Common Pathway Malfunction Found to Underlie CMT Subtypes 1C, 4J

A common pathway malfunction was found to underlie Charcot–Marie–Tooth (CMT) disease subtypes 1C and 4J, despite different disease-causing mutations, a cell-based study has discovered. 

Researchers showed that when cells carrying different CMT-linked mutations were treated with a certain single small molecule, disease-related processes were reduced in both subtypes. These findings support the development of therapies to treat more than one CMT subtype.

The study, “A dysfunctional endolysosomal pathway common to two sub-types of demyelinating Charcot–Marie–Tooth disease,” was published in the journal Acta Neuropathologica Communications.

Demyelinating forms of CMT result in the slow transmission of nerve signals due to the loss of the myelin sheath — the fatty coating surrounding nerve fibers.

Some genes associated with demyelinating CMT are involved in the endolysosomal pathway — a series of organelles (small sacs) within the cell that carry various molecules to be modulated or recycled for normal cell function. 

The first part of the pathway involves endosomes. These organelles surround materials and then fuse with lysosomes, or organelles that contain enzymes to digest materials for recycling, at the end of the pathway.

As endolysosomal pathway malfunction is common in some demyelinating CMT subtypes, there may be similar mechanisms underlying these different CMT subtypes, even if the mutated genes are different. Identifying common mechanisms may lead to single treatments for more than one CMT subtype.

CMT1C is a subtype of demyelinating CMT associated with mutations in the LITAF gene, which encodes a membrane-bound protein thought to play a role in the endolysosomal pathway.

CMT4J is caused by mutations in the FIG4 gene, which carries instructions for an enzyme also involved in endolysosomal function.

To find a common endolysosomal mechanism in these two CMT subtypes, researchers based at the University of Cambridge in the U.K. analyzed connective tissue cells (fibroblasts) isolated from the skin of two CMT1C patients, and then compared those results with fibroblasts carrying mutations in LITAF and FIG4 to search for common effects.

Using microscopes, the researchers found that both patients’ fibroblasts had enlarged late, pre-fusion endosomes and lysosomes compared to control fibroblasts isolated from age- and sex-matched controls. 

A more detailed microscopic analysis of the patients’ fibroblasts showed abnormal, large compartments (vacuoles) with interconnected membranes, which was not seen in the control cells. 

The LITAF gene was deleted (KO) in cells isolated from both patients and controls to confirm whether LITAF mutations caused these changes.

Loss of LITAF from control fibroblasts generated similar characteristics (phenotype) to those observed in CMT patient fibroblasts. Moreover, deleting the mutated LITAF genes from patient fibroblasts did not impact these traits, which showed the same swollen compartments.

“Taken together, these data support the hypothesis that expression of LITAF harbouring CMT1C-associated pathogenic mutations cause an overall loss of function phenotype,” the researchers wrote. 

Next, the FIG gene was deleted in control fibroblasts and examined under microscopes. The fibroblasts without FIG4 displayed enlarged compartments, some much larger than those seen in the CMT1C patients’ cells. Looking more closely, the researchers found some compartments were small, while others were very large. 

Previous studies of large lysosomes seen in FIG4-deleted mice fibroblasts showed that these reduced after treatment with a small molecule called ML-SA1, known to activate a protein channel (TRPML1) on lysosomes. 

Therefore, fibroblasts lacking LITAF and FIG4, as well as cells from one CMT1C patient, were treated with or without ML-SA1 for 36 hours and analyzed under the microscope. 

ML-SA1 treatment reduced the number of cells with enlarged compartments in LITAF-deficient fibroblasts (mean 14.89%) compared with untreated cells (mean 40.97%), the researchers found. Likewise, the proportion of FIG4-deficient cells with enlarged organelles (mean 97.22%) was also reduced following ML-SA1 treatment (mean 25.98%). 

More detailed examination confirmed the findings in these cell types and also in CMT1C fibroblasts incubated with ML-SA1.

“Our observation that the enlargement and vacuolation of late endosomal/lysosomal compartments in CMT1C and LITAF KO fibroblasts can also be partially reversed with ML-SA1 strongly suggests that LITAF functions on the same endolysosomal pathway as FIG4,” the researchers wrote. 

“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,” they added.