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First published online 1 February 2006
doi: 10.1242/dev.02270
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1 Renal Division, Washington University School of Medicine, St Louis, MO 63110,
USA.
2 Department of Cell Biology and Physiology, Washington University School of
Medicine, St Louis, MO 63110, USA.
3 CROET, Oregon Health Sciences University, Portland, OR 97239, USA.
* Author for correspondence (e-mail: minerj{at}wustl.edu)
Accepted 3 January 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Pierson syndrome, Human, LAMB2, Mouse, Rat
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All BMs contain members of four protein families: type IV collagen,
nidogen/entactin, sulfated proteoglycans and laminin (reviewed by
Sasaki et al., 2004
). The
particular protein isoforms present in a given BM are presumed to impart
unique functional properties important for behavior of the adjacent cells and
for organ development and function. Perhaps the best evidence for this is the
fact that mutations in genes encoding BM proteins cause human disease, despite
substitution by related isoforms in some cases. For example, Alport syndrome
is a type IV collagen disease of kidney and inner ear
(Kashtan, 2004); Knobloch
syndrome is a collagen XVIII (heparan sulfate proteoglycan) disease affecting
the eye (Suzuki et al., 2002);
and Herlitz's junctional epidermolysis bullosa and congenital muscular
dystrophy type 1A are laminin diseases of the skin and neuromuscular system,
respectively (Burgeson and Christiano,
1997; Patton,
2000).
Laminins exist in BMs as 
-ß-
heterotrimers. In mammals
there are five , four ß and three chains that assemble
nonrandomly to form at least 15 different heterotrimers [see Aumailley et al.
(Aumailley et al., 2005) for a
new laminin nomenclature] (reviewed by
Miner and Yurchenco, 2004).
Evidence from zebrafish supports the existence of a fourth ß chain
(Parsons et al., 2002).
Mutations in ten of the eleven known mouse laminin genes have been reported.
These studies have shown that laminin is absolutely required for BM formation
(Miner et al., 2004;
Smyth et al., 1999) and that
laminins play roles in diverse developmental and physiological processes
(reviewed by Miner and Yurchenco,
2004; Yurchenco et al.,
2004).
Recently, mutations in the laminin ß2 gene (LAMB2) were
reported in a rare, lethal human disease called Pierson syndrome
(Zenker et al., 2004a;
Zenker et al., 2005). Pierson
syndrome, also called microcoria-congenital nephrosis syndrome (OMIM 609049),
is an autosomal recessive disease with congenital nephrotic syndrome/diffuse
mesangial sclerosis, distinct ocular abnormalities including microcoria (small
pupil), and impairment of vision and neurodevelopment
(Zenker et al., 2004b). These
features are consistent with the phenotype of mice lacking laminin ß2,
which stop gaining weight at 1 week of age and die at 
3 weeks of age.
Lamb2-/- mice exhibit heavy proteinuria/congenital
nephrotic syndrome with podocyte foot process effacement
(Noakes et al., 1995b),
aberrantly formed and functionally impaired neuromuscular junctions
(Knight et al., 2003;
Nishimune et al., 2004;
Noakes et al., 1995a;
Patton et al., 1998), and both
structural and functional abnormalities in the retina
(Libby et al., 1999). These
phenotypes reflect the fact that laminin ß2 has been reported to be
concentrated in the kidney glomerulus and at the skeletal neuromuscular
junction (Hunter et al., 1989)
and to be deposited in the retina (Libby
et al., 2000; Libby et al.,
1997).
Although the eye abnormalities would not be expected to impair the growth
and longevity of Lamb2-/- mice, the glomerular and
neuromuscular defects probably contribute to the severity of the failure to
thrive phenotype. However, it has been difficult to determine the extent to
which each of these is involved. Moreover, laminin ß2 is expressed at
other sites quite widely throughout the body
(Sasaki et al., 2002), so
there may exist defects in other tissues that are being masked by the severity
of the kidney and muscle defects. To investigate these issues, which have
relevance in terms of understanding both basic laminin biology and Pierson
syndrome, we have used tissue-specific laminin ß2 transgenes to isolate
the neuromuscular and kidney defects, rescuing each one individually and both
together by mating the transgenes onto the Lamb2-/-
background. A similar approach was previously used to isolate skeletal muscle
defects from those in peripheral nerve in Lama2 mutant mice
(Kuang et al., 1998). Our
results suggest that the neuromuscular defect, rather than the kidney defect,
is responsible for the severe overt phenotype. Furthermore, a requirement for
laminin ß2 in tissues other than muscle and kidney to maintain normal
viability could not be demonstrated.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (a gift
from Joshua R. Sanes, Harvard University, Cambridge, MA) was inserted between
the 3.3 kb mouse muscle creatine kinase (MCK) enhancer/promoter and the SV40
large T-antigen transcription termination and polyadenylation signal sequences
(Jaynes et al., 1988) (a gift
from Jean Buskin and Stephen Hauschka, University of Washington, Seattle, WA).
To construct the NEPH-B2 transgene, the rat ß2 cDNA plus SV40 3'
processing signals from MCK-B2 were flanked with loxP sites and inserted
downstream of the 4.2 kb mouse nephrin promoter
(Eremina et al., 2002) (a gift
from Susan Quaggin, University of Toronto, Toronto, Canada).
Production of laminin ß2 knockout and transgenic mice
Production of mice carrying the targeted Lamb2 mutation (gift from
Joshua R. Sanes) has been previously described
(Noakes et al., 1995a).
Briefly, the MC1neo cassette was inserted into the AgeI site
in the second exon of mouse Lamb2. Antibodies to epitopes encoded by
mRNA sequences 3' of this insertion did not stain homozygous mutants
(Noakes et al., 1995b;
Sasaki et al., 2002),
indicating that the insertion generates a null mutation. Mice were genotyped
by PCR of DNA extracted from tail clips using the following primer pairs: for
the mutant allele (MC1neo-specific),
5'-CGAATTCGAACACGCAGATGCAG-3' and
5'-CCGGGCGCCCCTGCGCTGACAGC-3'; for the wild-type allele,
5'-TGACCCACTGTCCTCAGTGCTG-3' and
5'-GAGTGTAGGATAGGTACCTTAG-3'.
To produce transgenic mice, the transgenes were digested and gel-purified away from plasmid vector sequences and then microinjected individually into the pronuclei of single-celled B6CBAF2/J embryos by the Washington University Mouse Genetics Core. Founders and their transgenic offspring were identified by PCR of DNA extracted from tail clips using the following primers: for MCK-B2, 5'-CTGGCTAGTCACACCCTGTAGG-3' (MCK forward) and 5'-CTGGATAGCAGCTTCCTCGAG-3' (ß2 reverse); for NEPH-B2, 5'-GAAGCAGCAGAATGAGTTCACAC-3' (nephrin forward) and 5'-ATACGAAGTTATTCGAAGTCGAG-3' (vector/loxP sequences between the nephrin promoter and the ß2 cDNA).
Antibodies, immunostaining and histology
Mouse mAbs D5 and D7 that recognize the rat laminin ß2 C-terminal
coiled-coil domain (Hunter et al.,
1989; Sanes et al.,
1990) were gifts from Joshua R. Sanes and were also obtained from
the Developmental Studies Hybridoma Bank (Iowa City, IA). Polyclonal antisera
to the mouse laminin ß2 LF domain (formerly called domain IV)
(Sasaki et al., 2002), which
crossreact with rat ß2, were a gift from Takako Sasaki and the late
Rupert Timpl (Max Planck Institute for Biochemistry, Martinsried, Germany).
Rat mAb 5A2 to the mouse laminin ß1 LN domain (formerly VI)
(Abrahamson et al., 1989;
St John et al., 2001) was a
gift from Dale Abrahamson (University of Kansas Medical Center, Kansas City,
KS). Rabbit antiserum 8948 to the mouse laminin 5 LEb and L4b domains
(formerly IIIb and IVa) has been described
(Miner et al., 1997). Alexa
488- and Cy3-conjugated secondary antibodies were obtained from Molecular
Probes (Eugene, OR) and Chemicon (Temecula, CA), respectively.
For immunostaining, fresh tissues were immersed in OCT and quick-frozen in
2-methylbutane cooled in a dry ice-ethanol bath. Frozen sections were cut (7
µm) in a cryostat and air dried on gelatin-coated slides. Antibodies were
diluted in PBS with 1% BSA, applied to sections for 1 hour, and rinsed in PBS.
Secondary antibodies were then applied in a similar fashion. TRITC-conjugated
-bungarotoxin (Molecular Probes) was included to localize synaptic
sites in skeletal muscle. After rinsing, the sections were mounted in 90%
glycerol containing 0.1x PBS and 1 mg/ml p-phenylenediamine and
viewed under epifluorescence with a Nikon Eclipse 800 compound microscope.
Images were captured with a Spot 2 cooled color digital camera (Diagnostic
Instruments, Sterling Heights, MI).
For conventional light microscopy, tissues were fixed in 10% buffered
formalin, dehydrated through graded ethanols, embedded in paraffin, sectioned
at 4 µm and stained with periodic-acid-Schiff (PAS). For electron
microscopy, tissues were fixed, embedded in plastic, sectioned and stained as
previously described (Kikkawa et al.,
2003; Noakes et al.,
1995b).
Evans blue analysis
To assay the integrity of skeletal muscle fibers, Evans blue dye (Sigma, St
Louis, MO) at 10 mg/ml in 0.9% NaCl was injected intraperitoneally at 10
µl/g body weight. After 6 hours mice were perfused under anesthesia with 10
ml PBS and then 40 ml of 2% paraformaldehyde in PBS. The diaphragm with rib
insertions was dissected out and post-fixed overnight with 3% paraformaldehyde
and 1% glutaraldehyde in PBS at 4°C. The diaphragm was then detached from
the ribs and photographed with a QImaging Micropublisher digital camera
(QImaging, Burnaby, BC, Canada) attached to a stereomicroscope.
Urinalysis
Urine was collected from mice at various ages either by manual restraint,
by caging on raised wire floors (Ancare, Bellmore, NY) for several hours or by
bladder puncture under anesthesia at the time of sacrifice. Urine (1 µl)
was run on precast 4 to 20% SDS polyacrylamide gels (Invitrogen/Novex,
Carlsbad, CA), and gels were stained with Coomassie Blue and destained by
standard methods. For quantitation, urinary protein and creatinine
concentrations were measured with a Cobas Mira Plus analyzer (Roche,
Somerville, NJ).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Muscle-specific ß2 transgene
The reported developmental defects in Lamb2-/- muscles
are restricted to the synapse. Consistent with this, laminin ß2 is
concentrated in the synaptic region of the myofiber BM; it also accumulates at
myotendinous junctions (MTJs) (Patton et
al., 1997) and can be detected extrasynaptically with some
antibodies (Hunter et al.,
1989; Sasaki et al.,
2002; Wewer et al.,
1997). Previous studies have shown that ß2 is expressed by
skeletal muscle in vivo (Moscoso et al.,
1995) and is concentrated at synapse-like sites that form on
cultured myotubes in vitro even in the absence of neurons
(Martin et al., 1995).
Together, these data suggest that skeletal muscle is capable of concentrating
laminin ß2 at synaptic sites independent of motoneuron expression of
ß2, should there be any. We therefore constructed a ß2 transgene
(MCK-B2; Fig. 1A) that should
be expressed at high levels in skeletal muscle, using the well-characterized
mouse MCK enhancer/promoter. This element has also been shown to drive
expression in cardiac muscle with 100-fold less activity, but,
importantly, it is not significantly active in kidney
(Johnson et al., 1989). We
chose to use the rat ß2 cDNA because of the availability of mouse mAbs
that recognize rat but not mouse ß2
(Sanes et al., 1990). These
mAbs facilitated specific detection of transgene-derived ß2.
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-bungarotoxin,
which binds to acetylcholine receptors. Two of the seven founders expressed
rat ß2, and it was concentrated at synapses. These two were mated to
Lamb2+/- mice to generate Lamb2+/-;
MCK-B2 lines.
Analysis of offspring showed that only one of the two lines deposited the
transgene-derived ß2 consistently at all synapses, and this line was used
for subsequent studies. An immunohistochemical survey of tissues was performed
to determine whether expression was muscle specific. Rat ß2 was detected
at skeletal muscle synapses (Fig.
2C,D), but not in extrasynaptic regions of the myofiber BM. This
is in contrast to the endogenous mouse ß2, which is found in both
synaptic and extrasynaptic regions (Fig.
2) (Sasaki et al.,
2002). Expression was also observed in cardiac muscle and in some
visceral smooth muscle, but not in nerve, kidney, lung parenchyma, skin,
liver, retina, intestinal mucosa or brain
(Fig. 2E-H and data not shown).
Mosaic expression was observed in the vascular smooth muscle of arteries (data
not shown). Based on these results, we conclude that the MCK-B2 transgene
behaves in an appropriate tissue-specific fashion and that the presumed
expression of the transgene throughout the skeletal muscle fiber nevertheless
leads to concentration of ß2 at synapses, as previously shown in vitro
(Martin et al., 1995).
Podocyte-specific ß2 transgene
The known defects in Lamb2-/- kidney are restricted to
the glomerular filter, consistent with the fact that ß2 is highly
concentrated in the glomerular BM (GBM)
(Hunter et al., 1989). The GBM
is synthesized by two cells: the podocytes that are present in the urinary
space and lie on the outer aspect of the GBM, and the endothelial cells that
line the glomerular capillaries on the inner aspect of the GBM
(St John and Abrahamson,
2001). In order to restrict rat ß2 transgene expression to
the glomerulus, we chose to use a podocyte-specific promoter element isolated
from the Nphs1 gene (Eremina et
al., 2002), which encodes nephrin
(Kestila et al., 1998), to
make the NEPH-B2 transgene (Fig.
1B). For added flexibility in future experiments, the rat ß2
cDNA and the adjacent SV40 sequences were flanked by loxP sites so that
transgene expression could be halted by Cre-mediated recombination.
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Tissue-specific transgenic rescue of Lamb2-/- defects
Skeletal muscle rescue
To determine whether muscle-derived laminin ß2 was sufficient to
rescue the Lamb2-/- neuromuscular junction differentiation
defects, and to attempt to isolate the glomerular filtration defect,
Lamb2+/- mice were crossed with
Lamb2+/-; MCK-B2 mice to generate
Lamb2-/-; MCK-B2 mice. Lamb2-/-;
MCK-B2 mice were significantly more healthy than Lamb2-/-
mice and were usually indistinguishable from littermate controls with respect
to overall appearance and body weight during the pre-weaning period
(Fig. 4A). However, they
routinely died at 1 month of age, only 10 days later than
Lamb2-/- mice. SDS-PAGE analysis of urine showed massive
proteinuria before and after weaning, with albumin being the major urinary
protein (Fig. 4B; data not
shown). Quantitation of urinary protein to creatinine ratios showed that
proteinuria in Lamb2-/-; MCK-B2 mice was comparable with
that observed in Lamb2-/- mice. Consistent with the heavy
proteinuria, Lamb2-/-; MCK-B2 mice were edematous near the
time of death.
Immunohistochemical analyses showed that in skeletal muscle, rat ß2 was concentrated at synaptic sites in Lamb2-/-; MCK-B2 mice (Fig. 5). Interestingly, as discussed in detail in the Discussion, even with the more sensitive polyclonal antibody, little ß2 was detectable along the extra-synaptic regions of the myofiber. (One exception was the MTJ, as shown below.) Expression was also detected in cardiac muscle but not in kidney or in other non-muscle tissues, consistent with the data in Fig. 2.
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5,
the partner of laminin ß2 in the synaptic laminin 5ß21
(laminin-11 or Lm-521), and to increased synaptic ß1
(Patton et al., 1997), which
presumably compensates for the absence of ß2. Here, restoration of
ß2 to the synaptic cleft in Lamb2-/-; MCK-B2 mice led
to the reappearance of 5 and the disappearance of ß1
(Fig. 5C-F). Not only was the
molecular composition of the synaptic BM normalized, but the endplate also
acquired the normal pretzel-like shape
(Fig. 5B) that
Lamb2-/- synapses lack
(Noakes et al., 1995a).
Ultrastructural analyses further confirmed the rescue of synaptic
organization: the muscle endplate established junctional folds, and Schwann
cell processes did not aberrantly extend into the synaptic cleft
(Fig. 6A-C). We conclude that
muscle-derived ß2 is deposited into the synaptic cleft BM and is
sufficient to rescue synaptic structural and compositional defects in the
Lamb2 mutant. The overall improvement in health of the animal
suggests that synaptic function is also improved.
As mentioned previously, laminin ß2 is also normally concentrated at
MTJs, but developmental defects in Lamb2-/- MTJs have not
previously been reported. Injection of Evans blue, a dye that accumulates only
in damaged and regenerating skeletal and cardiac muscle fibers
(Straub et al., 1997), into
Lamb2-/- mice resulted in labeling of muscle fibers in the
diaphragm, but primarily at the ends, near MTJs
(Fig. 7A,B). (This pattern
contrasts with the labeling observed in typical dystrophic muscle, which
occurs along the entire length of muscle fibers.) Furthermore, transmission
electron microscopy showed that the BM associated with the MTJ was somewhat
discontinuous and less compact compared with control, and the folding at the
end of mutant muscle fibers exhibited reduced complexity
(Fig. 6E,F). Together, these
data suggest that in the absence of laminin ß2, MTJs are structurally and
functionally defective.
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We have previously shown that there is a laminin ß1 to ß2
developmental transition in the forming GBM
(Miner and Sanes, 1994). In
Lamb2-/- mice, ß1 remains in the GBM rather than
being eliminated, thus serving a structural role to maintain GBM integrity,
but nevertheless failing to maintain the glomerular barrier to protein
(Noakes et al., 1995b). Here,
restoration of podocyte-derived ß2 to the GBM was accompanied by proper
elimination of ß1 as glomeruli matured, though it was still detectable in
the mesangial matrix (Fig. 8).
Ultrastructural analysis showed normal podocyte foot process architecture in
Lamb2-/-; NEPH-B2 glomeruli at all stages, even at a time
near death (Fig. 6H,K; data not
shown). By contrast, analysis of Lamb2-/- and
Lamb2-/-; MCK-B2 glomeruli revealed widespread foot
process effacement (Fig. 6H-J),
consistent with the heavy proteinuria (Fig.
4B).
Combined transgenic rescue of muscle and kidney defects
We next generated Lamb2-/-; MCK-B2; NEPH-B2 mice
through appropriate crosses. Except for occasional mild proteinuria, these
mice have manifested no obvious defects and can live for well over 1 year,
some to 18 months. Both males and females have exhibited normal fertility and
are overtly indistinguishable from control littermates. Light microscopic
analysis of PAS-stained paraffin sections revealed no significant pathology in
either skeletal muscle or kidney in older mice
(Fig. 9), and ultrastructural
analysis of glomeruli and neuromuscular junctions showed no significant
abnormalities (data not shown). Interestingly, Evans blue only occasionally
labeled the ends of muscle fibers in the diaphragms of older doubly rescued
mice (Fig. 7D and data not
shown), and all MTJs contained ß2
(Fig. 7I-L), suggesting that
subsequent to any initial injury at developmental stages, as in
Fig. 7C, MTJs are apparently
repaired. Furthermore, no histopathology was observed in other tissues
reported to be rich in ß2, including lung, retina, pancreas or gut (data
not shown). Based on these results, a requirement for ß2 in BMs other
than those associated with muscle and the kidney glomerulus can not be
demonstrated, at least in the context of the controlled environment of a mouse
cage.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The growth of the laminin family during the evolution of
multicellular organisms has allowed a segregation of laminins into two
distinct groups in vertebrates: one group containing laminins absolutely
required during embryogenesis for basic developmental processes, and the
second containing laminins with more restricted, specialized functions in
postnatal animals.
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3 weeks of age with severe defects in both the
glomerular filter and the neuromuscular junction
(Noakes et al., 1995a;
Noakes et al., 1995b). Whether
there might be defects in other tissues (besides retina)
(Libby et al., 1999), and how
the kidney and muscle defects each contribute to the lethal failure to thrive
phenotype, have been important unanswered questions. The recent finding of
mutations in LAMB2 associated with Pierson syndrome
(Zenker et al., 2004a;
Zenker et al., 2005)
underscores the importance of these issues with regard to human development
and disease.
To investigate whether the kidney or the synapse defect is most detrimental
to the health of the animals, we used tissue-specific laminin ß2
transgenes to rescue each defect individually. We have clearly shown that the
defects in synaptic development and maturation are much more devastating:
rescuing the GBM defects with the podocyte-specific ß2 transgene
(NEPH-B2) did not improve the overt phenotype of the Lamb2 mutant,
whereas rescuing the structure and presumably the function of the
neuromuscular junction with the muscle-specific ß2 transgene (MCK-B2)
greatly improved weight gain and extended lifespan by 50%. The latter
mice must have died from nephrotic syndrome, because preventing it through
simple addition of the NEPH-B2 transgene allowed for a very long life (well
over 1 year) and normal fertility in Lamb2-/-; MCK-B2;
NEPH-B2 mice. Furthermore, we can conclude from this result that for normal
longevity of caged mice, laminin ß2 is only required in muscle and
glomerular BMs, the only sites where we could detect ß2 in
Lamb2-/-; MCK-B2; NEPH-B2 mice. Such an important
conclusion would not have been possible had we chosen to use the more
conventional Cre/loxP conditional knockout approach to mutate Lamb2
specifically in skeletal muscle or podocytes, because all tissues not
expressing Cre would contain their normal complement of ß2.
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;
Johnson et al., 1989), we
cannot be absolutely certain that the basis for improvement in the health of
Lamb2-/-; MCK-B2 mice is solely due to restored synaptic
architecture and function. But the fact that Lamb2-/-
hearts exhibit no obvious morphological or histopathological defects, such as
ventricular dilation or cardiomyocyte necrosis or fibrosis (J.H.M., B.L.P. and
G.J., unpublished), suggests that the improved health of
Lamb2-/-; MCK-B2 mice is not due to transgene expression
in the heart. Moreover, that cardiac defects are not generally associated with
Pierson syndrome (Zenker et al.,
2004b) is also consistent with the lack of a crucial role for
laminin ß2 in the heart.
So why do defects in synaptic structure and function kill
Lamb2-/- mice? The mice usually die at 21 days of
age, which is also the time of weaning. Together with the highly significant
50% decrease in mean quantal content at mutant nerve terminals
(Knight et al., 2003), the
most likely explanation for death is that the mice are simply unable to use
their muscles properly to obtain nutrition. Before weaning, this involves
suckling for many hours each day, in addition to competing with littermates
for access to teats; after weaning, it involves obtaining and chewing solid
food and reaching for water. Consistent with this explanation, we have found
that hand feeding Lamb2-/- mice can extend their life to
up to 40 days of age, though they remain very small and weak. A lack of proper
nutrition is fully consistent with the observed failure to thrive
phenotype.
Although laminin ß2 is concentrated at skeletal muscle synapses, it
can normally also be detected extrasynaptically all along the muscle fiber
with certain antibodies (Sasaki et al.,
2002; Wewer et al.,
1997), suggesting that the muscle deposits ß2 along its
entire length. Here, however, little MCK-B2 transgene-derived ß2 was
detected in the muscle fiber BM, other than at synapses and MTJs. Because
there is no reason to suspect that the MCK regulatory element is not active in
nuclei throughout the entire muscle fiber, these data suggest that skeletal
muscle fibers are programmed to deposit ß2 only in synaptic and MTJ BMs.
This was predicted in part by in vitro data demonstrating that the laminin
ß2 protein contains an inherent signal that targets it to synapse-like
sites on cultured myotubes (Martin et al.,
1995). Another cell type - perhaps the interstitial fibroblast -
is likely to be responsible for deposition of laminin ß2 along the
extra-junctional regions of the muscle fiber.
During kidney glomerular development, distinct BMs synthesized by the
epithelial podocytes and the invading endothelium fuse to form the immature
GBM (Abrahamson and Perry,
1986). The laminin component of the maturing GBM continues to be
synthesized by both the podocytes and the endothelial cells
(St John and Abrahamson,
2001). Our approach to restore laminin ß2 to the mutant GBM
by expressing it exclusively in podocytes via the nephrin promoter was
successful in several respects, including localization to the GBM, restoration
of the glomerular barrier to protein and restriction of laminin ß1 to the
mesangium. This indicates that though GBM laminin ß2 is normally
synthesized by both podocytes and endothelial cells, expression is podocytes
is sufficient for normal GBM structure and function.
Our results have important implications for Pierson syndrome, which is
caused by mutations in human LAMB2
(Zenker et al., 2004a;
Zenker et al., 2005).
Individuals with this disease present with congenital nephrotic syndrome,
diffuse mesangial sclerosis and microcoria (fixed narrowing of the pupils)
associated with absent dilator pupillae muscles in the iris. Other eye
abnormalities include an almost absent ciliary body, an enlarged cornea, an
abnormal lens shape, maldevelopment of the choroid and arrested retinal
development. No obvious structural abnormalities of the brain have been found,
but muscular hypotonia and abnormal movements have been reported
(Zenker et al., 2004b).
Affected individuals usually die within weeks of birth from kidney failure,
but some have lived for over 2 years. The kidney and retinal defects seem to
correlate quite well with defects in the Lamb2 mutant mouse, and the
muscular hypotonia may stem from similar defects in development and function
of the neuromuscular junction. However, cornea, lens and pupillary defects
have not been found in the mouse.
Because of the availability of renal replacement therapy, through either
dialysis or transplantation, the congenital nephrosis is the most treatable
aspect of Pierson syndrome. However, the results of our transgenic rescue
studies show that ameliorating the nephrotic syndrome on its own is not
significantly beneficial in extending the life of the
Lamb2-/- mouse. This calls into question whether renal
replacement therapy alone is warranted in individuals with Pierson syndrome.
Indeed, one individual who received chronic dialysis treatment and lived for
1.8 years exhibited significant neurological and developmental deficits
including severe hypotonia, psychomotor delay, and blindness
(Zenker et al., 2004a).
Recently, a missense mutation in LAMB2 was reported in a patient
exhibiting congenital nephrotic syndrome but not the other features of Pierson
syndrome (Hasselbacher et al.,
2005), demonstrating that there is heterogeneity in the severity
of disease resulting from LAMB2 mutations in humans. As more
individuals with LAMB2 mutations are discovered and
genotype-phenotype correlations are discerned, a better understanding of the
neurological and muscular defects will hopefully emerge and lead to the
development of extra-renal treatment paradigms.
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ACKNOWLEDGMENTS |
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