Molecular cloning and protein expression of Duchenne muscular dystrophy gene products in porcine retina.
ABSTRACT Due to the difference between rodent and human retinal circuitry, we characterize a new animal model of retinal perturbation in neurotransmission in Duchenne Muscular Dystrophy (DMD) patients. We investigated the expression and localization of dystrophin proteins and dystrophin associated proteins in porcine retina by reverse transcription polymerase chain reaction, Western blot analysis and immunohistochemistry. Homologues of human DMD gene products and alternative spliced isoforms of Dp71 were identified. We observed that dystrophins were expressed in the outer plexiform layer, around blood vessels and at the inner limiting membrane as previously described in human and mouse retinae. Moreover, by double immunostaining we showed that beta-dystroglycan co-localizes with dystrophin in the outer plexiform layer whereas alpha1-syntrophin labeling differs from that for dystrophins. Using confocal laser microscopy we observed that dystrophins labeling co-localizes with pre- and post-synaptic cell markers in the outer plexiform layer. We suggest that porcine retina constitutes a good model to study the role of dystrophins in retinal neurotransmission and should be used to investigate the physiological roles of dystrophins in signal transduction.
Cell 04/1995; 80(5):675-9. · 32.40 Impact Factor
Article: Cognitive functions in Duchenne muscular dystrophy: a reappraisal and comparison with spinal muscular atrophy.[show abstract] [hide abstract]
ABSTRACT: In order to clarify cognitive functions in Duchenne muscular dystrophy (DMD), we performed a new controlled neuropsychological study. IQ (WISC-R), verbal skills (fluency, confrontation naming and syntax comprehension) and memory abilities (BEM) were studied in two matched groups; 24 DMD children and 17 spinal muscular atrophy (SMA) children aged 12-16 yr. A significant difference appeared between the DMD and SMA patients: only in the DMD group were there significant disabilities in certain specific functions and normal scores in others. Despite similar education, the DMD children more often had significantly greater learning disabilities. There were more DMD left-handers. Verbal IQ was significantly low whereas performance IQ was at a normal level. DMD children also performed poorly in reading tasks and in some memory functions such as story recall and verbal recognition. Specific cognitive disabilities in certain DMD children, not seen in SMA children, suggest a relationship with a DMD genetic disorder.Neuromuscular Disorders 02/1992; 2(5-6):371-8. · 2.80 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Dystrophin, the Duchenne muscular dystrophy gene product, has been localized to the outer plexiform layer of normal human retina. The purpose of this study is to define completely the ocular phenotype associated with mutations at Xp21, the Duchenne muscular dystrophy gene locus. Twenty-one patients with a diagnosis of Duchenne muscular dystrophy and five patients with Becker muscular dystrophy had ophthalmologic examinations, including electroretinograms (ERGs). Electroretinogram results were correlated with respect to patient DNA analysis. Twenty-three (88%) patients had reduced scotopic b-wave amplitudes to bright-white flash stimulus, including nine with negative-shaped ERGs. Rod-isolated responses were reduced or not recordable above noise in 14 (67%) patients. Most isolated cone responses (92%) were normal. Flicker amplitudes were reduced in seven patients. Two of these patients with proximal (5' end) deletions had normal scotopic b-waves to dim blue and bright-white flash stimulus. Patients with deletions toward the middle of the gene had greater reductions in their scotopic b-wave amplitudes than patients with deletions located toward the 5' end. Most patients had normal color vision, extraocular muscle function, and Snellen visual acuity. Increased macular pigmentation was seen in 16 patients with Duchenne muscular dystrophy. Most patients with Duchenne or Becker muscular dystrophy have evidence of abnormal scotopic ERGs. Patients with deletions in the central region of the gene had the most severe ERG changes. This study supports previous suggestions that dystrophin may play a role in retinal neurotransmission. The presence of increased macular pigmentation and normal photopic ERGs distinguishes patients with Duchenne muscular dystrophy mutations from other X-linked retinal disorders with negative-shaped ERGs.Ophthalmology 06/1994; 101(5):856-65. · 5.45 Impact Factor
Molecular cloning and protein expression of Duchenne muscular
dystrophy gene products in porcine retina
Agne `s Bordaisa, Francisco Bolan ˜os-Jimenezb, Patrice Forta, Carolina Varelaa,
Jose ´-Alain Sahela, Serge Picauda, Alvaro Rendona,*
aLaboratoire de Physiopathologie Cellulaire et Mole ´culaire de la Re ´tine, INSERM U592, Ho ˆpital Saint-Antoine,
Ba ˆtiment Kourilsky, 184 rue du Faubourg Saint-Antoine, 75571 Paris, France
bInstitut National de la Recherche Agronomique, Unite ´ de Fonctions Digestives et Nutrition Humaine,
Rue de la Ge ´raudie `re, B.P. 71627, 44316 Nantes, France
Received 3 December 2004; received in revised form 17 March 2005; accepted 24 March 2005
Due to the difference between rodent and human retinal circuitry, we characterize a new animal model of retinal perturbation in
neurotransmission in Duchenne Muscular Dystrophy (DMD) patients. We investigated the expression and localization of dystrophin proteins
and dystrophin associated proteins in porcine retina by reverse transcription polymerase chain reaction, Western blot analysis and
immunohistochemistry. Homologues of human DMD gene products and alternative spliced isoforms of Dp71 were identified. We observed
that dystrophins were expressed in the outer plexiform layer, around blood vessels and at the inner limiting membrane as previously
described in human and mouse retinae. Moreover, by double immunostaining we showed that b-dystroglycan co-localizes with dystrophin in
the outer plexiform layer whereas a1-syntrophin labeling differs from that for dystrophins. Using confocal laser microscopy we observed that
dystrophins labeling co-localizes with pre- and post-synaptic cell markers in the outer plexiform layer. We suggest that porcine retina
constitutes agood model tostudythe role ofdystrophins inretinal neurotransmission andshould beused toinvestigate the physiologicalroles
of dystrophins in signal transduction.
q 2005 Elsevier B.V. All rights reserved.
Keywords: Dystrophins; DAPs; Retina; Porcine
Dystrophin is well known as the defective protein in
Duchenne Muscular Dystrophy (DMD). This X linked
disorder is characterized by a progressive degeneration of
skeletal muscular tissue  and several non-progressive
clinical manifestations including cardiomyopathy, mental
deficits, hearing deficits, gastric dilation and abnormalities
of the electroretinogram (ERG) [2,3]. Dystrophin is
a cytoskeletal-associated protein of 427 kDa, a member of
N-terminal actin binding domain, a rod-shaped spectrin-like
domain [4–6]. In addition to this, full-length dystrophin of
leads to the production of several short products named in
reference to their calculated molecular weight as Dp260 ,
Dp140 , Dp116  and Dp71 . Dp71 is the most
abundant DMD gene product in non-muscular tissue.
Dystrophin forms a linkage between cytoskeletal actin and
the extracellular matrix through a cluster of membrane and
cytoplasmic proteins (dystrophin-associated proteins, DAPs).
It has been suggested that in retina, dystrophins are key
elements in membrane stability, synaptic structure and
synaptic transmission. It has been shown that the dystro-
phin–DAPs complex is involved in the clustering of voltage-
and AQP4 [11–13]. It was also suggested that dystrophins
nervous system .
Although an abnormal electroretinogram (ERG) is
certainly one of the best characterized non-muscular
Neuromuscular Disorders 15 (2005) 476–487
0960-8966/$ - see front matter q 2005 Elsevier B.V. All rights reserved.
*Corresponding author. Tel.: C33 1 49 28 46 04; fax: C33 1 49 28 46
E-mail address: firstname.lastname@example.org (A. Rendon).
manifestations of the DMD disease, the precise functional
alterations underlying this phenotype remain unclear.
To date, a considerable advance in the understanding of the
pathogenic mechanisms in human visual diseases has come
from the study of animal models, specially using transgenic
and mutants mice. Several dystrophin-deficient mice strains
have been used to study the function of dystrophins in the
retina [15,16]. However, neither the anatomy nor the retinal
circuitry, which is mainly rod-driven in mice, is very
comparable between humans and mice. Moreover, whereas
a reduction of the b-wave amplitude of the ERG is observed
Dp427 form of dystrophin, the only mouse model reprodu-
cing this alteration is the mdx3cvstrain which has a severe
reduction of all the long and short forms of dystrophin. Other
animal models are, therefore, necessary to fully understand
the sequence of molecular events underpinning the ERG
phenotype of DMD patients and to assess the function of
dystrophins in retina. The pig is considered as an appropriate
animal model for biomedical research. In fact, among all the
non-primate mammals, the porcine retina is the most similar
to human ones. Not only the dimensions of the porcine and
human eyes are analogous, but also some anatomic and
physiological features are quite similar . Even if the pig
retina has no fovea and exhibits a rather homogenous
distribution of rods and cones, it has a typical primate-like
Since dystrophins are implicated in synaptic organization
of the retina, and there are several differences between the
retinal circuitry in current mouse models and the human
retina, the extrapolation of conclusions can be difficult. As
mentioned before, the porcine retina seems to be a closer
model to compare with the human than the mouse. In the
present study, we report the identification and molecular
characterization of the porcine homologues of dystrophin
and the short DMD gene products. Using specific antibodies
in immunoblot and immunohistochemical analysis we
further determined the presence and cellular localization
of dystrophins and their associated proteins in the porcine
retina. Our results indicate that pig can be an appropriate
animal model to study the role of dystrophins in retinal
neurotransmission as well as their involvement in the ERG
phenotype observed in DMD patients.
2. Materials and methods
2.1. Cloning by reverse transcription-polymerase
chain reaction (RT-PCR)
Adult pig eyes were collected from a local slaughterhouse
immediately after death. Retinae were recovered after
removal of the anterior segment. Total RNA from retinal
Technologies) according to the manufacturer’s instructions.
Total RNA (1 mg) was reverse transcribed using random
hexamers and SuperScript II (Invitrogen-Life Technologies)
in 20 ml final volume. PCR amplifications were performed
using 2 ml of the RT reaction and 25 pmol of primers specific
to the human (NM_004009) or (after initial characterization)
to the porcine mRNA sequences.
In order to generate a sequence contig of the entire pig
dystrophin coding region, porcine RT-PCR products were
subcloned into pGEM-t-easy vector (Promega), and
sequenced using T7 and SP6 primers and an automatic
sequencer Ceq8000 (Beckman Coulter, CA). Sequence reads
were assembled to generate a contig of dystrophin Dp427
and the CLUSTAL methods (Infobiogen, Evry, France). The
cDNA sequence for Sus scrofa dystrophin Dp427 (DMD
and registered under the accession number AJ865385.
To study alternative splicing, we performed RT-PCR.
After amplification with primers F1: 50-TGGGAAGCTCA
CTCCTCCACTCG-30and R1: 50-GGGACAGGCCTTTA
TGTTCA-30, 1 ml of the amplified product was used as
template for a second amplification with primers F2: 50-
CTGCAAGGAGTGTCCAATCA-30and R1. Amplified
products were purified, subcloned and sequenced as
Dystrophins were detected with the monoclonal pan-
specific antibody Dys2 (Novocastra, Newcastle-on-Tyne,
UK) which is directed against amino acids 3669–3685
of human dystrophin. b-dystroglycan was detected with
the polyclonal antibody previously characterized by Rivier
et al. : JAF. The distribution of a1-syntrophin was
analyzed using the polyclonal antibody raised against the
amino acids 191–206 of mouse a1-syntrophin (Sigma).
Cone photoreceptors and rod bipolar cells were, respect-
ively, identified with PNA coupled to Alexa 488 (Molecu-
lar Probes, Eugene, OR) and a polyclonal antibody against
PKC alpha (Santa Cruz Biotechnology, Inc., CA).
2.3. Protein extraction and Western-blot analysis
Retina samples from adult pig were homogenized in 10
volumes (wt/vol) of extraction buffer containing protease
inhibitors (Roche Diagnostics GmbH; Sigma), sonicated
and centrifuged at 1000g for 1 min. Supernatants were
recovered and protein concentrations were determined using
bovine serum albumin (BSA) as standard .
Protein extracts (40 mg) were resolved using NuPAGE
Tris–Acetate 3–8% gradient gels (Invitrogen) and electro-
to the manufacturer’s instructions. The efficiency of protein
transfer was controlled by both ponceau red staining of the
blot and Coomassie blue staining of the remaining gel.
Membranes were blocked in PBS containing 0.1% Tween
20, 1% BSA, 5% dry milk (BIO-RAD, Herts, UK) for 1 h at
A. Bordais et al. / Neuromuscular Disorders 15 (2005) 476–487 477
room temperature then incubated with the Dys2 antibody in
the same blocking buffer. Blots were then washed and
incubated with goat anti-mouse IgG secondary antibody
conjugated to horseradish peroxidase (Jackson Immunore-
Invitrogen). Chemiluminescence was performed using
ECLC plus Western blotting detection system (Amersham
Biosciences, UK) and documented on film (Kodak).
2.4. Tissue preparation and immunocytochemistry
Postmortem human eye tissue was obtained from the
Center for the Conservation of Corneal and Ocular Tissue
from Strasbourg, all procedures followed the principles
elicited in the Declaration of Helsinki. The retina was
separated from the posterior eye cup and fixed by immersion
in PBS 4% paraformaldehyde for 15 minutes and rinsed in
PBS before cryoprotection.
Adult pig eyes were opened along the ora serrata. The
retinae were isolated in phosphate buffer saline (PBS) and
fixed for 2 min in 4% paraformaldehyde. After cryoprotec-
tion retina samples were embedded in freezing medium
(cryoblock, Labonord) and frozen in liquid nitrogen.
Retinae were vertically sliced at 8 mm thickness in a
cryostat and sections were placed on gelatin-coated glass
Immunohistochemical labeling was carried out using
the indirect immunofluorescence method. After blocking
in PBS, containing 10% normal goat serum (NGS), 1%
bovine serum albumin (BSA) and 0.05% Triton X-100,
sections were incubated with primary antibody in PBS
supplemented with 3% NGS, 1% BSA and 0.1%
Tween20. Primary antibodies were detected using
secondary goat anti-rabbit or anti-mouse IgG antibody
coupled to Alexa (Molecular Probes, Eugene, OR) diluted
1:500 in PBS containing 3% NGS, 1% BSA, and 0.1%
Tween20. After mounting in Gel/Mount (biomeda, Foster
City, CA) the sections were examined and photographed
using a confocal laser scanning equipped with an
argon-kripton laser (TCS SP1 Leica, Lasertechnik
GmbH). The images were adjusted for contrast and
brightness using Adobe Photoshop 5.5 (Adobe Systems,
San Jose, CA). Controls were prepared by omitting the
primary antibody during the incubation; in these controls,
no specific staining could be detected.
3.1. Sequence analysis of swine dystrophin
To clone the porcine homologue of the dystrophin
gene, we designed several pairs of primers (Table 1) for
Oligonucleotides were chosen according to the sequences available in GenBank for human dystrophin mRNA (accession number NM_004009) or after initial
characterization, from porcine sequence (accession number AJ865385)
F, forward; R, reverse.
A. Bordais et al. / Neuromuscular Disorders 15 (2005) 476–487 478
Fig. 1. Comparison of the full length sequences of dystrophin from various species. Alignment of amino acid sequences of porcine, human (NM_004009) and
mouse (M68859) dystrophins. Stars indicate identity, dashes indicate gaps introduced to maximize similarity. Structural or functional domains are boxed and
shaded.X representsthe firstandlastaminoacidofthe central roddomain.Aminoacid residuesare numberedat theright ofthe sequence.Multiplesalignments
were conducted using Clustal W program (Infobiogen).
A. Bordais et al. / Neuromuscular Disorders 15 (2005) 476–487 479
PCR amplification of cDNA reverse-transcribed from total
RNA of porcine retina. The primers were chosen to span
the human dystrophin cDNA sequence (NM_004009) and
PCR products were cloned and sequenced. The predicted
porcine dystrophin protein shows a high degree of
sequence homology to human dystrophin (94% overall
amino acid identity). Alignment of the deduced amino
acid sequence of the protein encoded by the porcine gene
with full-length human and mouse dystrophin is shown in
Fig. 1. Moreover, the porcine cDNA sequence contains
several structural and functional domains originally
identified in human and mouse dystrophin: the WW
domain which is a protein-binding module found in
several signaling and regulatory molecules and thought
Fig. 1 (continued)
A. Bordais et al. / Neuromuscular Disorders 15 (2005) 476–487 480
to mediate the interaction of dystrophin with the
cytoplasmic domain of b-dystroglycan [20,21]; a
cysteine-rich domain containing two EF-hand motifs that
potentially bind calcium ; and a ZZ domain possessing
conserved cysteine residues . The COOH terminus of
human and porcine dystrophin comprise also two poly-
peptide stretches that are predicted to form a-helical
leucine zipper coiled-coil motifs  and has been named
the CC (coiled coil) domain. This domain forms the
binding site for dystrobrevin and may modulate the
interaction between syntrophins [24,25] and some other
dystrophin-associated proteins [23,26].
3.2. Cloning of the 50-end sequence of the shorter DMD
The activation of internal promoters of the DMD gene
leads to the production offour short products named Dp260,
Dp140, Dp116 and Dp71. PCR amplification using forward
primers spanning the 50UTR of Dp260, Dp140 or Dp71
human sequences in conjunction with reverse primers
spanning porcine full-length dystrophin previously
sequenced, allowed us to show that some of the
short products of the DMD gene are expressed in porcine
retina (Fig. 2).
Our results (Fig. 2(A)) agree with the initial descrip-
tion of Dp260 as a retinal-specific DMD gene product
with its first exon lying in intron 29 and producing 13
specific amino acids upstream of the common sequence
with dystrophin Dp427 . PCR amplification of the
Dp140 transcript (Fig. 2(B)) resulted from 50UTR region
with no specific coding sequence . Fig. 2(C) shows
alignment of the 50end region and the deduced seven
amino acids of the first specific exon of the Dp71
transcript. This protein is produced by the activation of an
internal promoter located between exons 62 and 63 of the
DMD gene [27,28].
3.3. RT-PCR analysis of alternative splicing
of Dp71 transcripts in porcine retina
It is known that the transcript encoding the Dp71
C-terminal product of the DMD gene can generate multiple
order to detect individual Dp71 mRNA populations, we
performed a cDNA amplification using a forward primer
corresponding to the specific 50-end of human Dp71 and a
reversed primerspecific tothe porcine Dp427 sequence. The
nested amplification of this PCR product with primers
led to the identification of three porcine Dp71 spliced
transcripts lacking either exon 71 (39 bp), exon 74 (159 bp),
or the region comprised between exons 71 and 74 (330 bp)
3.4. Detection of dystrophin and short protein
products in porcine retina
To investigate expression of the previously identified
porcine dystrophin transcripts at the protein level in retina,
total protein lysates from adult pig were examined by
Western blot analysis using the monoclonal pan-specific
antibody Dys2. As shownin Fig. 4,we detected the presence
offour bands in porcine retina homogenates. Comparison of
Fig. 2. Nucleotide and predicted amino acid sequences of the 50-end of short DMD gene products in various species. (A) The deduced amino acid sequence of
the N-terminus of porcine Dp260 is aligned with the previously reported 50-end sequences of human (NM_004012) and mouse (U15218) Dp260. (B) The
nucleotidesequencerepresentsthe specific50-endofDp140withno codingsequence.(C)ThededucedaminoacidsequenceoftheN-terminusofporcineDp71
is aligned with the previously reported 50-end sequences of human (NM_004015) and mouse (U00789) Dp71. The amino acids corresponding to the first and
specific exon of Dp71 are written in bold. The sequence used as forward primer in PCR experiments is underlined.
A. Bordais et al. / Neuromuscular Disorders 15 (2005) 476–487481
the apparent molecular masses of the detected proteins in
porcine retina with those of human and mouse homogen-
ates, clearly indicates that they correspond to full-length
dystrophin and the short products of the DMD gene Dp260,
Dp140 and Dp71. It should be noted that overexposure of
the same blot revealed, with an unavoidable background
increase, an additional band, with an apparent molecular
mass of 260 kDa (data not shown).
3.5. Localization of DMD gene products and DAPs
in the porcine retina
In order to determine, the cellular localization of DMD
gene products in porcine retina, serial retinal sections were
subjected toimmunohistochemistry with the exon78-specific
dystrophin antibody Dys2. For comparison, human retinal
sections were also included in our experiments. Dys2
produced a punctuate signal in the outer plexiform layer
(OPL) and staining around blood vessels, in both swine and
human retinae (Fig. 5(A) and (B)). In both cases, we also
observed a signal at the vitreal border of the retinae
corresponding to the inner limiting membrane (ILM),
which was less pronounced in the human sample. Close
analysis of the OPL labeling revealed the existence of two
differentpatterns ofstaining(Fig.5(C) and (D)). The firstone
consisted of a continuous line of dots aligned along the OPL,
whereas the second was formed by clusters of points grouped
in a row below the punctuate staining and positioned at
Fig. 3. AlternativesplicingwithinporcineDp71cDNA.(A)SchematicrepresentationoftheDp71cDNAandtheapproximatepositionsoftheoligonucleotideprimers
identity; dashes indicate gaps introduced to maximize similarity. Grey and white boxes represent coding and non-coding regions, respectively.
A. Bordais et al. / Neuromuscular Disorders 15 (2005) 476–487482
regular intervals along the OPL. Double staining experiments
with Dys2/PNA (a marker of cone photoreceptors) and Dys2/
PKCa (amarker ofrod bipolar cells)suggest that dystrophins
are presynaptically localized in porcine cone pedicles
(Fig. 5(F)) and also postsynaptically in bipolar cells
(Fig. 5(H)). In addition, we observed that antidystrophins
antibodies directed against different domains of the dystro-
phin molecule like the clone MANDYS8 (rod domain) or
H5A3 (peptide 3669–3685) revealed a similar staining to the
one observed with the Dys2 antibody (data not shown).
We further analyzed the localization of two dystrophin-
associated proteins involved in the DAPs complex: b-
dystroglycan and a1-syntrophin . Immunostaining with
polyclonal anti-b-dystroglycan produced a similar pattern of
staining to Dys2, i.e. numerous immunoreactive dots
scattered throughout the OPL, surrounding blood vessels
and along the ILM as reported previously in mouse [33–36],
human [35,37] and bovine retina . Considerable overlap
Fig. 5. Immunohistochemical analysis of the cellular localization of dystrophins in porcine and human retinae. Confocal fluorescence photomicrographs of
vertical sections through porcine (A), (C), (E)–(H) and human (B) and (D) retina immunolabeled with the Dys2 antibody. (C) and (D) Correspond to
enlargements of the OPL immunolabeled in (A) and (B), respectively. DMD gene products, detected by Dys2 antibody, are concentrated in the OPL, at the
ILM, and around blood vessels. Note the punctuate ((C) and (D), arrowhead) and row-like ((C) and (D), arrow) patterns of staining produced by dystrophin
antibodies in the OPL. Many Dys2 immunoreactive dots ((E), arrows) co-localize with PNA staining ((F), arrows) at the cone pedicles. Dys2 immunostaining
((G), arrows) co-localizes with PKCa immunostaining ((H), arrows) at the dendritic tips of rod bipolar cells. ONL, outer nuclear layer; OPL, outer plexiform
layer; INL, inner nuclear layer; IPL, inner plexiform layer; ILM, inner limiting membrane. Scale bars A–B: 25 mm; C–H: 2 mm.
Fig. 4. Immunoblot analysis of dystrophin gene products in porcine retina
usingthe pan-specificmonoclonalantibodyDys2(B).For comparison,DMD
gene products detected in human and mouse retinae with the same antibody
are also shown ((A) and (C), respectively). The apparent molecular mass in
kDa of dystrophin and short DMD gene products is indicated on the right.
Molecular weight markers are indicated on the left in kilodaltons.
A. Bordais et al. / Neuromuscular Disorders 15 (2005) 476–487483
of dystrophin and b-dystroglycan staining was observed
(Fig. 6(C) and (I)). Using an antibody directed against a1-
syntrophin, we detected a signal in the OPL, around blood
vessels, in the inner plexiform layer (IPL) and within the
photoreceptor outer segments. It can be observed (Fig. 6(F)
and (L)) that the OPL immunostaining for a1-syntrophin
differs from that of dystrophins. Simultaneous visualisation
of a1-syntrophin and PNA revealed the presence of a1-
syntrophin in cone outer segments (Fig. 6(O)).
Eighty percent of DMD patients present an abnormal
electroretinogram, when recorded in scotopic (dark-adapted)
conditions, characterized by a large diminution of the b-wave
amplitude. The ERG can be defined as the measure of the
global electrical activity of the retina in response to light
a functional role of DMD gene products in retinal
Fig. 6. Confocal micrographs of porcine retina double immunostained with antibodies against dystrophins and dystrophin-associated proteins. Vertical retinal
sectionswere double-immunostained withantibodieseitheragainstdystrophins(A)and (G)and b-dystroglycan(B)and (H)oragainst dystrophins((D)and (J))
and a1-syntrophin (E) and (K). (G)–(L) Represent magnified views of the OPL staining shown in (A)–(F). (M)–(O) Correspond to an enlarged views of
photoreceptor outer segments co-labeled by PNA (M) and a1-syntrophin (N). The images in (C), (F), (I), (L), (O) were superimposed to reveal areas of co-
localization which appear yellow. Dystrophin (A) and b-dystroglycan (B) are expressed in the OPL (filled arrowheads), and are concentrated at the ILM
(arrow)andaroundthe bloodvessels (star).a1-syntrophin (E)and(K) isconcentratedatthe OPL(filledarrowhead)and alsolocalizedin thecone matrixsheath
((E), (N), (O), open arrowheads). OS, outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform
layer; ILM, inner limiting membrane. Scale bars A–F: 25 mm; G–O: 5 mm.
A. Bordais et al. / Neuromuscular Disorders 15 (2005) 476–487 484
neurotransmission. Despite intense research in the field over
many years, the underlying mechanisms and the relevant
cellular interactions underlying this phenotype are not fully
Among the different animal models of muscular
dystrophy, the mdx3cvmouse is the only one to show a
scotopic phenotype involving reduced amplitude of the b-
wave and a long implicit time. The non-overlapping
distribution of dystrophins and the fact that only the
mdx3cvhave the ERG alteration suggest that each DMD
gene product may have discrete functions within the retina
[16,36,39]. In support to this idea, targeted deletion of
Dp71, which is exclusively expressed by Mu ¨ller glial cells,
has shown that this DMD gene product is not directly
involved in b-wave generation . This observation
further suggests that the DMD gene products localized in
the OPL are of critical importance in retinal visual
As mentioned before, reduction of b-wave amplitude is
found in most DMD patients that have a diminution
(absence) of Dp427, whereas in mice a severe reduction in
expression of all dystrophin products is necessary to
reproduce this phenotype . Thus, clear differences exist
in either the retinalcircuitry or the function ofdystrophins in
the retina between humans and mice, making the develop-
ment of other animal models necessary to determine the
cellular mechanisms underlying the visual defects of DMD
patients. The aim of this work was to establish the porcine
dystrophins and DAPs in human diseases.
We cloned and characterized the porcine homologues of
the dystrophin gene. Additionally, by RT-PCR, immuno-
histochemistry and immunoblot analysis, we demonstrated
the presence of all the transcripts and protein products of
pig DMD gene in the retina. Moreover, the 30end of the
dystrophin gene exhibits a complex pattern of alternative
splicing in different systems, tissues and throughout
different developmental stages [29,41,42]. We identified
three alternatively spliced transcripts that occur at the C-
terminal end of dystrophin that correspond to those
reported in human brain [30,31,43]. These differentially
spliced isoforms may regulate binding of dystrophins to
dystrophin-associated proteins at the membrane .
Indeed these observations point to the existence of Dp71
proteins that may not interact with syntrophins since the
essential binding region for a1 and b1-syntrophin closely
corresponds to the sequence encoded by exon 73–74
[24,25]. We not only identified the same DMD gene
products in porcine retina, but also Dp71 splicing
mechanisms that are quite similar to the human.
Finally, using the Dys2 antibody, we showed that in
both porcine (Fig. 5(A)) and human  (Fig. 5(B)) retina,
dystrophins are localized in the OPL, around blood vessels
and along the ILM. Previous studies in our laboratory with
the same antibody revealed a nearly identical pattern of
staining in wild type mice, whereas in Dp71-null mice
only the OPL signal was visible . Similarly, in human
and porcine retinae, full-length dystrophin, Dp260 and
Dp140 are localized in the OPL, and Dp71 is expressed in
Mu ¨ller glial cells. Uncertainly persists concerning the
cellular localization of DMD gene products in the OPL:
they have been localized to photoreceptor terminals in rats
and mice [46–48], but also to postsynaptic bipolar and
horizontal cells processes in rabbit and human [37,45,49].
Our present results in porcine retinae support the idea that
dystrophins are both pre- (Fig. 5(E) and (F)) and
postsynaptically located (Fig. 5(G) and (H)). Although
definitive proof of this hypothesis is still lacking, our
results demonstrate a much more similar distribution of
dystrophins between pig and humans than between mice
In muscle, the function of dystrophin is intimately linked
Thus, the dystrophin–DAPs complex links the extracellular
matrix tothe actin cytoskeleton.Ithasbeen suggested thatin
addition to its structural role, the DAPs complex also
to dystrophin’s functions, to date the composition of the
elucidated. In rodent Mu ¨ller glial cells, we previously
showed the existence of a DAPs complex composed of
Dp71, b-dystroglycan, d-sarcoglycan, a1-syntrophin, a-
dystrobrevin and PSD-93 , which is involved in
clustering of Kir4.1 potassium channels and AQP4 water
channels . Here we observed that the expression of a1-
syntrophin is independent of the presence of the DMD gene
products and/or b-dystroglycan. We observed a clear a1-
syntrophin signal in the photoreceptors outer segments
whereas, no immunostaining for dystrophins or b-dystrogly-
can could be detected. The identification of the binding
partners for either the dystrophins/b-dystroglycan complex
in the OPL or the a1-syntrophin cone photoreceptor outer
segments, would help to explain why the absence of
dystrophins in retina produces a relatively mild phenotype,
compared with the devastating muscle pathology. In this
for such biochemical and functional studies and would
greatly facilitate attempts to identify proteins and protein
interactions in photoreceptors and bipolar cells.
In vitro swine retina systems [51–53] also provide an
opportunity to study the molecular and cellular events
linked to the specific knock-down expression of each one of
the DMD gene products without any of the compensatory
mechanisms observed in vivo. In summary, our results
demonstrate that all DMD gene products and some DAPs
such as b-dystroglycan and a1-syntrophin are expressed in
the porcine retina. We also show that these products are
localized in pig retina at the same locations as previously
described in humans and many other species.
Given the large size of the retina, the minimal
ethical constraints concerning the use of this tissue
A. Bordais et al. / Neuromuscular Disorders 15 (2005) 476–487485
and the functional and structural similarities between human
and porcine retina, pig eyes can be considered a good model
for the study of the role of dystrophins in retinal physiology.
We thank Dr Hanneke Maas (Corneabank NORI,
Amsterdam) for generously supplying human retinae. We
thank Dr David Hicks for critical reading of the manuscript.
This work was supported by INSERM; University Pierre
and Marie Curie; Assistance Publique-Hopitaux de Paris;
Fe ´de ´ration des Aveugles de France, RETINA-France,
Association Franc ¸aise contre les Myopathies (AFM) and
the European Economic Community (RETRAINET:
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