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Eric
Pierce, M.D., Ph.D.
Associate
Professor of Ophthalmology
Genetics
and Gene Regulation Program
Address
F.M. Kirby Center for Molecular Ophthalmology
Scheie Eye Institute
304 Stellar-Chance Labs
422 Curie Boulevard
Philadelphia, PA 19104
Office tel.: 215 573-3919
Lab tel.: 215 573-7866
Fax: 215 573-8030
E-mail: epierce@mail.med.upenn.edu
Link(s)
Eric Pierce's FM Kirby website
Education
Dartmouth College, A.B. (Biochemistry)
1981
University of Wisconsin-Madison, Ph.D. (Biochemistry) 1986
Harvard Medical School and Massachusetts Institute of Technology,
M.D.1990
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Research
Interests
- Molecular bases of inherited retinal degenerations.
Key
words: Retinal degeneration, photoreceptor cell
biology, gene targeting, proteomics, RNA splicing
Search PubMed for articles
Description
of Research
Photoreceptor Sensory Cilia Biology
and Disease
Background
Primary cilia are present on most vertebrate cell types. These
structures are typically sensory organelles, and are involved
in many critical aspects of cell biology and development.
The sensory cilium elaborated by each rod and cone photoreceptor
cell of the retina is a classic example. Like other cilia,
photoreceptor sensory cilia (PSCs) contain an axoneme, which
begins at the basal bodies, passes through a transition zone
and into the outer segment (Figure 1). The basal bodies also
nucleate the ciliary rootlet, which extends into the inner
segment.
Figure 1. Photoreceptor
sensory cilia (right, top part of rod cell) are the light
sensitive parts of the rods and cones in the eye's retina
(middle and left). Like other cilia, the outer segment (OS)
contains an axoneme, which begins at the basal bodies, passes
through a transition zone (the so-called "connecting cilium")
and into the OS. The basal bodies also nucleate the ciliary
rootlet, which extends into the inner segment (IS). The PSC
complex comprises the OS and its cytoskeleton, including the
rootlet, basal body and axoneme. The membrane domain of the
OS is highly specialized, with discs (lamellar membranes)
stacked in tight order at 30 per micron along the axoneme.
The proteins required for phototransduction are located in
or associated with these discs.
Consistent with the importance of cilia in biology,
mutations in genes that encode cilia components are common
causes of disease. To date, mutations that cause inherited
retinal degenerations, which are common causes of blindness,
have been identified in genes encoding 38 photoreceptor sensory
cilium (PSC) proteins. These disorders are characterized by
PSC dysfunction, followed by degeneration and death of the
photoreceptor cells, resulting in loss of vision. In addition,
mutations in genes encoding proteins expressed both in photoreceptors
and other cilia result in systemic diseases, such as Usher
syndrome, Bardet-Biedl syndrome, and Joubert syndrome that
involve retinal degeneration along with other disorders consequent
to cilia dysfunction such as deafness and polycystic kidney
disease.
Despite the progress in identifying the genetic
causes of inherited retinal degenerations and other cilia
disorders, it is not known how the identified mutations lead
to PSC dysfunction photoreceptor cell death. Further, the
genes that harbor mutations which cause disease in 40-50%
of patients with inherited retinal degeneration remain to
be identified. The goals of my research program are to improve
our understanding of the molecular bases of inherited retinal
degenerations and related cilia disorders so that rational
therapies can be developed for these diseases.
Research Program
1. The Quantitative Biology of Photoreceptor Sensory Cilia
We are investigating how PSCs are built and maintained, and
how these processes are disrupted in disease. Our interest
in PSCs developed out of our work on the Retinitis Pigmentosa
1 (RP1) protein (see below). We are using several approaches
to study PSCs and related cilia. These include:
A. Proteomic analyses to identify and quantify the components
of PSCs.
B. Screening of novel PSC and primary cilia proteins for roles
in formation, maintenance and function of cilia.
C. Investigation of the quantitative biology of novel cilia
proteins, including characterization of protein turnover and
movement.
D. Screening of novel candidate cilia disease genes for mutations
that cause inherited retinal degenerations and other cilia
disorders.
Proteomics of Photoreceptor Sensory Cilia
As a first step toward studying photoreceptor sensory cilia,
we performed a detailed proteomic analysis of mouse PSC complexes.
This is the first proteome of a mammalian sensory or primary
cilium to be reported. The PSC complex proteome identified
by = 3 peptides contains 1968 proteins, including ~1500 proteins
not detected in cilia from lower organisms (Figure 2). This
includes 105 hypothetical proteins, and 60 proteins encoded
by genes that map within the critical intervals for 23 inherited
cilia-related disorders, increasing their priority as candidate
genes. The PSC complex proteome also contains many cilia proteins
not previously identified in photoreceptors, including 13
proteins produced by genes which harbour mutations that cause
cilia disease, and 7 intraflagellar transport (IFT) proteins.
Analyses of PSC complexes from rootletin knockout mice, which
lack ciliary rootlets, confirm that 1185 of the identified
PSC complex proteins are derived from the outer segment. We
used the mass spectrometry data, benchmarked by 15 well-characterized
outer segment proteins, to estimate the copy number of each
protein in a mouse rod outer segment. These results reveal
mammalian cilia to be several times more complex than the
cilia of unicellular organisms, and open novel avenues for
studies of how cilia are built and maintained, and how these
processes are disrupted in human disease.
Figure 2. Summary of proteomic
analyses of mouse photoreceptor sensory cilia (PSC) complexes.
A. LC-MS/MS analyses were performed on three protein preparations:
wild-type PSC complexes (1) and PSC complex-cytoskeletons
(2), and rootletin knockout (KO) PSC complexes (3). B. The
PSC complex proteome identified by 3 or more peptides contains
a total of 1968 proteins. C, D. Data from the rootletin KO
PSC preparation was used to differentiate proteins associated
with the inner (IS) and outer segment (OS) portions of the
PSC complex. For details, see Liu et al. Molecular Cellular
Proteomics, 2007.
2. The Pathogenesis of Retinal Degenerations
My lab is currently investigating the pathogenesis of several
types of retinal degeneration. These include RP caused by
mutations in the RP1 gene, and the spliceosome components
Pre-RNA Processing Factors 3 and 8 (PRPF3, PRPF8). We are
also studying an inherited form of macular degeneration to
gain insight into the pathogenesis of age-related macular
degeneration. The approach we use for each of these disorders
is to develop gene targeted mouse models that recapitulate
the human phenotype, and use the models to study the biochemical
and cell biologic details of the photoreceptor degeneration.
We also use the disease models for pre-clinical testing of
potential therapies.
Retinitis Pigmentosa 1 (RP1)
Mutations in RP1 gene are a common cause of RP, but the function
of the RP1 protein in vision, and how mutations in RP1 lead
to photoreceptor cell death, are not understood. Biochemical
studies in my lab have revealed RP1 to be a photoreceptor-specific
microtubule-associated protein or MAP. All 21 of the pathologic
mutations in RP1 found to date are nonsense or frame shift
mutations that cluster at the beginning of exon 4, and are
predicted to result in the production of truncated RP1 proteins
in the retinas of patients with RP1 disease. In mice with
a mutant Rp1 allele that produces a truncated Rp1 protein
analogous to that predicted to be produced in the retinas
of RP1 patients, outer segment discs of rods and cones are
generated, but fail to stack up into normal outer segments,
indicating that RP1 is required for the correct stacking of
discs into mature outer segments.
We are now working to identify proteins that
interact with RP1 in order to further define how it participates
in disc organization, and study how its mutations lead to
photoreceptor cell death. We are also beginning to test potential
therapies for RP1 disease, including gene augmentation and
stop codon suppression therapies, in point mutation Rp1 knockin
mice.
Inherited Macular Degeneration
Age-related macular degeneration (AMD) is one of the most
common cause of vision loss in developed countries. The most
characteristic clinical finding in the retinas of patients
with AMD is drusen, or extracellular deposits of protein,
lipid and debris that accumulate underneath the retinal pigment
epithelium (RPE). At present, the etiology of drusen in AMD
is not known, and there are only limited treatments are available
to prevent the progression of AMD. In order to gain insight
into the pathogenesis of AMD, we are studying an inherited
form of macular degeneration called Doyne honeycomb retinal
dystrophy (DHRD)/Malattia Leventinese (ML). Both DHRD and
ML are caused by a single mutation, Arg-345 to Trp (R345W),
in the EFEMP1 or Fibulin-3 gene. We have used gene targeting
techniques to introduce this mutation into the Efemp1 gene
of mice. We have found that the Efemp1-R345W knockin mice
develop AMD-like deposits under their retinas, and are now
studying the pathogenesis of these lesions.
3. Oligonucleotide-Directed and High Throughput
Gene Targeting
Our use of gene targeted mice as disease models has lead me
to be interested in developing improved techniques for gene
targeting in mouse embryonic stem (ES) cells. One effort is
directed toward developing the use of DNA oligonucleotides
to introduce point mutations into the genomic DNA of mouse
ES cells. We have made significant progress with this oligonucleotide-directed
gene targeting approach. In addition, as part of the NIH Knockout
Mouse Project (KOMP), I am working in collaboration with Dr.
Klaus Kaestner to develop C57BL/6 ES cell lines that are efficient
for high-throughput gene targeting. The goals of this work
include the use of gene expression profiling experiments to
identify the factors responsible for maintenance of ES cell
pluripotency, and application of this information to develop
conditions which permit feeder-independent growth of C57BL/6
ES cells.
TECHNIQUES
Molecular biology, cell biology, proteomics, gene targeting
to generate mouse models of disease.
Recent
Publications
Liu Q, Lyubarsky A, Skalet J, Pugh E and EA
Pierce. RP1 is required for correct stacking of outer segment
discs. Investigative Ophthalmology & Visual Sciences
44, 4171-4183, 2003.
Liu Q, Zuo J, and EA Pierce. The retinitis pigmentosa
1 protein is a photoreceptor MAP. Journal Neuroscience
24:6427-6432, 2004.
Murphy BR, Moayedpardazi HS, Gewirtz AM, Diamond
SL and EA Pierce. Delivery and mechanistic considerations
for the production of knock-in mice by single-stranded oligonucleotide
gene targeting. Gene Therapy 14:304-315, 2007.
Liu Q, Tan G, Levenkova N, Li T, Pugh EN, Rux
JJ, Speicher DW, and EA Pierce. The proteome of the mouse
photoreceptor sensory cilium complex. Molecular &
Cellular Proteomics 6:1299-1317, 2007.
Fu L, Garland D, Yang Z, Shukla D, Rajendran
A, Pearson E, Stone EM, Zhang K and EA Pierce. The R345W Mutation
in EFEMP1 is Pathogenic, and Causes AMD-Like Deposits in Mice.
Human Molecular Genetics 16:2411-2422, 2007.
Lab
Rotation
Projects
- Screening of novel PSC and primary cilia
proteins for roles in formation, maintenance and function
of cilia.
- Investigation of RP1 protein function and
potential therapies for RP1 disease
- Proteomic analyses of photoreceptor sensory
cilia sub-fractions
- Characterization of AMD-like deposits in
Efemp1 mutant mice
- Lab
personnel:
- Li Fu, M.D., Ph.D. – Research Project
Manager
Donna Garland, Ph.D. – Senior Research Investigator
John Graziotto – Graduate student, Neuroscience
Steve Hatfield, Ph.D. – Postdoctoral Fellow
Qin Liu, M.D., Ph.D. – Research Assistant Professor
Alex Saveliev – Research specialist
Jonathan Weiner – undergraduate
Qi Zhang, Ph.D. – Senior Research Investigator
last updated 1/2008
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