CSF proenkephalin as a biomarker for premotor Huntington's disease

The paper is titled, Cerebral Spinal Fluid Proenkephalin Predicts Striatal Atrophy Decades before Clinical Motor Diagnosis in Huntington's Disease. Mena, [00:01:00] congratulations on your paper, and thank you very much for joining us today.

Dr. Mena Farag: Thank you for the invitation and for having me on the Movement Disorder Society Podcast, Eduardo.

Dr. Eduardo de Pablo-Fernandez: So just before we dived in in your article what is proenkephalin and how is that linked to the pathophysiology of Huntington's disease?

Dr. Mena Farag: So we know that clinically Huntington's disease presents as a triage of progressive motor impairment, cognitive decline, and neuropsychiatric disturbance with aged onset, typically mid adulthood. Though with considerable inter-individual variability and at the neuropathological level, however, Huntington's disease is defined by early in regional selective neurodegeneration.

The striatum and medium spiny neurons represents the most vulnerable neuronal population. And striatal Atrophy is among one of the earliest most consistent imaging and post-mortem findings in the disease. And within this striatum neurons of the indirect pathway are particularly susceptible. These neurons are [00:02:00] characterized not only by their GABAergic phenotype, but also by expression of enkephalin derived neuropeptides.

And so proenkephalin, or PENK, is the precursor of enkephalins and is highly enriched in the indirect pathway, projecting to the globus pallidus of the striatum, and both animal and human transcript data demonstrate that PENK expression is concentrated within the basal ganglia and that supports biological specificity to the striatal circuitry. And prior to this work post-mortem studies and Huntington's have shown reduced enkephalin content across basal ganglion nuclide, which suggests that there could be early dysfunctional loss of enkephalin expressing neurons. And these findings really provide a strong mechanistic rationale for PENK as a candidate biomarker of striatal vulnerability in HD.

Over the last five years there's been cross-sectional studies that have shown that PENK can be reliably quantified in cerebral spinal fluid and that's CSF. [00:03:00] PENK concentrations are consistently reduced in Huntington's disease, and these reductions have actually been replicated across independent cohorts, which supports its role as a candidate disease stage biomarker.

And off note in neurology and compared to other neurodegenerative diseases, CSF PENK levels were lower in people with Huntington's compared not only with controls, but also with other neurodegenerative conditions, including Parkinson's, Alzheimer's, and ALS. And importantly, these lower concentrations of PENK have been shown to correlate with disease burden and motors of changes, which also supports its biological relevance to the pathology of HD.

Dr. Eduardo de Pablo-Fernandez: So it sounds like you have evidence for what sounds like an ideal biomarker. It's specific, it's linked to the biology. It changes over time. It's correlated with clinical changes and that's what your study tries to demonstrate whether this is more specific biomarker.

And in your study your hypothesis is that it may [00:04:00] be more specific than, other markers of neurodegeneration that has been used before in, in people with Huntington's or more generally in neurodegeneration, such as neurofilament light chain. And also you compare that with progression on the MRI and also progression on the biological staging of Huntington's disease.

Can you tell us a bit more about the hypothesis and the outcomes of this study?

Dr. Mena Farag: So despite the signal that that's been quite promising in the studies so far there, there have been some important gaps. Prior studies were largely cross-sectional. They had relatively small or predominantly clinically manifest cohorts, and they actually didn't address whether CSF PENK changes longitudinally or relates to regional brain atrophy. And in particular the actual neuro anatomical specificity of CSF PENK as a marker of striatal neuro degeneration had not been tested. So really the aim of the study was to evaluate CSF proenkephalin as a biologically informed biomarker of early [00:05:00] HD focusing specifically on the far from onset HD young adult study cohort.

And so we aim to investigate both cross-sectional and longitudinal associations between baseline CSF concentrations of PENK and regional specific neurodegeneration using structural MRI. And so our main hypothesis was that lower baseline CSF PENK concentrations would be associated with subsequent brain atrophy predominantly in the striatum.

And we also, like you said, sought to compare the regional patterns of PENK related structural associations with those observed for other biofluid biomarkers that showed disease related group differences, mainly neurofilament light, which we know reflects more global and generalized neuro axonal injury.

And we talk about staging and the potential for this work to have relevance for, stage stratification and clinical trial design. And so this might be a good point to introduce the HD Integrated Staging System or HD ISS, [00:06:00] which is a key framework which underlies the relevance of this work and.

Traditionally staging in HD has been grounded in clinical phenotypes, so broadly distinguishing between pre manifest and manifest disease. And while this is clinically useful, this approach does not really capture the underlying biological progression that precedes the symptoms that developed. And so the HD ISS was developed to address this limitation by providing more of a unified evidence-based framework that captures the natural history of HD across the whole continuum. And so there's four stages in the HD ISS and the work in this paper basically includes participants from the HD Young Adult Study who are at stage zero, which included. Participants that were gene expansion carriers with full penetrance, but no sort of detectable biological changes.

Stage one is actually defined by biomarker evidence of pathology, and it's categorized specifically by volumetric MRI measures of called date IAM [00:07:00] atrophy and stage two captures emergence of subtle motor and clinical signs, cognitive signs and stage three reflects functional decline. And a lot of the clinical trials at the moment enroll participants that are in HD ISS stage two and three.

And so early manifest disease. And so a lot of this work and a lot of the work for the HD young adult study, the whole premise of it really is to track the earliest biological changes at earlier cohorts that could potentially be used to inform preventative trial design.

Dr. Eduardo de Pablo-Fernandez: That's an excellent summary, and that's very interesting that your study is focusing on this very interesting cohorts at the very earliest stages of the disease process. Can you tell us a bit more about the study cohorts and also the methods that you used in this study?

Dr. Mena Farag: Of course. The HD Young Adult Study is a longitudinal observational cohort of young adults who carry the Huntington's disease gene [00:08:00] expansion and on average are about 20 years from predicted clinical motor diagnosis. And they were matched alongside unaffected controls, very closely matched in terms of age, sex, and education level.

And so actually the study was conceived and led by Professor Sarah Tabrizi and it started in 2017. And it's with, there's been so far, two time points. We've recently finished the third time point. And the interval between visit one and visit two was approximately four and a half years.

And so the cohort is really deeply phenotyped. So it included multimodal assessments including biofluid, sampling of both blood and spinal fluids imaging. So with three Tesla, MRI clinical examination, cognitive testing and neuropsychiatric profiling. And we've had over the four and a half years, we had 103 participants returned for follow up, and we also recruited 23 new participants. And in terms of the methods, particularly in, in this paper [00:09:00] it's predominantly image biofluid based analysis. So to analyze brain structure, we used whole brain voxel based morph geometry, which is known as VBM.

And so this is really, so rather than pre-selecting regions of interest, this approach allows us to assess gray and white matter volume across the whole brain in an unbiased approach. And it involves segmentation of the brain into gray matter, white matter and spinal fluids. And we use established methods, EBM methods to quantify within subject change over time in the brain.

And on a voxel level, it allows us to basically see maps of local tissue expansion or contraction between the first time point and the second time point to identify when neurodegeneration is occurring and what rates. And then what we did was we tested the first time point level baseline fluid levels of proenkephalin in CSF and also in plasma and CSF NfL. And we use those to [00:10:00] predict the, where the spatial pattern and magnitude of atrophy were found. And we adjusted this for age sex, the time interval between scans and also the disease burden. So the key point is really that we're not just asking where do these groups differ at a single time point, but we're asking where in the brain t issue loss is occurring over time and whether baseline biofluid biomarkers can predict the spatial pattern and magnitude of that loss.

Dr. Eduardo de Pablo-Fernandez: And the day did this biomarkers show the pattern, the anatomical pattern of atrophy on MRI.

Dr. Mena Farag: So the main finding of the study was that baseline CSF PENK strongly predicted brain atrophy with a predominantly striatal or spatial pattern specifically lower baseline CSF PENK concentrations were associated with greater longitudinal gray matter loss in the core date and putamen both on both sides with corresponding changes in white matter in the adjacent peri striatal white matter. And these associations remain [00:11:00] significant even after adjusting for the disease burden, which indicates that PENK provides information beyond age and CAG repeat length alone, which we know are known drivers of progression in Huntington's.

Whereas NfL showed quite a different spatial profile higher baseline CSF Neurofilament light concentrations were more predictive of g lobal and widespread white matter changes and diffuse cortical gray matter changes. Which this distinction fits well with what we actually know biologically about NfL.

'Cause we know that NfL is a structural axonal protein reflects global neuro axonal injury, whereas PENK appears to be more linked to striatal pathology. What was also quite interesting was actually we saw some differences between CSF and plasma NfL in these very early disease stages.

And in terms of the analysis, sort of baseline plasma NfL was associated with more of a restricted pattern [00:12:00] of brain volume loss, limited to small clusters in the cortical gray matter which was quite different compared to what we see with CSF NfL. And this finding particularly suggests that maybe plasma NfL could be less sensitive than CSF NfL for detecting early neurodegenerative changes in these far from onset cohorts.

But nonetheless, it's there is still utility and use in NfL in this context.

Dr. Eduardo de Pablo-Fernandez: So as you mentioned they are biomarkers of slightly different process. One more specific, one more link to the striatal neurons. One more general, just reflecting neuro axonal damage. How do these biomarkers can inform about the pathophysiology and the neurodegenerative process at the earliest stages.

Should we stick to proenkephalin? Should we use both to have complementary information? What do you think about the use of both biomarkers?

Dr. Mena Farag: That's a, it's a really good question, and rather than [00:13:00] thinking of them as competing biomarkers. I think they reflect complimentary disease processes. So PENK is more of a signal of striatal specific neuronal vulnerability, whereas NfL captures broader neuronal axonal injury. And when we actually looked at the disease staging, so I talked about how the cohort was predominantly stage zero in one before clinical science when we did receiver operator curves to look at the sensitivity of of PENK. We were comparing to basically see whether CSF PENK is similar or could outperform NfL in distinguishing between these two stages. And we know that stage one is defined by evidence of striatal or involvement, coordinating putamen atrophy loss.

So it would be biologically intuitive that striatally enriched biomarkers such as PENK could fit in with this transition. And while when we did this, we showed that PENK was actually more superior than NfL in distinguishing stage one from stage zero. And [00:14:00] when you combined both biomarkers, there was a modest improvement in this discriminatory performance.

So I think overall, I think one of the most key challenges in HD is that, by the time motor symptoms emerge, there may have been substantial, largely irreversible striatal neuronal loss, and when there's no clinical signs, for example, in stage zero and one, disease monitoring really depends on objective biological measures.

And that's where the biological and neuroimaging biomarkers come in. PENK has particular potential in this context because it reflects striatal neuronal integrity and it could predict future striatal atrophy. I think both NfL and PENK in combination offer a more sort of nuance and biologically grounded picture of these early Huntington's disease changes that are recurring.

Dr. Eduardo de Pablo-Fernandez: That leads to my next question. So I understand that the biological staging system of Huntington's, the progression between a stage zero when there is no detectable [00:15:00] neurodegeneration and the stage one when there is no symptoms but evidence of neurodegeneration, this is the demonstrated use usually with MRI and my understanding is that not what biomarkers are included in this staging system. So in terms of future clinical trials, how do you think the results of your study and in general biomarkers on NfL and proenkephalin and other potential wet biomarkers, how do you think they will informed the design of clinical trials? How do you see your results will be used in the near future?

Dr. Mena Farag: That's a really good question. From a sort of planning of future preventive clinical trial design and like you say the current HD ISS criteria don't incorporate wet biomarkers. So I think potentially the potential to expand the HD ISS criteria for stage zero and one to where we could potentially include.

Wet biomarkers such as PENK and NfL to further stratify and subdivide stage zero [00:16:00] one could be useful. And from a preventive trial design aspect having a adjusted norms and

More populative data of these biomarkers across the norm, we could potentially have staging enrichment which could be useful for clinical trial recruitment. 

Dr. Eduardo de Pablo-Fernandez: Excellent. Yeah. And I think recently the news have reached their. The general media and I think Huntington's disease in terms of prevention and this is modifying clinical trials is probably a few steps ahead of some of the other neurodegenerative movement disorders with genetic therapies and other successful, studies that have been published so far. Just my question, where do you think that the future of clinical trials in Huntington's disease is heading? And how do you see your results informing this? And what do you think will be the next highlight of Huntington's therapeutics?

Dr. Mena Farag: Yeah, Huntington's disease, the therapeutic landscape has expanded considerably over, over the last five to 10 [00:17:00] years. Lots of different approaches. So beyond Huntington lowering, there's splicing modulators. There's viral vector gene therapy, and more, more recently somatic CAG repeat expansion being a target as well for disease modifying therapies as in the pipeline.

I think while current clinical trials are in phase two with some in phase one I think the aim and hope would be for these programs to expand into phase three trials onwards. And hopefully with a disease modifying trials having approval via sort of regulatory bodies. But I think while all that's going on thinking ahead of future preventive trial design is really important.

If we want to try and prevent or slow down disease progression in clinically asymptomatic and presymptomatic cohorts, such as individuals that are in HD ISS stage zero and one. And I think in order to make steps towards that. That's where longitudinal [00:18:00] observational studies, like the young adult study are quite important because in order for regulatory bodies really to accept biomarkers as surrogate endpoints they need to be deemed reasonably likely to predict clinical benefit.

And. When you think about these very early stages, traditional clinical endpoints such as, the composite UHDRS, which is currently used as an endpoint in. In current clinical trials of stage two and three those trials are not those endpoints are not relevant for stage zero and one when there are no clinical signs.

And so that's really where objective biomarkers such as biofluid, biomarkers and neuroimaging markers are really important. 

Dr. Eduardo de Pablo-Fernandez: Thank you very much. I think it's like a very, promising landscape for Huntington's disease research in the next years ahead. Thank you very much Mena for your time today, I had Mena Farag from the UCL queen Square Institute of Neurology in the Huntington's Disease Center.

I hope you find the podcast interesting and I would encourage all [00:19:00] the readers to read the full paper published in Movement Disorders Journal. Bye-bye for now.

Dr. Mena Farag: Thank you. [00:20:00]