Upregulation of the endosomal-lysosomal pathway in the trembler-J neuropathy

Abstract

A nonconservative leucine to proline mutation in peripheral myelin protein 22 (PMP22) causes the Trembler-J (TrJ) neuropathy in mice and humans. The expression levels and localization of the PMP22 protein in the TrJ mouse have not been previously determined. The aim of our studies was to reevaluate the extent of myelin deficit in genotyped heterozygous and homozygous animals and to examine how the TrJ mutation alters the normal in vivo post-translational processing of PMP22. Morphological studies show evidence for primary dysmyelination and myelin instability in affected animals. As expected, Western blot analysis indicates that in adult heterozygous TrJ animals, the level of PMP22 is markedly decreased, similar to myelin basic protein and protein zero, whereas myelin-associated glycoprotein is largely unaffected. The decrease in myelin protein expression is associated with an increase in lysosomal biogenesis, suggestive of augmented endocytosis or autophagy. Double-immunolabeling experiments show the accumulation of PMP22 in endosomal/lysosomal structures of TrJ Schwann cells, and chloroquine treatment of nerve segments indicates that the degradation of protein zero, PMP22, and myelin basic protein is augmented in TrJ nerves. These studies suggest that the TrJ mutation alters myelin stability and that the mutant protein is likely degraded via the lysosomal pathway.

Fig. 1.

Identification of theTr^J genotype. TheTr^J mutation introduces a novelBanI restriction site in the pmp22sequence (A). A 103 bp fragment of thepmp22 gene, including the mutated site, was amplified by PCR. BanI restriction enzyme untreated (−) and treated (+) samples were analyzed on an ethidium bromide-stained 6% acrylamide gel (B). Homozygous (Tr^J/Tr^J) and heterozygous (Tr^J/+)Tr^J animals are identified by the cleavage of the 103 bp PCR-amplified fragment into two smaller pieces (70 and 33 bp). The numbers on the left of the gel indicate DNA fragment size in base pairs.

Fig. 2.

Morphological studies of normal andTr^J sciatic nerves. Microscopic sections at equivalent magnifications from normal (A,D), heterozygous Tr^J(B, E, F), and homozygous Tr^J(C) sciatic nerves from 10-d-old (A–C, F) and adult (D, E) mice are shown. Note the marked increase in axon caliber and myelin thickness during nerve development between normal (A, D) andTr^J/+ mice (B,E). Also note the increasing severity of dysmyelination, even at 10 d of age between the +/+,Tr^J/+, andTr^J/Tr^Jnerves (A–C, respectively). Homozygous Tr^J have only small amounts of myelin (C, arrows). AdultTr^J/+ animals have increased endoneurial connective tissue (E, brown material) and occasional tomaculae are observed (bold arrow). Myelinic debris (arrowhead) can be found in longitudinal sections from 10-d-old Tr^J/+ animals (F). Scale bar, 20 μm.

Fig. 3.

Scatterplot analysis of 10-d-old and adult nerves. Plots of fiber diameter (including axon and myelin) versus axon diameter for 10-d-old (A) and adult (B) wild-type (+) and Tr^J/+ (open circles) nerves reveal a marked tendency for theTr^J/+ myelinated fibers to have thinner myelin sheaths (fiber diameter) for a given axon size. The best-fit linear regression lines are shown for comparison purposes and do not necessarily imply a linear relationship between the two parameters.

Fig. 4.

PMP22, P0, and MBP protein levels are reduced significantly in Tr^J nerves, whereas MAG remains normal. Total sciatic nerve homogenates of 18-d-old (P 18) wild-type (+/+), heterozygous (−/+), homozygous (−/−), and adult wild-type (+/+) and heterozygous (−/+) mice were analyzed for the expression of four different myelin proteins, MAG, P0, PMP22, and MBP. All lanes contain 25 μg of total protein. Molecular weights are indicated in kilodaltons.

Fig. 5.

Increased proportion of PMP22 and P0 is endoH-resistant in Tr^J/+ nerves. Sciatic nerve lysates of 18-d-old and adult, wild-type (+/+), and heterozygous Tr^J (−/+) mice were treated with PNGase F (N) or endoH (H) and immunoblotted with anti-PMP22 and anti-P0 antibodies. Samples incubated without the addition of enzyme are analyzed in lanes C. Top open arrowsindicate endoH-resistant proteins, and bottom arrowsshow the migration position of the deglycosylated protein. Lanes for the wild-type samples (+/+) contain 25 μg of total protein, and lanes for the heterozygous (−/+) samples contain 75 μg of total protein. Molecular weights are indicated in kilodaltons.

Fig. 6.

Co-localization of PMP22 and LAMP1 inTr^J Schwann cells. Fresh-frozen sections of adult normal (A–C) andTr^J/+ (D–F) sciatic nerves were double-immunolabeled with polyclonal anti-PMP22 (A,D) and monoclonal LAMP1 (B,E) antibodies. Nuclei were stained with Hoechst dye (C, F). In the normal nerve, PMP22 is found in myelin (A) and is absent from the nodes of Ranvier (A, arrows). In comparison, PMP22 accumulates in the cell bodies of some of the Schwann cells inTr^J/+ nerve (D,arrowheads). A low level of LAMP1-like immunoreactivity is found in the normal nerve (B, arrows), which is markedly increased in theTr^J/+ nerve (E). Several Schwann cells are identified in theTr^J/+ sample and show co-localization of PMP22 and LAMP1 (arrowheads in D andE, respectively). In 10-d-old heterozygousTr^J nerve, PMP22-like immunoreactivity can be detected in the Schwann cell membrane (G). Scale bar (shown in G): 50 μm.

Fig. 9.

Chloroquine treatment enhances PMP22 protein levels in Tr^J/+ nerves. Chloroquine-untreated (−) and chloroquine-treated (+) sciatic nerve explants of adult wild-type (+/+) andTr^J/+ mice were analyzed for the levels of MAG, P0, PMP22, and MBP (A). All lanes contain 25 μg of total protein. Molecular weights are indicated in kilodaltons.

Fig. 7.

The Tr^J mutation alters the normal distribution pattern of MAG and P0. Fresh-frozen sections of adult normal (A, D) andTr^J/+ (B,C, E) sciatic nerves were examined for the distribution of MAG (A, B) and P0 (D, E). The normal localization pattern of MAG is shown in A. Intracellular MAG-like immunoreactivity (B, arrowheads) is co-localized with LAMP1 (C, arrowheads) in Tr^J/+ nerves. In the normal nerve, P0 is found in compact myelin (D). Reduced levels of myelin-like P0 immunoreactivity are present inTr^J/+ nerves (E). A nerve sample processed without primary antibody incubation is shown inF. Scale bar (shown in F): 80 μm.

Fig. 8.

Upregulation of the lysosomal pathway inTr^J/+ mice. Total sciatic nerve homogenates of 18-d-old (P 18) wild-type (+/+), heterozygous (+/−), homozygous (−/−), and adult wild-type (+/+) and heterozygous (+/−) mice were analyzed for the expression of LAMP1 and protein hydrolase CatD. Arrows point to LAMP1 and the 48 kDa precursor and 34 kDa mature forms of CatD. The open arrow indicates a nonspecific band that is immunoreactive against the polyclonal anti-CatD antibody. All lanes contain 25 μg of total protein. Molecular weights are indicated in kilodaltons.