Tiffany Hough

The N-terminal region of centrosomal protein 290

(CEP290) restores vision in a zebrafish model of human

blindness.

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.

OutlineOutline Background

Objectives

Experimental Approach & Results

Conclusions

Critique and Future Research

BackgroundBackground 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

30% of LCA

Most common mutation results in stop codon giving alternative splicing

ObjectivesObjectives Observe developmental and functional roles

of CEP290 in zebrafish

Test different regions of CEP290 for localization and ability to restore knockout function

Test ability to bind NPHP2 (Senor-Loken)

Experimental Design & Results: Experimental Design & Results:

OutlineOutline

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: Experimental Approach & Results:

Developmental ExpressionDevelopmental Expression RT-PCR demonstrates maternal

inheritance and presence throughout all stages of development (A)

Whole mount in situ hybridization show: Ubiquitous expression at

8-somite 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 Experimental Approach & Results: Gene

Targeting & Gross Morphant PhenotypesTargeting & Gross Morphant Phenotypes

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

RT-PCR with cDNA Shows some wild type

present: hypomorphic (C)

Percentages of embryos with body curvature defects based on MO doseExperimental group Phenotype (percentage) n

Straight Bent Curly Wild-type 100 0 0 129cep290 MO (5 ng) 60 6 34 205cep290 MO (8 ng) 40 5 55 282cep290 MO (10 ng) 13 7 80 208

Experimental Approach & Results: BBS Experimental Approach & Results: BBS

PhenotypesPhenotypes

10 ng MO dose

All BBS genes have these characteristics

Same size or smaller KV than notochord (C,D) occurred in dose dependent manner (D)

Treatment of dark adapted 5dpf embyos with epinephrine Delayed

melanosome transport (H)

Experimental Approach & Results: Experimental Approach & Results:

Characteristics of LCACharacteristics of LCA 8ng MO dose

Histological analysis using hematoxylin & eosin staining

Regardless of curly/straight phenotype at 3dpf: Fully laminated retina

(C,D) Photorecerptor outer

segments in central retina normal (G,H)

At 5dpf outer segments disorganized (wavy, K,L)

Decreased visual acuity Wt = 3.53 responses crx positive control = 2.00

responses Morphant = 2.46 responses

Experimental Approach & Results: Experimental Approach & Results:

Human truncated constructs Human truncated constructs

localize normallylocalize normally

N-terminal fragment (A blue, 1059AA) – spans CEP290 mutation being studied

C-terminal fragment (A green, 1765-2479AA) – spans mutations in rd16 mouse and rdAC cat

Injected mRNA encoding myc-tagged fusion constructs & immunohistochemistry at 50% epiboly(B,E,H)

Centrin fused with gfp for centrioles (C,F,I)

Paracentriolar & cytoplasmic localization (D,G)

Experimental Approach & Results: Experimental Approach & Results:

Vision RescueVision Rescue No change in KV

defects

C-terminal treatment significantly reduced melanosome transport delays

Experimental group KV (%) MT (min) ± SEVision (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: Experimental Approach & Results:

Binding with NPHP2Binding with NPHP2

NPHP5 shown to interact with N-terminal region of CEP290

Mutations in NPHP2 and NPHP5 have been implicated in vision impairment

Transient transfection and co-immunoprecipitation assays followed by SDS-PAGE and Western blot

N-terminal region interacts with NPHP2

ConclusionsConclusions

CEP290 is present throughout development and is expressed in multiple tissues

The mutation model resulted in abnormal body curvature, decreased KV size, delayed melanosome transport, and vision impairment

Both constructs localize normally

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

Supported hypothesis of the mutation being hypomorphic: presence of wild type in models and dose dependence of effects

Future Research & CritiqueFuture 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 N-terminus

Identify the specific mechanism of N-terminal region of CEP290

Test other models, such as a mouse, with this mutation for rescue.

Negates other research in other models (rd16 mouse and rdAc cat)

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.