Research Bieke Bekaert

Abstract

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy, affecting 1 in 2.500 people worldwide. CMT causes length-dependent degeneration of motor and sensory peripheral nerves, resulting in progressive muscle weakness, sensory loss, and pain. CMT is highly genetically heterogeneous, with causative mutations in over 100 different genes. CMT2J, caused by the T124M mutation in myelin protein zero (P0/MPZ; the main myelin protein), is a subtype that presents as a late-onset axonal neuropathy, characterised by severe axonal degeneration with minimal myelin damage. While previous studies in a CMT2J mouse model identified a defect in axonal transport, the slow onset and mild phenotype of these mice make preclinical studies challenging. Recent data suggest that inhibiting histone deacetylase 6 (HDAC6) could mitigate axonal transport defects, making HDAC6 inhibition a promising therapeutic target for CMT2J. However, there is currently no human-specific in vitro model to investigate this therapeutic potential. In this project, we aim to develop a human-relevant cellular model by the creation and characterisation of induced pluripotent stem cells derived neuromuscular assembloids (NMAs) specific to CMT2J. NMAs are created by fusing a motor neuron sphere with a muscle sphere to achieve a posterior-anterior cellular organisation. This adds directional growth to the neuronal cell structures allowing for a more accurate modelling of the peripheral nervous system in vitro. Immunohistochemical staining will be used to characterise the myogenic, neuronal, and Schwann cell markers, while advanced techniques such as transmission electron microscopy (TEM) will be employed to identify signs of axonal degeneration. In addition, live-cell imaging will assess axonal transport dynamics, and multi-electrode array (MEA) assays will evaluate the electrophysiological parameters. The ultimate goal of the project is to use these NMAs to investigate the therapeutic potential of HDAC6 inhibition in CMT2J. We will treat both isogenic control and CMT2J-specific NMAs with Tubastatin A, a known HDAC6 inhibitor, to assess its effects on axonal transport defects and axonal degeneration. Live-cell imaging will be repeated to determine if axonal transport is restored, and immunohistochemical staining and TEM will be used to examine any changes in axonal integrity. The end objective is to validate HDAC6 as a pharmacological target for CMT2J and to establish a human-based model that can be used for future therapeutic screening. This research will provide essential insights into the underlying pathomechanisms of CMT2J and support the development of novel treatment strategies for this currently untreatable disease.

Researcher(s)

• Promoter: Bekaert Bieke

Research team(s)

• Peripheral Neuropathies Group

Project type(s)

• Research Project

Abstract

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy. Although multiple animal models have been created, the low animal-to-human translational success demands the need of new disease models originating from human cells. In this project, I will develop and characterize induced pluripotent stem cell (iPSC)-derived organoids for CMT1A and CMT2A, the most common CMT types. These organoids consist of neuronal cells surrounded by myelinating Schwann cells, demonstrating pathological signatures (e.g. myelin disruption) when derived from CMT1A-iPSCs. Notwithstanding the mimicking potential of neuromuscular organoids, they do not grow in a directional manner which complicates the investigation of various CMT-related features, including the understudied disruption of neuromuscular junctions which is a hallmark for CMT. Therefore I will additionally generate iPSC-derived assembloids which have an anterior-to-posterior organisation, highly relevant to study neuromuscular junctions in CMT. For both organoids and assembloids, an in-depth investigation of CMT-related structural and molecular characteristics will be performed and a comparison between both CMT types will be undertaken. Furthermore, I will conduct an in-depth assessment of the response to therapeutic interventions in these models. In general, these CMT models have the potential to facilitate the development of new treatments and to diminish the necessity for animal experiments.

Researcher(s)

• Promoter: Timmerman Vincent
• Fellow: Bekaert Bieke

Research team(s)

• Peripheral Neuropathies Group

Project type(s)

• Research Project