By Lisa Munsie, PhD
1) STUDIES ON MODULATING TRANSCRIPTIONAL DYSREGULATION
There is substantial data to support that there are many changes to how normal gene — and thus proteins — are expressed in cells of HD patients. This is called transcriptional dysregulation. Work still needs to be done to parse cause vs. effect of this transcriptional dysregulation. We need to determine if the transcriptional alterations can be tracked over the course of HD, and if modulating them may be therapeutic.
Epigenetic Alterations Explored
Most reports looking at changes in gene expression focus broadly on transcription, with fewer studies focusing on epigenetic alterations. Epigenetic alterations are a kind of change that happens to the DNA without changing the DNA. A study published in Nature Communications investigates temporal dynamics and mechanisms underlying epigenetic changes contributing to HD1.
Q140 heterozygous mice were used because they are a slow-progressing disease model, and therefore useful for examining early disease markers. The main technique used to detect epigenetic changes is called ChIP-Seq. ChIP-Seq was used to detect certain DNA changes, specifically H3K27ac, RNAPII and H3K27me3. These markers keep DNA in different structures that lead to different expression of genes. ChiP-Seq data for these changes were generated at two and six months in neuronal and non-neuronal populations in this mouse model.
This extensive and detailed study indicated that there are age-related epigenetic and transcriptional striatal changes that are exacerbated by mHtt. The group shows a structural change of the DNA in the nucleus (called chromatin) that correlates with the transcriptional changes. This data indicates that mHtt leads to an accelerated epigenetic aging phenomenon.
Impact of Histone Deacetylases Alterations on Somatic Expansion
A phenomenon called somatic expansion occurs in HD. This is where the mutation that causes HD actually increases over time in certain cells of HD patients and is a hallmark of disease. Histone deacetylases (HDACs) are chromatin-remodeling enzymes responsible for transcriptional regulation and epigenetic modifications. HDACs have been shown to correlate with disease-causing gene expression alterations in HD.
A group led by Dr. Vanessa Wheeler investigated whether alterations in HDACs and their chromatic remodeling abilities had any effect on the increased somatic expansion seen in HD.2 Using the HD Q111 mouse model, and conditionally knocking out Hdac2 or Hdac3 from the striatum, the group found a trend towards decreased somatic expansion and altered nuclear pathology. This supports the theory that HDACs may enhance CAG expansion in striatal neurons, and warrants further studies.
Lamin Proteins Track with HD Pathology
Lamin proteins assist in maintaining nuclear structure and function. They are critical for RNA nuclear export which, with transcription, are altered in HD. A recent paper looked at lamin alterations in the R6/2 mouse model and post mortem HD tissue.3 There are many different lamin isoforms, but this study highlighted how lamin B1 affected the brain in both models. They found increased levels of lamin B1 tracking with pathology, and these changes were not age-related.
The group generated Lamin B1 ChIP-seq data in the mouse and showed changes in the nuclear lamina heterochromatin organization and lamin B1 chromatin binding. This data shows another avenue that can be explored with respect to transcription, chromatin and epigenetic-related pathophysiology in the brain during HD.
1Alcala-Vida, R., et.al. (2021) Age-Related and disease locus-specific mechanisms contribute to early remodeling of chromatin structure in Huntington’s disease mice. Nature Communications 12(364)
2Kovalenko, M., et.al. (2020) Histone deacetylase knockouts modify transcription, CAG instability and nuclear pathology in Huntington disease mice. Elife DOI: https://doi.org/10.7554/eLife.55911
3Alcala-Vida R., et.al. (2021) Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington’s Disease EMBO Mol Med 13:e12105
2) BASIC RESEARCH IMPACTING HUNTINGTIN LOWERING THERAPY
Huntingtin (Htt) is the protein, that when mutated (mutant huntingtin (mHtt)), causes Huntington’s Disease (HD). Lowering the levels of Htt in the brain is a promising therapeutic avenue. To track success of these therapies, it is important to have the ability to detect if the levels of Htt in the brain are decreasing after the therapy has been administered. Ideally,of scientists and doctors would like to have non-invasive procedures, like blood collection, to achieve this.
The CNS-CFS Connection
This effort has been assisted by a recent study published in Neurobiology of Disease, which describes an investigation of how Htt moves from the central nervous system (CNS), or the brain, to cerebrospinal fluid (CSF), a fluid that can be easily collected and analyzed by doctors1. Using ultrasensitive protein detection techniques, researchers were able to detect mHtt in CSF at levels that correlate to that in the CNS.
Using a variety of cellular and animal models and a sensitive protein detection technique called immunoprecipitation flow cytometry (IP-FCM), the group finds that the neurodegeneration that causes HD leads to increased mHtt in CSF. However, interestingly, in the absence of neurodegeneration, mHtt is still detectable in the CSF.
Findings showed that both wildtype Htt (wtHtt) and mtHtt are secreted from neurons, the cells in the brain that are affected by Htt, indicating the movement of Htt from CNS to CSF may be a normal function. However, during neurodegeneration, an increase of mHtt is found to be actively moved into the CSF by a cellular system called the glymphatic system. These findings tell researchers that Htt is found in the CSF normally, but in HD is also actively moved into the CSF. This will help them understand how and why they see changes in Htt levels in the blood and correlate this back to treatments.
Finding the Best Methods to Detect Htt
To assess how best to detect low levels of Htt using immunoassays, another manuscript systematically assesses how different molecules and antibodies used to detect Htt perform. This is done through using one of the best detection methods for proteins: the single molecular counting (SMC) assay2.
This assay requires antibodies that detect a certain part of the Htt protein called the N-terminus, to be immobilized on magnetic particles that capture the protein. Then detection of Htt is based on another antibody that detects a different part of the protein conjugated to a fluorphore – a molecule that can light up and be quantitated by researchers.
This paper tests different antibodies and produces a summary table which displays their ability to detect different Htt fragments and whether there is specificity for the assay to detect mHtt . The paper makes a reference table for other researchers to use when designing their assays that tells them the specificity of the antibody, the ability to pick up Htt in different species and how sensitive the antibody is. The group also makes different recommendations on which antibodies to use or avoid in certain situations.
Neurofilament Light as Predictive Tool
While detecting mHtt levels is important for tracking the success of Htt knockdown therapies, finding other markers in the blood (biomarkers) that indicate whether lowering Htt is having a positive outcome on disease progression is also required.
Neurofilament light (NfL), is an axonal protein indicative of brain injury or neuronal injury, which is what happens to individual with HD. NfL levels have been shown to track with HD progression in blood, however NfL spikes in the CSF of patients who have undergone Htt-lowering therapy.
Dr. Ed Wild’s group examined the correlation of Htt and NfL levels in HD patients3. Both NfL and mHtt increased in this cohort of patients over a two-year period, and this correlated with clinical prognosis. NfL proved to be a more sensitive readout than just looking at levels of mHtt, and has potential to be sensitive enough for clinicians to track in the blood and determine when a patient is moving from PreHD (no symptoms) to manifest HD (starting to have symptoms).
1 Caron, N., et.al (2021) Mutant Huntingtin Is Cleared from the Brain via Active Mechanisms in Huntington’s Disease. Neurobiology of Disease 41(4) 780-796
2 Fodale, V., et. al. (2021) Analysis of mutant and total huntingtin expression in Huntington’s disease murine models. Scientific reports 10(22137)
3 Rodrigues, F.B., et. al. (2020) Mutant Huntingtin and Neurofilament Light have Distinct Longitudinal Dynamic in Huntington’s Disease Sci.Transl.Med 12(eabc2888)
3) IDENTIFYING PERIPHERAL MARKERS FOR HUNTINGTON’S DISEASE
Because Huntingtin (Htt) is difficult to detect peripherally (in blood or other non-invasive tests) as a marker for Htt knockdown in the brain. It is important to have other markers that correlate with disease. Additionally, these are important for future therapies that do not involve knockdown of Htt, as clinicians will need ways to know their therapy is working in patients.
BDNF Level Differences Ruled Out as Biomarkers
A study published in Scientific Reports investigates if Brain Derived Neurotropic Factor (BDNF), a protein whose expression levels in the brain are known to be associated with HD progression, can be detected in CSF and track with disease1. Conventional ELISA assays, which are standard protein detection assays, were not able to detect BDNF in the blood.
Using an ultra-sensitive detection method, the group was able to detect BDNF in plasma and CSF. However, when examining HD patient cohorts of premanifest and manifest HD, no differences in BDNF levels were detectable in the blood between control and HD patients, nor between patients at different stages of disease. Although BDNF levels decrease over the course of HD in the brain, this decrease cannot be detected peripherally. Therefore, BDNF can be ruled out as a peripheral biomarker.
Dynein Light Chain Expression Correlates to Disease Progression
There is a lot of genome-wide data from HD mouse models, providing clues to pathways and genes/proteins that may be affected during disease. Large studies on this data lead to many potential biomarkers. Many changes in gene and protein expression have been validated as up- or down-regulated in HD patients. Dynein light chain Tctex type 1 (DYNLT1) is a motor protein with a gene expression that is regulated by mHtt.
A group aimed to determine if changes in DYNLT1 could be detected in human blood2. The patient cohort studied included patients at different stages of disease. Some patients were sampled twice, three years apart. The group shows that expression of DYNLT1 by qPCR, a sensitive technique that detects changes in level ofgene expression, is correlated with disease progression. The study was not powered enough to make any correlations to age of onset or disease stage. However, it does indicate that DYNTL2 should be further studied as a biomarker in larger cohorts.
Prodynophin as Potential Biomarker
Prodynorphin (PDYN) is a gene that is highly enriched in the striatum, the area of the brain affected in HD. It has been shown to have decreased expression in HD brains as neurodegeneration occurs. Based on this, a group set out to see if PDYN-derived peptides could be correlated with HD onset in CSF3.
The detection of PDYN protein in CSF was performed using a detection technique called liquid chromatography mass spectrometry. The levels of PDYN-derived peptides was significantly decreased in the HD cohort compared with controls and compared to patients with other neurodegenerative disorders. This decrease was trending down with disease severity and did not correlate with age, indicating this is another potential biomarker for HD progression and treatment.
1Andy Ou, Z., et. al. (2021) Brain-derived neurotrophic factor in cerebrospinal fluid and plasma is not a biomarker for Huntington’s Disease. Scientific Reports (11)3481
2Rosseto, S.M., et.al. (2020) DYNTL1 gene expression is downregulated in whole blood of patients at different Huntington’s disease stages. Neurological Sciences doi/s10072-020-04772-0
3Al Shweiki, R., et.al (2020) Cerebrospinal Fluid Levels of Prodynorphin-Derived Peptides are Decreased in Huntington’s Disease. Movement Disorders doi.org/10.1002/mds.28300