which measures motor impairment in the disease, as well as some assessments like symbol digit modalities and Stroke Ward Reading Test indicate an a broader cognitive decline.
Furthermore on the neuroimaging side, higher skin tau levels were closely linked with atrophy in specific brain regions known to be affected in AD. And a voxel based analysis revealed that increased tau levels correlated with gray matter volume reduction. in the caudate and putamen, which are key subcortical regions implicated in HD.
And then furthermore volumetric analysis showed significant correlations between skin tau and [00:11:00] volume reductions in the pallidum, hippocampus, and insula that are not the traditional parts that are affected in the brain in HD,
and these regions are associated with the cognitive and behavioral symptoms of HD. Then these correlations held independently of age and gender, underscore the potential of skin tau to serve as a reliable indicator of uh, neurodegeneration in HD.
[00:11:34] Dr. Eduardo de Pablo-Fernandez: So these results suggests that tau pathology has a great impact on the clinical outcomes. clinical presentation of Huntington's disease and progression of the disease in terms of motor cognitive, but also in terms of neurodegeneration in the central nervous system. And as you mentioned, not just in the typical areas associated to Huntington's disease, but other also cortical areas that are involved in cognitive function. One [00:12:00] other question I wanted to Discuss about the results is how do you interpret that? , pre manifest mutation carriers didn't have an increased tau in the skin? Do you think that tau deposition is like a later process that is driven by huntingtin and manifest a bit later? Or do you think that this process can start also in the sort of preclinical phases of the disease?
[00:12:26] Dr. Jaime Kulisevsky: yeah the reason it was demonstrated an interaction between huntingtin and Tau in the sense that the more huntingtin, then the more Tau you can have, as it happens with other proteinopathies. And then perhaps this is not uniform in all patients and we can characterize. Diverse phenotypes, independence of how this interaction is more or less active.
And then beyond [00:13:00] have a marker of tau pathology in the periphery indicating that the disease is active perhaps we can quantify this in different people and have a biomarker that perhaps may serve not only for diagnostic purposes in the sense that, okay, you have tau in your biopsy, then the process is now active because we found that it differentiate pre manifest from manifest.
And perhaps it may serve as as a biomarker for follow up or to characterize diverse groups to include into clinical studies to enrich populations in dividing them in whether they have more or less tau pathology indicating more severe forms of the disease, both from a cognitive and also from a behavioral and motor point of [00:14:00] view.
[00:14:00] Dr. Eduardo de Pablo-Fernandez: So there is increasing in research interest in the role of copathologists and people with Huntington's disease I'll be obviously present at a relatively early age, so they don't have pathological changes associated with age that we can see in other diseases. But perhaps, as you suggested, there is a proportion of patients that there is also associated Tau deposition, that they have this more aggressive phenotype and that you mentioned the biomarker as tool to classify people and there is also interesting results from your study when you try to analyze the role of tau quantification in a skin to define subgroups and you use cluster analysis to analyze skin tau in isolation and then when you include also in the model other prognostic factors And can you discuss a bit your results about on this topic?
[00:14:54] Dr. Ingio Ruiz-Barrio: Our cluster analysis based on tau levels in skin biopsies revealed some fascinating [00:15:00] insights into subgroups within Huntington's disease patients. By analyzing tau concentrations in skin tissue, we identified distinct clusters that appear to align with different stages and characteristics of the disease.
This analysis allowed us to group patients based on their tau levels, providing a new way to define subgroups beyond just genetic markers or clinical symptoms. To refine our analysis, we incorporated additional variables like CAG, repeat length, motor symptoms from the Unified Huntington Disease Rating Scale, and age.
And then we used principal component analysis to see if adding skin tau levels could enhance subgroup stratification. This approach confirmed that skin tau levels add significant value, helping to create clearer and more distinct clusters based on disease progression, cognitive decline and motor symptoms.
So essentially this suggests that peripheral tau would serve as an important marker for disease severity, improving how we define and track subgroups within Huntington's disease. So these findings suggest that Huntington's may [00:16:00] not be one uniform condition as we discussed previously, but could have distinct subtypes or progression patterns that might be tracked through tau levels in the skin.
This approach offers a potential tool for more personalized monitoring of the disease.
[00:16:15] Dr. Eduardo de Pablo-Fernandez: So, um
We have to bear in mind that, yeah, tau may be an important element in the clinical presentation of Huntington's. Obviously a skin is very accessible and it's a relatively simple procedure to have a skin biopsy. But there has been previous studies attempting to evaluate tau protein in other tissue samples or fluid biomarkers. How do your results from your study compared to those previous studies?
[00:16:44] Dr. Jaime Kulisevsky: Yeah, compared to previous research on Tau as a biomarker in Huntington's disease, our approach using skin biopsies perhaps offers a fresh perspective. Most studies have looked of Tau in blood [00:17:00] or cerebrospinal fluid to track disease progression, but blood based Tau studies have shown limited correlations with clinical variables.
making it challenging to use for tracking disease stage reliably. And CSF Tau, on the other hand, does correlate well with cognitive symptoms, as we have shown in our previous study and with disease severity, but obtaining CSF samples is invasive and less practical for routine monitoring. And then our study focusing on Tau levels in skin tissue provides a minimally invasive alternative with distinct advantages.
We found that skin tau levels correlated with key markers of disease progression, including cognitive and motor symptoms, and allowing us to identify clear subgroups within patient population. And this [00:18:00] suggests that skin tau could be a stable and accessible tool for monitoring HD severity. Of course, this has to be confirmed with, for other groups and other studies.
[00:18:17] Dr. Eduardo de Pablo-Fernandez: What do you think will be the next steps and how do you see the skin tau as a biomarker in clinical practice or future research?
[00:18:27] Dr. Jaime Kulisevsky: Yes, for next steps, there is still a lot to explore and we need prospective research. We'll need to validate these findings in larger, more diverse groups. to confirm that telling the skin reliably reflects disease progression across different stages and also different patient backgrounds. We studied in a very specific population in Spain.
It would also be interesting to see [00:19:00] if combining skin tau levels with other markers like imaging or genetic data. could create a more comprehensive profile for each patient. It will also be fascinating to dive deeper into the connection between tau accumulation in the peripheral and central nervous system.
And understanding these relationships could shed light on the broader pathology of Huntington's disease as a multi system disorder that isn't limited to the brain.
[00:19:36] Dr. Eduardo de Pablo-Fernandez: I think the results of your study, I think it sheds some light already and that there's demonstrate that Tau may be a piece in the jigsaw of Huntington's disease. So I hope the listeners enjoyed the discussion and I encourage them to read the full article published in Movement Disorders. Thank you very much Jaime and Inigo for joining us today and thank you very much to the [00:20:00] listeners.
Bye bye for now.
[00:20:01] Dr. Jaime Kulisevsky: bye. Thank you to you.