A variant-specific insight is revealed into CMT2A disease mechanisms and HDAC6 is confirmed as a promising target for further therapeutic development by showing that mitochondrial trafficking defects could be alleviated by treatment with an HDAC6 inhibitor.
Abstract
Charcot-Marie-Tooth disease (CMT) is a group of inherited progressive conditions affecting distal motor and sensory neurons, leading to muscle weakness, pain, and loss of sensation in limbs. CMT type 2A (CMT2A) is the most common form of axonal CMT and is associated with a more severe clinical manifestation. However, there are no treatments currently available. To investigate disease mechanisms and facilitate treatment discovery, we developed an in vitro model for CMT2A by introducing the patient-specific MFN2R94Q/+ variant into human embryonic stem cells (hESCs). Isogenic variant and wild-type hESCs differentiated into spinal motor neurons with similar efficiency and gave rise to functional motor neurons in vitro. However, MFN2R94Q/+ spinal motor neurons displayed impaired mitochondrial trafficking, resulting in altered distribution of mitochondria in axons. Unbiased quantitative proteomic profiling of the endogenous MFN2 interactome revealed dose-dependent remodelling by the R94Q variant across 412 proteins, highlighting candidate mechanisms in disease pathology. Importantly, we showed that mitochondrial trafficking defects could be alleviated by treatment with an HDAC6 inhibitor. Chemical inhibition of HDAC6 also rescued the motor phenotype in a zebrafish CMT2A model. Taken together, our study reveals a variant-specific insight into CMT2A disease mechanisms and confirms HDAC6 as a promising target for further therapeutic development.
Dominant loss-of-function mutations in DNM2 cause Charcot-Marie-Tooth neuropathy characterized by sensory and motor deficits associated with myelin and/or axonal abnormalities and muscle atrophy. Increasing DNM2 activity from embryogenesis has been reported to ameliorate neuromuscular phenotypes in the Dnm2K562E/+ Charcot-Marie-Tooth mouse; however, this model displays predominantly muscle pathology and limited nerve involvement, precluding rigorous evaluation of neuropathic mechanisms and potential therapies. Here, we performed comprehensive behavioral, electrophysiological, histological and molecular analyses to characterize the Dnm2K562E/SC- mouse, which combines systemic heterozygosity for the common K562E mutation together with Schwann cell-specific deletion of wild-type Dnm2. This model faithfully reproduces key clinical and pathological features of DNM2-Charcot-Marie-Tooth, including motor deficits, reduced general force and coordination, and severe sensory and motor conduction deficits associated with axonal loss, demyelination, and inflammation. Mechanistically, we delineate a coherent pathological sequence that explains the profound functional deficits. In particular, a downregulation of the transcription factor EGR2, a master regulator of myelin gene expression, and of the myelin protein MPZ correlates with demyelination. To evaluate the therapeutic potential of DNM2 supplementation, post-symptomatic intrathecal delivery of AAV9-DNM2 driven by the Schwann cell-specific MPZ promoter was performed at 4 weeks. Although DNM2 expression increased in peripheral nerves (∼1.9-fold), no significant improvements were observed across behavioral, electrophysiological, structural, or molecular parameters. Together, these findings establish the Dnm2K562E/SC- mouse as a robust preclinical model, recapitulating key features of DNM2- Charcot-Marie-Tooth, and provides crucial insight into the biological and temporal constraints that must guide future therapeutic strategies for DNM2-Charcot-Marie-Tooth.
Marie Goret, T. Arbogast, Jocelyn Laporte· Brain Communications· 0 citations
Charcot-Marie-Tooth disease type 2D (CMT2D) results from gain-of-function mutations in GARS1, which encodes glycyl-tRNA synthetase (GlyRS), the enzyme responsible for charging transfer RNA (tRNA) with glycine. There are several CMT2D mouse models, but GarsΔETAQ/+ is the only one that bears a patient-sourced mutation. Created using CRISPR/Cas9 to model a 12-nucleotide de novo GARS1 deletion identified in an unusually severe CMT2D patient, GarsΔETAQ/+ mice have previously been shown to display several neuromuscular phenotypes; motor axon loss, denervated neuromuscular junctions (NMJs) and reduced muscle function. Here, we extend these analyses to provide a more comprehensive understanding of both motor and sensory nerve deficits across hind- and fore-limbs. At 3 months, GarsΔETAQ/+ mice possess sex-independent alterations in the levels of neuropathy biomarkers – including decreased NfL and increased periaxin – alongside reduced muscle endurance and strength, and impairments in the sensory modalities of mechanosensation, proprioception and nociception. Underpinning these dysfunctions, we identified site-specific defects comprising altered sensory neuron populations, muscle spindle loss, reduced motor neuron size, disrupted NMJ innervation and maturation, and reduced axonal transport of signalling endosomes in vivo. Together, these experiments show that GarsΔETAQ/+ mice display robust and selective peripheral nerve pathology that manifests in a general distal-to-proximal fashion, priming this CMT2D allele for testing treatments and evaluating mechanisms underlying peripheral nerve vulnerability.
R. Simkin, A. Paulo-Ramos, Qiuhan Lang et al.· bioRxiv· 0 citations
Charcot-Marie-Tooth (CMT) disease type 2A is a rare heritable disorder caused by pathogenic variants of mitofusin (MFN) 2 that suppress mitochondrial fusion and motility in peripheral nerves, culminating in denervation myoatrophy. The rarity of this condition and the limited choice of animal models preclude pre-clinical evaluation of many tests that could be translated to human trials. Here, we introduced the CMT2A pathogenic variant MFN2 T105M into the rat genome for phenotype characterization and evaluation of disease response to a third-generation mitofusin activator, 8015-P2. CMT2A rats exhibited peripheral motor and sensory neuron dysfunction. Functional, histological, neuroelectrophysiological and magnetic resonance imaging testing readily distinguished between wild-type (WT) and mutant rats via axonopathy and myoatrophy. Compound 8015-P2 reversed CMT2A-linked neuromuscular degeneration in a dose- and time-dependent manner; at 10 mg/kg/d, normalization occurred at 4 weeks. The minimal effective 8015-P2 dose was 2 mg/kg/day. Rapidity of phenotype reversal and primary muscle abnormalities are consistent with extra-neuronal effects of the causal MFN2 DNA variant. These data demonstrate unprecedented utility of the Mfn2 T105M rat as a model of CMT2A, expand the menu of clinically applicable tests that may have use in future human trials, and establish a strong foundation for exploration of extra-neuronal consequences of pathogenic mitofusin variants in non-mouse models.
J. Weigele, A. Franco, Gerald W. Dorn· International Journal of Mol...· 0 citations
Simple Summary Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons, leading to progressive muscle weakness and severe muscle wasting. In addition to neuronal loss, increasing evidence suggests that problems in mitochondrial function, the process by which cells produce energy, may contribute to disease development and progression. In this study, we investigated the effects of Mdivi-1, a drug that inhibits mitochondrial fragmentation, in a mouse model of ALS. Treatment began before symptoms appeared in order to assess whether it could slow disease progression. Our findings showed that Mdivi-1 partially protected motor neurones in the spinal cord, but this did not result in improved motor performance. Moreover, treated animals developed more severe muscle wasting and changes in muscle fiber composition. We also observed cellular alterations associated with impaired neuromuscular function. Overall, these results highlight that mitochondrial dynamics play a complex and context-dependent role in the disease. A better understanding of these mechanisms will be important for developing more effective therapeutic strategies for ALS.
Maria Ciuro, Chantal Rovetto, Angela A. Sirna et al.· Biology· 0 citations
Astrocyte-specific mitochondrial dysfunctions and calcium dysregulation are identified as key features of LRRK2- and PRKN-related pathology, providing new insights into how glial metabolic alterations contribute to neurodegeneration in PD.
Giovanna C. Cavalcante, Camille C. Caldeira da Silva, É. Vogt et al.· bioRxiv· 0 citations
This work generates human induced pluripotent stem cell-derived midbrain dopaminergic neurons using both a 2D differentiation protocol and a recently developed 3D method based on the microencapsulation of hiPSCs in small alginate/fibronectin beads.
A. Zanon, E. Kerschbamer, D. Riekschnitz et al.· Communications Biology· 0 citations
