(Investigative Ophthalmology and Visual Science. 1999;40:2795-2802.)
© 1999
by The Association for Research in Vision and Ophthalmology, Inc.
Retinal Degeneration in tulp1-/- Mice: Vesicular Accumulation in the Interphotoreceptor Matrix
Stephanie A. Hagstrom1,
Mabel Duyao2,
Michael A. North2 and
Tiansen Li3
1 From the Ocular Molecular Genetics Institute and
3 Berman–Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston; and
2 AxyS Pharmaceuticals, La Jolla, California.
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Abstract
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PURPOSE. The Tulp1 gene is a member of the tubby gene
family with unknown function. Mutations in the human
TULP1 gene cause autosomal recessive retinitis
pigmentosa. To understand the pathogenic mechanism associated with
TULP1 mutations and to explore the physiologic function
of this protein, we examined tissue distribution of the Tulp1 protein
in normal mice and the photoreceptor disease phenotype in
Tulp1–ablated mice.
METHODS. Tissue distribution of the Tulp1 protein in normal mice was examined by
immunoblotting and immunocytochemistry. The disease phenotype in
tulp1-/- mice was studied by light and
electron microscopy, electroretinography (ERG), and
immunocytochemistry. These results were compared with another mouse
model of retinal degeneration carrying a rhodopsin mutation.
RESULTS. Tulp1 is found exclusively in photoreceptors, localizing predominantly
in the inner segments. It is a soluble protein with an apparent
molecular weight of approximately 70 kDa. Photoreceptor degeneration
developed in tulp1-/- mice, with early
involvement of both rods and cones. At the early stage of degeneration,
rod and cone opsins, but not peripherin/RDS, exhibited prominent
ectopic localization. Electron microscopy revealed massive accumulation
of extracellular vesicles surrounding the distal inner segments.
CONCLUSIONS. The function of Tulp1 is required to maintain viability of rod and cone
photoreceptors. Extracellular vesicular accumulation is not a common
phenomenon associated with photoreceptor degeneration but appears to be
a distinct ultrastructural feature shared by a small group of retinal
disease models. The defect in tulp1-/- mice
may be consistent with a loss of polarized transport of nascent opsin
to the outer segments.
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Introduction
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The tubby gene family has at least four members
that are defined by the highly conserved carboxyl-terminal half of
their primary sequences.1
2
These include
tubby, the prototype of this family, and Tulp1,
Tulp2, and Tulp3, for tubby-like proteins 1, 2,
and 3, respectively. The physiological functions of any member of this
family are not known, but two have been linked to neurosensory
disorders. A recessive mutation in the tubby gene causes
photoreceptor and cochlear degeneration, and adult-onset obesity in
mice.3
4
5
6
Recently, genetic mutations in the human
TULP1 gene were found to cause autosomal recessive retinitis
pigmentosa (RP).7
8
9
Most of these mutations reside in the
conserved C-terminal half of the coding sequence.
RP is a genetically and phenotypically heterogeneous group of
inherited retinal diseases that lead to the eventual degeneration of
the rod and cone photoreceptor cells. Early stages of typical RP are
characterized by progressive night blindness and loss of midperipheral
visual field. Later in the disease, patients lose far peripheral field
and central vision as well. Clinically, most RP patients have a primary
rod defect and later involvement of the cones. It is believed that in
these cases the cone defect is secondary to the loss of rods. A large
number of gene mutations have been identified as causes of RP in the
past decade (http://www.sph.uth.tmc.edu/RetNet). Most of
these genes encode proteins that reside in the photoreceptor outer
segments and participate in the rod phototransduction cascade, which
begins with photon capture and leads to membrane hyperpolarization and
neural signaling. Visual transduction has been intensely investigated
for years. Functions of the components in this pathway are therefore
known, and the consequences of their defects are often understood. More
recently, a number of RP-related genes have been identified that do not
appear to participate in phototransduction but that have functions that
are otherwise unknown. Among this group of genes is Tulp1.
To begin to understand the physiological function of Tulp1, we
have determined the normal protein distribution and analyzed the
retinal disease in tulp1-/- mice in
comparison with another mouse model of retinal degeneration due to a
dominant rhodopsin mutation affecting primarily rod photoreceptors. We
describe the distinct histopathologic features in the
tulp1-/- retina.
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Methods
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Animals and Genotyping Analysis
Heterozygous tulp1 knockout mice, in which the coding
sequence was disrupted by the insertion of a neomycin-selectable
marker, were generated at AxyS Pharmaceuticals (La Jolla, CA; M. North
and M. Duyao, unpublished data). Homozygotes
(tulp1-/-) were derived from heterozygous
mating and identified by PCR amplification of genomic DNA using primer
pairs in exon 8 of the Tulp1 sequence:
5'-AAGGAGGAGAGAGCCTCTTC, sense, and 5'-TTCTCAGTGTCCAGGTGCAG, antisense;
and a pair of primers for the neoR sequence,
5'-ACAATCGGCTGCTCTGATGC and 5'-GTCACGACGAGATC-ATCGC. All four
primers were used together in a 20-µl polymerase chain reaction (PCR)
buffer (pH 8.6) containing approximately 100 ng DNA, 20 picomoles of
each primer, 200 µM each dNTP, 1.5 mM MgCl2, and 10%
dimethyl sulfoxide. PCR reactions were cycled 35 times at an annealing
temperature of 55°C and an elongation temperature of 72°C. A 167-bp
product and a 450-bp product identified the wild-type (wt) and the
knockout alleles, respectively. A transgenic mouse line carrying a
dominant rhodopsin mutation T17M was created by pronuclear injection of
a DNA construct containing the full length of human rhodopsin gene with
4.8 kb of 5' and 6.2 kb of 3' flanking sequences and an engineered
threonine-to-methionine mutation in its coding sequence. This line of
mice had been used in a previous study of therapeutic modalities for
retinal degeneration10
and was used in this study to
compare the degeneration phenotypes between the two genetically
distinct mutants. The Fvb/n mice homozygous for the rd
allele were obtained from the Charles River Laboratory (Wilmington,
MA). The rd allele carries a nonsense mutation in the
gene encoding the ß-subunit of cGMP phosphodiesterase.11
Homozygous rds mice in the Balb/c background were
obtained from Maisy Tang at the New England Primate Research Center
(Southborough, MA). The rds allele carries a mutation in
the gene encoding the peripherin/RDS protein, a structural protein
located in the outer segment disc membrane.12
13
All
experiments were performed in accordance with the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research.
Immunoblot Analysis and Immunocytochemistry
Two antibodies specific for the Tulp1 protein were used in this
study. A 17-mer synthetic peptide was made and used to generate peptide
antibodies in rabbits, which were then affinity purified (Quality
Controlled Biochemicals, Hopkinton, MA). The peptide sequence
KKPETPDSLESKPRKAG, corresponding to amino acid residues 44–60 of the
mouse Tulp1 sequence (GenBank accession AF085681), was chosen based on
its divergence from other members of the tubby gene family,
as well as on predicted surface probability and antigenicity. An
antibody against a fusion protein corresponding to the divergent
N-terminal half of the human TULP1 protein was raised in rats (AxyS
Pharmaceuticals1
) and used as crude serum. For immunoblot
analysis, isolated retinas and other somatic tissues were solubilized
in Laemmli buffer, separated on 10% sodium dodecyl
sulfate–polyacrylamide gels, transferred to polyvinylidene difluoride
membranes, incubated with primary antibodies and peroxidase-conjugated
secondary antibodies, and detected using chemiluminescent reagents
(Pierce, Rockford, IL). An anti-actin antibody was used to probe the
same blots as approximate loading controls (Cat. No. A-2066; Sigma, St.
Louis, MO). To determine whether the Tulp1 protein is present in the
cytosol or whether it exhibits membrane-cytoskeleton association,
isolated retinas were disrupted by one cycle of freeze-thaw; suspended
in a buffer containing 50 mM Tris [pH 7.5 ], 150 mM NaCl, 4 mM
MgCl2; and centrifuged at 160,000g for
1 hour at 4°C. Supernatant and pellet were then subjected to
immunoblot analysis as above. For immunocytochemistry, mouse eyes were
fixed in 4% formaldehyde in phosphate-buffered saline for 1 hour. The
anterior segments and lens were removed. The eyecups were cryoprotected
in 30% sucrose, shock frozen in liquid nitrogen, and sectioned at
10-µm thickness. All sections were cut along the superior to inferior
axis so that the density gradient of cones could be visualized along
this axis.14
Sections were incubated with primary
antibodies against Tulp1, rod opsin (rho-4D2 for the N terminus and
rho-1D4 for the C terminus,15
) blue (short-wavelength
sensitive) and green (middle-wavelength sensitive) cone
opsins,16
and peripherin/RDS.17
This was
followed by incubation with Cy3-conjugated secondary antibodies.
Sections were viewed and photographed under a 40x objective on an
epifluorescence microscope. Higher magnification images were acquired
under a 100x objective on a confocal laser scanning microscope (TCS4D;
Leica, Deerfield, IL).
Electroretinography
Corneal electroretinograms (ERGs) were recorded from dark- and
light-adapted mice as previously described.10
Dark-adapted
ERGs measure rod function in response to a flash of light.
Light-adapted ERGs, performed in the presence of background lighting,
isolate the cone responses.18
Light and Electron Microscopy
Microscopic examination of mouse retinas were performed
essentially as described previously.10
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Results
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To determine the tissue distribution of Tulp1, immunoblotting
analyses were performed using both Tulp1 antibodies on a number of
different tissues. As shown in Figure 1
(upper left), only retina displayed a single band migrating at an
apparent molecular weight of approximately 70 kDa, which is slightly
larger than the calculated molecular weight for the mouse Tulp1
polypeptide (~60 kDa). Both the peptide antibodies and the fusion
protein antibodies recognized a band of the same molecular weight on
immunoblots. Thus, Tulp1 expression was restricted to the retina. To
further localize Tulp1 within the retina, retinal homogenates from wt
and adult rd/rd mice were probed together on immunoblots
with the Tulp1 antibodies. In adult rd/rd retinas, the
photoreceptor layer, but not the other layers of the neural retina, had
degenerated to completion. As shown in Figure 1
(upper right), the
Tulp1 signal was absent in this photoreceptorless retina, indicating
that Tulp1 is expressed specifically in photoreceptor cells. The Tulp1
signal was also greatly diminished in 2-month old rds
(retinal degeneration slow) mouse retinas12
in line with
the extent of photoreceptor cell loss in these retinas (data not
shown). Retinal homogenate from a 3-week-old
tulp1-/- mouse, which had retained most
of its photoreceptors (see below), was found not to contain Tulp1,
thereby confirming the disrupted expression of Tulp1 in these mice.
After a high-speed centrifugation of retinal homogenates from wt mice,
to pellet all membranous and cytoskeletal fractions, Tulp1 was found
exclusively in the supernatant (Fig. 1
, lower left). Thus, there was no
stable association between Tulp1 with any particulate fractions under
our conditions of homogenization.
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Figure 1. Immunoblot analyses of Tulp1 expression using Tulp1 antibodies. Actin
was included to control for loading equivalency. Top
left: multiple tissue blot showing specific expression in
retina. Top right: retinal homogenates from mice of
different genotypes. Tulp1 is detected in wt retina but not in
photoreceptorless (rd/rd) or
tulp1-/- retinas. Lower
left: Tulp1 is present in a high speed supernatant (S) but not in
the pellet (P) fraction of retinal homogenates.
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The photoreceptor-specific expression of Tulp1 and the absence of Tulp1
in the tulp1-/- photoreceptors were
further confirmed by immunocytochemical staining of retinal sections
(Fig. 2)
, using both Tulp1 antibodies. These data show that Tulp1 was
distributed predominantly in the inner segments of the photoreceptor
cells of wt retinas. To a much lesser extent, there was also staining
in the perinuclear and synaptic regions (Fig. 2
, left and middle; wt
retina at 3 weeks of age). The outer segments showed no Tulp1 staining.
An age-matched tulp1-/- retina showed no
expression of Tulp1 in the photoreceptor layer or any retinal layers
(Fig. 2 , right). The staining pattern in older wt retinas was the same
as that at 3 weeks of age (not shown).
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Figure 2. Localization of Tulp1 by immunofluorescence. Left: wt
retina at 3 weeks of age. Staining is most prominent in the inner
segments. Middle: higher magnification of the same wt
retina focusing on the inner segment layer. Right:
tulp1-/- retina at 3 weeks of age
(littermate of the wt mouse shown in the left and
middle panels) showing no significant staining in the
photoreceptor layer by the Tulp1 antibodies. Staining of vascular
profiles (seen in the choroidal layer just above the retinal pigment
epithelium and as bright dots in the inner neural retina) was due to
cross-reactivity to the circulating mouse IgG with the secondary
antibody (goat anti-rat). IS, inner segment; OS, outer segment; ONL,
outer nuclear layer.
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Mutant mice lacking Tulp1 expression showed a progressive retinal
degeneration that was already apparent by 3 weeks (Fig. 3)
. At this age, the outer and inner segments were shortened, and the
outer segments were disorganized. The outer nuclear layer was close to
normal thickness but contained frequent pyknotic nuclei indicating
ongoing photoreceptor cell death. The retinal pathology became
progressively severe and appeared to reach end stage before 5 months of
age. Retinal function, revealed by ERG (Fig. 4)
, also declined with age in parallel to the deterioration of retinal
morphology. Both rod and cone ERG amplitudes were markedly reduced at 4
weeks, and cone ERG became unrecordable by 8 weeks of age. Heterozygous
animals as old as 4 months of age retained normal retinal function as
shown by ERG testing (Fig. 4)
.
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Figure 3. Photoreceptor degeneration shown by light microscopy. Shortened and
disorganized photoreceptor outer segments were apparent at 3 weeks of
age, and degeneration was complete by 5 months. RPE, retinal pigment
epithelium; OS, outer segment; IS inner segment; ONL, outer nuclear
layer; INL, inner nuclear layer.
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Figure 4. ERGs of tulp1-/- and
tulp1+/- mice. Dark-adapted ERGs measure rod
function, whereas light-adapted ERGs isolate the cone response. From
top to bottom: a tulp1+/-
mouse at 4 months of age exhibited normal waveforms and amplitudes for
rods and cones. The rod and cone ERG amplitudes of
tulp1-/- mice were severely diminished at 4
and 8 weeks and declined to an undetectable level by 5 months.
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To follow the histopathologic changes in cone photoreceptors, we
stained tulp1-/- retinas ranging from 17
days to 4 weeks (not shown) of age with antibodies specific for blue
and green cone opsins (Fig. 5)
. These were compared with the T17M rhodopsin mutant and wt retinas
(Figs. 5A
5B
5C
5D
5E
5F)
. A similar pathologic condition was observed
between the blue and green cones in
tulp1-/- retinas. There was ectopic
staining of the inner segment and nuclear and synaptic layers by the
cone opsin antibodies. There was only rudimentary outer segment
formation at 17 days of age, the earliest time point examined (Figs. 5A
, 5D
). At 4 weeks, cones became sparse in the
tulp1-/- retinas (not shown). In
contrast, the T17M retinas had nearly normal-appearing cones at 3 weeks
(not shown). In the T17M retinas, even at 7 weeks when a substantial
number of rods had been lost, both blue and green cone opsins still
exhibited a predominant outer segment localization, and the cones were
present in approximately normal densities (Figs. 5B
, 5E
). This is
consistent with cones being affected secondary to rod disease in the
rhodopsin mutant retinas. It appears that cones were affected earlier
and more severely in tulp1-/- mice.
Thus, Tulp1 function may be essential in both rods and cones.
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Figure 5. Immunofluorescent localization of cone opsins, rod opsin, and
peripherin/RDS in tulp1-/-, T17M, and wt
retinas. The genotypes of mice are indicated at the left and
the target proteins recognized by the antibodies are marked at the
top. (a) Ectopic localization of blue cone opsin
in inner segments, nuclei, and synapses of
tulp1-/- photoreceptors;
(d) similar staining pattern for green cone opsin in
tulp1-/- photoreceptors as in
(a); (g) ectopic localization of rod opsin in
tulp1-/- photoreceptors, indicated by
equal staining intensities in outer and inner segments and heavy
staining of the nuclear layer. Shown are sections stained with rho-4D2,
which are identical with those stained with rho-1D4 (not shown);
(j) normal outer segment localization of RDS
protein in tulp1-/- photoreceptors. The ages
of the animals were: tulp1-/-, 17 days; T17M
(b, e), 7 weeks; T17M (h,
k), 4 weeks; and wt, 6 weeks. The
tulp1-/- mice were shown at the earlier
age for best photoreceptor preservation. Photoreceptor outer segments
and cell bodies will degenerate further at older ages. Older T17M
retinas were shown to illustrate the differences in cone involvement in
these two mutants.
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Staining for rod opsin (Figs. 5G
, 5H
, 5I
) showed similar signal
intensities in the outer and inner segments and prominent staining in
the nuclear layer of the tulp1-/-
retina, both at 17 days and at 4 weeks of age (not shown). Thus, at the
level of light microscopy the predominant outer segment staining
pattern for opsin seen in wt retinas was lost in the
tulp1-/- retina. In comparison, the T17M
retinas at an early stage of degeneration (4 weeks) preserved
predominant outer segment localization of rod opsin, with only slightly
increased staining of the inner segments and nuclear layer compared
with a wt retina. Therefore, ectopic localization of both rod and cone
opsins was an early hallmark of retinal disease in the
tulp1-/- retinas. Interestingly,
staining for another outer segment–specific membrane protein,
peripherin/RDS, showed partitioning of this protein in the outer
segments of the tulp1-/- retinas,
similar to that found in the T17M or the wt retinas (Figs. 5J
, 5K
, 5L
).
Electron microscopy revealed that the
tulp1-/- retinas had disorganized and
shortened outer segments and shortened inner segments, consistent with
the findings by light microscopy. At 3 weeks of age, there was a
massive accumulation of vesicular profiles in the interphotoreceptor
matrix (Fig. 6A
). The highest abundance was around the ellipsoid (distal) inner
segments, tapering off toward the proximal inner segments and the outer
segments. The vesicles were relatively uniform in size averaging 0.1 to
0.2 µm in diameter and bounded by a single membrane. No such vesicles
were observed in the degenerating T17M retina or the wt retina (Figs. 6B
, 6C
). Vesicle accumulation quickly diminished after peaking at 3
weeks of age, and few vesicles were observed in
tulp1-/- retinas at 5 or 8 weeks (data
not shown). The extracellular location of the vesicles could be better
visualized in tangential sections through distal inner segments (Fig. 6D)
. Packets of vesicles were found clearly in the extracellular space
among the profiles of several distal inner segments (as indicated by
the abundance of mitochondria within them). Cross-sectional views of
connecting cilia (Fig. 6D)
had the normal 9 + 0 arrangement of
microtubule pairs. Scanning through a number of tangential sections did
not reveal any abnormal connecting cilia (from ~50 examined). At
higher magnification (Fig. 7)
, the inner segment plasma membranes were marked by numerous
protrusions (blebbing), some of which appeared to be in the process of
pinching off. It is not yet clear whether these plasma membrane
protrusions are in fact sites of origin of the accumulated vesicles,
because membrane blebbing often accompanies apoptotic cell death. There
was also an abundance of cytoplasmic vacuoles of varying sizes in the
distal inner segments of tulp1-/-
photoreceptors.
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Figure 6. A distinct ultrastructural feature of tulp1-/-
photoreceptors revealed by electron microscopy. (A,
B, C) longitudinal sections; (D)
tangential section. (A) Three-week-old
tulp1-/- retina showing the region
between the distal inner segment and basal outer segment. Three packets
of extracellular vesicles (*) are seen between adjacent inner
segments. (B) Degenerating T17M retina is completely free
of the extracellular vesicles. (C) Three-week-old wt retina.
(D) Tangential section of a
tulp1-/- retina at the level of distal
inner segments. Packets of accumulated vesicles (*) are seen in the
interphotoreceptor matrix between adjacent inner segments. Three
connecting cilia (arrowheads) have the typical 9 + 0
arrangement of microtubules. M, mitochondria; IS, inner segment. Bar
(A, B, C) 1 µm; (D) 0.4
µm.
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Figure 7. Higher magnification electron micrographs of 3-week-old
tulp1-/- photoreceptors.
(A) Longitudinal section showing the basal outer segment
(OS) and distal inner segment (IS) of one photoreceptor cell
(left), connecting cilium (CC) of a neighboring
photoreceptor, and a collection of extracellular vesicles surrounding
the distal inner segment. Protrusions from the inner segment plasma
membrane (arrowheads) were frequently observed.
(B) Tangential section through distal inner segments. Plasma
membrane protrusions are marked by arrowheads.
Low-density intracellular vesicles (*) bounded by a single membrane
were also present in this region. M, mitochondria. Bar, 0.2 µm.
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Discussion
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Tulp1 is a photoreceptor protein of unknown function.
Search of sequence databases did not reveal any significant homology
with known proteins or functional motifs, other than members of the
tubby family. Early-onset progressive retinal degeneration develops in
mice deficient in Tulp1, affecting both rods and cones. In typical
retinal degenerations, cone photoreceptors eventually die, even though
the primary disease may affect only rods, because of an undefined
secondary effect. In tulp1-/- mice,
there is primary involvement of both rods and cones. This is indicated
by the abnormal cone opsin distribution, a near absence of cone outer
segments, and diminished cone ERG responses in young animals. The loss
of cone ERG responses occurred much earlier than that observed in mice
carrying the T17M rhodopsin mutation (unpublished data) or a P23H
rhodopsin mutation19
; both mutations primarily affect the
rods only. By analogy with the tulp1-/-
murine model, involvement of cone photoreceptor cells is likely also to
be a part of the primary retinal disease in human patients with RP due
to TULP1 mutations. These patients manifest an early-onset
retinal disease and have a more severe visual handicap than the average
patient with RP due to other genetic defects. By their early thirties,
the patients’ rod ERG responses are undetectable, and cone ERG
responses are minimal.7
This explains the severity of
visual deficit, because only cones are essential for visual acuity and
daytime vision.
A marked feature in the tulp1-/- retina
is the massive accumulation of vesicles in the interphotoreceptor
matrix surrounding the distal inner segments. We do not believe these
to be debris from degenerating photoreceptors. That would imply that
accumulation of extracellular vesicles is an expected feature in models
of photoreceptor degeneration as long as the severity of disease is at
or above a certain level. In fact, many rodent models of photoreceptor
degeneration do not exhibit this phenotype.20
21
22
23
Vesicular accumulation was first described in the rds mice
in which vesicular elements amass in the subretinal space just distal
to the rudimentary connecting cilia.24
25
These vesicular
profiles are immunopositive for opsin.26
27
28
The
rds photoreceptors never elaborate outer segments, because
of the absence of a disc membrane structural protein,
peripherin/RDS.29
Therefore, the vesicular elements in the
rds retinas are likely to be the product of aborted outer
segment formation. Treatment of isolated Xenopus retinas
with tunicamycin also induced vesicular accumulation at the
extracellular space between the rod inner and outer segments,
accompanied by an arrest of new disc formation.30
These
findings may be explained by the suggestion that nonglycosylated opsin
and/or other membrane proteins are incompetent to support disc
morphogenesis. Thus, in both the rds and the
tunicamycin-induced models, it appears that nascent disc-building
materials could be targeted to the approximately correct location, but
disc morphogenesis could not be completed. Three other genetic murine
models of retinal degeneration, in which outer segment formation
occurs, have also been reported to accumulate extracellular vesicles
accompanying photoreceptor degeneration. The pcd mice have
photoreceptor and cerebellar Purkinje cell
degeneration31
32
; the identity of the pcd gene
is not yet known. The tubby mice, equivalent to
rd5, have both retinal and auditory defects.5
A
point mutation at the C terminus of rhodopsin, P347S, results in a
dominant photoreceptor degeneration in transgenic mice.33
All three exhibit extracellular vesicle accumulation in the
interphotoreceptor matrix surrounding the distal inner segments that
appear similar in size, density, and location to that seen in
tulp1-/- mice. Thus, the available data
indicate that vesicular accumulation is a distinct phenotype
attributable to a subset of gene mutations that cause photoreceptor
degeneration.
In the vertebrate retina, disc membranes in the photoreceptors are
continually renewed by the addition of new membranes at the base and
the loss of packets of older discs from the tip. Rhodopsin, the most
abundant membrane protein in the outer segments, and other outer
segment-bound proteins must be continually sorted and trafficked
through transport vesicles from the trans-Golgi network toward the
outer segments.34
35
This is an essential, but poorly
understood, aspect of photoreceptor cell biology.36
A
number of small guanosine triphosphatases appear to play a prominent
role.37
Polarized protein trafficking in general requires
a large number of cytoplasmic components as well as interactions with
signal sequences present on the cargo proteins.38
A single
defect in these complex interactions could result in a loss of polarity
in protein trafficking. There is biochemical evidence that the C
terminus of rhodopsin may serve as a signal sequence in this
process.39
40
In transgenic mice, a mutation affecting the
conserved penultimate proline residue (P347S) of rhodopsin causes a
vesicular accumulation phenotype,33
the severity of which
varies with levels of transgene expression. This is in contrast to mice
bearing rhodopsin mutations not known to affect the trafficking signal
and that do not exhibit this phenotype.10
21
23
It can be
argued that in the tulp1-/- mice, as
well as in the P347S mice, the accumulated vesicles may represent
misrouted transport carriers for opsin. In support of this notion, both
rod and cone opsins were ectopically localized early in the
tulp1-/- photoreceptors, suggesting that
it is part of the primary defect in the
tulp1-/- photoreceptors. The vesicle
accumulation phenotype in tulp1-/- mice
may be consistent with a loss of polarized trafficking of certain outer
segment-bound membrane proteins. We speculate that in the
tulp1-/-, tubby, P347S, and pcd mice,
the mutations affect functions that regulate the polarity of normally
outer segment–bound vesicular traffic. This results in a reduced
number of vesicles reaching the correct target membranes; therefore,
the outer segments are shortened, even though the nascent membrane may
not be defective in its disc morphogenesis potential. Ectopically
targeted vesicles may bud from lateral inner segment plasma membranes
and accumulate in the interphotoreceptor matrix. This hypothesis
implies that tubby, Tulp1, and pcd
genes encode essential functions in this pathway. Interestingly, the
RDS protein has a normal localization in the
tulp1-/- photoreceptors, supporting the
notion that rhodopsin and the RDS protein are sorted to different
transport vesicles, as noted previously.41
It is also
interesting to note that two members of the tubby gene
family are now correlated with this phenotype, suggesting that this
gene family may participate in polarized protein trafficking in
different cell types. In-depth investigation into their functions and
those of any newly found genotypes that produce the distinct phenotype
discussed earlier may provide a genetic dissection of the process of
polarized trafficking in photoreceptor cells. It also promises to
broaden our understanding of the pathogenesis of RP and allied retinal
degenerative diseases.
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Acknowledgements
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The authors thank Gary Yue for expert technical assistance, Robert
Molday for rod opsin antibodies, Jeremy Nathans for cone opsin
antibodies, Gabriel Travis for peripherin/RDS antibodies, and Thaddeus
Dryja for discussions.
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Footnotes
|
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Supported by National Institutes of Health Grant EY10309, the
Foundation Fighting Blindness, and the Massachusetts Lions Eye Research
Fund. SAH is the recipient of postdoctoral training Grant EY06915. TL
is the recipient of a career development award from Research to Prevent
Blindness.
Submitted for publication April 7, 1999; revised June 28, 1999; accepted July 14, 1999.
Commercial relationships policy: N.
Corresponding author: Tiansen Li, Berman–Gund Laboratory,
Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA
02114. E-mail: tli{at}meei.harvard.edu
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References
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|---|
-
North, MA, Naggert, JK, Yan, Y, Noben–Trauth, K, Nishina, PM (1997) Molecular characterization of TUB, TULP1, and TULP2, members of the novel tubby gene family and their possible relation to ocular disease Proc Natl Acad Sci US 94,3128-3133[Abstract/Free Full Text]
-
Nishina, PM, North, MA, Ikeda, A, Yan, Y, Naggert, JK (1998) Molecular characterization of a novel tubby gene family member, TULP3, in mouse and human Genomic 54,215-220[Medline][Order article via Infotrieve]
-
Kleyn, PW, Fan, W, Kovats, SG, et al (1996) Identification and characterization of the mouse obesity gene tubby: a member of a novel gene family Cel 85,281-290[Medline][Order article via Infotrieve]
-
Noben–Trauth, K, Naggert, JK, North, MA, Nishina, PM (1996) A candidate gene for the mouse mutation tubb Natur 380,534-538[Medline][Order article via Infotrieve]
-
Heckenlively, JR, Chang, B, Erway, LC, et al (1995) Mouse model for Usher syndrome: linkage mapping suggests homology to Usher type I reported at human chromosome 11p15 Proc Natl Acad Sci US 92,11100-11104[Abstract/Free Full Text]
-
Ohlemiller, KK, Hughes, RM, Mosinger–Ogilvie, J, Speck, JD, Grosof, DH, Silverman, MS (1995) Cochlear and retinal degeneration in the tubby mous Neurorepor 6,845-849[Medline][Order article via Infotrieve]
-
Hagstrom, SA, North, MA, Nishina, PM, Berson, EL, Dryja, TP (1998) Recessive mutations in the gene encoding the tubby-like protein TULP1 in patients with retinitis pigmentos Nat Gene 18,174-176[Medline][Order article via Infotrieve]
-
Banerjee, P, Kleyn, PW, Knowles, JA, et al (1998) TULP1 mutation in two extended Dominican kindreds with autosomal recessive retinitis pigmentos Nat Gene 18,177-179[Medline][Order article via Infotrieve]
-
Gu, S, Lennon, A, Li, Y, et al (1998) Tubby-like protein-1 mutations in autosomal recessive retinitis pigmentos Lance 351,1103-1104[Medline][Order article via Infotrieve]
-
Li, T, Sandberg, MA, Pawlyk, BS, et al (1998) Effect of vitamin A supplementation on rhodopsin mutants threonine-17
methionine and proline-347serine in transgenic mice and in cell culture Proc Natl Acad Sci US 95,11933-11938[Abstract/Free Full Text]
-
Pittler, SJ, Baehr, W. (1991) Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mous Proc Natl Acad Sci US 88,8322-8326[Abstract/Free Full Text]
-
Travis, GH, Brennan, MB, Danielson, PE, Kozak, CA, Sutcliffe, JG (1989) Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds Natur 338,70-73[Medline][Order article via Infotrieve]
-
Arikawa, K, Molday, LL, Molday, RS, Williams, DS (1992) Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration J Cell Bio 116,659-667[Abstract/Free Full Text]
-
Szel, A, Rohlich, P, Caffe, AR, Juliusson, B, Aguirre, G, Van Veen, T. (1992) Unique topographic separation of two spectral classes of cones in the mouse retin J Comp Neuro 325,327-342[Medline][Order article via Infotrieve]
-
Molday,/SNM>, R. (1988) Monoclonal antibodies to rhodopsin and other proteins of rod outer segment Osborne, N Chader, G eds. Progress in Retinal Researc Vol 8,174-209 Pergamon New York.
-
Wang, Y, Macke, JP, Merbs, SL, et al (1992) A locus control region adjacent to the human red and green visual pigment gene Neuro 9,429-440
-
Kedzierski, W, Bok, D, Travis, GH (1998) Non-cell-autonomous photoreceptor degeneration in rds mutant mice mosaic for expression of a rescue transgen J Neurosc 18,4076-4082[Abstract/Free Full Text]
-
Peachey, NS, Goto, Y, al-Ubaidi, MR, Naash, MI (1993) Properties of the mouse cone-mediated electroretinogram during light adaptatio Neurosci Lett 162,9-11[Medline][Order article via Infotrieve]
-
Goto, Y, Peachey, NS, Ripps, H, Naash, MI (1995) Functional abnormalities in transgenic mice expressing a mutant rhodopsin gen Invest Ophthalmol Vis Sc 36,62-71[Abstract/Free Full Text]
-
Cagianut, B, Sandri, G, Zilla, P, Theiler, K. (1985) Studies on hereditary retinal degeneration: the rd gene in the mouse Graefes Arch Ophthalmo 223,16-22
-
Olsson, JE, Gordon, JW, Pawlyk, BS, et al (1992) Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa Neuro 9,815-830
-
Lem, J, Flannery, JG, Li, T, Applebury, ML, Farber, DB, Simon, MI (1992) Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subuni Proc Natl Acad Sci US 89,4422-4426[Abstract/Free Full Text]
-
Liu, X, Wu, T-H, Stowe, S, et al (1997) Defective phototransductive disk membrane morphogenesis in transgenic mice expressing opsin with a mutated N-terminal domai J Cell Sc 110,2589-2597[Abstract]
-
Cohen, AI (1983) Some cytological and initial biochemical observations on photoreceptors in retinas of rds mic Invest Ophthalmol Vis Sc 24,832-843[Abstract/Free Full Text]
-
Jansen, HG, Sanyal, S. (1984) Development and degeneration of retina in rds mutant mice: electron microscopy J Comp Neuro 224,71-84[Medline][Order article via Infotrieve]
-
Nir, I, Papermaster, DS (1986) Immunocytochemical localization of opsin in the inner segment and ciliary plasma membrane of photoreceptors in retinas of rds mutant mic Invest Ophthalmol Vis Sc 27,836-840[Abstract/Free Full Text]
-
Usukura, J, Bok, D. (1987) Changes in the localization and content of opsin during retinal development in the rds mutant mouse: immunocytochemistry and immunoassay Exp Eye Re 45,501-515[Medline][Order article via Infotrieve]
-
Jansen, HG, Sanyal, S, de Grip, WJ, Schalken, JJ (1987) Development and degeneration of retina in rds mutant mice: ultraimmunohistochemical localization of opsin Exp Eye Re 44,347-361[Medline][Order article via Infotrieve]
-
Molday, R. (1994) Peripherin/rds and rom-1: molecular properties and role in photoreceptor cell degeneration Osborne, N Chader, G eds. Progress in Retinal and Eye Researc Vol 13,272-297 Pergamon New York.
-
Fliesler, S, Rayborn, M, Hollyfield, J. (1985) Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin J Biol Che 100,574-587
-
Blanks, J, Mullen, R, LaVail, M. (1982) Retinal degeneration in the pcd cerebellar mutant mouse, II: electron microscopic analysis J Comp Neuro 212,231-246[Medline][Order article via Infotrieve]
-
Blanks, JC, Spee, C. (1992) Retinal degeneration in the pcd/pcd mutant mouse: accumulation of spherules in the interphotoreceptor space Exp Eye Re 54,637-644[Medline][Order article via Infotrieve]
-
Li, T, Snyder, WK, Olsson, JE, Dryja, TP (1996) Transgenic mice carrying the dominant rhodopsin mutation P347S: evidence for defective vectorial transport of rhodopsin to the outer segments Proc Natl Acad Sci US 93,14176-14181[Abstract/Free Full Text]
-
Papermaster, DS, Schneider, BG, Besharse, JC (1985) Vesicular transport of newly synthesized opsin from the Golgi apparatus towards the rod outer segmen Invest Ophthalmol Vis Sc 26,1386-1404[Abstract/Free Full Text]
-
Besharse, JC, Wetzel, MG, Janssen, JJM, Boveegeurts, PHM, Merkx, M, Degrip, WJ (1995) Immunocytochemical localization of opsin in rod photoreceptors during periods of rapid disc assembl J Neurocyto 24,371-388[Medline][Order article via Infotrieve]
-
Deretic, D, Papermaster, DS (1995) The role of small G-proteins in the transport of newly synthesized rhodopsi Osborne, N Chader, G eds. Progress in Retinal and Eye Researc Vol 14,249-265 Pergamon New York.
-
Deretic, D, Huber, LA, Ransom, N, Mancini, M, Simons, K, Papermaster, DS (1995) Rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesi J Cell Sc 108,215-224[Abstract]
-
Aroeti, B, Okhrimenko, H, Reich, V, Orzech, E. (1998) Polarized trafficking of plasma membrane proteins: emerging roles for coats, SNAREs, GTPases and their link to the cytoskeleton Biochim Biophys Act 1376,57-90[Medline][Order article via Infotrieve]
-
Deretic, D, Puleo–Scheppke, B, Trippe, C. (1996) Cytoplasmic domain of rhodopsin is essential for post-Golgi vesicle formation in a retinal cell-free syste J Biol Che 271,2279-2286[Abstract/Free Full Text]
-
Chuang, JZ, Sung, CH (1998) The cytoplasmic tail of rhodopsin acts as a novel apical sorting signal in polarized MDCK cell J Cell Bio 142,1245-1256[Abstract/Free Full Text]
-
Fariss, RN, Molday, RI, Fisher, SK, Matsumoto, B. (1997) Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathway J Comp Neuro 387,148-156[Medline][Order article via Infotrieve]
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Abstract
Introduction
