Lecture #17

The visual cycle + eye disease4/2/13

Spatial resolution – last timeB/W did have highest resolution

Patrick – what do photoreceptors see?

• Visualization

tan θ and θ

0.1

10

EEEEEEEEEE

• Calculate the image size on your retina for one line of the small E in the front of the room

• Write the image size of one line on the board according to where you are sitting

• How many receptors does one line of the small E cover on your retina?

Next few lectures

• Visual cycle - todayMuch of this pathway has been identified through disease studiesVisual cycle in rods Visual cycle in cones

• Eye diseases – next 3 lecturesRetinal diseaseOther diseases (glaucoma, cataracts etc)Treatments

Visual cycle: recycling 11-cis retinal through isomerization and pigment regeneration

George Wald showed 11-cis retinal isomerizes to start vision

G protein pathway in rod disc

R + hv g R* Rhodopsin absorbs photon g excitedR* + Gαβγ g R* + Gα*-GTP + Gβγ Rhodopsin activates G protein

Rhodopsin G protein

R* shutoff: phosphorylation and arrestin binding

Inactive R has all trans retinal bound to it – need to regenerate 11-cis retinal and reform visual pigment

Photoreceptors at the very back of the retina next to retinal pigment epithelium (RPE)

http://webvision.med.utah.edu/

Pigment epithelium

Some facts about pigment regeneration after pigment absorbs light (bleached)

Rods Cones

Retina plus RPE Regenerate Regenerate

Remove RPE None Still regenerate

Remove cones from retina

None

Phototransduction: 11-cis + photon = all transVisual cycle: all trans + enzymes = 11-cis

Visual cycle Phototransduction

Retinal pigment epithelium (RPE) - plays a big role in 11-cis retinal regeneration in rods

Retinal dissociates from opsin, diffuses out of photoreceptor, goes to RPE, gets regenerated and comes back

Both 11-cis and all-trans retinal are isomers of the same aldehyde : retinaldehyde

R-CHO R H

C

O

11

11

Retinoid biochemistry

• Aldehyde RCHO -al

• Alcohol ROH -ol

• Ester R-CO-O-R’

R HC

O

R OH

R OC

O

R’

Opsin + retinal = Schiff base

Schiff base is a carbon nitrogen double bond, C=NR3

In the case of rhodopsin, the N comes from the lysine and C comes from the retinal

+ X-(CH2)4NH2Lysine

Schiff base

Schiff base can often be protonated NH+

Visual pigment =opsin + retinal

11-cis retinal

membrane

In rod, visual pigment is called rhodopsin

11-cis in retinal binding pocket

Phototransduction occurs in retina: rods and cones

Phototransduction occurs in retinaVisual cycle occurs in retina and RPE

Retinoids(Bound to opsin)

Moving retinoids around takes chaperones

They protect retinoids from oxidation and isomerizationThey help transfer fat soluble retinoids across aqueous layers

Moving retinoids around takes chaperones

IRBP - interphotoreceptor retinoid binding proteinCRBP - cellular retinol binding proteinCRALBP - cellular retinaldehyde binding protein

Slightly different version

First steps to retinal regeneration

ABCR = ATP binding cassette transporter

ABCR binds to all trans retinal (RAL) and helps it diffuse out of discs

This leaves free opsin which is ready to make new visual pigment

Disc

Apo-protein = protein without prosthetic group which is needed for its function

Rod

First steps to retinal regeneration

Disc

All-trans retinol dehydrogenase (RDH) converts all trans retinal to all trans retinol

Rod

Retinal(dehyde) retinol

RDH12

C=O

H

C-OH

HHNote: all-trans-ROL=Vitamin A!!

Moving retinoids around takes chaperones

IRBP - interphotoreceptor retinoid binding protein

Transport of retinol to RPE

RPEInterphotoreceptor retinol binding protein (IRBP) helps transport all-trans retinol to the RPE

Moving retinoids around takes chaperones

In RPE, CRBP binds retinol 100x greaterCRBP = cellular retinol binding protein

Conversion of all trans retinolLRAT = lecithin retinol acyl transferase

Converts all trans retinol to all trans retinal ester

Works best on retinol bound to CRBP

Convert to ester

LRAT = adds acyl group

O

IsomerizationIsomerase RPE65 converts all trans retinyl ester to 11 cis retinol

RPE65 is 65 kilodalton protein which occurs in RPE

RGR - retinal G protein coupled receptor is enzyme that may help in isomerization

Forms 11-cis retinalRetinal dehydrogenase converts 11-cis retinol to 11-cis retinal

CRALBP is cellular retinal binding protein

Can bind ROL or RAL to help move either

Can store 11-cis ROL as ester till needed.

Conversion

RDH5

Conversion

RDH5 RDH12

In RPE In rod

Summary

IRBP takes 11cis-RAL back to photoreceptor

Reforms the active visual pigment

RPE65

Visual cycle in rods

• Needs RPEAll trans retinol leaves retina and goes to RPE to be regeneratedWithout RPE, no isomerization and no visual pigment regenerationTakes time for retinal to be transported around so slow process

Retinal regeneration in cones

• Cones do NOT require RPEIsolated retina can regenerate pigment

• Cones can use 11 cis retinol to regenerate pigmentIf add 11 cis retinol, cones can convert this to 11 cis retinal and regenerate pigment

• In bright sun, need more 11-cis than can regenerate with known rod visual cycle

Cone visual cycle is different - uses Muller cells

Cone visual cycle

Cone visual cycleDon’t yet know the isomerase

Use ARAT to form retinal ester

BIG DIFFERENCE - 11cis ROL goes back to cone outer segment where it is converted to 11-cis RAL

Why have two visual cycles in same retina?

11-cis RAL to rods 11-cis ROL to cones

Theories

• Deficiency / survival - independence of rod and cone pathways

• Sensitivity - so rods can detect lower levels of light

• Energy needs of cones are greater• Cones / rods process different information• Cones function at hi light, rods at low

Cone pathway

• Cones work at higher light levelsOpsin bleached more rapidlyCone visual cycle is 20 times faster

• Muller cells send 11-cis retinol to conesRods can’t convert this so can’t “steal” from cone pathwayImportant for rod dominated retina

20-100x more rods than cones

Macula - pigmented layers coincident with fovea

Macula is key to high quality vision

Where all the cones are!

Gene mutations cause disease

ABCA4 mutations = Stargardt macular degeneration

Stargardt macular degeneration

RecessiveShows up in people 7-12 yrs old

Most common juvenile macular degeneration

Loss of central vision Eventual blindness

Affects 1 : 10,000 children

Gene mutations cause disease

RDH12 mutants = Leber congenital amaurosis

Leber congenital amaurosis

• Discovered by Leber in 1800sCongenital - present from birthAmaurosis - vision loss without abnormal tissuePhotoreceptors don’t develop

• Affects 3 : 100,000 newbornsCaused by several genesRDH12

- 13 known mutations

Gene mutations cause disease

LRAT mutations = retinitis pigmentosa or Leber congenital amaurosis

Many causes of Retinitis pigmentosa

Pigmentation and loss of photoreceptors through time

Usually starts with night blindness

2 LRAT mutations S175R 2 bp del 396AA

Gene mutations cause disease

RPE65 mutations = Leber congenital amaurosis

Gene mutations cause disease

RGR mutations = recessive retinitis pigmentosa

Gene mutations cause disease

CRALBP mutations = recessive retinitis pigmentosa

Mutations in visual cycle genes cause 3 types of retinal disease

• Stargardt macular degenerationABCA4

• Leber congenital amaurosisRDH12, RPE65, LRAT

• Retinitis pigmentosaLRAT, RGR, CRALBP

Online mendelian inheritance in man (OMIM)

• NCBI database of genetic basis of disease http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim

• Describes gene causing diseaseHow discoveredGene function

• Keep track of mutations identified to cause disease

Mouse knockouts for all key genes

• Stargardt macular degenerationabca4-/-

• Leber congentical amaurosisRdh12-/- rpe65-/-

• Retinitis pigmentosaLrat-/- rgr-/- cralbp-/-

Retinal disease

• Have isolated gene mutants in rod pathway• Developed animals models

Have gene mutation built inUseful to follow progression of diseaseUseful to test different treatments

• Will discuss treatment options in a few lectures