Charcot MarieTooth Disease Cx32

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Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy, affecting about 1 in 2500 individuals. The disease causes progressive degeneration of the peripheral nerves. It presents in childhood or adolescence, generally beginning with a weakness in the legs, which causes difficulty in walking, and progressing later to the arms. CMT is also characterized by foot deformities, muscle wasting, ataxia, decreased tendon reflexes and distal sensory loss. The disease is both pathologically and genetically heterogeneous. Traditionally, two main forms of CMT have been distinguished, based on electrophysiological differences (Vance, 1991). CMT1 is associated with a decreased nerve conduction velocity which results from demyelination of the peripheral nerves, whereas CMT2 is a non-demyelinating disease in which nerve conduction velocity is nearly normal. The CMT1 form of the disease is genetically heterogeneous and has been linked to chromosomes 17 (CMT1A), 1 (CMT1B) and X (CMTX). The first two result from mutations in the genes encoding peripheral myelin protein and myelin protein zero, respectively (Patel and Lupski, 1994). The X-linked form of Charcot-Marie-Tooth disease, however, is a disease of gap junction channels. It shows incomplete dominant inheritance, with heterozygous females being affected less severely than hemizygous males. X chromosome inactivation in heterozygous females probably accounts for the fact that the dominance is not complete. The phenotype may vary from mild, in which the patient has a normal gait, to a severe form which may necessitate the use of a walking stick or wheelchair.

CMTX results from mutations in the gene encoding connexin 32 (Bergoffen et al., 1993). Nearly 90 of these have now been identified and they occur in all domains of the channel (Fig. 20.3; Table 20.1). Although not all mutations have been examined electrophysiologically, it appears that they fall into two main groups: those in which the protein never reaches the plasma membrane and those where the protein reaches the membrane but forms connexins with altered functional properties. The former primarily result from deletions, insertions and nonsense mutations which introduce premature stop codons and give rise to a severe phenotype. Missense mutations may be associated with either mild or severe phenotypes, according to whether they partially or completely disrupt channel function (Ionasescu et al., 1996).

CMTX mutations provide insight into the structure of gap junction channels

One mutation, a leucine-for-serine substitution at position 26 (S26L) in Cx32, has proved useful in defining the region of the protein that forms the

TABLE 20.1 SOME Cx32 MUTATIONS ASSOCIATED WITH CHARCOT-MARIE-TOOTH DISEASE

Mutation

Phenotype

Reference

W3S

Moderate/Severe

Ionasescu et al. (1996)

W3R

Mild

Ionasescu et al. (1996)

V13L

Bone et al. (1995)

R22G

Moderate/Severe

Ionasescu et al. (1996)

R22stop

Severe

Ionasescu et al. (1996)

S26L

Moderate

Oh et al. (1997)

M34T

Oh et al. (1997)

Y65C

Bone et al. (1995)

W77S

Mild

Ionasescu et al. (1996)

G80R

Mild

Ionasescu et al. (1996)

V95M

Bone et al. (1995)

E102G

Mild

Ionasescu et al. (1996)

111-116 del

Mild/Moderate

Ionasescu et al. (1996)

W133R

Bone et al. (1995)

V139M

Bone et al. (1995); Bergoffen et al. (1993)

R142W

Mild

Ionasescu et al. (1996)

L156R

Bone et al. (1995); Bergoffen et al. (1993)

R164W

Moderate/Severe

Ionasescu et al. (1996)

E186stop

Severe

Ionasescu et al. (1996)

C217stop

Severe

Ionasescu et al. (1996)

R220stop

Bone et al. (1995); Ionasescu et al. (1996)

pore of the gap junction channel (Oh et al., 1997). This residue lies within the first transmembrane domain (Fig. 20.3). When the mutated connexin is expressed, the gap junction channels conduct Cs+ ions normally but their permeability to large molecules is dramatically reduced. Glycerol, for example, is no longer permeant. The conductance to Li+ ions is also decreased. As both the hydrodynamic radius of glycerol and the radius of the hydrated Li+ ion are ~3 A, the radius of the mutant channel must be reduced to less than 3 A (the normal radius is ~6-7 A). Leucine has a larger side chain than serine and its substitution for serine at position 26 may introduce a constriction at the mouth of the channel and so lower the pore diameter and permeability to large molecules. Importantly, the mutant channels are likely to be impermeable to Ca2+ and to second messengers such as cAMP and IP3, which have diameters greater than 3 A. The demonstration that mutation of S26 affects the single-channel conductance suggests that the first TM contributes to the pore of the gap junction channel. A similar conclusion has been reached by cysteine scanning mutagenesis (Zhou et al., 1997) (see Chapter 4 for an explanation of this method).

Another CMTX mutation that lies within TM1 (M34T) forms functional channels which have a near normal 70 pS conductance in the fully open state: however, most of the time the channel resides in a low conductance (15 pS) substate so that the macroscopic conductance is greatly reduced (Oh et al., 1997). The latter also shows a marked shift in voltage dependence. Although M34 is predicted to face the channel lumen, the similarity in the singlechannel conductance of the fully open state of the mutant channel with that of the wild-type channel is not surprising as the side chain of threonine is actually smaller than that of methionine.

Some CMTX missense mutations produce non-functional gap junctions because the protein is not inserted correctly into the plasma membrane. It has been reported that several of these mutations (eg., V139M, R215W) have a dominant negative effect, as in coexpression studies they are able to combine with wild-type Cx32 and suppress functional gap junctional formation (Omori et al., 1996). This effect is unlikely to be of physiological or clinical significance, however, because the Cx32 gene is located on the X chromosome: thus, both males and females will have only a single functional copy of the gene in any one cell. More significantly, connexin 32 expressing the mutation R142W, which lies within the third TM, prevented gap junction coupling when it was coexpressed with Cx26 (Bruzzone et al., 1994). Since Cx32 forms heteromeric connexons with Cx26, the effect presumably results from a dominant negative action of the mutant Cx32. This suggests that mutant Cx32 may interfere with the formation of Cx26 junctions in those tissues where both Cx32 and Cx26 are expressed, such as hepatocytes, pancreatic acinar cells and mammary gland cells.

Why does the lack of functional Cx32 give rise to Charcot-Marie-Tooth disease?

The Cx32 protein is expressed at high levels in myelinated peripheral nerve, where it appears to be located in the Schwann cells at the nodes of Ranvier and at Schmidt-Lanterman incisures. In these regions, the myelin is not completely compacted but instead there is a thin layer of cytoplasm between each of the enveloping turns of the Schwann cell. This suggests that Cx32 may form channels within a single Schwann cell (rather than between different cells) and connect the folds of cytoplasm between adjacent turns of myelin (Fig. 20.6). Nutrients and other substances would therefore not need to diffuse through the long thin cytoplasmic spirals of the Schwann cell to reach the innermost layers of the cell. Failure of the gap junctions may therefore lead to impaired Schwann cell function and thus to demyelina-tion. The CMTX mutation S26L, in which the pore diameter is narrowed, is particularly illuminating in this respect. Its ability to cause CMT indicates that electrical coupling is not sufficient to prevent neuropathy and that the

Charcot Marie Tooth Disease Cx32

FIGURE 20.6 CONNEXIN 32 MAY FORM CONNECTIONS BETWEEN SCHWANN CELL LAYERS

Diagram illustrating a single Schwann cell wrapped many times around an axon. Gap junctions are depicted connecting adjacent layers of cytoplasm at Schmidt-Lantermann incisures. They therefore provide a shortcut for the diffusion of nutrients, regulatory molecules and trophic substances to the inner part of the Schwann cell. After Paul (1995).

FIGURE 20.6 CONNEXIN 32 MAY FORM CONNECTIONS BETWEEN SCHWANN CELL LAYERS

Diagram illustrating a single Schwann cell wrapped many times around an axon. Gap junctions are depicted connecting adjacent layers of cytoplasm at Schmidt-Lantermann incisures. They therefore provide a shortcut for the diffusion of nutrients, regulatory molecules and trophic substances to the inner part of the Schwann cell. After Paul (1995).

supply of nutrients and second messengers (between 3 and 7 A in radius) is critical for the integrity of the Schwann cell. Likewise, a decrease in the flux of such molecules may be expected with those mutations that reduce the channel open probability. Axonal degeneration also occurs in CMTX, suggesting that the Schwann cell gap junctions may also be involved in providing nutrients and trophic signals to the axon itself.

One somewhat surprising finding is that Cx32 is also present in liver, epithelial cells and brain, yet the effects of CMTX appear to be confined to the peripheral nervous system. The reason for this is unknown. One possibility is that different connexins may be able to substitute for Cx32 in other tissues; alternatively, the loss of coupling may not be deleterious in most cells.

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