Huntington’s Disease (HD) is an dominant inherited neurodegenerative disorder with the combined symptoms of Alzheimer, Parkinson and ALS. HD is caused by a mutation in the gene encoding the huntington protein. Due to the expansion of the CAG repeat in the gene an N-terminal Htt exon1 fragment is generated with a long repeat of glutamine amino acids (polyQ) in the mutant HTT exon1 (mHtt) fragment. These fragments initiate aggregation in neuronal cells, leading to their dysfunction, with consequences for memory and movement and psychiatric problems. There is no cure for the disease, yet various ongoing clinical trails aim to reduce HTT synthesis using various anti-sense approaches.

mHtt lowering by improving the degradation of the  mHtt fragments prior to aggregation would be an alternative therapeutic strategy for this devastating disease. The main protein degradation machinery in cells is the Ubiquitin-Proteasome System (UPS) which is present in both the cytoplasm and nucleus. The role of the UPS in HD was initial controversial as UPS impairment had been observed in various HD models, which could be due to sequestration of proteasomes into aggregates (inclusion bodies, IB) that are initiated by mHTT. In addition, proteasomes were reported to be unable to cleave within the polyQ repeat in mHTT that is caused by the CAG repeat expansion in the mutated gene.

Our research group has addressed various research questions with relation to the UPS and its role in the degradation of mHtt, and examines different strategies to improve selective degradation of mHtt.

We examined whether proteasomes are indeed unable to degrade polyQ-expanded mHtt fragments in living cells. When mHtt was exclusively targeted to proteasome, these fragments were efficiently and completely degraded. The degradation may however occur in subsequent steps, as initially the flanking amino acids were degraded, while the remaining polyQ fragment was degraded in time. Here different proteasome activating complexes, chaperones like DnaJB6, and peptidases like Insulin-Degrading-Enzyme (IDE) are involved.

• Expanded Polyglutamine-containing N-terminal Huntingtin Fragments Are Entirely Degraded by Mammalian Proteasomes (2013)
• The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides (2013)
• Reduction in PA28αβ activation in HD mouse brain correlates to increased mHTT aggregation in cell models (2022)
• Insulin-Degrading Enzyme Efficiently Degrades polyQ Peptides but not Expanded polyQ Huntingtin Fragments (2024)

To examine the reported sequestration of proteasomes into polyQ-expanded htt aggregates in more detail, we performed fluorescent pulse-chase experiments in living cells to study the dynamics of proteasomes over longer time spans. In contrast to earlier reports, we observed that proteasomes are reversibly recruited into aggregates, and in addition are still active and accessible for substrates. This would explain the absence of UPS impairment in HD models when aggregates are observed, and indicates that proteasomes are actively recruited and remain active.

Using synthesized TAMRA-labeled ubiquitin moieties, we observed that intracellular TAMRA-ubiquitin is reversibly recruited to mHtt IBs and is incorporated into poly-ubiquitin chains of intracellular substrates including mHtt. This is due to catalytically active (de)ubiquitinating enzymes present in IBs, as demonstrated using novel activity-based probes. In contrast to TAMRA-Ub, the larger GFP-ubiquitin reporter used in earlier studies becomes irreversibly sequestered, suggesting a methodical disadvantage of GFP-tagged ubiquitin.

• Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies (2014)
• Dynamic recruitment of ubiquitin to mutant huntingtin inclusion bodies (2018)
• Visualizing Proteasome Activity and Intracellular Localization Using Fluorescent Proteins and Activity-Based Probes (2019)

Ubiquitination of proteins can be a signal for degradation and relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated Htt fragments offers an important possibility to understand Htt degradation and aggregation processes within the cell. For the identification of aggregated Htt and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. We generated methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt, and showed that mHtt is poly-ubiquitinated when it is aggregated.

While aggregated mHtt is poly-ubiquitinated but no longer accessible for proteasomal degradation, soluble mHtt is poorly ubiquitinated. We could not detect soluble, uniquitinated mHtt by biochemistry, and needed diGly Ips and targeted massspec to detect ubiquitinated mHtt. In addition, we observed that mHTT is differently ubiquitinated compared to wildtype HTT, with ubiquitination of the N17 domain of mHtt exon1 but not wtHtt. In addition we identified various E3 ligases and DUBs associating with mHtt exon1, and are exploring the role of these enzymes in mHtt turnover.

• The ubiquitin proteasome system in glia and its role in neurodegenerative diseases (2014)
• Detection of ubiquitinated huntingtin species in intracellular aggregates (2015)
• Global Proteome and Ubiquitinome Changes in the Soluble and Insoluble Fractions of Q175 Huntington Mice Brains (2019)
• Strategies to Investigate Ubiquitination in Huntington’s Disease (2020)
• Identification of Full-Length Wild-Type and Mutant Huntingtin Interacting Proteins by Crosslinking Immunoprecipitation in Mice Brain Cortex (2021)
• Ubiquitin-modifying enzymes in Huntington’s disease (2023)

Using immortalized striatal neurons expressing inducible untagged Htt fragments we aim to identify involved (de)ubiquitinating enzymes and modify their activities to improve mHtt degradation. Here, untagged mHtt ensures the absence of unwanted effects of tags like GFP on half-life and degradation, and an independent Q16-GFP protein is used as reporter for mHtt aggregates. We use this setup to screen with compounds libraries (including collaborations with biotech companies like MSD), DUB inhibitor libtraries and both crispr and RNA knockdown screens to improve degradation of (untagged) mHtt fragments.

Within our national NWA consortium CureQ (www.cureQ.nl) we will use isogenic iPSC derived neurons with varying polyQ repeats to examine polyQ-dependent differences in metabolism and activity using a variety of assays in order to create a cell-based landscape model. This model will be used to validate therapeutic strategies, and used to improve determination of onset and progression of disease, as well as determine the optimal time to start treatment.
Researchers and physicians from various Dutch universities, different HBO programs, ethicists, biotechnology companies, patient associations, Hersenstichting and Proefdiervrij work together in this study, aiming to enable a personalized approach and treatment for Huntington’s and SCAs.
Our lab focuses on exploring the HD isogenic lines, and validate new therapeutic strategies for HD.

Since the UPS appear to work properly in HD, with proteasomes and ubiquitin being dynamically recruited to mHtt aggregates, and the proteasome able to degrade mHtt when it is marked for destruction by ubiquitin, we are currently addressing the following questions:

• Using intrabodies to target mHtt for degradation, and to detect mHtt oligomerization in living cells as we need assays to detect different stages of mHtt aggregation
• Examine cross-disease mechanisms: expanding our research to the polyglutamine disorders SCA1 and SCA3, with focus on involved (de)ubiquitination enzymes
• Advanced microscopy techniques to detect and predict mHtt oligomerization and aggregation.

The research group is part of the Amsterdam Neuroscience.

The described research is currently funded by the Campagneteam Huntington and CHDI, and previously by NWO (VIDI), Hersenstichting, Prinses Beatrix Fondsn, de Vereniging van Huntington, an AMC PhD scholarship, and earlier by a grant from the KWF, HDF and a NWO-VENI-grant.

The core facility Cellular Imaging at the AMC harbors all the advanced fluorescence microscopy, electron microscopy and flow cytometry in one facility and is currently facilitating almost 600 active users. The facility is headed by Eric Reits and has a staff of 10. Techniques include:

• Confocal microscopy
• Superresolution microscopy
• FLIM imaging
• Automated live cell microscopy
• Lightsheet microscopy
• Inverted and upright fluorescence microscopy

• Transmission EM
• Scanning EM
• Correlative Light and Electron Microscopy (CLEM)
• Immuno EM
• Cryo EM

• Cell analysis (up to 18 different colours)
• Cell sorting

While Alzheimer and Parkinson are in general not inherited diseases and occur ‘spontaneously’ while aging,  Huntington’s Disease (HD) is a dominant inherited disease and children of a parent with the HD-related mutation (gene-carrier) have 50% chance to inherit the disease. Due to the symptoms, the absence of a cure but also the direct consequences for someone’s live (e.g. for relations, choices in life like studying or not, mortgage, insurances) only 10-15% of these so-called risk-carriers want to know whether they are in fact gene-carrier. As a result of this taboo, the mutated gene is silently passed on within families. The taboo also limits the willingness to raise awareness by public campaigns, and therefore also to raise funding for research.

In 2016 the Campagneteam Huntington was founded by many volunteers from families with Huntington’s Disease (HD), with the goal to raise awareness for the disease and to raise funds for research aiming for a cure for HD. There were various reasons including:

• Funding for HD research evaporated when the Prinses Beatrix Foundation (movement disorders) decided to focus on muscle diseases only, which excluded diseases like HD and ALS that affect movement
• In contrast to ALS, Parkinson and Alzheimer there was no awareness campaign for HD, partly due to the huge taboo on the disease. Being a 100% genetic disorder with 50% chance of inheritance, with various symptoms ranging from psychiatric problems and depression to chorea, and the absence of any treatment, most persons at risk do not want a genetic test. One can test at the age of 18, but many prefer not to know what may lay ahead and will affect relations, career and insurances.
• There were various promising research lines by different Dutch HD groups aiming to reduce mutant huntingtin levels by either lowering the synthesis of the protein, prevent aggregation of the mutant protein, or improve its degradation. These research lines  were jeopardized when funding dropped.

With the free help of the advertising agency Artica and marketing professionals like Roger Verdurmen the volunteers of the Campagneteam Huntington worked on a campaign including a television commercial, street commercials and flyers, and a website. Designing a slogan to describe the disease was not easy, as how to visualize a disease with the combined symptoms  of ALS, Alzheimer and Parkinson, and the huge taboo in most families affected by the disease? It turned out that HD cannot be visualized but can better be described:

The word ‘doodgezwegen’ stands for being dead silence,  not speaking about it at all, being a taboo.

The foundation Campagneteam Huntington was raised on March 22 2016 in order to acquire an ANBI bank account (Public benefit organization, allowing people and companies to deduct their gift from their taxable income). Together with the HD researchers in the Netherlands (Huntingtonresearch.nl) the Campagneteam Huntington aims to finance (fundamental) research needed to find a cure for HD and has the following objectives:

• Organise fundraising activities for research aiming for a cure for HD.
• Organising a national campaign to create awareness for HD by the general public.
• Finance research projects that are selected and approved by the independent Scientific Advisory Board

The foundation explicitly does not aim to make a profit, and is run by volunteers including the board. All raised funds will entirely be spent on funding  research projects that are chosen by the independent Scientific Advisory Board comprised of neuroscientists from related research fields that are not working on HD themselves. The board is formed by Melanie Kroezen (chair), Marijke Heefer (secretary), Ivo Braakhuis (treasurer) and Eric Reits (as chair of the Dutch HuntingtonResearch network).

The campaign was launched in May 2016, and raised much attention in the national and local media. Hundreds of fundraising activities have been initiated by even more volunteers throughout the country, and these activities are listed on the website. Besides these heart-warming activities also large donations were made by families and foundations, including gifts of 40.000 to 250.000 euro’s to finance complete research lines. Within 2 years more than 2 million euros were donated, which enabled the financing of 6 research lines already and an open call in 2018 for more scientists to work on a cure for HD.

For decades most research was aiming for treating symptoms, and studying the various consequences of mutant huntington protein expression in cells (that affects many cellular processes such as vesicle transport and mitochondrial functioning). Various potential compounds were tested in trials but none were effective, or can only be used to treat particular symptoms (e.g. depression). However, more recent research focuses on the cause of disease: the mutant huntington protein itself. The central aim is to decrease synthesis of the mutant protein, prevent aggregation of the mutant protein, and/or improve degradation of the mutant protein.

There are various research lines thinkable (and ongoing) that aim for a cure for HD, including:

1. Inhibition of synthesis of the mutant huntington protein: making use of so-called interference or antisense RNA the signal between de corresponding mutated gene and the protein synthesis machinery is intercepted. There are various strategies that can be exploited in HD, and the most well-known is the IONIS phase I clinical trial that is now continued by Roche.
2. Preventing aggregation of the mutant huntington protein. Or cells express so-called chaperones that assist in protein folding, and the prevention of protein aggregation. Some chaperones were shown to prevent aggregation of the mutant huntington protein, and a therapeutic strategy would be to induce these chaperones in the brain. It was recently published that overexpression of one of these chaperones delayed onset of HD in animal models.
3. Improving recognition and degradation of mutant huntington proteins. All proteins are degraded sooner or later by a protease, an enzyme that recycles proteins. In order to be recognized for degradation these proteins need to be labelled by a marker, and preliminary data indicates that due to the mutation the huntington protein is inefficiently labelled, and therefor hardly recognized for degradation. Identifying involved enzymes and stimulating proper labelling and degradation would be a therapeutic strategy to accelerate mutant huntington protein degradation. Interestingly, while the mutant huntington protein is synthesized in most cells, only particular cells are affected, while others seem more efficient in degrading mutant huntington proteins. This could be due to differences in the levels of involved enzymes, or chaperones.
4. Screening with existing medicines/compounds. Using large libraries of existing compounds (used/developed for other disease studies) their effect on reducing mutant huntington protein levels can be tested. Effective compounds must be verified, and their mechanism explored (how, via what enzyme, specificity).

Note that all these research lines aim to reduce the level of mutant huntington protein, which is the direct cause of disease.