Transcript Tiffany Hough Presentation

Baye, L.M., Patrinostro, X., Swaminathan, S., Bck, J.S., Zhang, Y., Stone, E.M., Sheffield, V.C.,
& Slusarski, D.C. (2011). The N-terminal region of centrosomal protein 290 (CEP290) restores
vision in a zebrafish model of human blindness. Human Molecular Genetics. 20 (8): 1467-1477.
Tiffany Hough
The N-terminal region of
centrosomal protein 290 (CEP290)
restores vision in a zebrafish model of
human blindness.
Outline

Background

Objectives

Experimental Approach & Results

Conclusions

Critique and Future Research
Background
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Cilia-based Disorders





*Lebers congenital
amaurosis
Meckel Gruber
Syndrome
Joubert Syndrome
Senor-Loken
Syndrome
Bardet-Biedl
Syndrome

CEP290: 12q21.33

54 exons, 2479 AA, 25
predicted domains, motifs,
& localization signals
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30% of LCA
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Most common mutation
results in stop codon giving
alternative splicing
Objectives
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Observe developmental and functional roles of
CEP290 in zebrafish
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Test different regions of CEP290 for localization and
ability to restore knockout function
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Test ability to bind NPHP2 (Senor-Loken)
Experimental Design & Results: Outline
1.
Developmental Expression
2.
Gene Targeting & Gross Morphant Phenotypes
3.
BBS Phenotypes
4.
Characteristics of LCA
5.
Human Truncated Constructs Localize Normally
6.
Vision Rescue
7.
Binding with NPHP2
Experimental Approach & Results:
Developmental Expression
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RT-PCR demonstrates maternal
inheritance and presence throughout
all stages of development (A)
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Whole mount in situ hybridization
show:
 Ubiquitous expression at 8somite stage especially in KV
(B,C)
 5dpf: gut, notochord, brain, eye,
ear (F)
 5 dpf retina: ganglion cell layer,
inner nuclear layer, photoreceptor
cell layer (H)
Experimental Approach & Results: Gene Targeting &
Gross Morphant Phenotypes
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Most common mutation in LCA

Used antisense oligonucleotide
(Morpholino) MO to disrupt splicing and
result in intron inclusion.(A)
 Body curvature defects (B) in dose
dependent manner
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RT-PCR with cDNA
 Shows some wild type present:
hypomorphic (C)
Percentages of embryos with body curvature defects based on
MO dose
Experimental group Phenotype (percentage)
n
Straight Bent Curly
Wild-type
100
0
0
129
cep290 MO (5 ng)
60
6
34
205
cep290 MO (8 ng)
40
5
55
282
cep290 MO (10 ng) 13
7
80
208
Experimental Approach & Results: BBS
Phenotypes
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10 ng MO dose
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All BBS genes have these
characteristics
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Same size or smaller KV
than notochord (C,D)
occurred in dose dependent
manner (D)
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Treatment of dark adapted
5dpf embyos with
epinephrine
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Delayed melanosome
transport (H)
Experimental Approach & Results:
Characteristics of LCA
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8ng MO dose
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Histological analysis using
hematoxylin & eosin staining
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Regardless of curly/straight
phenotype at 3dpf:
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Fully laminated retina (C,D)
Photorecerptor outer segments
in central retina normal (G,H)
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At 5dpf outer segments
disorganized (wavy, K,L)
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Decreased visual acuity
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Wt = 3.53 responses
crx positive control = 2.00
responses
Morphant = 2.46 responses
Experimental Approach & Results: Human
truncated constructs localize normally
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N-terminal fragment (A blue,
1059AA) – spans CEP290
mutation being studied
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C-terminal fragment (A green,
1765-2479AA) – spans mutations
in rd16 mouse and rdAC cat
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Injected mRNA encoding myctagged fusion constructs &
immunohistochemistry at 50%
epiboly(B,E,H)
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Centrin fused with gfp for
centrioles (C,F,I)
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Paracentriolar & cytoplasmic
localization (D,G)
Experimental Approach & Results: Vision
Rescue
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No change in KV defects
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C-terminal treatment
significantly reduced
melanosome transport
delays
Experimental group
KV (%)
MT (min) ± SE
Vision (responses) ±
SE
Wild-type
5.2
1.51 ± 0.07
3.53 ± 0.17
control MO (10 ng)
8.3
1.52 ± 0.06
3.48 ± 0.18
cep290 MO (8 ng)
18.2++
2.10 ± 0.10**
2.46 ± 0.12**
cep290 MO (8 ng) +
N terminus (1.8 ng)
22.6++
2.05 ± 0.10**
3.48 ± 0.13
cep290 MO (8 ng) +
C terminus (0.6 ng)
15.4++
1.82 ± 0.07
2.44 ± 0.22**
Experimental Approach & Results:
Binding with NPHP2
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NPHP5 shown to interact
with N-terminal region of
CEP290
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Mutations in NPHP2 and
NPHP5 have been implicated
in vision impairment
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Transient transfection and coimmunoprecipitation assays
followed by SDS-PAGE and
Western blot
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N-terminal region interacts
with NPHP2
Conclusions
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CEP290 is present throughout development and is
expressed in multiple tissues
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The mutation model resulted in abnormal body
curvature, decreased KV size, delayed melanosome
transport, and vision impairment
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Both constructs localize normally
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The N-terminal region is sufficient to rescue vision
and can bind with NPHP2, suggesting domain and
tissue specificity for this region of the protein
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Supported hypothesis of the mutation being
hypomorphic: presence of wild type in models and
dose dependence of effects
Future Research & Critique


Identify NPHP complexes and
proteins with which they interact
during proper vision: Molecular
pathway examined in a tissue
specific manner
Identify other proteins that
interact specifically with CEP290
to help explain rescue by Nterminus
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Identify the specific mechanism
of N-terminal region of CEP290
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Test other models, such as a
mouse, with this mutation for
rescue.
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Negates other research in other
models (rd16 mouse and rdAc cat)
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Didn’t explain how this mutation
models BBS or why this was
important. What mutations result
in BBS?

Otherwise a very strong paper
which demonstrated known
phenotypes for this mutation and
introduces a promising target for
retroviral gene therapy.