Single molecule fingerprinting of mutant Httex1 oligomers for early detection of Huntington’s Disease
Description: Huntington’s disease (HD) is a neurodegenerative disorder characterized by an increasing number of CAG trinucleotide repeats in the first exon of the human HTT gene that leads to progressive deterioration in both cognitive function and motor control. A potential biomarker for HD is urgently needed to determine the disease onset and monitor the progression of the disease. Currently, there are no disease-modifying treatments for HD and an effective biomarker will improve the discovery and development of potential therapies. The principal pathogenic molecule of HD, the mutant HTT exon 1 (Httex1) protein is identified in cerebrospinal fluid (CSF), and blood, and is also detectable in saliva potentially representing a convenient and non-invasive biomarker for HD. Aggregation of the biomarker, Httex1 protein with long polyglutamine (polyQ) expansion is the hallmark of the disease in the brains of HD transgenic mice. Growing evidence indicates that early-stage oligomeric aggregates of amyloid-like protein biomarkers such as a-synuclein and Tau are the key drivers of neuronal toxicity in Parkinson's and Alzheimer's disease. Quantification of mutant Huntingtin proteins in CSF and related body fluids in several studies promises both biomarker development to monitor disease progression and HD-lowering therapeutic approaches. However, existing single-molecule immunoassays for mutant HTT protein detection from blood and saliva do not provide any insight into the size or shape of Httex1 protein oligomers. This constraint limits the information of quantitative Huntingtin’s studies because some sizes and forms of oligomeric Httex1 aggregates could be more toxic than others. Measuring the size and shape of Httex1 protein oligomers represents an essential step in biomarker development for clinical and diagnostic relevance. Therefore, a new approach that detects the size-, and the shape-distribution of heterogeneous Httex1 oligomers in real-time from microliter samples of saliva in solution would be a significant development. Here, we propose to utilize a biological nanopore-based sensing technique to detect and quantify Httex1 oligomers in human saliva samples, as a relevant and non-invasive HD biomarker. We will utilize the ability of a pore-forming toxin, pneumolysin (PLY), to spontaneously self-assemble upon contact with a lipid membrane and generate a stable biological nanopore with a large diameter (˜ 20 nm; own unpublished data); and conjugate it with Httex1-specific high affinity-nanobodies to bind, and detect Httex1 oligomers from the saliva samples by changes in pore resistance during the passage of each molecule. The use of PLY-nanopore (3.6 times large in diameter compared to any existing biological nanopore) sensing with specific nanobodies will ensure high-sensitivity quantification of the Httex1 protein and its oligomers in extracellular fluid samples for the first time. Single-molecule detection with biological nanopores will facilitate targeted sensing of Httex1 protein in limited sample volumes and it will also fingerprint Httex1 soluble oligomers with respect to size, and shape without any clogging of the pore. The proposed strategy of toxic Httex1 oligomer subpopulation detection through low-noise biological nanopores will provide the true size and shape distributions, allowing the implementation of all oligomeric species for both biomarkers’ development and clinical research application.
Amount: CHF 99,680
Funding type: grant
Funder scheme: Spark
Start date: 2024-04-01
End date: 2025-03-31
Investigator: Mukhopadhyay, Anasua
Institution: University of Fribourg – FR
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Funded outputs count: 1
Funder award ID: 221078
OpenAlex ID: G1301461582