Aberrant Mitochondrial Dynamics and Exacerbated Response to Neuroinflammation in a Novel Mouse Model of CMT2A

Abstract

Charcot-Marie-Tooth disease type 2A (CMT2A) is the most common hereditary axonal neuropathy caused by mutations in MFN2 encoding Mitofusin-2, a multifunctional protein located in the outer mitochondrial membrane. In order to study the effects of a novel MFN2K357T mutation associated with early onset, autosomal dominant severe CMT2A, we generated a knock-in mouse model. While Mfn2K357T/K357T mouse pups were postnatally lethal, Mfn2+/K357T heterozygous mice were asymptomatic and had no histopathological changes in their sciatic nerves up to 10 months of age. However, immunofluorescence analysis of Mfn2+/K357T mice revealed aberrant mitochondrial clustering in the sciatic nerves from 6 months of age, in optic nerves from 8 months, and in lumbar spinal cord white matter at 10 months, along with microglia activation. Ultrastructural analyses confirmed dysmorphic mitochondrial aggregates in sciatic and optic nerves. After exposure of 6-month-old mice to lipopolysaccharide, Mfn2+/K357T mice displayed a higher immune response, a more severe motor impairment, and increased CNS inflammation, microglia activation, and macrophage infiltrates. Overall, ubiquitous Mfn2K357T expression renders the CNS and peripheral nerves of Mfn2+/K357T mice more susceptible to mitochondrial clustering, and augments their response to inflammation, modeling some cellular mechanisms that may be relevant for the development of neuropathy in patients with CMT2A.

Mitofusin 1 overexpression rescues the abnormal mitochondrial dynamics caused by the Mitofusin 2 K357T mutation in vitro

“…The CMT2A‐causing 1070A>C (p.K357T) mutation 20 was introduced by PCR site‐directed mutagenesis to the human MFN2 WT CDS in the previously generated mThy1.2‐flag‐MFN2 WT plasmid 22 . MFN2 WT (Figure 1A) and MFN2 K357T (Figure 1B) were fused with flag ‐tag at their N‐termini as a means to verify their expression, which is dictated by the mouse Thy1.2 promoter driving postnatal, restricted expression in long‐projection neurons of the PNS and CNS 22,27 …”

“…The previously characterized novel MFN2 K357T mutation 20 was further studied here in differentiated SH‐SHSY cells. Cells overexpressing MFN2 WT depicted widely distributed mitochondria from the cell body down to the axon‐like processes, while MFN1 WT overexpression resulted in highly interconnected mitochondria throughout the cell body and the axon‐like processes, along with mitochondrial aggregates.…”

“…At the molecular level, numerous MFN2 mutations have been shown to mediate disruption of mitochondrial fusion giving rise to mitochondria that are smaller in size and appear fragmented due to the ongoing fission process, accompanied by the formation of mitochondrial clusters 10,15–19 . In a recently characterized novel knock‐in (KI) mouse model, expression of the novel Mfn2 K357T mutant also induced clustering of mitochondria and arrested the fusion of their OMMs 20 …”

“…10,[15][16][17][18][19] In a recently characterized novel knock-in (KI) mouse model, expression of the novel Mfn2 K357T mutant also induced clustering of mitochondria and arrested the fusion of their OMMs. 20 Before mitochondrial fusion, MFN1 and MFN2 interact with each other on the OMMs of juxtaposed mitochondria and form heterotypic (MFN1-MFN2) and homotypic (MFN1-MFN1, MFN2-MFN2) complexes, 15,21 a process termed mitochondrial tethering. 18,22 Among these, the MFN1-MFN2 heterotypic interaction is the most favored during the GDP bound state, 21 and in mouse embryonic fibroblasts (MEFs), MFN1 WT better complements various MFN2 mutants in regard to mitochondrial fusion than MFN2 WT itself.…”

The immune system as a driver of mitochondrial disease pathogenesis: a review of evidence

“…Mice lacking Gdap(-/-) develop astrocytosis and microgliosis in the spinal cord, with increased expression of pro-inflammatory TNF-α and Cxcl10 [126]. Mice heterozygous for a disease-causing Mfn2 mutation also present with microgliosis in the optic nerves and in the lumbar spinal cord [127]. When immunologically challenged with LPS, these mice have a greater increase in serum TNFα and IL-6, and an exacerbated immune response in the lumbar spinal cord and optic nerve after LPS challenge, compared to control animals [127].…”