Investigation of axonal transport deficits in Charcot-Marie-Tooth disease type 1A and modification through selective inhibition of histone deacetylase 6

Charcot-Marie-Tooth disease (CMT) is the most common inherited neurological disorder of the peripheral nervous system (PNS). Patients suffer from mild-to-severe muscle loss at the most distal regions of their body in a ‘stock-glove’ distribution. Duplications in the gene coding for peripheral myelin protein 22 (PMP22) causes the most common form of CMT, CMT type 1A (CMT1A). PMP22 is an essential protein for the initiation and maintenance of Schwann cell myelination in the PNS. Myelin is a multilamellar lipid dense structure Schwann cells produce to enables fast and efficient ‘signalling’ of PNS sensory and motor axons so that we may take in sensory information and respond to our environment. In CMT1A, the duplication PMP22 is known to cause lipid metabolism defects that ultimately impar CMT1A Schwann cells in their ability to myelinate PNS axons, however, how PMP22 duplications caused this is unknown.

In the first part of this thesis, we investigated whether inhibiting known negative regulator of myelination, histone deacetylase inhibitor 3 (HDAC3), was a valuable treatment strategy for CMT1A using the C3 CMT1A mouse model. We demonstrated that early treatment of CMT1A mice with the selective HDAC3 inhibitor (HDAC3i) increased myelination and myelin g-ratios, and improved electromyography recordings. However, a high dose of the HDAC3i caused a decline in rotarod performance and a decline in overall grip strength. Additionally, macrophage presence in peripheral nerves was increased in HDAC3i treated CMT1A mice. We conclude that HDAC3 does not only play a role in regulating myelination but is also important in neuroimmune modulation. Overall, our results indicate that correct dosing of HDAC3 inhibitors is of crucial importance if translated to a clinical setting for demyelinating forms of CMT, or other neurological disorders.

In the second part of the thesis, we aimed to investigate the molecular mechanisms that lead to the a lipid metabolic defect in CMT1A. Bulk RNA-sequencing data of sciatic nerves from the C3 and the C22 CMT1A mouse models, which overexpress 5 and 8 copies of human PMP22 respectively, demonstrated that cholesterol metabolism is the most dysregulated pathway throughout their development. PMP22 overexpression had a dose-dependent effect on the suppression of cholesterol metabolism. Lipidomic analysis of sciatic nerves of 5 weeks old C3 mice revealed dysregulated relative expression of lipids regulating lipid storage/droplet homeostasis, plasma membrane identity, and very-long-chain polyunsaturated fatty acids (VLC-PUFAs). To model this lipid metabolism phenotype in patient and patient-corrected (isogenic) derived cells, we developed an induced pluripotent stem cell (iPSC) to Schwann cell lineage protocol. We differentiated iPSCs to Schwann cell precursors (iPSC-SCPs) and showed via bulk RNA-sequencing that CMT1A patient-derived iPSC-SCPs have dysregulated expression of plasma membrane-associated genes in comparison to their isogenic control iPSC-SCPs. Lipidomic analysis of these cells similarly revealed alteration in the relative expression of lipids regulating plasma membrane identity, VLC-PUFAs, and lipid storage/droplet homeostasis. Using electron microscopy, we identified lipid accumulations in late endosome-lysosomes in the CMT1A iPSC-SCPs. Moreover, Di-4-ANEPPDHQ flow cytometry analysis highlighted increased CMT1A iPSC-SCP membranes are more disordered compared to their isogenic iPSC-SCP. We further investigated these alterations via high-content confocal immunofluorescence imaging to analyze cholesterol distribution as well as LD and lysosome dynamics. We discovered that CMT1A iPSC-SCPs have reduced membrane cholesterol and, upon oleic acid stimulation, generate a larger size and quantity of LDs. This LD/storage phenotype was confirmed using Western blot analysis which showed alterations in DGAT1, perilipin-2, seipin, LAMP1, and NPC1. As a proof-of-concept, we showed that forskolin or progesterone antagonist treatment can modulate and improve the deficits in lipolysis/autophagy in CMT1A iPSC-SCPs. Moreover, we show that lipolysis/autophagy modulation in CMT1A iPSC-SCPs attenuates lipid recycling homeostasis and restores their membranes’ cholesterol content.

In conclusion, in this thesis, we highlight the therapeutic potential of HDAC3i for the treatment of CMT1A and shed light on some of the molecular mechanisms underlying lipid metabolic phenotype in CMT1A. Further research is needed to better understand how lipids are processed by CMT1A Schwann cells, as this may lead to more effective therapies being developed for this currently untreatable disease.