Curcumin Treatment Abrogates Endoplasmic Reticulum Retention and Aggregation-Induced Apoptosis Associated with Neuropathy-Causing Myelin Protein Zero–Truncating Mutants

Article (PDF Available)inThe American Journal of Human Genetics 77(5):841-50 · December 2005with43 Reads
DOI: 10.1086/497541 · Source: PubMed
Abstract
Mutations in MPZ, the gene encoding myelin protein zero (MPZ), the major protein constituent of peripheral myelin, can cause the adult-onset, inherited neuropathy Charcot-Marie-Tooth disease, as well as the more severe, childhood-onset Dejerine-Sottas neuropathy and congenital hypomyelinating neuropathy. Most MPZ-truncating mutations associated with severe forms of peripheral neuropathy result in premature termination codons within the terminal or penultimate exons that are not subject to nonsense-mediated decay and are stably translated into mutant proteins with potential dominant-negative activity. However, some truncating mutations at the 3' end of MPZ escape the nonsense-mediated decay pathway and cause a mild peripheral neuropathy phenotype. We examined the functional properties of MPZ-truncating proteins that escaped nonsense-mediated decay, and we found that frameshift mutations associated with severe disease cause an intracellular accumulation of mutant proteins, primarily within the endoplasmic reticulum (ER), which induces apoptosis. Curcumin, a chemical compound derived from the curry spice tumeric, releases the ER-retained MPZ mutants into the cytoplasm accompanied by a lower number of apoptotic cells. Our findings suggest that curcumin treatment is sufficient to relieve the toxic effect of mutant aggregation-induced apoptosis and may potentially have a therapeutic role in treating selected forms of inherited peripheral neuropathies.
Am. J. Hum. Genet. 77:841–850, 2005
841
Curcumin Treatment Abrogates Endoplasmic Reticulum Retention and
Aggregation-Induced Apoptosis Associated with Neuropathy-Causing
Myelin Protein Zero–Truncating Mutants
Mehrdad Khajavi,
1
Ken Inoue,
5
Wojciech Wiszniewski,
1
Tomoko Ohyama,
1
G. Jackson Snipes,
2
and James R. Lupski
1,3,4
Departments of
1
Molecular and Human Genetics,
2
Pathology, and
3
Pediatrics, Baylor College of Medicine, and
4
Texas Children’s Hospital,
Houston; and
5
Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of
Neurology and Psychiatry, Tokyo
Mutations in MPZ, the gene encoding myelin protein zero (MPZ), the major protein constituent of peripheral
myelin, can cause the adult-onset, inherited neuropathy Charcot-Marie-Tooth disease, as well as the more severe,
childhood-onset Dejerine-Sottas neuropathy and congenital hypomyelinating neuropathy. Most MPZ-truncating
mutations associated with severe forms of peripheral neuropathy result in premature termination codons within
the terminal or penultimate exons that are not subject to nonsense-mediated decay and are stably translated into
mutant proteins with potential dominant-negative activity. However, some truncating mutations at the 3
end of
MPZ escape the nonsense-mediated decay pathway and cause a mild peripheral neuropathy phenotype. We examined
the functional properties of MPZ-truncating proteins that escaped nonsense-mediated decay, and we found that
frameshift mutations associated with severe disease cause an intracellular accumulation of mutant proteins,primarily
within the endoplasmic reticulum (ER), which induces apoptosis. Curcumin, a chemical compound derived from
the curry spice tumeric, releases the ER-retained MPZ mutants into the cytoplasm accompanied by a lower number
of apoptotic cells. Our findings suggest that curcumin treatment is sufficient to relieve the toxic effect of mutant
aggregation-induced apoptosis and may potentially have a therapeutic role in treating selected forms of inherited
peripheral neuropathies.
Introduction
Mutations in the gene encoding myelin protein zero
(MPZ) cause dominantly inherited peripheral neuropa-
thies that range in severity from adult-onset Charcot-
Marie-Tooth disease (CMT) type 1B (CMT1B [MIM
118200]) to childhood-onset Dejerine-Sottas neuropa-
thy (DSN [MIM 145900]) or congenital hypomyelinat-
ing neuropathy (CHN [MIM 605253]) (Lupski and Gar-
cia 2001). CMT1B, a demyelinating neuropathy that
results in progressive distal muscle atrophy, is charac-
terized by a symmetrically slowed motor nerve conduc-
tion velocity (Shy et al. 2005). DSN is a more severe
form of CMT that has an earlier onset of clinical symp-
toms, evidenced by the delayed achievement of motor-
skill milestones. Clinical findings include hypertrophied
nerves, increased cerebrospinal fluid protein, and, com-
Received July 11, 2005; accepted for publication August 25, 2005;
electronically published September 30, 2005.
Address for correspondence and reprints: Dr. James R. Lupski, De-
partment of Molecular and Human Genetics, Baylor College of Med-
icine, One Baylor Plaza, Room 604B, Houston, TX 77030. E-mail:
jlupski@bcm.tmc.edu
2005 by The American Society of Human Genetics. All rights reserved.
0002-9297/2005/7705-0013$15.00
pared with CMT, more-significant slowing of nerve con-
duction velocity, more-pronounced demyelination, and
neuropathology significant for more-numerous onion
bulbs (Hayasaka et al. 1993; Rautenstrauss et al. 1994).
The related CHN presents at birth and is distinguished
from DSN by its congenital manifestations and, in some
cases, the absence of myelin (Harati and Butler 1985).
We previously documented that the nonsense-medi-
ated degradation of mRNA carrying premature termi-
nation codons in upstream exons is a mechanism for
MPZ haploinsufficiency alleles (Inoue et al. 2004). The
nonsense-mediated decay pathway is an mRNA sur-
veillance system that specifically recognizes and de-
grades erroneous mRNA that harbors premature ter-
mination codons often resulting from abnormal splicing
or frameshifts (Sun and Maquat 2000). Failure to elim-
inate mRNA with premature termination codons may
result in the translation of aberrant proteins that can
be toxic to cells through dominant-negative or gain-of-
function effects (Wong and Filbin 1996; Frischmeyer
and Dietz 1999; Mendell and Dietz 2001; Holbrook et
al. 2004; Inoue et al. 2004). Essentially all nonsense and
frameshift mutations that are associated with a rela-
tively mild peripheral neuropathy phenotype have their
mRNA degraded because of premature stop codons that
842 Am. J. Hum. Genet. 77:841–850, 2005
are detected by nonsense-mediated decay. Therefore,
haploinsufficiency is the disease mechanism (Inoue et
al. 2004). Most late-occurring premature termination
codons that result in mRNA that escapes nonsense-me-
diated decay encode apparent dominant-negative or
gain-of-function proteins that convey a more severe neu-
ropathy phenotype. However, a subset of truncating
mutations at the 3
end of MPZ escapes the nonsense-
mediated decay surveillance pathway and causes a mild
CMT phenotype (fig. 1A) (Inoue et al. 2004; Shy et al.
2004; Inherited Peripheral Neuropathies Mutation Da-
tabase). Thus, the question remains: How can the same
types of mutation—frameshift mutations that escape
nonsense-mediated decay—result in distinct clinical
phenotypes?
We noted an apparent correlation between the type
of frameshift mutations occurring after the transmem-
brane domain and the associated phenotype. Mutations
associated with CMT retain their transmembrane do-
main and have a net addition of a 2 frameshift mu-
tation (a 2-bp insertion or a 1-bp deletion), mainly in
downstream exons (fig. 1A). We hypothesized that
frameshift mutations associated with severe disease may
lead to a mutant protein with a gain-of-function that
causes a more deleterious effect by being misprocessed
in the cell. To examine this hypothesis, we selected four
mutations for functional analyses. These all represent
frameshift alleles that, as we documented elsewhere (In-
oue et al. 2004), escape nonsense-mediated decay and
consist of two mutations from each of two categories:
severe DSN- or CHN-associated mutations (MPZ
506delT and 550del3insG) and relatively mild CMT-
associated mutations (MPZ 554delG and 676insCA)
(fig. 1B). Notably, both 550del3insG and 554delG were
located after the transmembrane domain but differed in
frame and associated phenotype. We introduced such
disease-associated mutations (fig. 1B) in an expression
vector and used both a transiently transfected HeLa cell
line and human embryonic kidney cells (HEK293) to
study the effects of wild-type MPZ cDNA and each
disease-causing mutation on its intracellular processing.
The severe alleles that cause DSN and CHN appear to
be retained in the endoplasmic reticulum (ER) and in-
duce increased apoptotic cell death that can be partially
mitigated by pretreatment with curcumin.
Material and Methods
Recombinant Constructs
Full-length human MPZ cDNA (IMAGE: 3926008)
was obtained (OpenBiosystems) and was subcloned into
pcDNA3.1 (Invitrogen) to generate pcDNAMPZ. Mu-
tations were generated in each construct with use of the
QuikChange site-directed mutagenesis kit (Stratagene).
Clones were verified by direct double-strand DNA se-
quencing with use of the DyePrimer chemistry and
ABI377 sequencer (Applied Biosystems).
Tissue Culture and Transfection
HeLa cells and HEK293 cells were grown in Dul-
becco’s modified Eagle medium (BioWhittaker), supple-
mented with 10% fetal bovine serum, and were trans-
fected with use of FuGENE 6 transfection reagents
(Roche Applied Science). Cells were incubated for 24 h
at 37C in a humidified incubator containing 10% CO
2
.
Immunostaining
Cells were fixed with 2% paraformaldehyde in PBS
at room temperature for 10 min. Cells were then washed
and permeabilized with 0.1% Triton X-100 in PBS on
ice for 2 min. Cells were rinsed twice with PBS and were
blocked with 5% normal goat serum for 1 h at 37C.
Fixed cells were incubated with primary antibodies di-
luted in PBS with 5% normal goat serum at appropriate
concentrations for 1 h at 37C. Antibodies used in this
study include the polyclonals directed against MPZ pro-
tein (Trapp et al. 1979) and mouse monoclonal protein
disulfide isomerase (PDI; 1:1,000 [Affinity Bioreagents]).
This incubation was followed by two washes in PBS and
another incubation with Alexa Fluor goat anti-mouse or
anti-rabbit antibody (1:1,000 [Molecular Probes]) for 1
hat37C. For visualizing the nuclei, SlowFade Light
Antifade Kit with 4
, 6-diamidino-2-phenylindole (DAPI)
(Molecular Probes) was used in accordance with the
manufacturer’s instructions. Fluorescently labeled cells
were visualized by standard fluorescence microscopy.
Apoptosis Assay
TUNEL staining was performed with use of an in situ
cell death detection kit, Flourescein (Roche Applied Sci-
ence). Cells were grown in four chamber slides and were
fixed with 4% paraformaldehyde in PBS at room tem-
perature for 10 min. Cells were then washed and per-
meablized with 0.1% Triton X-100 in PBS on ice for 2
min. TUNEL staining was performed in accordance with
conditions recommended by the supplier (1 h at 37C).
The average numbers of TUNEL-positive and DAPI-pos-
itive cells were calculated, and the standard deviation of
the ratio was determined for each slide. Student’s ttests
comparing wild-type and MPZ mutants were performed.
Statistical significance was defined as .P!.05
Flow-Cytometric Analysis
Cells were transiently transfected for 48 h and then
harvested. Annexin V–fluorescein isothiocyanate (FITC)
and propidium iodide (PI) staining (BD Biosciences
Pharmingen) was performed by the incubation of cells
(1#10
6
cells/ml) in the dark for 15 min at room tem-
perature in a binding buffer (10 mM HEPES, 140 mM
Figure 1 Genotype-phenotype correlation of MPZ. A, Published MPZ-truncating mutations that escape nonsense-mediated decay and
are associated with inherited peripheral neuropathies. The six coding exons of MPZ are indicated by shades of blue, and the transmembrane
(TM) domain is encoded by exon 4. Filled triangles, 1 Frameshift mutations. Open triangles, 2 Frameshift mutations. Arrows, Altered region
incorporated after frameshift mutations, with Xdemarcating the new stop codon. Green, CMT1B phenotype. Red, More severe DSN/CHN
phenotype. Note that mutations associated with a severe DSN/CHN phenotype have either an early frameshift that disrupts the transmembrane
domain or a net addition of a 1 frameshift in downstream exons while retaining the transmembrane domain, whereas CMT-associated
mutations have a 2 frameshift in downstream exons. B, Wild-type and mutant MPZ protein sequences investigated in this study. Green, CMT-
associated frameshift mutations. Red, DSN/CHN-associated mutations. Black, Wild-type sequence, with dots representing identity to the wild-
type sequence. The TM domain (in rectangle) and RSTK motif (in boldface) are demarcated in wild-type MPZ.
844 Am. J. Hum. Genet. 77:841–850, 2005
Figure 2 Subcellular localization of mutated MPZ proteins in HeLa cells. The HeLa cell line transiently transfected with wild-type MPZ
shows the expression of wild-type MPZ protein on the plasma membrane. Red, DSN/CHN-associated mutations (MPZ mutants 506delT and
550del3InsG) are extensively retained in the ER, as evidenced by colocalization with PDI (fand i, arrows). Note that cells transiently transfected
with these mutants display distinctive apoptotic morphology, including cell shrinkage. Green, CMT-associated MPZ mutations (554delG and
676insCA) are detected on the cell surface as well as in the ER by colocalizing with PDI (land o, arrows).
NaCl, and 2.5 mM CaCl
2
, at pH 7.4) containing a sat-
urating concentration of annexin V–FITC and PI. After
incubation, the cells were washed, pelleted, and analyzed
in a FACScan analyzer (Becton Dickinson).
Curcumin Treatment
Curcumin was purchased from Sigma (catalogue num-
ber C7727). Curcumin stock was dissolved in dimethyl
sulfoxide (DMSO) in accordance with conditions rec-
ommended by the supplier. Cells were pretreated with
curcumin (10 mM, unless otherwise indicated) for 3 h
before transfection.
Results
Mutant MPZ Is Retained in the ER
We transiently transfected wild-type MPZ cDNA and
disease-associated mutations (fig. 1B) in different cell
lines to visualize the effects of each disease-causing mu-
tation on its intracellular processing. We performed real-
time PCR on cell lines after transfection and confirmed
comparable levels of gene expression for the mutant and
wild-type constructs (data not shown). In our control
experiments, we verified that HeLa cell lines transfected
with wild-type MPZ expressed the MPZ protein on the
plasma membrane. We observed similar expression pat-
terns in the HEK293 cell line (data not shown). Of note,
all disease-causing mutants tested had a noticeably ab-
normal intracellular distribution in both cell lines (fig.
2 and data not shown). The distribution of CMT-as-
sociated mutants (MPZ 554delG and MPZ 676insCA)
was different from the wild-type control, in that not only
are the mutant proteins expressed on the cell surface but
they are also detectable in the ER, as shown by colo-
calizing with the ER marker-protein PDI (fig. 2). Al-
though these mutant proteins are expressed on the cell
surface, they are likely to behave as null alleles, because
of the disruption of the protein structure of the basic
cytoplasmic domain (fig. 1B). Interestingly, MPZ mu-
Khajavi et al.: Abrogation of MPZ Mutant–Induced Apoptosis 845
tants 506delT and 550del3insG were detected only in
the ER, and the staining pattern observed is clearly dif-
ferent from that seen for wild-type and CMT-associated
mutants (fig. 2). The toxic effects of these mutant pro-
teins perhaps do not require translocation to the cell
membrane surface, where normal tetrameric MPZ pro-
teins localize and function.
Severe Disease-Associated MPZ Mutants Cause
Increased Apoptosis
When visualized by light microscopy, cells transfected
with the severe MPZ mutant alleles, 506delT and
550del3insG, display distinctive apoptotic morphology,
including cell shrinkage and detachment from the sur-
face of the plate (data not shown). These observations
led us to consider the possibility that such MPZ early
frameshift mutations have a deleterious effect on cells
and may induce apoptosis. To determine whether cell
death of cells expressing disease-causing MPZ mutants
are apoptotic, we performed TUNEL labeling on cells
transfected with wild-type and MPZ mutants. We quan-
tified the number of positive cells by counting all TU-
NEL-positive cells and DAPI-positive nuclei in 10 sec-
tions of the chamber slides. We found a large number
of positive cells among those transfected with MPZ
506delT and 550del3insG (DSN- and CHN-associated
mutations) compared with cells expressing either wild-
type or CMT-associated mutations ( andPp.015
, respectively) (fig. 3A).Pp.018
To exclude the possibility that the apoptosis may
represent a nonspecific consequence of protein overex-
pression, we used the green fluorescent protein (GFP)/
PI assay (Lamm et al. 1997) to analyze the DNA
fragmentation in transiently transfected cells, with GFP
as a marker. Coexpression of MPZ 506delT and
550del3insG resulted in an increase of apoptotic cells to
a level 20% higher than that seen with wild-type MPZ
or CMT-associated mutations (fig. 3B). These findings
are indicative of a mutation-specific increase inapoptosis
and are inconsistent with a nonspecific effect from pro-
tein overexpression.
We also used a combined annexin V–PI staining to
quantify cell apoptosis by an independent method. This
objective assay measures apoptosis via the binding of
FITC-labeled annexin V to phosphatidylserine, as ana-
lyzed by flow cytometry. Phosphatidylserine is normally
confined to the inner leaflet of the plasma membrane
and is externalized during apoptosis of many cell types
(Preobrazhensky et al. 2001). The PI staining enables
simultaneous determination of the associated loss of
membrane integrity. Our analyses confirmed that MPZ
506delT and 550del3insG induce apoptosis, with the
annexin V–positive cells reaching 25.6% and 17.1%,
respectively (fig. 3C). In contrast, cells transfected with
wild-type MPZ or CMT-associated mutations contained
a minimal fraction of !7% (fig. 3C). These experiments
clearly support the contention that the latter mutations
conveying a milder CMT phenotype cause a less toxic
effect on cells and, thus, likely act as loss-of-function
alleles.
Curcumin Enhances Mutant MPZ Processing in the ER
These data are consistent with the contention that
MPZ early frameshift mutations associated with severe
disease (fig. 1A) convey a more deleterious effect to the
cell. The severity of the DSN/CHN phenotype is mainly
caused by the more pronounced demyelination or dis-
ruption of the axon–Schwann cell interactions that leads
to axonal loss. This could be due to a misfolding and
mislocalization of MPZ mutants in the ER, possibly by
mechanisms involving certain ER chaperones or a de-
crease in the amount of MPZ available for myelin com-
paction in Schwann cells.
To evaluate a potential reagent’s ability to relieve the
toxic effect associated with severe disease-causing MPZ
frameshift mutations, we investigated the effect of cur-
cumin, a dietary supplement, on MPZ mutants. Cur-
cumin modulates a number of cellular messenger path-
ways, including NF-kB and intracellular calcium (Egan
et al. 2004; Sarkar and Li 2004). Recently, Egan et
al. (2004) have shown that curcumin can apparently
rescue misfolded proteins in both cell cultures and a
homozygous DF508-CFTR mouse model by presumably
interfering with the function of ER calcium-dependent
chaperones (Egan et al. 2004). We hypothesized that cur-
cumin could have a protective effect by causing MPZ-
misfolded and MPZ-aggregated mutants to be released
from the ER, potentially relieving the toxic effect as-
sociated with these mutations in cells. This hypothesis
was experimentally addressed by treating cells express-
ing mutant and wild-type control MPZ with curcumin.
When curcumin was present at low doses (2 mM
and 5 mM), we observed a limited number of cells hav-
ing partial ER release of the severe disease-associated
mutants (MPZ 506delT and 550del3insG) (data not
shown). However, after increasing the concentration of
curcumin to 10 mM, we observed a greater apparent
release from ER retention, mostly to the cytoplasm and
the cell surface (fig. 4A). Notably, we also observed a
significant decrease in apoptosis after treating cells with
10 mM of curcumin (fig. 4Band 4C). The percentage of
apoptotic cells after curcumin treatment is essentially the
same as that observed for the control wild-type MPZ
protein and is significantly different from that observed
for these mutants without curcumin treatment (fig. 3C).
These data indicate that curcumin treatment abrogates
the ER retention and aggregation-induced apoptosis as-
Khajavi et al.: Abrogation of MPZ Mutant–Induced Apoptosis 847
Figure 3 Increased cell death in HeLa cell lines after transfection with mutant MPZ. A, Apoptosis induced in HeLa cell lines after transient
transfection with MPZ 506delT (c) and 550del3insG (d). Cell lines transfected with wild-type MPZ (b), MPZ 554delG (e), and MPZ 676insCA
(f) show a significantly lower number of positive cells. Data from TUNEL assays revealed the presenceof more positive cells onlyafter transfection
with DSN/CHN-associated mutations (cand d). A negative control (a) is also shown. B, DSN/CHN-associated mutations (MPZ 506delT and
550del3insG) increased the number of apoptotic cells, compared with wild-type MPZ. Bar graph shows results for severe (red) and relatively
mild (green) mutant alleles SD for each of four independent experiments ( ). C, Representative study of the flow-cytometric analysis ofnp4
apoptosis after transfecting cells with wild-type MPZ and disease-associated mutations. Significant differences were observed in the percentage
of cells undergoing apoptosis when transfected with 506delT and 550del3insG (DSN/CHN-associated mutations), whereas CMT-associated
mutations show a less toxic effect on cells. Note that overexpression of wild-type MPZ induces measurable apoptosis, compared with the
negative control (representative data from one of three independent experiments with comparable results; FS pflow-sorted cells and PI p
propidium iodide).
sociated with neuropathy-causing MPZ-truncating mu-
tants observed in cells.
Discussion
More than 90 different point mutations in MPZ that
result in a spectrum of inherited demyelinating neurop-
athies, including CMT, DSN, and CHN (Inherited Pe-
ripheral Neuropathies Mutation Database), have been
identified. The identification and evaluation of these mu-
tations in patients with different clinical severities not
only has provided insights into the role of MPZ in myelin
structure (Warner et al. 1996) but has also enabled ge-
notype/phenotype correlations (Inoue et al. 2004; Shy
et al. 2004). Various nonsense and frameshift mutations
in MPZ result in both mild and severe forms of neu-
ropathy, and we previously demonstrated that mutations
in ORFs could dictate disease severity by mechanisms
other than effects on the protein product (Inoue et al.
2004).
Most MPZ-truncating mutations associated with
a more severe form of peripheral neuropathy result
in premature termination codons within the terminal
or penultimate exons and are thus not detected by the
nonsense-mediated decay surveillance pathway. Such
mRNA is translated into mutant proteins with potential
dominant-negative activity. However, a subset of pre-
mature termination codon mutations at the 3
end of
MPZ escapes the nonsense-mediated decay pathway
and the mRNA is translated into mutant protein, but
a mild peripheral neuropathy phenotype results. Here,
we provide experimental evidence that the escape from
nonsense-mediated decay does not necessarily result in
a more severe phenotype and that the phenotypic out-
come depends on the function of mutant proteins. Fur-
thermore, we demonstrate that curcumin, a dietary sup-
plement, apparently stimulates the translocation of
intracellularly retained mutant MPZ from the ER to the
plasma membrane, clearly reducing cytotoxicity of the
mutant protein, as evidenced by a decreased percentage
of apoptotic cells (fig. 4).
CMT-associated mutants are expressed on the cell
surface but convey only a minor toxic effect, as evi-
denced by apoptosis assays, and, thus, may function as
loss-of-function alleles. The mild phenotype produced
by these mutants suggests that they do not interfere with
tetramer formation and do allow wild-type MPZ mol-
ecules to partly restore myelin function. CMT-associ-
ated mutants thus likely act as null alleles or possess a
reduced level of activity because of the disruption of the
protein structure of the basic cytoplasmic domain. The
cytoplasmic domain of MPZ is extremely basic and has
been shown to stabilize adhesion between the intracel-
lular components of the plasma membrane in myelin by
interacting with the apposing anionic lipid bilayer to
help in the formation of the major dense line (Ding and
Brunden 1994; Martini et al. 1995a). The cytoplasmic
domain contains a PKC target motif (RSTK), and this
property of the intracellular domain can be extensively
affected by changes in some amino acids critical to hom-
ophilic interactions (Xu et al. 2001).
In contrast, mutations associated with severe DSN/
CHN cause a more deleterious effect to the cells by
being associated with ER retention (fig. 2) and apparent
aggregation-induced apoptosis (fig. 3). Such MPZ mu-
tants may also indirectly affect wild-type MPZ cell-tar-
geting to myelin. In fact, Mpz
/
heterozygous knockout
mice show normal myelination at an early age because
of the partial expression of wild-type Mpz molecules,
whereas a lack of MPZ expression could account for
the severe phenotype, which has been observed in null
Mpz
/
mice (Giese et al. 1992; Martini et al. 1995b).
The gene dosage dependence of wild-type MPZ for
proper myelin maintenance has also been observed in
heterozygous MPZ-truncated mutations in parents who
presented with a CMT1 phenotype, whereas the ho-
mozygous children had a more severe DSN phenotype
(Warner et al. 1996).
We provide evidence that, by treating cells with cur-
cumin, we could abrogate the ER retention of selected
MPZ mutants enough to ameliorate the toxic effect, as
determined by apoptosis assays, associated with these
mutations (fig. 4). Previous studies have shown that
some mutant MPZ aggregates colocalize with BiP, an
HSP70 chaperone in the lumen of the ER, but not with
calnexin (Matsuyama et al. 2002; Shames et al. 2003).
Figure 4 Release of ER-retained MPZ mutants with curcumin treatment. A, Curcumin treatment of HeLa cells rescues MPZ 506delT
(d) and 550del3insG (f) mutants (in green) retained in the ER (PDI markers in red) to the cytoplasm. Interestingly, mutant MPZ 506delT (d,
arrow) was detectable only in the cytoplasm, mainly because of the disruption of the transmembrane domain in this mutant protein. Note that
cells treated with 10 mM curcumin showed reduced ER retention and mostly cytoplasmic localization for MPZ mutants (dand f, arrows). B,
Apoptosis analysis of cells transiently transfected with wild-type MPZ mutations 506delT and 550del3insG after curcumin treatment. C, Bar
graph of wild-type (black) and severe (red) alleles treated with curcumin, showing average SD from three independent experiments, as shown
in panel B. Cells undergo less apoptosis when treated with curcumin (np3). DMSO alone, at the same concentration used in our curcumin
preparation, had no toxic effect on cells (data not shown).
Khajavi et al.: Abrogation of MPZ Mutant–Induced Apoptosis 849
The involvement of mutant proteins with BiP is often
associated with activation of the “unfolded protein re-
sponse,” which leads to the upregulation of certain ER
chaperones and interference with folding of newly syn-
thesized proteins that, under stress conditions, could be
detrimental to cell growth and survival (Little and Lee
1995; Gething 1999). Although the mechanism through
which curcumin corrects the processing and function of
misfolded or aggregated mutant proteins in cells is not
yet determined, it is hypothesized that it may interfere
with the function of the ER calcium-dependent chap-
erones by altering the calcium levels in the ER (Egan et
al. 2004). However, one study brings into question this
purported mechanism for the curcumin effect (Song et
al. 2004). Nevertheless, other recent reports document
the correction of impaired folding mutations (Teijido et
al. 2004) and the inhibition of aggregation formation
(Yang et al. 2004) after curcumin treatment. We now
document in cell culture the apparent release of proteins
with severe MPZ alleles from ER retention, and we
demonstrate less apoptosis for curcumin-treated mis-
folding mutants. These findings suggest that curcumin
treatment may be sufficient to relieve the toxic effects
associated with severe disease-causing MPZ mutations
in whole animals.
Our observations could potentially be relevant for
patients with severe peripheral neuropathies that result
from protein misfolding in the ER, including those
caused by MPZ, Cx32 (Deschenes et al. 1997), PMP22
(Naef et al. 1997; D’Urso et al. 1998; Dickson et al.
2002; Ryan et al. 2002), or a host of other proteins
encoded by disease-associated CMT genes (Saifi et al.
2003; Szigeti and Lupski, in press). Although curcumin
shows utility in cell culture, further studies are required
in transgenic mouse models to evaluate its potential use
for therapy of peripheral neuropathy disorders.
Acknowledgments
We thank Dr. Bruce Trapp for providing P0 antibody. This
study was supported in part by the Japanese Ministry of
Health, Labor, and Welfare research grant 16B-1 for Nervous
and Mental Disorders (to K.I.); by the Japanese Ministry of
Education, Culture, Sports, Science and Technology grant-in-
aid for scientific research 17390102 (to K.I.); by the U.S. Na-
tional Institute for Neurological Disorders and Strokes, U.S.
National Institutes of Health, grant R01 NS27042 (to J.R.L.);
and by the Muscular Dystrophy Association (to J.R.L. and
G.J.S.).
Web Resources
The URLs for data presented herein are as follows:
Inherited Peripheral Neuropathies Mutation Database, http://
www.molgen.ua.ac.be/CMTMutations/
Online Mendelian Inheritance in Man (OMIM), http://www
.ncbi.nlm.nih.gov/Omim/ (for CMT1B, DSN, and CHN)
References
Deschenes SM, Walcott JL, Wexler TL, Scherer SS, Fischbeck
KH (1997) Altered trafficking of mutant connexin32. J Neu-
rosci 17:9077–9084
Dickson KM, Bergeron JJ, Shames I, Colby J, Nguyen DT,
Chevet E, Thomas DY, Snipes GJ (2002) Association of cal-
nexin with mutant peripheral myelin protein-22 ex vivo: a
basis for “gain-of-function” ER diseases. Proc Natl Acad
Sci USA 99:9852–9857
Ding Y, Brunden KR (1994) The cytoplasmic domain of myelin
glycoprotein P0 interacts with negatively charged phospho-
lipid bilayers. J Biol Chem 269:10764–10770
D’Urso D, Prior R, Greiner-Petter R, Gabreels-Festen AA, Mul-
ler HW (1998) Overloaded endoplasmic reticulum-Golgi
compartments, a possible pathomechanism of peripheral
neuropathies caused by mutations of the peripheral myelin
protein PMP22. J Neurosci 18:731–740
Egan ME, Pearson M, Weiner SA, Rajendran V, Rubin D,
Glockner-Pagel J, Canny S, Du K, Lukacs GL, Caplan MJ
(2004) Curcumin, a major constituent of turmeric, corrects
cystic fibrosis defects. Science 304:600–602
Frischmeyer PA, Dietz HC (1999) Nonsense-mediated mRNA
decay in health and disease. Hum Mol Genet 8:1893–1900
Gething MJ (1999) Role and regulation of the ER chaperone
BiP. Semin Cell Dev Biol 10:465–472
Giese KP, Martini R, Lemke G, Soriano P, Schachner M (1992)
Mouse P0 gene disruption leads to hypomyelination, ab-
normal expression of recognition molecules, and degener-
ation of myelin and axons. Cell 71:565–576
Harati Y, Butler IJ (1985) Congenital hypomyelinating neu-
ropathy. J Neurol Neurosurg Psychiatry 48:1269–1276
Hayasaka K, Himoro M, Sawaishi Y, Nanao K, Takahashi T,
Takada G, Nicholson GA, Ouvrier RA, Tachi N (1993) De
novo mutation of the myelin P0 gene in Dejerine-Sottas dis-
ease (hereditary motor and sensory neuropathy type III). Nat
Genet 5:266–268
Holbrook JA, Neu-Yilik G, Hentze MW, Kulozik AE (2004)
Nonsense-mediated decay approaches the clinic. Nat Genet
36:801–808
Inoue K, Khajavi M, Ohyama T, Hirabayashi S, Wilson J,
Reggin JD, Mancias P, Butler IJ, Wilkinson MF, Wegner M,
Lupski JR (2004) Molecular mechanism for distinct neu-
rological phenotypes conveyed by allelic truncating muta-
tions. Nat Genet 36:361–369
Lamm GM, Steinlein P, Cotten M, Christofori G (1997) A
rapid, quantitative and inexpensive method for detecting
apoptosis by flow cytometry in transiently transfected cells.
Nucleic Acids Res 25:4855–4857
Little E, Lee AS (1995) Generation of a mammalian cell line
deficient in glucose-regulated protein stress induction
through targeted ribozyme driven by a stress-inducible pro-
moter. J Biol Chem 270:9526–9534
Lupski JR, Garcia CA (2001) Charcot-Marie-Tooth peripheral
neuropathies and related disorders. In: Scriver CR, Beaudet
AL, Sly WS, Valle D (eds) The metabolic and molecular basis
of inherited disease. McGraw-Hill, New York, pp 5759–
5788
850 Am. J. Hum. Genet. 77:841–850, 2005
Martini R, Mohajeri MH, Kasper S, Giese KP, Schachner M
(1995a) Mice doubly deficient in the genes for P0 and myelin
basic protein show that both proteins contribute to the for-
mation of the major dense line in peripheral nerve myelin.
J Neurosci 15:4488–4495
Martini R, Zielasek J, Toyka KV, Giese KP, Schachner M
(1995b) Protein zero (P0)-deficient mice show myelin de-
generation in peripheral nerves characteristic of inherited
human neuropathies. Nat Genet 11:281–286
Matsuyama W, Nakagawa M, Takashima H, Osame M (2002)
Altered trafficking and adhesion function of MPZ mutations
and phenotypes of Charcot-Marie-Tooth disease 1B. Acta
Neuropathol 103:501–508
Mendell JT, Dietz HC (2001) When the message goes awry:
disease-producing mutations that influence mRNA content
and performance. Cell 107:411–414
Naef R, Adlkofer K, Lescher B, Suter U (1997) Aberrant pro-
tein trafficking in Trembler suggests a disease mechanism
for hereditary human peripheral neuropathies. Mol Cell
Neurosci 9:13–25
Preobrazhensky S, Malugin A, Wentz M (2001) Flow cyto-
metric assay for evaluation of the effects of cell density on
cytotoxicity and induction of apoptosis. Cytometry 43:199–
203
Rautenstrauss B, Nelis E, Grehl H, Pfeiffer RA, Van Broeck-
hoven C (1994) Identification of a de novo insertional mu-
tation in P0 in a patient with a Dejerine-Sottas syndrome
(DSS) phenotype. Hum Mol Genet 3:1701–1702
Ryan MC, Shooter EM, Notterpek L (2002) Aggresome for-
mation in neuropathy models based on peripheral myelin
protein 22 mutations. Neurobiol Dis 10:109–118
Saifi GM, Szigeti K, Snipes GJ, Garcia CA, Lupski JR (2003)
Molecular mechanisms, diagnosis, and rational approaches
to management of and therapy for Charcot-Marie-Tooth
disease and related peripheral neuropathies. J Investig Med
51:261–283
Sarkar FH, Li Y (2004) Cell signaling pathways altered by
natural chemopreventive agents. Mutat Res 555:53–64
Shames I, Fraser A, Colby J, Orfali W, Snipes GJ (2003) Phe-
notypic differences between peripheral myelin protein-22
(PMP22) and myelin protein zero (P0) mutations associated
with Charcot-Marie-Tooth–related diseases. J Neuropathol
Exp Neurol 62:751–764
Shy ME, Jani A, Krajewski K, Grandis M, Lewis RA, Li J,
Shy RR, Balsamo J, Lilien J, Garbern JY, Kamholz J (2004)
Phenotypic clustering in MPZ mutations. Brain 127:371–
384
Shy ME, Lupski JR, Chance P, Klein CJ, Dyck PJ (2005) He-
reditary motor and sensory neuropathies. In: Dyck PJ, Tho-
mas PK (eds) Peripheral neuropathy. Elsevier Science, Phil-
adelphia, pp 1623–1658
Song Y, Sonawane ND, Salinas D, Qian L, Pedemonte N, Gal-
ietta LJ, Verkman AS (2004) Evidence against the rescue of
defective DF508-CFTR cellular processing by curcumin in
cell culture and mouse models. J Biol Chem 279:40629–
40633
Sun X, Maquat LE (2000) mRNA surveillance in mammalian
cells: the relationship between introns and translation ter-
mination. RNA 6:1–8
Szigeti K, Lupski JR. Hereditary motor and sensory neurop-
athies. In: Rimoin DL, Connor JM, Pyesitz RE, Korf BR
(eds) Principles and practice of medical genetics. 5th ed.
Harcourt, London (in press)
Teijido O, Martinez A, Pusch M, Zorzano A, Soriano E, Del
Rio JA, Palacin M, Estevez R (2004) Localization and func-
tional analyses of the MLC1 protein involved in megalen-
cephalic leukoencephalopathy with subcortical cysts. Hum
Mol Genet 13:2581–2594
Trapp BD, McIntyre LJ, Quarles RH, Sternberger NH, Webster
HD (1979) Immunocytochemical localization of rat periph-
eral nervous system myelin proteins: P2 protein is not a
component of all peripheral nervous system myelin sheaths.
Proc Natl Acad Sci USA 76:3552–3556
Warner LE, Hilz MJ, Appel SH, Killian JM, Kolodry EH, Kar-
pati G, Carpenter S, Watters GV, Wheeler C, Witt D, Bodell
A, Nelis E, Van Broeckhoven C, Lupski JR (1996) Clinical
phenotypes of different MPZ (P0) mutations may include
Charcot-Marie-Tooth type 1B, Dejerine-Sottas, and congen-
ital hypomyelination. Neuron 17:451–460
Wong MH, Filbin MT (1996) Dominant-negative effect on
adhesion by myelin Po protein truncated in its cytoplasmic
domain. J Cell Biol 134:1531–1541
Xu W, Shy M, Kamholz J, Elferink L, Xu G, Lilien J, Balsamo
J (2001) Mutations in the cytoplasmic domain of P0 reveal
a role for PKC-mediated phosphorylation in adhesion and
myelination. J Cell Biol 155:439–446
Yang F, Lim GP, Begum AN, Ubeda OJ, Simmons MR, Am-
begaokar SS, Chen PP, Kayed R, Glabe CG, Frautschy SA,
Cole GM (2004) Curcumin inhibits formation of Abeta olig-
omers and fibrils and binds plaques and reduces amyloid in
vivo. J Biol Chem 280:5892–5901
    • "However, in evolution of species, any change in DNA may produce a negative rather than positive effect on the organism as a whole, with the PMP22 gene being an prime example of this phenomenon. Almost every level of the cellular machinery has been shown to be involved at some level in CMT16171819202122232425262728293031323334, including mitochondrial function, disruption of axonal transport, membrane fusion and fission, protein transport, protein misfolding, endoplasmic reticulum retention, and RNA processing16171819202122232425262728293031323334. "
    [Show abstract] [Hide abstract] ABSTRACT: Charcot-Marie-Tooth (CMT) disease, which encompasses several hereditary motor and sensory neuropathies, is one of the most common neuromuscular disorders. Our understanding of the molecular genotypes of CMT and the resultant clinical and electrophysiological phenotypes has increased greatly in the past decade. Characterized by electrodiagnostic studies into demyelinating (type 1) and axonal (type 2) forms, subsequent genetic testing often provides an exact diagnosis of a specific subtype of CMT. These advancements have made diagnostic paradigms fairly straightforward. Still, the nature and extent of neuromuscular disability is often complex in persons with CMT, and no curative treatments are yet available. Genotypically homologous animal models of CMT have improved exploration of disease-modifying treatments, of which molecular genetic manipulation and stem cell therapies appear to be the most promising. Research is also needed to develop better rehabilitative strategies that may limit disease burden and improve physical performance and psychosocial integration. Clinical management should be multidisciplinary, including neurologists, physiatrists, neurogeneticists, neuromuscular nurse practitioners, and orthopedists, along with physical and occupational therapists, speech-language pathologists, orthotists, vocational counselors, social workers, and other rehabilitation clinicians. Goals should include maximizing functional independence and quality of life while minimizing disability and secondary morbidity.
    Full-text · Article · Jan 2014
    • "Although the proband's father harboring the same mutation was asymptomatic, the finding implicates a variable modifier, and strong candidates include factors related to the ER stress response. The association of apoptosis with certain neurological disorders was reported recently (Inoue et al., 2004; Khajavi et al., 2005 ), although the association of apoptosis with epilepsy has not yet been confirmed. Electrophysiological assays were performed on living cells in this study, and not on cells that had undergone apoptosis . "
    [Show abstract] [Hide abstract] ABSTRACT: Mutations in GABRG2, which encodes the γ2 subunit of GABAA receptors, can cause both genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. Most GABRG2 truncating mutations associated with Dravet syndrome result in premature termination codons (PTCs) and are stably translated into mutant proteins with potential dominant-negative effects. This study involved search for mutations in candidate genes for Dravet syndrome, namely SCN1A, 2A, 1B, 2B, GABRA1, B2, and G2. A heterozygous nonsense mutation (c.118C>T, p.Q40X) in GABRG2 was identified in dizygotic twin girls with Dravet syndrome and their apparently healthy father. Electrophysiological studies with the reconstituted GABAA receptors in HEK cells showed reduced GABA-induced currents when mutated γ2 DNA was cotransfected with wild-type α1 and β2 subunits. In this case, immunohistochemistry using antibodies to the α1 and γ2 subunits of GABAA receptor showed granular staining in the soma. In addition, microinjection of mutated γ2 subunit cDNA into HEK cells severely inhibited intracellular trafficking of GABAA receptor subunits α1 and β2, and retention of these proteins in the endoplasmic reticulum. The mutated γ2 subunit-expressing neurons also showed impaired axonal transport of the α1 and β2 subunits. Our findings suggested that different phenotypes of epilepsy, e.g., GEFS+ and Dravet syndrome (which share similar abnormalities in causative genes) are likely due to impaired axonal transport associated with the dominant-negative effects of GABRG2.
    Full-text · Article · Jan 2014
    • "While an effective therapy for this disease has not been discovered, researchers are trying to discover compounds that could either inhibit the formation of aggregates or release these aggregates from the ER. Curcumin negates the ER retention of myelin protein aggregates, which is caused by mutations in myelin genes, and inhibits the aggregation-induced apoptosis and neuropathy [100] , indicating that it may serve as a potential therapeutic candidate for RP. The effects of curcumin on RP have been investigated using the transgenic rats with the P23H rhodopsin muta- tion [101]. "
    [Show abstract] [Hide abstract] ABSTRACT: Curcumin, the major extraction of turmeric, has been widely used in many countries for centuries both as a spice and as a medicine. In the last decade, researchers have found the beneficial effects of curcumin on multiple disorders are due to its antioxidative, anti-inflammatory, and antiproliferative properties, as well as its novel function as an inhibitor of histone aectyltransferases. In this review, we summarize the recent progress made on studying the beneficial effects of curcumin on multiple retinal diseases, including diabetic retinopathy, glaucoma, and age-related macular degeneration. Recent clinical trials on the effectiveness of phosphatidylcholine formulated curcumin in treating eye diseases have also shown promising results, making curcumin a potent therapeutic drug candidate for inflammatory and degenerative retinal and eye diseases.
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