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The Journal of Neuroscience, January 15, 1998, 18(2):731-740
Overloaded Endoplasmic Reticulum-Golgi Compartments, a Possible
Pathomechanism of Peripheral Neuropathies Caused by Mutations of the
Peripheral Myelin Protein PMP22
Donatella
D'Urso1,
Reinhard
Prior1,
Regine
Greiner-Petter1,
Anneke A. W. M.
Gabreëls-Festen2, and
Hans Werner
Müller1
1 Molecular Neurobiology Laboratory, Department of
Neurology, Heinrich-Heine-University, 40225 Düsseldorf, Germany,
and 2 Institute of Neurology, University Hospital Nijmegen,
6500 Nijmegen, The Netherlands
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ABSTRACT |
Nonconservative point mutations of the peripheral myelin protein 22 (PMP22) are associated with Charcot-Marie-Tooth type 1A disease, the
most common inherited peripheral neuropathy in humans, and with the
Trembler J (TrJ) and Trembler (Tr) alleles in mice. We investigated the
intracellular transport of wild-type PMP22 and its TrJ and Tr mutant
forms in Schwann cells and in a non-neuronal cell line. In contrast to
wild type, mutant proteins were not inserted into the plasma membrane
and accumulated in the endoplasmic reticulum and Golgi compartments.
Coexpression of each mutant with wild-type PMP22 confirmed the
different intracellular distribution of the mutant forms, indicating
that neither the TrJ nor Tr protein has a dominant-negative effect on
the cellular distribution of wild-type PMP22. Accumulation of PMP22
immunoreactivity in the cell body of myelinating Schwann cells was also
observed in nerve biopsies obtained from CMT1A patients carrying the
TrJ point mutation. We propose that impaired trafficking of mutated
PMP22 affects Schwann cell physiology leading to myelin instability and
loss.
Key words:
Schwann cell; PMP22; Trembler J; Trembler; Charcot-Marie-Tooth disease; transfection; chimeric protein; cellular
sorting; myelin
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INTRODUCTION |
Peripheral neuropathies comprise a
large and heterogeneous group of disorders, and the molecular
mechanisms involved in these pathologies are not yet clearly
understood. A subset of these neuropathies is hereditary, and the most
common of these is Charcot-Marie-Tooth disease type 1 (CMT1), a motor
and sensory neuropathy with an estimated prevalence of 1:2500. The
first symptoms of CMT1 diseases appear mostly during the second or
third decade of life and are characterized by progressive weakness of
the distal muscles, mild sensory impairment, and reduction of
peripheral nerve conduction velocities. Nerve biopsies of CMT1 patients
show demyelination, remyelination, onion bulb formation, and loss of
myelinated axons. CMT1A, the most frequent form, appears to be caused
by different mechanisms, such as altered gene dosage or point mutations
of the peripheral myelin protein 22 (PMP22). In particular, the
majority of CMT1A patients presents a duplication of a 1.5 megabase
region of chromosome 17 containing the PMP22 gene, and several distinct nonconservative point mutations of PMP22 (L16P, H12Q, M69K, S72L, S72W,
S76I, S79C, S79P, L80P, G100R, G107V, T118M, L147R, G150D) (De Jonghe
et al., 1997 ) have been found in families affected by different
subtypes of the disease, displaying phenotypes with varying degrees of
disease severity.
The PMP22 gene encodes a transmembrane glycoprotein that contains four
transmembrane hydrophobic regions and two extracellular domains, and
the amino and C termini are exposed to the cytosol (D'Urso and
Müller, 1997). It is highly expressed by myelinating Schwann
cells and at lower levels also in several non-neural tissues (Spreyer
et al., 1991; Welcher et al., 1991; Baechner et al., 1995). PMP22 is
localized mainly in the compact portion of myelin, and it is involved
in regulating myelin stability and cell growth (Suter and Snipes, 1995;
Zoidl et al., 1995; D'Urso et al., 1997). Point mutations in the PMP22
gene have been found in the spontaneous mouse mutants Trembler J (TrJ)
(Suter et al., 1992a) and Trembler (Tr) (Suter et al., 1992b). Both
mutants carry nonconservative amino acid changes, leucine to proline
(L16P) in TrJ and glycine to aspartic acid (G150D) in Tr, that
introduce a helix-breaking amino acid or a charged residue into the
first and fourth transmembrane domains of the protein, respectively.
Such mutations could modify the tertiary structure of the molecule and
interfere with its membrane interactions. TrJ and Tr mice display a
disease phenotype very similar to CMT1, with progressive impairment of
the hind limbs, severe hypomyelination in the peripheral nervous system (PNS) associated with aberrant Schwann cell growth, and a slow progression of the disease. PNS myelin alterations are more severe in
homozygous TrJ and Tr animals compared with heterozygous, suggesting a
gene dosage effect. Furthermore, the identical TrJ single mutation has
been found in a family affected by a severe form of CMT1A disease
(Valentijn et al., 1992), whereas the Tr amino acid substitution was
detected in two patients suffering from Dejerine-Sottas demyelinating neuropathy (Ionasescu et al., 1997). Thus, Tr and TrJ mice are regarded
as animal models for CMT1A neuropathy.
In vivo studies have shown that PMP22-deficient mice, or
mice with only one functional copy of the PMP22 gene, are affected by
delayed onset of myelination, tomacula formation at young age, and
subsequent severe demyelination (Adlkofer et al., 1995). Transgenic rats (Sereda et al., 1996) or mice (Magyar et al., 1996) that carry
additional copies of the PMP22 gene present peripheral hypomyelination and a phenotype similar to that described in CMT1A patients.
Furthermore, it has been reported that rat Schwann cells engineered to
overexpress PMP22 (Zoidl et al., 1995) or Schwann cells isolated from
nerve biopsies of CMT1A patients (Hanemann et al., 1997) are delayed in
their growth rates compared with controls.
Taken together, it seems that PMP22 is involved in PNS myelin formation
and maintenance and in regulating Schwann cell proliferation and
differentiation (for review, see Müller et al., 1997).
We set out to investigate how single point mutations may affect the
cellular fate of the PMP22 protein and whether the mutated molecules
interfere with the function of the wild-type form. In this study we
provide experimental evidence that Schwann cells are not able to target
the TrJ and Tr proteins to the plasma membrane. TrJ and Tr PMP22 are
abundantly present in the endoplasmic reticulum (ER) and the Golgi
apparatus. Coexpression of each mutant with the wild-type protein
showed that native and mutated forms display a different cellular
distribution. Only the wild-type PMP22 is present in the cytoplasm and
the plasma membrane. Similar results were obtained both in Schwann
cells and in HeLa cells, a non-neural cell line. Immunostaining and
confocal microscopy of sural nerve sections obtained from CMT1A
patients carrying the TrJ point mutation are consistent with the
in vitro studies, showing accumulation of PMP22
immunoreactivity in the cell body of myelinating Schwann cells. Our
observations raise the intriguing possibility that an intracellular
accumulation of PMP22 could activate a cascade of events that lead to
functional impairment of Schwann cells and destabilize the myelin
structure.
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MATERIALS AND METHODS |
cDNA constructs of PMP22 wild type and mutants. The
PMP22 open reading frame sequence, obtained from the pGDSV7-PMP22
vector (Fabbretti et al., 1995), was used to generate the wild-type and mutant constructs subcloned into the pRc-CMV vector (Invitrogen, San
Diego, CA). The cDNAs encoding the PMP22 mutants were constructed using
PCR site-directed mutagenesis (Ho et al., 1989; Fabbretti et al.,
1995). We used two pairs of complementary inverse primers containing
the mutated codons 5 -TCGCGGTGCCGGTGCTGCT-3 and
5-AGCAGCACCGGCACCGCGA-3 for the L16P mutation and
5-TTCTCAGCGATGTCATCTA-3, 5-TAGATGACATCGCTGAGAA-3 for the G150D
mutation. We engineered an eight-amino acid Flag epitope in frame at
the 3 end of the wild-type and mutant PMP22 sequence by PCR. To
generate the C terminus Flag constructs, the 5 primer contained an
initiation consensus sequence and 27 nucleotides of the PMP22 cDNA
starting at the initiation codon
(5-TGCCGCCAGAATGCTCCTCCTGTTGCTGAGTATCA TCGTC-3), and the 3
primer included the Flag sequence (underlined) and 12 nucleotides of
the 3 end PMP22 coding region (5-AGATCTTCA CTTGTCATCGTCGTCCTTGTAGTCTTCGCGTTTCCG-3). Tagged
constructs were also subcloned into the pRc-CMV vector. All constructs
were sequenced in both directions using an ABI automated sequencer.
Cell cultures and transfections. HeLa cells were routinely
cultured in DMEM supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and streptomycin, and 2 mM
L-glutamine.
Schwann cells were isolated from sciatic nerves of newborn rats
according to the method of Brockes et al. (1979). To eliminate contamination of fibroblasts, cultures were maintained in DMEM, 10%
FCS, and 10 mM cytosin arabinoside for 1 week and treated twice with fibroblast-specific anti-Thy1.1 antibody (Serotec, Oxford,
UK) and baby rabbit complement (Cedarlane, Hornby, Ontario, Canada).
Cells were then expanded and cultivated in DMEM supplemented with 10%
FCS, 2 mM forskolin (Sigma, St. Louis, MO), and 100 mg/ml bovine glial growth factor (GGF) (Upstate Biotechnology, Lake Placid,
NY). Before transfection, HeLa cells or Schwann cells were plated on
poly-L-lysine-coated coverslips in 24-multiwell dishes and
grown in the appropriate medium to 60-70% confluence. For
transfection, cells were washed three times with serum-free medium
(OPTI MEM I, Life Technologies, Gaithersburg, MD) and incubated at
37°C for 6 hr in 0.5 ml OPTI MEM I containing 2.5 µg plasmid DNA
and 4 mg liposomes (LipofectAMINE, Life Technologies). Cells were
cultured further in complete DMEM until they were processed for
immunostaining. To select stable transformants of HeLa PMP22 expressors, cells were split once the day after transfection and maintained in complete DMEM containing 400 µg/ml of Geneticin G418
(Life Technologies).
Cotransfections were performed following the same procedure described
above using equal amounts of each plasmid DNA.
Northern blot analysis. Total RNA was prepared from
confluent monolayers of controls and PMP22-expressing HeLa cells using the guanidinium isothiocyanate acid-phenol method (Chomczynski and
Sacchi, 1987). Ten micrograms of total RNA for each sample were loaded
per lane and electrophoresed on a formaldehyde agarose gel. RNA was
transferred to a nylon membrane (Schleicher & Schuell, Dassel, Germany)
and UV-fixed. Northern blots were analyzed with a random primed
(32P)dCTP-labeled PMP22 cDNA or GAPDH cDNA probes. Blots
were prehybridized with 0.5% sodium phosphate, pH 7.0, and 7% SDS at
60°C for 30 min, and hybridized with labeled probes in the same
buffer at 60°C overnight. Filters were washed twice in 2× SSC/1%
SDS at 62°C for 10 min and in 0.1× SSC/1% SDS at 62°C for 20 min,
and then exposed to x-ray film (Agfa).
Immunofluorescence and confocal microscopy. For single and
double immunostaining of cultures grown on glass coverslips, cells were
washed with PBS, fixed in Bouin's solution or 4% paraformaldehyde in
PBS for 10 min at room temperature, permeabilized with 0.05% Triton
X-100 in PBS for 5 min, and incubated for 30 min in PBS containing 3%
normal goat serum to block nonspecific binding. Incubation with primary
antibodies was performed for 1 hr at room temperature followed by
extensive washes in PBS and incubation with the appropriate
fluorescein- or Cy3-conjugated secondary antibodies. To detect
extracellular immunoreactivity, live cultures were incubated with the
primary antibody diluted in medium before fixation, and no
permeabilization was performed. Finally, samples were rinsed in PBS,
and coverslips were mounted on slides in a solution of 2.5% Dabco
(Sigma) in PBS-buffered glycerol. PMP22 expression was detected using
rabbit polyclonal PMP22 antibodies that recognize the extracellular
domains of the molecule (1:200) (prepared as described by D'Urso et
al., 1997), or a mouse monoclonal anti-Flag-M2 (1:100; IBI, Kodak).
For subcellular localization we used mouse monoclonal anti-BiP (1:300;
StressGen), mouse monoclonal anti-58K Golgi protein (1:50; Sigma), and
mouse monoclonal anti-LAMP (1:25; H4B4, Developmental Studies Hybridoma
Bank, Iowa) antibodies. The secondary antibodies used to detect primary
monoclonals were an affinity-purified goat anti-mouse coupled to
fluorescein (1:25; Southern Biotechnology, Birmingham, AL) and a
Cy3-conjugated goat anti-mouse IgG (1:500; Dianova, Hamburg, Germany).
PMP22 polyclonal antibodies were visualized with Cy3-conjugated goat
anti-rabbit (1:300; Dianova) or with a biotin-conjugated goat
anti-rabbit (1:100) combined with a avidin-FITC (1:400) (both Vector
Laboratories, Burlingame, CA). Controls treated with secondary
antibodies alone showed no staining.
Human sural nerve biopsies were fixed in 4% paraformaldehyde and
embedded in paraffin. Tissue sections (5-10 µm) were treated with
xylol and ethanol solutions (100, 70, and 50%) before incubation with
primary and secondary antibodies according to the procedure described
above. For immunostaining of neurofilaments and S-100 we used mouse
monoclonal antibodies [1:800 (Affinity) and 1:100 (Boehringer
Mannheim, Mannheim, Germany), respectively] (data not shown).
All labeled probes were analyzed using a confocal laser scanning
microscope system (MRC 1024, Bio-Rad, Munich, Germany) supplied with an
argon-krypton laser. For double-immunostained samples, data from two
channels were collected simultaneously. After collection, data from
each channel were analyzed in merged images, where colocalization of
the green (fluorescein) and the red (Cy3) fluorochromes appear yellow,
or individually to show the localization of each antigen.
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RESULTS |
Expression and cellular sorting of wild-type, TrJ, and Tr PMP22 in
transfected HeLa cells
To investigate the effect of TrJ and Tr mutation on the cellular
sorting of the proteins we used an in vitro model system. Sibling HeLa cell cultures were transiently transfected with plasmids coding the wild-type (wt), the TrJ, or the Tr forms of PMP22. Twenty-four or thirty-six hours after transfection, PMP22 expression was analyzed by immunofluorescence using antibodies directed against the extracellular domains of the protein (D'Urso and Müller, 1997). In permeabilized cells, wt-PMP22 was found at the plasma membrane and widely distributed in the cytoplasm (Fig.
1A), as expected for an
integral membrane glycoprotein. In contrast, both mutant proteins were
highly concentrated in the perinuclear regions (Fig. 1C,E).
To look exclusively for PMP22 inserted into the plasma membrane,
immunofluorescence was performed on nonpermeabilized cells. PMP22
immunoreactivity was detected only in the plasma membrane of
transfectants expressing wt-PMP22 (Fig. 1B). In
contrast, PMP22 immunoreactivity was absent from the plasma membrane
surfaces of cells expressing either TrJ or Tr PMP22. The intracellular pools of the different forms of PMP22 proteins could be localized in
subcellular compartments along their cellular sorting pathway. To
identify the intracellular structures with which the mutated proteins
were associated, we used specific antibodies directed against BiP, a
resident luminal protein of the ER, or a 58 kDa protein of the Golgi
membranes. Double-immunostaining experiments revealed the presence of
all three PMP22 forms within the ER compartments (Fig.
2A,D,G) and also a
consistent amount of the proteins concentrated in the Golgi (Fig.
2B,E,H). Again, only the wt-PMP22 reached the plasma membrane (Fig. 2A). Moreover, merged confocal
images showed that a portion of the intracellular pool of all PMP22
forms was degraded through a lysosomal pathway, as indicated by
colocalization with the lysosomal-associated membrane protein LAMP2
(Fig. 2C,F,I).
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Figure 1.
Expression of wild-type, TrJ-, and Tr-PMP22 in
transfected HeLa cells. Mutants are not inserted in the plasma membrane
and accumulate in the perinuclear regions. PMP22 immunostaining of permeabilized cultures expressing wild type (A),
TrJ (C), and Tr (E) PMP22.
In nonpermeabilized transfectants (B, D, F), a
PMP22 antibody directed against an extracellular domain of the protein recognizes only the wild-type form on the membrane
(B). No signal was obtained in cultures
transfected with TrJ-PMP22 (D) or Tr-PMP22 (F). Scale bar: 10 µm in A,
B, C, E; 20 µm in
D, F.
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Figure 2.
Cellular sorting of wild-type, TrJ-, and Tr-MP22
proteins in transfected HeLa cells. Merged confocal images of
transfected cultures immunostained for PMP22
(green; red only in
a) and the ER protein BiP (a, d, g, red;
green only in a), the 58 kDa Golgi protein (b, e, h, red), and the lysosomal associated
protein LAMP2 (c, f, i, red). Colocalization of the
antigens appears yellow. Scale bar, 50 µm.
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For both mutants no membrane staining was obtained when transfected
cultures were analyzed at different time points after transfection.
Coexpression of wt-PMP22 and PMP22 mutants in HeLa cells
Because the PNS myelin of TrJ/+ and Tr/+ animals as well as CMT1A
patients carrying PMP22 mutations is severely affected, one hypothesis
could be that the mutant proteins can interfere with the membrane
targeting of the wild-type form. Clonal lines of PMP22 expressors were
selected from HeLa cells transfected with a plasmid containing the
wt-PMP22 cDNA and the neomycin resistance gene. PMP22 expression was
monitored by Northern blotting (Fig. 3A) and immunofluorescence
(Fig. 3B). PMP22 mRNA and protein were detected only in
cells transfected with a PMP22 expression plasmid; no signals were
obtained from nontransfected cells (Fig. 3A, lane 1) or from cultures transfected with the plasmid containing only the neo-gene (Fig. 3A, lane 2). Confocal
microscopy of PMP22 expressors showed PMP22 immunofluorescence
localized at the cell-cell interfaces and in the cytoplasm (Fig.
3B). In nonpermeabilized cultures, staining was found
exclusively at the plasma membranes (data not shown). We examined the
sorting of mutants when coexpressed with the wt-PMP22. We inserted a
Flag octapeptide epitope tag at the C terminus of the TrJ- and
Tr-PMP22. This epitope does not contain any targeting sequence that
might participate in specific cellular or membrane translocation. In
addition, we have recently shown that the Flag peptide does not
interfere with the cellular sorting and membrane insertion of the
wild-type PMP22 protein in HeLa cells (D'Urso and Müller, 1997).
Tagged TrJ- and Tr-PMP22 were expressed in stable wt-PMP22 expressors,
and their localization was analyzed using a monoclonal antibody that
specifically recognizes the Flag epitope. Confocal images of
cotransfected cultures double-immunostained using anti-Flag and
anti-PMP22 antibodies showed a clear difference in the subcellular
localization of the antigens. Both TrJ-PMP22 (Fig.
4B) and Tr-PMP22 (Fig.
4D) were present only in the cytoplasmic compartments, in contrast to wild-type protein, which was mostly targeted to the plasma membranes along the cell-cell borders of the
PMP22 expressors (Fig. 4A,C). In perinuclear areas of
double-expressors, colocalization of Flag and PMP22 staining was
observed, indicating that the two PMP22 forms coexisted in the same
subcellular compartments (data not shown). In these experiments no
evidence for induced apoptosis was observed. When stable PMP22
expressors were transfected with another transmembrane protein of
peripheral myelin, protein zero (P0), both proteins were sorted
correctly and inserted into the plasma membrane (data not shown). Taken
together, the results obtained in the HeLa cells demonstrate that the
TrJ and Tr mutant proteins are not inserted into the plasma membrane,
indicating an impairment in their cellular sorting. We further
investigated the cellular distribution of the wt and mutated proteins
in the natural cellular environment for PMP22, the Schwann cells.
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Figure 3.
Stable transfection of PMP22 in HeLa cells.
wt-PMP22 expression in clonal lines was analyzed by
(A) Northern blotting. PMP22 mRNA was present
only in cells transfected with the expression vector (lane
3). No signal was detectable in the untransfected HeLa cells
(lane 1) or cells transfected with the neomycin
resistance gene (lane 2). B, In PMP22
expressors the protein was localized in the plasma membrane and in the
perinuclear regions, as detected by immunofluorescence and confocal
microscopy. Scale bar, 10 µm.
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Figure 4.
Membrane targeting of wt-PMP22 is not impaired by
coexpression of mutant PMP22. A clonal line of HeLa cells that stably
expressed wt-PMP22 were transiently transfected with tagged Flag-TrJ
and Flag-Tr PMP22, and the localization of PMP22 was determined by immunofluorescence using PMP22 and anti-Flag antibodies. PMP22 mutants
were identified by their C-terminal Flag-epitope (B, D). The membrane targeting of the wt-PMP22 (A, C) is not
affected in cells that coexpress TrJ (B) or Tr
(D) mutants. Examples of coexpressing cells are
marked by arrows.
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TrJ- and Tr-PMP22 are not inserted into the plasma membrane of
Schwann cells but accumulate in the ER-Golgi compartments
In vivo, PMP22 protein is expressed by myelinating
Schwann cells and once synthesized is readily inserted into compact
myelin. In myelinating cocultures of Schwann cells and dorsal root
ganglia neurons, PMP22 is targeted to the myelin membrane where it
colocalizes with other myelin proteins, such as P0 and myelin basic
protein (MBP), and is also distributed within the cytoplasm of
myelinating Schwann cells (D'Urso et al., 1997). Normally, PMP22
protein is not detectable by immunohistochemistry in nonmyelinating
Schwann cells or in pure Schwann cell cultures, and this is consistent with its axonally regulated expression. We wanted to determine whether
the TrJ and Tr proteins were impaired in their transport to the plasma
membrane of Schwann cells. First we performed a transient transfection
of all PMP22 forms into cultured rat Schwann cells. PMP22
immunostaining of permeabilized transfectants showed that the wild-type
protein was distributed within the entire cytoplasm of expressors (Fig.
5A), whereas both mutants
appear to be concentrated exclusively in the perinuclear areas (Fig.
5C,E). Confocal optical sections of live stained transfected
cells showed that a portion of wt-PMP22 was inserted into the plasma
membrane (Fig. 5B). The signal was quite weak compared with
that obtained in myelinating cultures (D'Urso et al., 1997),
confirming that the presence of axons enhances the amount of protein
targeted to the cell surface of Schwann cells. In nonpermeabilized
cells, no TrJ or Tr proteins were detected at the plasma membrane at
any time point after transfection (Fig. 5D,F).
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Figure 5.
TrJ and Tr proteins concentrate in the
perinuclear regions of transfected Schwann cells and do not reach the
plasma membrane. PMP22 immunostaining of permeabilized Schwann cells
transfected with wt- (a), TrJ-
(c), Tr-PMP22 (e). When
cells were not permeabilized, a weak signal was obtained for PMP22
immunoreactivity on the surface of cells expressing wild-type protein
(b, arrows). No membrane staining was visible in TrJ
(d) and Tr
(f) transfectants. Scale bar, 10 µm.
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To identify the subcellular compartments where the mutant proteins were
trapped in Schwann cells, we performed double immunostaining combining
PMP22 antibody together with antibodies against BiP or the 58 kDa Golgi
protein. Merged confocal images showed that most of the mutant proteins
were retained within the endoplasmic reticulum (Fig.
6C,E). However, a reproducible
fraction of the proteins was further transported into the Golgi
apparatus (Fig. 6D,F). In cells expressing the
wild-type form, the protein was transported through the ER (Fig.
6A) and Golgi (Fig. 6B)
compartments and localized along the cellular processes (Fig.
6A,B), clearly showing a wider distribution than the
mutants. A minimal amount of the proteins colocalized with the
lysosomal marker LAMP2 (data not shown), indicating that in the case of
Schwann cells lysosomal degradation is not a major route of PMP22
metabolism.
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Figure 6.
TrJ and Tr proteins accumulate in the ER and Golgi
compartments of transfected Schwann cells and do not show a
dominant-negative effect on the cellular distribution of wt-PMP22.
Confocal optical sections of transfected Schwann cells
double-immunostained for PMP22 (red) and the ER marker
BiP (a, c, d, green), or the 58 kDa Golgi protein
(b, d, f, green). In merged images, the yellow signals show colocalization of the antigens. g
and h are superimposed confocal micrographs of Schwann
cells cotransfected with wt-PMP22 (red) and Flag-TrJ
(g, green) or Flag-Tr (h, green).
Anti-flag and anti-PMP22 immunostaining colocalize
(yellow) only in the perinuclear regions. Mutants
do not interfere with the sorting of wt-PMP22.
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TrJ and Tr proteins do not impair the transport of wt-PMP22 when
coexpressed in Schwann cells
When tissue sections obtained from mutant animals or from
patients' sural nerve biopsies are used to analyze the distribution of
PMP22, it is not possible to study the cellular localization of
wt-PMP22 versus that of its mutant forms. This is so because antibodies
able to distinguish between mutant and wt-PMP22 are not available, and
in vivo it is not feasible to follow the
post-transcriptional pathways of the newly synthesized molecules. To
overcome this problem, we generated an in vitro system by
coexpressing a Flag-tagged TrJ- or Tr-PMP22 with the wild-type protein
in cultured Schwann cells, an approach already tested successfully on
HeLa cell transfectants. Cotransfected Schwann cells were immunostained
with anti-PMP22 and anti-Flag antibodies and analyzed by simultaneous
confocal microscopy. The distribution of the two antigens clearly
demonstrated that neither the TrJ nor the Tr protein has a
dominant-negative effect on the cellular distribution of wt-PMP22. The
latter was widely present throughout the cytoplasm of double-positive
cells (Fig. 6G,H), in contrast to TrJ (Fig.
6G) and Tr (Fig. 6H) proteins, which did
not leave the cytoplasm surrounding the nucleus. This area was the only
region in which the Flag and PMP22 staining colocalized.
Schwann cell bodies overloaded with PMP22 immunoreactivity are
present in CMT1A nerve
In the majority of cases, CMT1A disease is associated with a 1.5 Mb DNA duplication on chromosome 17p11.2-p12, which contains the PMP22
gene. Valentijn and colleagues (1992) have described a family affected
by CMT1A, which showed a complete segregation of a point mutation in
the PMP22 coding region. This mutation, L16P, is identical to the
mutation found in TremblerJ mice (Suter et al., 1992a). We analyzed the
distribution of PMP22 immunoreactivity in a sural nerve biopsy obtained
from a patient displaying clinical and electrophysiological symptoms of
CMT1A and carrying the L16P point mutation as detected by DNA analysis.
Serial paraffin sections (5-10 µm) were double-stained with PMP22
polyclonal antibody combined with anti-neurofilament or anti-S100
monoclonal antibodies and analyzed by confocal microscopy. Although
PMP22 immunostaining was present in myelin, it was mostly localized in
the cytoplasm of myelinating Schwann cells (Fig.
7A-C), as confirmed by
colocalization with S-100 (data not shown). Enlarged cell bodies of
Schwann cells that wrapped axons were a frequent feature observed in
the CMT1A sections analyzed. These observations are consistent with
recent similar studies performed on CMT1A patients carrying a PMP22
duplication (Nishimura et al., 1996) and on Trembler mice (Naef et al.,
1997). In control nerves, PMP22 immunoreactivity was confined mostly to
the myelin membranes surrounding axons of different sizes (Fig. 7D) and did not exhibit enlarged Schwann cell bodies.
Similar results were obtained in tissue sections from patients with an acquired inflammatory demyelinating neuropathy (data not shown). These
observations, together with our in vitro data, suggest that also in CMT1A nerves the mutated PMP22 protein accumulates in the
cytoplasmic compartments, whereas the native form accounts for the
immunoreactivity found in compact myelin. So far, however, it is
impossible to distinguish between the two forms of the protein in
vivo.
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Figure 7.
PMP22 is abundant in the cytoplasm of myelinating
Schwann cells in CMT1A sural nerve. A, PMP22
immunostaining in CMT1A tissue section highlights cell bodies of
Schwann cells. Two examples are enlarged in B
(open arrow in A) and C
(full arrow in A). In B and C, open arrows
indicate the position of dislocated axons, and
arrowheads the position of the nuclei. D,
PMP22 immunoreactivity in control sural nerve. Magnification 60× in
A and D; 100× in B and
C.
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DISCUSSION |
The mouse mutants Trembler J and Trembler carry point mutations
identical to those found in a CMT1A family (Valentjin et al., 1992) and
in two Dejerine Sottas Syndrome patients (Ionasescu et al., 1997),
respectively, and they are considered to be animal models of CMT1A
disease. Indeed, CMT1A is the most frequent form of hereditary
neuropathies in humans, and perturbed peripheral myelin is a common
feature of the pathology of these diseases. In our in vitro
model we show that both the TrJ- and Tr-PMP22 forms, when expressed in
Schwann cells, accumulate in the ER and are also present in the Golgi
apparatus. The combination of live immunostaining and confocal analyses
clearly demonstrates that the mutant proteins are not targeted to the
plasma membrane, in contrast to the wt-PMP22, which is always
detectable on the cell surface of expressing cells. To investigate the
functional consequence of the expression of the mutant PMP22 on
wt-PMP22 we have tagged PMP22 at the C terminus with the Flag epitope.
In this way we were able to distinguish between the wild-type and the
mutant forms in the same cell. We have reported previously that the C- or N-terminal Flag epitope does not interfere with the intracellular sorting of PMP22, and that the fusion protein is glycosylated and
correctly transported to the plasma membrane (D'Urso and Müller, 1997). We have shown here that wt-PMP22 reaches the plasma membrane even when PMP22 mutants are coexpressed. Interestingly, the impaired transport of the mutated proteins does not influence the correct subcellular distribution of the native form, and it does not exhibit a
dominant-negative effect on the cellular distribution of wt-PMP22. Furthermore, this shows that overloading of the ER and the presence in
the Golgi of the PMP22 mutants do not impair the function of these
organelles, as indicated by the correct sorting of the native protein.
wt-PMP22 obviously colocalizes to some extend with BiP and Golgi
staining during its transport to the plasma membrane. In parallel, the
similar results obtained in transfected HeLa cultures demonstrate that
the inability of the mutant proteins to be transported to the cell
surface is a general bona fide phenomenon that is independent from the
cell type. In CMT1A nerve, PMP22 immunostaining was mostly but not
exclusively concentrated in the cell body of myelinating Schwann cells.
Thus, it is likely that also in vivo the mutated and
wild-type proteins are segregated differently, and that the PMP22
immunoreactivity associated with myelin sheaths is caused by the native
protein expressed in these cells. Accumulation of PMP22 in the
perinuclear regions of Schwann cells has been reported also in Trembler
J (Notterpek et al., 1997) and Trembler mice (Naef et al., 1997). Thus,
this subcellular gathering of PMP22 appears to be a common feature
observed in Schwann cells expressing the mutated PMP22. Interestingly,
both TrJ and Tr mutations seem to have the same defect in intracellular trafficking of the protein. However, it remains unclear whether the
underlying mechanism is the same. Moreover, both mutations are located
in transmembrane domains of the protein, and because these are the most
conserved regions and probably relevant for a proper function of the
protein, it is not surprising that such mutations affect PMP22
severely.
During the preparation of this manuscript, a paper was published on the
intracellular trafficking of Tr-PMP22 in transfected COS-7 cells (Naef
et al., 1997). In part, our results are in close agreement with these
data, because they show that the mutated PMP22 protein localizes mainly
in the ER. However, in contrast to our results in COS-7 cells it is
reported that Tr protein impairs the correct transport of the wild-type
form to the plasma membrane. Our data have been generated in Schwann
cells, the "natural host" for this protein, as well as in HeLa
cells. It might be that the dominant-negative effect shown in COS-7
cultures can be observed under certain conditions but that it is not a
general phenomenon. Furthermore, these results are also in contrast to
the in vivo observations showing PMP22 immunoreactivity in
the myelin sheath of Tr/+ mice.
We propose that the disease phenotype associated with the TrJ-
and Tr-PMP22 mutations involves the combination of different mechanisms. With a heterozygous genetic background, only the product of
the wild-type allele is correctly targeted and inserted into the myelin
membrane, whereas the mutated PMP22 is retained in the cytoplasmic
compartments. A decreased amount of the functional protein present in
myelin may cause a gene dosage effect. Additionally, there may be a
minimum amount of PMP22 needed to maintain compact myelin. If this
hypothesized critical amount is not realized, there may also be a
consequent abnormal interaction of PMP22 and another protein, or
complex of proteins, in the lipid bilayer. Thus a reduced level of
myelin-associated PMP22 may lead to an unstable myelin structure. At
the same time, most of the mutated PMP22 proteins accumulate in the ER.
Overloading of the ER with membrane proteins may have pronounced
effects on cell signaling, which can induce a cell response involving
different second messenger pathways and transcription factors (Thomas
et al., 1995; Pahl and Baeuerle, 1997a,b). An ER-overloaded response
has been associated with different genetic diseases caused by naturally
occurring mutations. In cystic fibrosis the mutated cystic fibrosis
transmembrane conductance regulator (CFTR) is entirely retained in the
ER together with a portion of the wild type (Cheng et al., 1990; Yang
et al., 1993; Lukacs et al., 1994). In Alzheimer's disease, wild-type and mutant forms of presenilins, which are responsible for the majority
of early onset familial forms of the disease, are localized in the ER
(Kovacs et al., 1996). In both cases upregulation of specific
transcription factors has been described (Pahl and Baeuerle, 1997a). It
is likely that distinct sets of genes are activated, and this may
change the phenotype of the affected cell in different ways. Our
experimental evidence raises the interesting hypothesis that the
disease phenotype associated with PMP22 mutations is attributable not
only to an impaired membrane targeting but also to secondary
intracellular changes effected by ER-Golgi overloading. Transfection
of primary Schwann cells with epitope-tagged protein offer a useful
model to further substantiate this hypothesis.
| |
FOOTNOTES |
Received Sept. 8, 1997; revised Oct. 29, 1997; accepted Oct. 7, (null).
This work was supported by Deutsche Forschungsgemeinschaft (Mu
630/5-3). We are grateful to Dr. C. Schneider for kindly providing the
pGDSV7-hgas3/PMP22 plasmid used in preliminary studies.
Correspondence should be addressed to Dr. Donatella D'Urso, Molecular
Neurobiology Laboratory, Department of Neurology,
Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf,
Germany.
| |
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Top
Abstract
Introduction
