Charcot-Marie-Tooth disease-linked protein SIMPLE functions with the ESCRT machinery in endosomal trafficking

Mutations in small integral membrane protein of lysosome/late endosome (SIMPLE) cause autosomal dominant, Charcot-Marie-Tooth disease (CMT) type 1C. The cellular function of SIMPLE is unknown and the pathogenic mechanism of SIMPLE mutations remains elusive. Here, we report that SIMPLE interacted and colocalized with endosomal sorting complex required for transport (ESCRT) components STAM1, Hrs, and TSG101 on early endosomes and functioned with the ESCRT machinery in the control of endosome-to-lysosome trafficking. Our analyses revealed that SIMPLE was required for efficient recruitment of ESCRT components to endosomal membranes and for regulating endosomal trafficking and signaling attenuation of ErbB receptors. We found that the ability of SIMPLE to regulate ErbB trafficking and signaling was impaired by CMT-linked SIMPLE mutations via a loss-of-function, dominant-negative mechanism, resulting in prolonged activation of ERK1/2 signaling. Our findings indicate a function of SIMPLE as a regulator of endosomal trafficking and provide evidence linking dysregulated endosomal trafficking to CMT pathogenesis.

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Connexin 32 mutations from X-linked Charcot-Marie-Tooth disease patients: functional defects and dominant negative effects.

Molecular Biology of the Cell ◽

10.1091/mbc.7.6.907 ◽

1996 ◽

Vol 7 (6) ◽

pp. 907-916 ◽

Cited By ~ 114

Author(s):

Y Omori ◽

M Mesnil ◽

H Yamasaki

Keyword(s):

Hela Cells ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Base Substitution ◽

Cell Contact ◽

Wild Type ◽

Gap Junctional Intercellular Communication ◽

Charcot Marie Tooth ◽

Charcot Marie Tooth Disease ◽

Tooth Disease

We have characterized the function of connexin (Cx) 32 gene mutations found in X-linked dominant Charcot-Marie-Tooth disease with respect to their ability to form functional gap junctions among themselves and to inactivate wild-type Cx32 by a dominant negative mechanism. We prepared four types of Cx32 mutant cDNAs and transfected them into HeLa cells, which do not show detectable levels of gap junctional intercellular communication (GJIC), nor expression of any connexins examined. Cells transfected with the wild-type Cx32 gene, but not those transfected with three different base substitution mutations (i.e. Cys 60 to Phe, Val 139 to Met, and Arg 215 to Trp), restored GJIC. Unexpectedly, in cells transfected with a nonsense mutant at codon 220, there was also restored GJIC. When we double-transfected these mutant constructs into the HeLa cells that had already been transfected with the wild-type Cx32 gene and thus were GJIC proficient, three base substitution mutants inhibited GJIC, suggesting that these three mutants can eliminate the function of wild-type Cx32 in a dominant negative manner. The nonsense mutation at codon 220 did not show such a dominant negative effect. Since both mutant and wild-type Cx32 mRNAs were detected, but only poor Cx32 protein expression at cell-cell contact areas was observed in the double transfectants, it is suggested that certain mutants form nonfunctional chimeric connexons with wild-type connexins, which are not properly inserted into the cytoplasmic membrane.

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Axonal Transport Defects in a Mitofusin 2 Loss of Function Model of Charcot-Marie-Tooth Disease in Zebrafish

PLoS ONE ◽

10.1371/journal.pone.0067276 ◽

2013 ◽

Vol 8 (6) ◽

pp. e67276 ◽

Cited By ~ 32

Author(s):

Anna L. Chapman ◽

Ellen J. Bennett ◽

Tennore M. Ramesh ◽

Kurt J. De Vos ◽

Andrew J. Grierson

Keyword(s):

Axonal Transport ◽

Loss Of Function ◽

Function Model ◽

Mitofusin 2 ◽

Charcot Marie Tooth ◽

Charcot Marie Tooth Disease ◽

Tooth Disease

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SIRT2-knockdown Rescues GARS-induced Charcot-Marie-Tooth Neuropathy

10.1101/854588 ◽

2019 ◽

Author(s):

Chao Shen ◽

Qian Qi ◽

Yicai Qin ◽

Dejian Zhou ◽

Xinyuan Chen ◽

...

Keyword(s):

Trna Synthetase ◽

Nerve Degeneration ◽

Loss Of Function ◽

Wild Type ◽

Partial Loss ◽

Charcot Marie Tooth ◽

Charcot Marie Tooth Disease ◽

Underlying Mechanisms ◽

Tooth Disease

AbstractCharcot-Marie-Tooth disease is the most common inherited peripheral neuropathy. Dominant mutations in glycyl-tRNA synthetase (GARS) gene cause peripheral nerve degeneration and lead to CMT disease type 2D. Mutations in GARS (GARSCMT2D) show partial loss-of-function features, suggesting that tRNA-charging deficits play a role in disease pathogenesis, but the underlying mechanisms are not fully understood. In this study we report that wild-type GARS tightly binds the NAD+-dependent deacetylase SIRT2 and inhibits its deacetylation activity, resulting in the hyperacetylated α-tubulin, the major substrate of SIRT2. Previous studies showed that acetylation of α-tubulin protects microtubules from mechanical breakage and keep axonal transportation. However, CMT2D mutations in GARS can not inhibit SIRT2 deacetylation, which leads to decrease acetylated α-tubulin and severe axonal transport deficits. Genetic reduction of SIRT2 in the Drosophila model rescues the GARS–induced axonal CMT neuropathy and extends the life span. Our findings demonstrate the pathogenic role of SIRT2-dependent α-tubulin deacetylation in mutant GARS-induced neuropathies and provide new perspectives for targeting SIRT2 as a potential therapy against hereditary axonopathies.

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Connexin32 Mutations Associated with X-Linked Charcot–Marie–Tooth Disease Show Two Distinct Behaviors: Loss of Function and Altered Gating Properties

Journal of Neuroscience ◽

10.1523/jneurosci.18-11-04063.1998 ◽

1998 ◽

Vol 18 (11) ◽

pp. 4063-4075 ◽

Cited By ~ 74

Author(s):

Catherine Ressot ◽

Danielle Gomès ◽

André Dautigny ◽

Danielle Pham-Dinh ◽

Roberto Bruzzone

Keyword(s):

Loss Of Function ◽

Charcot Marie Tooth ◽

Charcot Marie Tooth Disease ◽

Tooth Disease

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Fly model causes neurological rethink

eLife ◽

10.7554/elife.01820 ◽

2013 ◽

Vol 2 ◽

Author(s):

Madhumala K Sadanandappa ◽

Mani Ramaswami

Keyword(s):

Neurological Disorder ◽

Therapeutic Approach ◽

Loss Of Function ◽

Partial Loss ◽

Gain Of Function ◽

Charcot Marie Tooth ◽

Charcot Marie Tooth Disease ◽

Drosophila Model ◽

Tooth Disease

A Drosophila model for a neurological disorder called type 2B Charcot-Marie-Tooth disease reveals that it has its origins in a partial loss of function, rather than a gain of function, which points to the need for a new therapeutic approach.

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A dysfunctional endolysosomal pathway common to two sub-types of demyelinating Charcot–Marie–Tooth disease

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01043-z ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

James R. Edgar ◽

Anita K. Ho ◽

Matilde Laurá ◽

Rita Horvath ◽

Mary M. Reilly ◽

...

Keyword(s):

Mammalian Cells ◽

Human Fibroblasts ◽

Cell Types ◽

Loss Of Function ◽

Charcot Marie Tooth ◽

Charcot Marie Tooth Disease ◽

Recessive Mutations ◽

Late Endosomes ◽

Demyelinating Peripheral Neuropathy ◽

Tooth Disease

Abstract Autosomal dominant mutations in LITAF are responsible for the rare demyelinating peripheral neuropathy, Charcot–Marie–Tooth disease type 1C (CMT1C). The LITAF protein is expressed in many human cell types and we have investigated the consequences of two different LITAF mutations in primary fibroblasts from CMT1C patients using confocal and electron microscopy. We observed the appearance of vacuolation/enlargement of late endocytic compartments (late endosomes and lysosomes). This vacuolation was also observed after knocking out LITAF from either control human fibroblasts or from the CMT1C patient-derived cells, consistent with it being the result of loss-of-function mutations in the CMT1C fibroblasts. The vacuolation was similar to that previously observed in fibroblasts from CMT4J patients, which have autosomal recessive mutations in FIG4. The FIG4 protein is a component of a phosphoinositide kinase complex that synthesises phosphatidylinositol 3,5-bisphosphate on the limiting membrane of late endosomes. Phosphatidylinositol 3,5-bisphosphate activates the release of lysosomal Ca2+ through the cation channel TRPML1, which is required to maintain the homeostasis of endosomes and lysosomes in mammalian cells. We observed that a small molecule activator of TRPML1, ML-SA1, was able to rescue the vacuolation phenotype of LITAF knockout, FIG4 knockout and CMT1C patient fibroblasts. Our data describe the first cellular phenotype common to two different subtypes of demyelinating CMT and are consistent with LITAF and FIG4 functioning on a common endolysosomal pathway that is required to maintain the homeostasis of late endosomes and lysosomes. Although our experiments were on human fibroblasts, they have implications for our understanding of the molecular pathogenesis and approaches to therapy in two subtypes of demyelinating Charcot–Marie–Tooth disease.

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The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein

Biochemical Journal ◽

10.1042/bcj20160657 ◽

2016 ◽

Vol 473 (21) ◽

pp. 3965-3978 ◽

Cited By ~ 10

Author(s):

Wenxia Qin ◽

Lydia Wunderley ◽

Anne L. Barrett ◽

Stephen High ◽

Philip G. Woodman

Keyword(s):

Membrane Protein ◽

Multivesicular Body ◽

Integral Membrane Protein ◽

Membrane Topology ◽

Zinc Binding ◽

Charcot Marie Tooth ◽

Charcot Marie Tooth Disease ◽

Tooth Disease ◽

In Cells

LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61β, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins.

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