Human Molecular Genetics 2005 14(Review Issue 2):R259-R267; doi:10.1093/hmg/ddi272
© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Insights into X-linked retinitis pigmentosa type 3, allied diseases and underlying pathomechanisms
Paulo A. Ferreira*
Departments of Ophthalmology and Molecular Genetics and Microbiology, Duke University Medical Center, Erwin Road, Durham, NC 27710, USA
* To whom correspondence should be addressed. Tel: +1 919-684-8457; Fax: +1 919-684-3826; Email: ferre044{at}mc.duke.edu
Received May 6, 2005; Accepted August 1, 2005
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ABSTRACT
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In the past decade, we have witnessed great advances in the
identification of genes underlying numerous neurodegenerative
diseases and the stark complexity determining genotype–phenotype
relationships that lead to the impairment, and ultimately, premature
death of neurons. However, significant challenges lie ahead
in understanding the pathobiological and spatiotemporal processes
triggered by genetic lesions underlying neurodegenerative disorders.
Neuroretinal dystrophies occupy a prominent place among neurodegenerative
diseases, because of the large number and prevalence of disease-causing
genes, the diverse functions, the wealth of allelic, non-allelic
and clinical heterogeneities determining the phenotypic expressivity
and penetrance of the disease and the ease of use of animal
models to probe gene function and disease pathogenesis in a
well-defined neuroretinal circuitry. Retinitis pigmentosa (RP)
has a prevalence of about one in 4000. RP is a retinal dystrophy
leading primarily to the progressive death of photon-capturing
neurons—the rod photoreceptors. X-linked retinitis pigmentosa
type 3 (XlRP3) accounts up to 14% of all RP cases, higher than
any other single RP locus identified to date, and considered
to be the most severe of all RP cases. The
XlRP3 encodes the
retinitis pigmentosa GTPase regulator (RPGR). RPGR interacts
with the RPGR interacting protein-1 (RPGRIP1). Mutations in
RPGRIP1 cause Leber's congenital amaurosis. This review highlights
the progress devoted to understand the pathogenesis associated
with XlRP3 and allied disorders and, concepts, trends and discrepancies
emerging as molecular, subcellular and physiological processes
linked to RPGR and RPGRIP1-protein network begin to be elucidated,
and that may serve as a paradigm for other biological processes
and neurodegenerative diseases.
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NEURORETINAL CIRCUITRY
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The retina is a stratified neuronal tissue that lines the posterior
eyecup. The retina is an integral part of the central nervous
system but it is often portrayed as an appendix organ of the
brain in light of its ‘distant’ connection via a
dense bundle of axons—the optic nerve. The lamination
of the retina comprises three major neuronal (nuclear) layers
with a diverse population of classes of neurons (Fig.
1)
(1
). The polarized, compartmentalized and ciliated (9+0) photon-capturing
neurons, the photoreceptors, represent the primary sensory neurons,
whose cell bodies are located in the outer nuclear layer. They
consist of rod and cone photoreceptors, with the latter being
further subdivided into red, green and blue cone photoreceptors
(Fig.
1). The rods mediate low-acuity vision and operate
under dim-light conditions, and the cones mediate color perception
and high acuity vision and operate under daylight conditions.
The spatial distribution and relative preponderance of the subclasses
of photoreceptors across the retina confer a distinct organization
to this that varies among vertebrate species (e.g. fovea, macula,
etc.). Genetic lesions in photoreceptors impart distinct electrophysiological
and cellular phenotypes, which are determined by what type of
photoreceptor(s) is(are) primarily affected. Photoreceptors
synapse with the second-order neurons, rod or cone bipolar neurons
and horizontal neurons, whose cell bodies are located in the
inner nuclear layer. The bipolar neurons synapse with the ganglion
neurons and the synaptic input to the latter is modulated by
the amacrine neurons located mainly in the inner nuclear layer.
The axons of ganglion neurons coalesce and form the optic nerve,
which projects into the brain (thalamus).
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Figure 1. Laminar organization of the neuroretinal circuitry. The vertebrate retina is a well-defined and stratified neuronal tissue comprising several classes of the photosensory neurons—the photoreceptors (1). These are highly compartmentalized and polarized and are comprised by the OS and inner segment (IS) compartments, respectively, a cell body (nucleus, ONL) and a synaptic terminal. Photoreceptors synapse with second-order neurons, cone or rod bipolar neurons and horizontal neurons, at the outer plexiform layer. The synaptic input of these to the ganglion neurons at the inner plexiform layer is modulated by the amacrine neurons. IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer; CC, connecting cilium; H, horizontal neurons; RB, rod bipolar neurons; CB, cone bipolar neurons; A, amacrine neurons.
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X-LINKED RETINITIS PIGMENTOSA TYPE 3
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RP is a genetically heterogeneous disease affecting primarily
the rod photoreceptor neurons, although cone photoreceptors
are affected subsequently by the loss of rod photoreceptors
and die, hence leading to complete blindness (2
,3
). X-linked
retinitis pigmentosa type 3 (XlRP3) (MIM312610) stands out among
other RP disorders for several reasons. First, XlRP3 accounts
up to 14% of all RP cases in North America, higher than any
other RP locus identified to date (4
). Secondly, it is considered
to be the most severe form of RP, often with an onset during
the first decade of life (5
). Thirdly, there is a tremendous
amount of allelic-dependent phenotypic expressivity and (clinical)
variability in XlRP3. Mutations in
XlRP3 locus are implicated
in RP (4
,6
–10
), cone–rod (11
) and cone dystrophies
(12
) and recessive atrophic macular degeneration (13
). Finally,
XlRP3 is also a systemic disorder, because two distinct mutations
in a highly conserved domain of retinitis pigmentosa GTPase
regulator (RPGR) were linked to several non-ocular diseases
such as hearing loss, sinusitis and chronic recurrent respiratory
tract and ear infections (14
–17
).
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MUTATION AND TRANSCRIPTIONAL HETEROGENEITY OF XlRP3 AND FUNCTIONAL IMPLICATIONS
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The human
XlRP3 locus was initially identified with 19 constitutive
exons and it encodes a protein, hereafter called RPGR
1–19,
with a predicted molecular mass of 90 kDa (6
). Exons 2–10
encode a domain, the RCC1-homologous domain (RHD), homologous
to the nuclear guanine nucleotide exchange factor (GEF) of RanGTPase,
RCC1, whereas the protein sequence encoded by the remaining
exons exhibits no significant homology to other proteins in
databases (6
,8
) (Fig.
2). The RHD contains the first six
of the seven tandem repeats present in RCC1 (18
). RPGR
1–19 has a functional, C-terminal isoprenylation motif (19
). Initially,
mutations found in RPGR were clustered in the RHD, but they
accounted for only
20–30% of patients with XlRP3, a frequency
at odds with the linkage data analysis, which supported that
XlRP3 is responsible for about two-thirds of XlRP (5
,6
). No
mutations were ever found in exons 16–19. Further, mutation
screening of
XlRP3 led to the discovery of an alternative splice
exon derived from the extension of constitutive exon 15 into
intron 15, upon skipping of the splicing donor site of exon
15 (9
). This generates a highly purine-rich, repetitive and
terminal exon, ORF15, encoding repeats with G and E residues
and repeat-like sequence such as ‘EEEGEGEGE’. The
resulting RPGR isoform has a predicted molecular mass of 127 kDa
and it is designated hereafter as RPGR
ORF15 (Fig.
2). Strikingly,
ORF15 was identified as a mutational hot spot, which accounts
for the remaining XlRP3 cases (9
). The ORF15 mutations comprise
mostly frame-shift and/or small out-of-frame deletion mutations,
but not a single missense mutation has been identified (4
,9
,10
).
Surprisingly, the ORF15 can accommodate diverse and concomitant
residue changes (e.g. insertions and in-frame deletions) and
still be non-pathogenic (20
). This is in striking contrast to
those found in the exons encoding the RHD (and up to exon 14),
which consisted of all types of mutations including over 30
point mutations (missense and nonsense) and these are the most
severe clinically (4
,9
,10
,21
). One of these mutations, G436D,
falls well outside the RHD, implicating a functional role for
putative domain(s) outside RHD and ORF15 (4
). Two other mutations,
G173R and 845–846delTG, within RHD (exon 8) lead intriguingly
to RP and systemic phenotypes, as previously described (14
,16
).
In contrast, ORF15 mutations have been suggested to constitute
hypomorphic alleles because they are associated with milder
disease states (10
). Moreover, there appears to be a correlation
between the location of the mutations within ORF15 and the expressivity
and penetrance of the phenotype in the human (10
), an observation
that seems to be corroborated by dogs harboring mutations in
ORF15 (22
). Mutations towards the 3' end of ORF15 (and with
shorter frame-shifted protein sequence) produce milder RP phenotypes
(better retention of rod function). Interestingly, mutations
downstream of codon 445 of ORF15 lead to preferential loss of
cone function, although affecting much less rod function and
hence generating cone–rod and cone dystrophies (10
–12
).
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Figure 2. Primary structure of RPGR isoforms. RPGR1–19 is encoded by the constitutive exons 1–19 of RPGR. RPGR1–19 contains a functional, C-terminal isoprenylation site. RPGRORF15 is encoded by the constitutive exons 1–15 and intron 15. The constitutive exon 15 and intron 15 encode ORF15. The ORF15 contains a repetitive and very acidic domain (AD) and a small C-terminal basic domain (BD). The two putative and conserved GTP-binding motifs (NB) located at the N-terminus of RPGR are shown. RHD is the conserved RCC1-homologous domain and it contains six RCC1 repeats. The seventh repeat present in RCC1 is poorly conserved in RPGR (or unique to RPGR). Amino acid numbering is shown above the constructs. Note the reported numbering of ORF15 often begins with the encoded sequence of exon 15.
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Although
RPGR1–19 and
RPGRORF15 transcripts represent
two splice variants shown to have translated products in the
retina and elsewhere (but the apparent molecular masses appear
to differ among laboratories), transcriptional studies indicate
that the
XlRP3 locus is subjected to an exceptionally high degree
of complex and heterogeneous splicing, mostly downstream of
RHD, and some splice variants are species and tissue specific
(9
,23
,24
). These observations raise interesting questions on
what are the molecular species that are physiologically and/or
pathologically relevant and whether these contribute to (or
confer) unique species- and tissue-specific effects. For example,
the ORF15 undergoes additional and complex alternative splicing
in a tissue-specific fashion (24
). In the mouse, a murine RPGR
ORF15 isoform lacking an in-frame 654 bp deletion of the purine-rich
repetitive region appears to rescue RPGR function in null
RPGR mice (25
), although intriguingly, an equivalent transgenic variant
seems to exhibit a gain-of-function (26
). Moreover, this truncated
ORF15 variant is expressed in C57Bl/6, but not in 129/Sv, mice,
because the former has a
90 bp deletion in the genomic
region of ORF15 (25
,26
). In the mouse retina, intron 14 is not
spliced out (9
,23
). In addition, among other variants, exons
13 and 16 appear to be capable of splicing together in the mouse
and human retinas (23
,24
). Interestingly, an alternative and
terminal exon, exon 15a (encoding only 11 residues), downstream
and upstream of ORF15 and exon 16, respectively, by
6.4 and
2 kb, was reported with specific expression in the human
and mouse retinas (23
). A 6.4 kb deletion with proximal
and distal breakpoints 1.5 kb and 70 bp downstream
of exon 15 and splice donor site of exon 15a, respectively,
lead to cone–rod dystrophy (CRD) in a patient (7
,23
).
It is unclear whether a small distal segment of ORF15 is affected,
but such finding would help to shed additional light on the
role of ORF15 and/or exon 15a in
XlRP3.
In contrast to the transcriptional variants identified, far fewer RPGR isoforms have been detected at protein level. Our laboratory has detected only one major isoform for RPGR1–19 and another for RPGRORF15 in the human retina, with antibodies specific towards unique domains of these (27). Another antibody against the conserved and complete RHD also detects only two isoforms in lymphoblasts, which are absent in patients with truncations, Q236X and 468:RNQIICX, but not with the 6.4 kb deletion of intron 15, as previously described (P.A. Ferreira, unpublished data). In addition, equivalent RPGR isoforms appear to exhibit differences in molecular masses, depending on whether they are ectopically expressed in the retina or transfected cells (24,26). Observations like these appear to be supported by reports from other laboratories, independent of the apparent discrepancies in molecular masses (22,24,28). Altogether, it is likely that many of the splice variants are not translated (biologically relevant) and that the ORF15 plays a dual role by modulating the proposed splicing efficiency (e.g. exonic splicing enhancer) (24) of this genomic region, although concomitantly causing the production of pathogenic protein variants and a change in the ratio between the RPGR1–19 and RPGRORF15 isoforms upon genetic lesions. Differences in the structure of the constitutive intron 15 among species may also determine the ratio of these isoforms between species, production of novel RPGRORF15 isoforms that contribute to unique species-specific phenotypes and significant differences in the phenotypic penetrance observed between the human/dog and the murine species. The effect of mutations in changing the ratio of protein isoforms is known to occur in other genes such as the MAPT gene, which is implicated in frontotemporal dementia with parkinsonism (FTDP-17) (29,30).
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MOLECULAR PARTNERS OF RPGR:  PDE AND RPGRIP1
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To elucidate the molecular pathogenesis of XlRP3 and the function
of RPGR, several groups set out independently to find molecular
partners of RPGR. The
PDE was the first RPGR partner identified
from a yeast two-hybrid screen. Linari
et al. (31
) reported
that the interaction of RPGR with the
PDE is mediated by the
RHD (amino acids 1–392) of RPGR and the few human missense
mutations tested in RHD impaired its interaction with
PDE
in vitro and
in vivo. Although the C-terminal domain of RPGR
1–19 (amino acids 579–815) failed to interact with
PDE, this
construct lacked about 190 residues located between the RHD
and C-terminal domains tested. A number of questions remain
unanswered to impart biological relevance to these findings.
In particular, it needs elucidation whether
PDE is a natural
partner of RPGR
1–19 and/or RPGR
ORF15 and whether these
colocalize in photoreceptors. Finally, no human mutations were
found in the ubiquitously expressed
PDE to date and the findings
fail to explain the basis of the retina-specific effects of
genetic lesions in
XlRP3 locus, because the RPGR isoforms are
also expressed across several tissues (9
,23
). The interaction
of
PDE with RPGR may be significant in light of the findings
that the
PDE associates with the GTPase, Rab13, to retrieve
this from the cell membranes (acting similar to a GDP-dissociation
inhibitor) (32
) and it functions as a prenyl-binding protein
(33
). This raises the possibility that one or more RPGR isoforms
may be implicated in vesicular trafficking.
Subsequently, we reported the isolation of a novel partner for RPGR, RPGR interacting protein-1 (RPGRIP1) (34), from yeast two-hybrid screenings, a finding independently corroborated by two other groups (35,36). In contrast to PDE (and RPGR), RPGRIP1 is predominantly expressed in the retina of the human, bovine and mouse (albeit restricted expression of specific isoforms is found also in other tissues), thus leading to a rational for the mainly retina-restricted effects of mutations in RPGR (34). With the exception of another protein with unknown function in the GenBank database (NM_015272
[GenBank]
/KIAA1005) (34), RPGRIP1 does not exhibit strong overall homologies to other proteins. RPGRIP1 consists of N-terminal and coiled coil domains followed by a C2 domain and a C-terminal RPGR-interacting domain (RID) (Fig. 3A). Mutations in RHD of RPGR abolish its interaction with RID of RPGRIP1 in vivo (34,35). RPGR and RPGRIP1 form a complex in vivo in the retina, partially colocalize to the outer segment (OS) compartment of photoreceptors, and associate directly with each other in vitro (27,34).
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Figure 3. (A) Nomenclature and primary structure of RPGRIP1 isoforms. A new nomenclature is employed herein for the classification of all RPGRIP1 isoforms identified to date and derived from the alternative splicing of RPGRIP1. This classification is based on the variable primary architecture (structural/functional domains) of RPGRIP1 isoforms. RPGRIP1 1 is found in the bovine and human retina (formerly named b/hRPGRIP1). RPGRIP12 is like RPGRIP11 but it contains a long stretch of acidic residues between the C2 and RID domains, and it is unique to the murine. RPGRIP1 is expressed only in the murine. RPGRIP1, RPGRIP1ß, RPGRIP1, RPGRIP1 and RPGRIP1 were formerly named, respectively, mRPGRIP1b, bRPGRIP1b, bRPGRIP1c, hRPGRIP1a/b and bRPGRIP1a/hRPGRIP1d. Letters in italics note the species where expression was found (m, murine; b, bovine; h, human). RPGRIP1 has two subtypes, which differ by a short stretch of residues at the N-terminus. Numbering above the RPGRIP12 isoform refers to the residues of the human sequence delimiting the boundaries of structural/functional domains. (B) Human mutations found in RPGRIP11. Some of the human mutations identified to date and domains thereby affected are depicted. R827L causes CRD and all others lead to LCA. SMC/CC, -helical coiled coil–hinge coiled coil protein interaction motif of members of the structural maintenance of chromosomes (SMC) superfamily; C2, protein kinase C conserved region 2 and Ca2+-binding motif (C2); RID, RPGR-interacting domain; ND, nuclear domain.
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TRANSCRIPTIONAL HETEROGENEITY OF RPGRIP1
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Molecular analysis of the
RPGRIP1 transcripts revealed that
RPGRIP1 undergoes extensive alternative splicing in the human
and bovine (34
,35
) and, to a less extent, in the murine (36
,37
),
and some of the splice variants are species specific (37
). These
observations are reminiscent to those described for RPGR. The
heterogeneous splicing of
RPGRIP1 leads to the production of
RPGRIP1 transcript variants lacking single structural domains
or combination of these. In an attempt to establish clarity
and uniformity to the nomenclature of
RPGRIP1 isoforms identified
to date (34
–38
), we reclassify these into different types
(and subtypes) in this review on the basis of key features of
their primary structure deduced from the translation of the
cognate transcripts (Fig.
3A). RPGRIP1
1 (formerly b/hRPGRIP1)
contains all structural domains and it seems to be the predominant
isoform in the retina of the bovine and human (34
,38
). RPGRIP1
2 is like RPGRIP1
1 with the exception of an insertion of a long
acidic poly(E) (glutamate) stretch of about 80 residues between
the C2 and RID domains (36
). This feature is only present in
the mouse and it would be interesting to find whether it confers
distinct biological properties to RPGRIP1
2. RPGRIP1
formerly
mRPGRIP1b) is produced from the retention of intron 13 (37
).
This isoform has three unique C-terminal residues, it lacks
the C2 and RID domains and there are no counterparts in other
species but it is the most abundant isoform in the mouse retina
(37
). The biological properties of RPGRIP1
appear distinct from
all other isoforms because of its unique subcellular distribution
in the retina and cultured cells. It is found only in the cytosolic
compartment, where it partially colocalizes with a subpopulation
of lysosomes (discussed subsequently) (37
). More importantly,
the role of this isoform is likely not linked to RPGR, because
of the absence of RID, or to other RPGRIP1 isoforms, because
it does not colocalize with these in the retina (37
). RPGRIP1ß
(formerly bRPGRIP1b), RPGRIP1
(formerly bRPGRIP1c) and RPGRIP1
(formerly hRPGRIP1a/b) lack the C2, comprise only the RID and
contain only the C2 and RID domains, respectively (34
,38
). Finally,
RPGRIP1
(formerly bRPGRIP1a and hRPGRIP1d) and RPGRIP1
(formerly
hRPGRIP1c) are truncated at the N-terminus and, in addition,
have the C2 domain deleted, respectively (34
,35
,38
). The expression
of these isoforms was established from the isolation of full-length
transcripts (cDNAs) in the retina, but not all of these may
have translated products in the retina. For example, antibodies
against the SMC/CC domain only detect a major isoform (RPGRIP1
1)
of
175 kDa in the retina (38
). In contrast, a high-affinity
antibody against the conserved RID of RPGRIP1 recognizes three
major isoforms (including RPGRIP1
1 in the (bovine) retina, with
two of these specific to the retina, and a single isoform specifically
expressed in the kidney, spleen and liver (34
,38
). As explained
subsequently, the determination of the relative protein expression
levels of the various RPGRIP1 isoforms may also be hampered
partly by the fact that RPGRIP1
1 undergoes limited proteolysis
(38
). In any event, RPGRIP1
1 is highly enriched in the OS compartment
of the photoreceptor neurons and may be the only RPGRIP1 isoform
expressed in these neurons (38
).
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GENETIC LESIONS IN RPGRIP1 CAUSE LEBER'S CONGENITAL AMAUROSIS
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The discovery of
RPGRIP1 led to the finding that genetic lesions
in RPGRIP1 cause Leber's congenital amaurosis (LCA) (39
–41
)
(Fig.
3B). LCA (MIM204000) is a genetically heterogeneous
retinal dystrophy, it is the leading cause of blindness in children
and it has been variably linked to several systemic phenotypes
(e.g. mental retardation, nephronophthisis, etc.) (42
–47
).
LCA is a severe retinopathy typically characterized for extinguished
electroretinogram (ERG) responses normally during the first
year of life (42
–47
). In a recent study, four distinct
clinical subtypes of LCA were identified on the basis of the
phenotypic variations of molecularly well-defined LCA patients
(41
). The phenotypic expression of patients harboring
RPGRIP1 [and guanylate cyclase 2D (GUCY2D) and aryl hydrocarbon receptor-interacting
protein-like 1 (AIPL1)] mutations was synonymous to congenital
or very early onset of CRD, wherein extremely strong cone dysfunction
was invariably present (41
). In addition, allelic mutations
in
RPGRIP1 and
GUCY2D do not seem epistatic (41
). Among the
mutations found in
RPGRIP1, those affecting the RID and C2 domains,
are of particular interest in light of the structural/functional
relevance of these domains and RPGRIP1 isoforms containing these
(Fig.
3B). For example, mutations leading to small C-terminal
(premature) truncations of RID and the non-conservative substitution,
D1114G (39
–41
), are consistent with a loss-of-function
of RPGRIP1, because we have shown that the complete RID domain
and D
1114 residue are critical for RPGR and RPGRIP1 interaction
in vivo (34
,48
). An interesting mutation is the in-frame deletion
of E1279 (
E1279) (40
). This mutation is found only in the monoallelic
state and it exhibits variable penetrance, because the
E1279
is found in carriers and an LCA patient (40
). The molecular
basis of the disease expressivity of this allele is unclear,
but a recent report of ours supports that the
E1279 likely represents
a dominant epistatic allele (48
). The RID
E1279 exhibits
in vivo stronger binding activity towards RPGR than wild-type RID, and
in contrast to this, it is extremely tolerant to physicochemical
stresses (48
). This observation is in line to that found for
other photoreceptor genes with known epistatic activity, such
as the retinal degeneration slow/pheripherin (
RDS), the rod
OS membrane protein-1 (
ROM1), the cone–rod homeobox (
CRX)
and the
GUCY2D (41
,49
,50
). The G746E mutation (40
), just upstream
of the C2 domain, may likely affect the (relative) conformation
of this domain and/or defines a novel domain, hence placing
these with a critical physiological role in photoreceptor and
retinal functions. Recently, the mutation, R827L, in the C2
domain was found to cause CRD (51
), which is in concordance
with the new classification proposed by Hanein
et al. (41
),
and further implicating a role of the C2 domain in cone photoreceptors.
Finally, other mutations in
RPGRIP1 located upstream of these
lead to premature truncations of RPGRIP1 and possibly represent
null alleles (39
–41
).
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SUBCELLULAR LOCALIZATION OF RPGR AND RPGRIP1
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Numerous antibodies have been generated and shared by different
laboratories against unique domains of RPGR and RPGRIP1 isoforms
(22
,24
,27
,28
,34
,36
–38
,52
,53
). The localization of RPGR
and RPGRIP1 in the retina by these antibodies, particularly
within the subcellular compartments and structures of photoreceptors,
appears to be ambiguous to some extent. Our laboratory has colocalized
RPGR and RPGRIP1 isoforms to the OS of cone and rod photoreceptors
in the human and bovine retinas, which is consistent with the
disease phenotypes observed (27
,34
,38
). These observations are
also supported by the immunopurification of RPGRIP1
1 from OS,
upon extensive purification of these by subcellular fractionation
and confirmation of RPGRIP1
1 identity by Edman sequencing (38
).
The localization of RPGR to the OS has also been reported in
the retina of the dog (22
), human (14
) and mouse (28
) by other
groups. In addition, a recent comprehensive proteomic analysis
of the human ciliary axoneme, where over 200 of the 250 proteins
residing in the axoneme were identified, failed to identify
RPGR and RPGRIP1, but not RP1, as a component of the axoneme
(54
). In the mouse however, our studies support that RPGR is
localized to the connecting cilium in the photoreceptors, whereas
all but one RPGRIP1 isoform, RPGRIP1
, which is specific to the
mouse, are prominently, but not exclusively, localized to the
connecting cilium (27
,37
,38
). This observation is concordant
to that observed by Li and coworkers (24
,36
,53
), but they found
this localization restricted solely to the connecting cilium
and across species (52
). In contrast to RPGR, we also find RPGRIP1
isoforms expressed in the inner retinal neurons, where they
strongly localize to the perinuclear rim of the cell bodies
of amacrine neurons and branching processes thereof (27
,37
,38
).
These observations are further strengthened by the identification
of RPGRIP1 isoforms and/or proteolytic products thereof in homogenate
of OS-depleted retinal preparations, and highly purified nuclear
fractions (38
).
We have proposed that the apparent and partial discrepancies for the localization of RPGR and RPGRIP1 in the photoreceptors may reflect known variations in the structural organization of the OS of photoreceptors among species (rodents versus other ciliated species) and contribute to variations of disease expressivity of genetic lesions in RPGR and possibly, RPGRIP1, among species (discussed subsequently) (27). Likewise, expression of RPGRIP1 in inner retina neurons may underlie the distinct phenotypes observed in patients with mutations in RPGR and possibly in RPGRIP1. Hence, the genetic variations among the RPGR and RPGRIP1 genes, the unique features of the cognate proteins described previously, the variations in isoform ratios and species-specific expression of some of these may underlie the molecular basis for some of the unique structural and circuitry organization of retinal neurons among species and prove to constitute excellent molecular tools to probe further these distinct variations. Finally, the systemic phenotype (e.g. progressive hearing loss) linked to specific allelic mutations in XlRP3 is concordant with RPGR localization in non-ciliated cochlear tissues (14) and hints towards an enhanced effect of these mutations in cell function. Altogether, the localization results mentioned earlier are reminiscent to the apparently discrepant localization results obtained for the complex subcellular distribution pattern of polycystin-1 (PKD1) (55), which is implicated in autosomal dominant polycystic kidney disease (ADPKD) (56), and that has been localized to the cilium (57) and to several membrane domains (55,58,59). In this regard, it is interesting to note that ocular–renal phenotypes may also be linked to RPGR and RPGRIP1 (60) (R. Roepman, unpublished data). The complex subcellular distribution pattern observed for PKD1 extends also to other proteins such as PDE (61) and nephrocystin-4 (62), with PDE having also varied subcellular localization between species (61).
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ANIMAL MODELS OF RPGR AND RPGRIP1
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The canine model with mutations in ORF15 of RPGR
ORF15 (designated
as
XLPRA in the dog) closely mimics the human
XlRP3 in terms
of disease progression, severity, ERG abnormalities and cellular
pathology (22
,63
,64
). These dogs begin to exhibit defects in
the organization of the OS of the rod photoreceptors, followed
by their rapid fragmentation, intradiscal vesicle formation,
and ultimately, neuronal death via apoptosis (64
). Abnormalities
in membrane turnover of OS and phagocytosis by the retina pigment
epithelium are absent during the disease process (64
). A mouse
model with a disrupted
rpgr locus has been generated and this
presents retinal phenotypes that are considerably milder when
compared with those observed in the human and canine (65
). Interestingly,
hemizygous transgenic mice with a murine-specific ORF15 variant
harboring an in-frame deletion of the purine-rich region in
the RPGR
null background lead to a dramatic increase in the
disease progression (26
). This increase was not significantly
prevented by the coexpression of the wild-type allele, hence
suggesting that the murine ORF15 variant exerts a gain-of-function
role in the retina (26
). Subsequently, the same group reported
the rescue of a
null RPGR mouse with an equivalent transgenic
and murine ORF15 construct variant, supporting that this RPGR
isoform may suffice to preserve photoreceptor function (25
)
but raising questions on the dominant phenotypes obtained with
their previous transgenic work (26
).
A mouse model harboring a disrupted rpgip1 locus has also been reported (53). In contrast to rpgr ko model, these exhibit extremely severe and very early dysplasia of the OS compartment of rod photoreceptors and expansion of their disks (53). Like with the null and transgenic rpgr mouse models (26,65), no ultrastructural defects were observed in the connecting cilium of rpgip1–/– mice (53), a ultrastructural phenotype observed in 97% of genetic lesions leading to primary ciliary dykinesia (immotile cilia syndrome) (66). Moreover, a strong word of caution should be noted in interpreting the phenotypes obtained with the rpgip1–/– mice (53). These arouse from the partial homologous recombination of the right arm of the recombination construct and insertion (instead of homologous recombination) of the left arm, comprising exons 4 and 5, downstream of exon 13 of the rpgrip1 locus (the numbering of murine exons described herein assumes the translation initiation codon in exon 1 for the sake of consistency to that described for the human gene). Hence, it is likely that exon 13 splices with out-of-phase exons 4 and 5, and this may contribute to ‘off-target’ phenotypes. In addition, the expression of the 63 kDa and murine-specific RPGRIP1 isoform is likely not affected (37). To this end, new genetically targeted rpgrip1 null models will be required to establish the role of the various RPGRIP1 isoforms, mutations therein and epistatic interactions, in retinal and photoreceptor function and possibly in syndromic phenotypes.
|
PHYSIOLOGICAL FUNCTION OF RPGR AND RPGRIP1
|
|---|
Although a wealth of information has been collected with the
clinical/phenotypic characterization of genetic lesions in
RPGR and
RPGRIP1, the role of these in molecular and subcellular
processes underlying photoreceptor and retinal function and
the pathogenesis of (neuroretinal) disorders linked to RPGR
and RPGRIP1 remains elusive. In an attempt to bridge this gap,
the findings suggesting that RPGRIP1
1 undergoes limited proteolysis
selectively in subcellular compartments of retinal neurons (38
)
was instrumental, in combination with the genetic data available,
to probe further the biological role of RPGRIP1 and RPGR and
set the stage of subsequent experiments in our laboratory. Quantitative
phenotypic analysis of cells transfected with RPGRIP1
1, RPGRIP1ß
and RPGRIP1
(Fig.
3A) supported that the combination of
the C2 and RID domains determined the subcellular localization
and fate of the RPGRIP1 and domains thereof (48
). In particular,
the lack of C2 and RID from RPGRIP1 confers cytosolic localization
and stability to RPGRIP1
(37
,48
), whereas RPGRIP1
1 and RPGRIP1ß
undergo constitutive and limited proteolysis (48
). This leads
to the production of a stable and small N-terminal fragment
(ND) that relocates to and is retained apparently in the nucleus
and most prominently in the nucleolus, whereas the remaining
downstream domains are short lived and remain in the cytoplasmic
compartment (48
). These results imply that factors interacting
with the C2 and RID domains (e.g. RPGR) very likely modulate
the specificity and/or activity of the limited processing and
nuclear signaling pathway of RPGRIP1. This will have important
implications on the role of RPGRIP1 in retinal neurons with
and without expression of RPGR (or its variants). Likewise,
LCA-mutations in the RID of RPGRIP1
1 strongly affect the biological
properties of RPGRIP1. A LCA and frame-shift mutation (Q893X)
leading to the premature truncation of RID enhance dramatically
the localization of ND solely to the nuclear compartment, whereas
the D1114G mutation completely abolishes the immunoreactivity
of the SMC/CC domain towards two cognate antibodies in the cytosol
compartment (48
). This latter effect is likely due to the misprocessing
of RPGRIP1
1 within the SMC/CC domain, which is reflected by
the accumulation of a pathological misprocessed and larger byproduct
of RPGRIP1
1 containing the ND. Finally, the ‘dominant’
E1279 mutation causes a significant increase in the retention
of ND in the cytosolic compartment and interestingly, it leads
to a decrease in the electrophoretic mobility of the unprocessed
RPGRIP1
1 (48
).
The limited proteolysis of RPGRIP11/ß is reminiscent to the regulated intermembrane proteolysis of several proteins (67), such as PKD1 (55,68), which C-terminus translocates to the nucleus upon proteolytic cleavage to activate the AP-1 pathway (55). In addition, LCA-linked mutations in RPGRIP1 may share pathomechanisms to various diseases, wherein the pathological relocation to the nucleus of misprocessed proteolytic fragments underlies the molecular pathogenesis of neurodegenerative (69,70) and other diseases (71,72). Altogether, these data raise a new set of questions. Among these, it will be of particular importance to address the identity of the components and signaling mechanisms/pathways that mediate (i) the limited proteolytic processing of RPGRIP1, (ii) the retention and role of the unprocessed RPGRIP1 in the cytosolic compartment, (iii) the nuclear translocation of the ND from the cytosolic compartment to components of the nuclear pore complexes (e.g. RanBP2) and from these to the nucleoli and (iv) its function in the nucleolus (e.g. mode of gene regulation). In addition, the effect(s) of human RPGR/RPGRIP1 mutations in these processes will be critical to further understand the disease pathomechanisms linked to the cognate genes. Recently, nucleophosmin, a nucleocytoplasmic shuttling and multifunctional molecular chaperone with nucleolar localization in interphasic cells, was found to interact with RPGRORF15 through its small C-terminal basic domain (Fig. 2) (28), hence raising the possibility that it could act as a chaperone for the nuclear translocation of ND of RPGRIP1. In any event, novel partners identified towards the ND and SMC/CC domains of RPGRIP1 and the generation of RPGRIP1 mouse models are bound to provide new insights into the role of the RPGRIP1 interactome in retinal function and disease (unpublished data).
|
CONCLUDING REMARKS
|
|---|
The
RPGR and
RPGRIP1 genes are likely to represent additional
targets of positive selection during evolution and contribute
to variations in the retina among species. To this effect, they
join a growing list of evolving genes determining human traits,
disease phenotypes and allied pathogenic processes and therapeutic
efficacies (73
,74
). In any event, the RPGRIP1 interactome will
unravel novel biological processes of fundamental significance
to the understanding of retinal function and pathogenesis of
severe and allied neurodegenerative diseases. This will pave
the way for the design of novel therapeutic strategies for the
treatment and/or delay the onset of these maladies, which severely
impair neuronal function.
|
ACKNOWLEDGEMENTS
|
|---|
In light of space constraints, the author apologizes for not
referencing many reports from other colleagues, who contributed
for the mutation analysis of XlRP3 and LCA. This work was supported
by grants NIH EY11993 and EY012665 to P.A.F. P.A.F. is the Jules
and Doris Stein Research to Prevent Blindness Professor.
Conflict of Interest statement. None declared.
|
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TOP
ABSTRACT
NEURORETINAL CIRCUITRY