HD Research: Profiles on Current HD Research

Lise Munsie, PhD, is Senior Development Manager and Project Leader, Pluripotent Stem Cell Therapies at CCRM, a Canadian, not-for-profit group that is focused on developing and commercializing regenerative medicine, cell and gene therapy technologies.

1. Discovery of Binding Sites Needed for mtHTT Aggregation and Folding into Amyloid Fibrils
Understanding the normal function of huntingtin (HTT), how it interacts with other proteins and how it functions in cellular pathways is important for understanding the natural history of HD and may inform pathways for drug discovery and future therapeutics.

When HTT is mutated (mtHTT) it causes aggregation of mtHTT into amyloid fibrils in the brain. These aggregates are a hallmark of HD and are caused by the misfolding of the protein. Chaperones are proteins that are responsible for the proper folding of proteins, and a trimeric complex of three chaperones, Hsc70, DNAJB1 and Apg2, have the ability to bind mtHTT and suppress or reverse aggregation. Modulating the expression of these chaperones can be beneficial in models of HD.

Recently, a research group mapped out and modeled how this trimeric complex binds mtHTT, allowing it to suppress or reverse mtHTT aggregation. Using a technique called cross-linking mass spectrometry, the group found that mtHTT specifically binds the DNAJB1 protein in the complex. They homed in on a binding region on the protein, and when they mutated that binding region,  they found that the complex could no longer bind mtHTT, but no other normal protein interactions were inhibited. The group concludes that this binding site in DNAJB1, H244, is specific for HTT binding and required for the trimeric complex to act on amyloid fibrils in HD.

Ayala Mariscal, S.M. et.al. (2022) Identification of a HTT-specific binding motif in DNAJB1 essential for suppression and dis-aggregation of HTT. Nature Communications: 13:4692

2. Using Stem Cells to Create Blood-Brain Barrier Mimicker
The blood-brain barrier (BBB) is part of the human central nervous system (CNS) and is a structural and functional barrier to organisms entering the brain. Changes associated with cell types other than neurons, like those in the BBB, are hypothesized to contribute to neurological disease progression.

The BBB can be mimicked for research by differentiation of iPSC into neuro-endothelial cells. In a study, scientists took iPSC derived from individuals with 33 CAG (wildtype), 77 CAG. and 109 CAG (HD), and differentiated these cells into a BBB model. All three cell lines were able to successfully differentiate into the BBB.

The group found that the HD model led to higher leakage in BBB, indicating compromised BBB morphology. By analyzing protein expression, the authors found that a particular protein involved in the creation of this barrier, claudin 5, was downregulated. Whole genome sequencing indicated that many other transporter proteins were also dysregulated in the HD models.

In response to an immune stimulus, HD cells had less ability to respond compared to wildtype cells.

This model allows for the evaluation of different transport routes in the brain for which there is no other in vivo or in vitro model. This iPSC-derived BBB model will serve as a tool to support studies for CNS drug discovery and delivery, which is critical for finding treatments for HD.

Vignone, D. et.al. (2022) Modelling the human blood-brain barrier in Huntington’s Disease. International Journal of Molecular Sciences: 23: 7813

3. Knock Down Model Shows HTT’s Crucial Role in Cytoskeletal Regulation
Defining HTT function in neurodevelopment is important. Since HTT knock down is a promising therapeutic avenue for treating HD, it is critical to know at what point in the course of disease HTT can be knocked down. A new study investigated this by designing a mouse model that has HTT knocked down in just the cortical excitatory neurons. The researchers investigated the consequences of HTT knock down in this HD-vulnerable cell type during mouse development.

The group found that depletion of HTT in the excitatory neurons led to enlarged neuronal spines (sites on neurons where they communicate to one another). Normally enlarged spines leads to increased neuronal activity, however in the case of HTT knock down, the group found there was reduced neuronal activity.

The authors found that proteins involved in the cytoskeleton of the cell, which are responsible for the size and shape of the spines, were not being properly regulated when HTT was knocked down. The authors determined that HTT is involved in the activation of a protein called cofilin which regulates the cytoskeleton. When HTT is not present, cofilin is less active and the morphology of neuronal spines and functions becomes impaired and decoupled.

Synaptic dysfunction is well-defined in HD, and this study indicates that HTT involvement in the actin cytoskeleton both in development and during the course of disease could be a major factor contributing to HD progression.

Wennagel, D. et.al (2022) Huntingtin coordinates dendritic spine morphology and function through cofilin-mediated control of the actin cytoskeleton. Cell Reports: 40, 111261

4. Disrupting mHTT-CaM Binding is a Potential Complementary Therapy
There are many promising therapeutic avenues in the field of gene therapy which involve delivering drugs to patients to stop the huntingtin (HTT) gene from being expressed. There are still a lot of unknowns with this radical approach for disease treatment, however, so it is important to continue to try to find different ways to treat HD using more traditional drug discovery or treatment approaches.

Understanding mutant HTT (mHTT) function in HD is a way to find drugs to treat HD. It has been established that mHTT protein binds other proteins abnormally. One protein is called calmodulin (CaM), and the inappropriate binding of mHTT-CaM causes cellular perturbations that are hypothesized to lead to cellular dysfunction that causes HD.

Recently a research group used this abnormal binding in a screening assay to find small molecules that would stop mHTT from binding CaM. Initially, the group screened 3,951 compounds. Using a cell-based assay, they were able to find multiple compounds that inhibit mHTT-CaM binding. These were narrowed down to four compounds that disrupt mHTT-CaM but do not cause cellular toxicity.

When treating cells with these compounds, they found three of them could rescue mHTT-induced cellular toxicity. The group then found that two of the compounds showed positive effects on CaM activity in cells expressing mHTT, and these two compounds became the lead compounds. Neither had effects on cellular aggregate number or sizing or on the expression level of mHTT, indicating that these aspects of disease are not affected by mHTT-CaM interaction. The group concluded that these small molecules could be used as a complementary therapeutic in the treatment of HD.

Kapadia, K. et.al. (2022) Small-molecule disruptors of mutant huntingtin-calmodulin protein-protein interaction attenuate deleterious effects of mutant huntingtin. ACS Chemical Neuroscience: 13:2315-2337

5. Trial Outcomes for Immunotherapy Drug Pepinemab
Pepinemab, an antibody-based compound that has been shown to have positive effects in HD mouse models, was moved into human clinical trials for HD. This SEMA4D protein is known to have functions specific to the immune response in the brain and is upregulated in stressed or damaged neurons, like those in HD. It is hypothesized that inhibiting the action of SEMA4D may reduce neuronal loss in HD.

Pepinemab is known to have a good safety profile in humans, and therefore moved into the Signal-HD phase II  HD clinical trial (sponsored by Vaccinex Inc. and conducted by the HSG)  in 265 patients with preclinical or early clinical signs of HD. The pepinemab treatment was well tolerated. While clinical outcomes did not reach significance in this trial for HD patients, the treatment did decrease brain atrophy and have potential cognitive benefit, suggesting further trials or development of this drug may lead to positive clinical outcomes.

This treatment seemed to have more benefit in patients who were further into their clinical progression compared with pre-clinical patients, which means patients showing clinical signs of HD should be the focus group for future clinical trials using this SEMA4D immunotherapy-based approach.

Feigin, A. et.al. (2022) Pepinemab antibody blockage of SEMA4D in early Huntington’s disease: a randomized, placebo-controlled, phase 2 trial. Nature Medicine: http://doi.org/10.1038/s41591-022-01919-8

6. Stem Cells Used to Probe HD Effect on Striatal Neurons

Scientists can take cells from an adult—generally skin or blood cells—and derive stem cells. These cells are called induced pluripotent stem cells (iPSC) and are the best tools scientists have to model human disease, since stem cells have the ability to become any cell type in the body.

Stem cells made from Huntington’s disease (HD) patients allow scientists to study any cell type or system from not only human cells, but the specific cell types, like striatal neurons, that are uniquely affected by mutant huntingtin (mHTT).

New HD iPSC lines continue to be derived. Most recently a line from a patient containing 77 CAG repeats has been derived, characterized, and is available for research.

iPSC cells can be changed into neurons using specific protocols and growth conditions. A recent study used this unique property of iPSC to study how mHTT affects human neurons. The group analyzed how neurons develop when mHTT is present in the cell. They found that neurons expressing mHTT had altered morphology compared with wildtype neurons, which was indicative of a delayed maturation.

To support this, the researchers found a decrease in protein levels associated with mature neurons in the cells expressing mHTT. Neurons signal to each other through pathways called synaptic connections, which are critical for normal brain function. Using image-based techniques, the group found that there were fewer of these connections in cells expressing mHTT and there was a delay in the development of the neurons’ ability to perform their normal function, which is measured through electrophysiology.

This study confirms HTT involvement in neuronal development and indicated the need to understand the consequences of these changes during development.

Grigor’eva, E.V. et.al. (2022) Generation of induced pluripotent stem cell line, ICGi033-A, by reprogramming peripheral blood mononuclear cells from a patient with Huntington’s disease. Stem Cell Research: 63: 102868

Dinamarca, M.C. et.al. (2022) Synaptic and functional alterations in the development of mutant huntingtin expressing hiPSC-derived neurons Frontiers in molecular biosciences: 10.3389/fmolb.2022.916019