Understanding Charcot-Marie-Tooth Pathogenesis Using in vivo and Gene Networking Studies

Charcot-Marie-Tooth disease (CMT) is a common, hereditary, length-dependent peripheral neuropathy roughly classified as either demyelinating (CMT1) or axonal (CMT2). CMT has no known cure, with patients relying on corrective surgeries and braces, as well as low-stress strength training, for support. Though the disease is highly heterogeneous, many of the most severe axonal cases are caused by dominant mutations in mitofusin 2 (MFN2). MFN2 (and its paralog, MFN1) reside in the outer mitochondrial membrane, where they participate in various mitochondrial functions, some of which are complimentary (including mitochondrial fusion and transport). The mechanism by which MFN2 mutations cause CMT are unknown. A robust animal model could inform our pathologic understanding of the disease and could be used in pre-clinical trials for novel therapies. We have characterized the first knock-in mouse model using a common mutation seen in human patients, Mfn2R94W (Chapter 2). Homozygous inheritance of the mutation was found to be lethal within a day of birth, with concurrent deficits in oxygen consumption, ATP production, and mitochondrial network morphology. Heterozygous animals showed only mild changes, including nerve morphology, age-dependent movement and rearing behaviors, and mitochondrial network morphology. In order to produce a more severely affected model, we took a number of approaches to stress the surviving heterozygous animals (Chapter 3, 4). Three different genetic approaches and two different chemical approaches (tested at multiple doses) were unable to elicit a pronounced neuropathic phenotype in these mice. One specific challenge, however, revealed decreased nerve regenerative capabilities: optic nerve crush with ciliary neurotrophic factor (CNTF) treatment (Chapter 4). In humans, the R94W mutation has been linked to optic nerve degeneration that presents after onset of peripheral neuropathy, which is an uncommon phenotype in CMT patients. Therefore we reasoned this tissue may be particularly susceptible to insult. Wild type animals that underwent nerve crush with CNTF treatment showed robust axonal regeneration that was not matched by heterozygotes. Over 80 genes are known to cause CMT, with wide-ranging cellular functions and proposed mechanisms. MFN2 is involved in mitochondrial fusion, axonal transport, mitochondrial tethering to the ER, mitophagy, and mitochondrial metabolism, further confounding pathological understanding of the disease. To better understand the pathways that might contribute to MFN2-induced neuropathy, we performed transcriptome analysis using RNA-sequencing data from neural tissue isolated from newborn mice (Chapter 5). Differential expression between genotypes did not include significant levels of genes involved in any known MFN2 functions, however, we did see significant downregulation of known CMT genes, of genes transcriptionally regulated by EGR2, and of genes differentially expressed in other mouse models of CMT and delayed peripheral nerve regeneration. In conclusion, we have described the first Mfn2 knock-in model of CMT, which displays homozygous lethality, mostly mild heterozygous phenotypes, and a unique vulnerability to optic nerve crush. Expression analysis revealed differential expression of known CMT genes and genes involved in Schwann cell functions, indicating cohesive forces among the broad genotypic and phenotypic variance of this disease. Future studies may reveal the nature of these interactions

Characterization of the mitofusin 2 R94W mutation in a knock‐in mouse model

Charcot‐Marie‐Tooth disease (CMT) comprises a group of heterogeneous peripheral axonopathies affecting 1 in 2,500 individuals. As mutations in several genes cause axonal degeneration in CMT type 2, mutations in mitofusin 2 (MFN2) account for approximately 90% of the most severe cases, making it the most common cause of inherited peripheral axonal degeneration. MFN2 is an integral mitochondrial outer membrane protein that plays a major role in mitochondrial fusion and motility; yet the mechanism by which dominant mutations in this protein lead to neurodegeneration is still not fully understood. Furthermore, future pre‐clinical drug trials will be in need of validated rodent models. We have generated a Mfn2 knock‐in mouse model expressing Mfn2R94W, which was originally identified in CMT patients. We have performed behavioral, morphological, and biochemical studies to investigate the consequences of this mutation. Homozygous inheritance leads to premature death at P1, as well as mitochondrial dysfunction, including increased mitochondrial fragmentation in mouse embryonic fibroblasts and decreased ATP levels in newborn brains. Mfn2R94W heterozygous mice show histopathology and age‐dependent open‐field test abnormalities, which support a mild peripheral neuropathy. Although behavior does not mimic the severity of the human disease phenotype, this mouse can provide useful tissues for studying molecular pathways associated with MFN2 point mutations

De novo PMP2

We performed whole exome sequencing on a patient with Charcot–Marie–Tooth disease type 1 and identified a de novo mutation in PMP2, the gene that encodes the myelin P2 protein. This mutation (p.Ile52Thr) was passed from the proband to his one affected son, and segregates with clinical and electrophysiological evidence of demyelinating neuropathy. We then screened a cohort of 136 European probands with uncharacterized genetic cause of Charcot–Marie–Tooth disease and identified another family with Charcot–Marie–Tooth disease type 1 that has a mutation affecting an adjacent amino acid (p.Thr51Pro), which segregates with disease. Our genetic and clinical findings in these kindred demonstrate that dominant PMP2 mutations cause Charcot–Marie–Tooth disease type 1

Characterizing the molecular phenotype of an Atp7a(T985I) conditional knock in mouse model for X-linked distal hereditary motor neuropathy (dHMNX)

ATP7A is a P-type ATPase essential for cellular copper (Cu) transport and homeostasis. Loss-of-function ATP7A mutations causing systemic Cu deficiency are associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome. We previously identified two rare ATP7A missense mutations (P1386S and T994I) leading to a non-fatal form of motor neuron disorder, X-linked distal hereditary motor neuropathy (dHMNX), without overt signs of systemic Cu deficiency. Recent investigations using a tissue specific Atp7a knock out model have demonstrated that Cu plays an essential role in motor neuron maintenance and function, however the underlying pathogenic mechanisms of ATP7A mutations causing axonal degeneration remain unknown. We have generated an Atp7a conditional knock in mouse model of dHMNX expressing Atp7a(T985I), the orthologue of the human ATP7A(T994I) identified in dHMNX patients. Although a degenerative motor phenotype is not observed, the knock in Atp7a(T985I/Y) mice show altered Cu levels within the peripheral and central nervous systems, an increased diameter of the muscle fibres and altered myogenin and myostatin gene expression. Atp7a(T985I/Y) mice have reduced Atp7a protein levels and recapitulate the defective trafficking and altered post-translational regulatory mechanisms observed in the human ATP7A(T994I) patient fibroblasts. Our model provides a unique opportunity to characterise the molecular phenotype of dHMNX and the time course of cellular events leading to the process of axonal degeneration in this disease

A novel mutation in VCP causes Charcot–Marie–Tooth Type 2 disease

Mutations in VCP have been reported to account for a spectrum of phenotypes that include inclusion body myopathy with Paget’s disease of the bone and frontotemporal dementia, hereditary spastic paraplegia, and 1–2% of familial amyotrophic lateral sclerosis. We identified a novel VCP mutation (p.Glu185Lys) segregating in an autosomal dominant Charcot–Marie–Tooth disease type 2 family. Functional studies showed that the Glu185Lys variant impaired autophagic function leading to the accumulation of immature autophagosomes. VCP mutations should thus be considered for genetically undefined Charcot–Marie–Tooth disease type 2

Mutation screen reveals novel variants and expands the phenotypes associated with DYNC1H1

Dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) encodes a necessary subunit of the cytoplasmic dynein complex, which traffics cargo along microtubules. Dominant DYNC1H1 mutations are implicated in neural diseases, including spinal muscular atrophy with lower extremity dominance (SMA-LED), intellectual disability with neuronal migration defects, malformations of cortical development (MCD), and Charcot-Marie-Tooth disease, type 2O (CMT2O). We hypothesized that additional variants could be found in these and novel motoneuron and related diseases. Therefore we analysed our database of 1,024 whole exome sequencing samples of motoneuron and related diseases for novel single nucleotide variations. We filtered these results for significant variants, which were further screened using segregation analysis in available family members. Analysis revealed six novel, rare, and highly conserved variants. Three of these are likely pathogenic and encompass a broad phenotypic spectrum with distinct disease clusters. Our findings suggest that DYNC1H1 variants can cause not only lower, but also upper motor neuron disease. It thus adds DYNC1H1 to the growing list of spastic paraplegia related genes in microtubule-dependent motor protein pathways

Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder

Dominant optic atrophy (DOA)(1,2) and axonal peripheral neuropathy (Charcot-Marie-Tooth Type 2 or CMT2)(3) are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively(4). In yeast, homologs of OPA1(Mgm1) and MFN2(Fzo1) work in concert with Ugo1(5,6), which has no human equivalent to date(7). By whole exome sequencing patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein, mitofilin(Fcj1). Loss-of-function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2, while exemplifying a novel class of modified solute transporters linked to mitochondrial dynamics