Photoreceptor function and replacement

Marius Ader

The Eye and Retina

The retina contains seven intrinsic cell types

RPE

rod

cone

Müller glia

horizontal cell

bipolar cells

amacrine cell

RGC

ONL

INL

RGL

Karl et al.,

Cell-typ specification during retinal development

The sequence of cell generation is remarkably conserved among vertebrates

All intrinsic cell-types of the retina are generated from a common multipotent retinal progenior cell

Marquardt & Gruss, 2001

Signal transduction

Photoreceptors: main light sensing cells of the retina

Photoreceptors: rods and cones

very sensitive> dim light

no colors

less sensitive> day light vision> focused vision

colors

rods cones120 million 6 million

1um

40 um

outer segment

inner segment

connecting cillium

nucleus

axonal terminal synapse

outer process

appr. 1000 discs

9

Photoreceptor Function

light induced hyperpolarization is passively transported over the plasma membrane to axonal terminal

Light induces a hyperpolarization of the photoreceptor

mem

bran

e po

tent

ial

light impulse

time (s)

dark: cation-specific channels are open-> sodium ions (Na+) flush into outer segment -> electrochemical gradient for Na+ is build by Na/K-ATPase pump (localised in inner segment) and Na/Ca, K exchanger (NCKX, localised in outer segment)

cyclic nucleotide gated channels (CNG)

dark current: -40mV

cGMP molecules in cytosol

light: cation-specific channels (CNG) close -> sodium ions (Na+) can’t flush into outer segment -> hyperpolarization (i.e. inside more “negative” than outside)

a single absorbed photon closes hundreds of cation-specific channels (in dark-adapted rods) therefore stopping appr. 1 million Na+ ions leading to a hyperpolarization of 1mV that induces changed synaptic transmission

cGMP molecules in cytosol

light decreasescGMP level

the decreasedcGMP level

leads to closing of

Na+ channels

light

resting potential :-40mV hyperpolarization

14

A rod can be activated by a single photon!How is this possible?

Light detection takes place in the outer segments of photoreceptors

16

modified from Kennedy et al., 2009

retina

ONL

INL

GCL

rhoGFP

OS

Photopigments are located in disc membranes

17

Rhodopsin: structure

Light detector - chromophore:

11-cis-retinal

11-cis-retinal: generated from all-trans-retinol (vitamin A) that can NOT be syntethysed de novo. Decreased levels of vitamin A leads to night blindness and vision loss

all-trans-retinal

light

Light detection

11-cis-retinal is bound to opsin by “Schiff base”:aldehyde group is bound to epsilon amino group of a specific lysine

rhodopsin: 40-kD membrane protein with seven transmembrane helices1. protein: opsin2. chromophore: 11-cis-retinal

Chromophore + Opsin = Photopigment

n-terminus of rhodopsin: inner disc spacec-terminus of rhodopsin: cytoplasm -> binding side for transducin (initiation of second messenger cascade)

cytosol

cytosol

intradisk space

binding side for transducin,

rhodopsin-kinase and arrestin

The origin of vision: isomerization of 11-cis-retinal to all-trans-retinal

distance of Schiff-base to chromophore ring increases by 0.5nm- photon has been changed into atomic movement!

Isomerization of retinal leads to conformational changes of rhodopsin (intermediates)

-> induces enzymatic cascade

Phototransduction cascade

rhodopsin (R) -> activated rhodopsin (R*) -> G-protein - transducin-GTP -> activated phosphodiesterase (PDE*) -> hydrolysis of cGMP to 5’-GMP -> closing of cation-specific channels

cGMP molecules in cytosol

light decreasescGMP level

the decreasedcGMP level

leads to closing of

Na+ channels

light

transducin

inhibitory subunits

CNG, cyclic nucleotide gated channels

1 R* activates appr. 500 transducin molecules

PDE* has a very high catalytic power: PDE* hydrolyses cGMP at a rate close to the limit set by aqueous diffusion!(single PDE* hydrolyses 1000 cGMP molecules)

Amplification of light impulse:

A rod can be activated by a single photon!Amplification by second messenger cascade!

1 Tα* activates 1 PDEγ subunit

26

Deactivation

27

1. rhodopsin-kinase catalyses phosphorylation of R* 2. binding of arrestin, an inhibitory molecule that stops binding of R* to transducin

R* termination is a two step process

29

Cones allow focused and color daylight vision

31

Color vision with cones: different spectral sensitivity

S-cones (short wavelength receptor) appr. 12% - blue

M-cones (medium wavelength receptor) variabel - green

L-cones (long wavelength receptor) variabel - red

How do cones develop different light absorption peaks?

-> same chromophore: 11-cis-retinal!

Groups of the opsin change -> the absorption maximum of the bound 11-cis-retinal changes

cytosol

extracellular side

33

Phototransduction Initiation:In all visual pigment opsins 11-cis retinal is joined to a lysine in the 7th transmembrane region of the opsin by a Schiff base linkage. Light forms an all-trans retinal which actuates a cascade of events leading to cell polarisation change. The opsin is regenerated via the visual cycle. If this Schiff base is proteated the pigment has an absortion maxima > 440nm. Such positively charged Schiff bases are modulated by a counterion which is a glutamate residue from the 3rd transmembrane region of the opsin. A weak interaction delocalises the positive charge through the π system of the retinal causing more red shift Further modification of the electronic dipolar environment is possible by neutral side chains of the opsin. In man the spectral shift between the red and green cone opsins is 95% accounted for by the aminoacid differences at AA180 (alanine/serine) in the 4th transmembrane segment and at AA277(tyrosine/phenylalanine) and AA285 (alanine/threonine) in the 6th transmembrane segment.

red green blindness

mostly males affected

red and green genes are located in close proximity on X-chromosome

Isihara test

Focused vision: Cones are concentrated in the macula

36

Macula formation

365

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15

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a

A group of cells that are all the same type have certain special properties. The retina is a two-dimensional array and most cell types tile the retina in a consistent manner so that an even coverage is obtained. Cells of the same type are usually not adjacent. The position of the cells relative to others of the same class is also an indicator of cell type because unique popula-tions form nonrandom mosaics.41 Consider a handful of marbles dropped on the floor. Some will stop close by but others may roll into a corner. If a measure is taken between the nearest neighbors, it will be found to have a high variation. In contrast, for a nonrandom mosaic of neurons, the spacing is controlled such that the distance to the nearest cell of the same type is relatively constant and the variance of this measure is low. The ratio of mean nearest neighbor distance to the variance gives an index of regularity for a cell population. We would expect the ratio to be low for the randomly scattered marbles, but indices of three or higher are common for the typical non-random, orderly mosaics of retinal neurons. The most satisfying results for classification come from the convergence of these methods to indicate the function of a retinal cell in the process of vision.

PhotoreceptorsConesThe mammalian retina contains two types of photoreceptor, rods and cones.45 Rods account for 95% of all photoreceptors. They are numerous, with slender outer segments, densely packed and specialized for high sensitivity under dark or starlight condi-tions. Cones are larger, with tapering outer segments, and they are found in the top row of the ONL (see Fig. 15.3). Cones make up only ~5% of photoreceptors28,45 but they provide high-acuity color vision in daylight conditions when photons are abundant. This versatile combination of rods and cones and their associated circuits covers an intensity range of around 10 log units from the darkest night to bright sunlight.46 While the average visual scene has a range of intensities covering 2–3 log units, the continual adaptation of retinal sensitivity slides this operating range through the entire range of light intensities. This is a critical function of the retina because outside the normal operating range we are functionally blind. Common examples include the inability to see momentarily when entering a dark cinema or driving into the setting sun. Much of the adaptation takes place in photoreceptors but, as we shall see below, this is accompanied by major changes in the neural pathways through the retina. This is arguably the most important function of the retina, after light detection itself.

There are approximately 5 million cones in the human retina and 190 000 in the mouse retina.47,48 Cones make up approxi-mately 5% of the total photoreceptors in humans, compared to 2.8% in the mouse,49 so we are all rod-dominated in terms of absolute numbers. One exception is the ground squirrel, which is truly cone-dominated. Importantly, cones are not evenly dis-tributed. In human retina, there is a massive peak at the fovea (Fig. 15.3) where the density reaches around 200 000/mm2, approximately 100 times the density in the periphery.28,47 This is the region of maximum acuity, although the peak density slightly exceeds experimentally measured visual acuity due to blurring by the optics of the eye.50–52 Where the ganglion cell axons gather to form the optic nerve there are no photoreceptors and their absence from this location is the cause of the blind spot (Fig. 15.3).

Fig. 15.6 The mosaic of red, green, and blue cones in the human retina. This image, taken from a human subject using adaptive optics, shows the distribution of the three cone classes. Blue cones make up a small fraction, <10%, but make a regular mosaic. Red and green cones have a clumpy, random distribution. In this subject, the red cones outnumber the green cones but this ratio is highly variable, even in subjects who have normal color vision. (Courtesy of Austin Roorda; after Roorda A, Williams DR. The arrangement of the three cone classes in the living human eye. Nature 1999;397:520–2.)

Cones support color vision and, in old-world primates and humans, there are three classes: blue, green and red. They are maximally sensitive to 430, 530, and 561 nm light, respectively.53–55 Other mammals, including cats, rabbits, and rodents, have an evolutionary ancient form of color vision based on green and blue cones only. The presence of red and green cone opsins in a tandem array on the X chromosome is thought to be due to a recent gene duplication and underlies the preponderance of color blindness among males.56 Using adaptive optics to correct for blurring in the lens and cornea, the distribution of red, green, and blue cones can be mapped in the living human eye.57 Sur-prisingly, the distribution of cones was random and clumpy (Fig. 15.6). In addition, there appears to be enormous variation in the red/green cone ratio among individuals with normal color vision.

Blue cones are present as a minority: they make up approxi-mately 10% of the cone population (Fig. 15.6). This is not enough to support high acuity but calculations show that the blue cone density is sufficient to support the reduced resolution caused by visual aberration at the blue end of the spectrum.30 At the very center of the fovea, blue cones are absent.29,58 The fact that this is not readily apparent is due to the relatively poor spatial acuity of color vision compared to the luminance-driven pathways.

Red and green cones cannot be differentiated morphologically and the red and green cone opsins are so closely related that they are also indistinguishable. However, blue cones can be mapped

Roorda et al., 1999

Pseudocolour image of the trichromatic cone mosaic

periphery central

37

Retinal pigment epithelium (RPE)

The tips of outer segments are shedded daily – complete renewal in 9 days

The RPE is essential for photoreceptor function

Outer segments are removed by the RPE and recycled

> visual cycle(all-trans retinal > 11-cis retinal)

39

40

Transmitter release at synaptic terminal

resting potential :-40mV hyperpolarization

Hyperpolarization of photoreceptors results in decreased release of glutamate

> depolarization (ionotropic receptor) or hyperpolarization (metabotropic receptor)

Photoreceptors release less neurotransmitter when stimulated by light!

Consequences of Hyperpolarization:

> voltage-gated calcium channels close

> decreased influx of calcium ions

> intracellular calcium ion concentration falls

> reduced release of glutamate

> either excite or inhibit bipolar cells

The rod spherule The cone pedicle

Signal transduction

Common neurotransmitters in the retina are glutamate, GABA, glycine, dopamine, and acetylcholine

photoreceptors release glutamate

Main excitatory neurotransmitter in the retina is glutamate

46

How can the same signal (reduction in glutamate release) lead to a hyperpolarization or depolarization of bipolar cells?

Different receptors! - inhibitory (metabotropic glutamate receptor - mGluR6)- excitatory (AMPA, NMDA, kainate receptors)

light: decrease in glutamate release!

Synaptic connection of photoreceptors to bipolar cells

48

Summary

Photoreceptors are the main light sensing cells of the retina

Isomerization of 11-cis-retinal is the first step in light detection

The light signal is strongly amplified by a G-protein coupled second messenger cascade

The light sensing photopigments are located in the outer segment

Light results in hyperpolarization of the photoreceptor by closing cyclic nucleotide gated ion channels

Hyperpolarization leads to a reduced glutamate release at synaptic terminal

> check out: http://webvision.med.utah.edu/

The signal is transfered via bipolar cells and retinal ganglion cells to the brain

50

Therapeutic approaches for the treatment of retinal degeneration

RetinalDegeneration:

adapted from: http://webvision.med.utah.edu/book/

The$eye

Re'nal$degenera'on$due$to$photoreceptor$loss:

Most$common$reason$for$disability$in$industrialized$countries!

Re#ni#s'pigmentosa'(RP):'inherited$diseaseloss'of'rods3'million'worldwide'30,000':'40,000'in'Germany216'in'Dresden

(Age:related)'Macula'degenera#on'(AMD):'mul'factorial$diseaseloss'of'cones>34'million'worldwide2'million'in'Germany12,400'in'Dresden

No$intrinsic$regenera'on$of$photoreceptors$in$mammalian$re'na!

Gene$Therapy

Re'nal$Implants

‘Intrinsic’$Regenera'on

Cell$Transplanta'on

Approaches*to*Cure*Blindness

disease

mut. DNAmut. DNA

mut. mRNAmut. mRNA

No\not functional protein

mut. DNAmut. DNA

healthy

mut. mRNAmut. mRNA

corr. RNA

corr. DNA

GeneBTherapy$for$recessive$IRD DNA RNA ProteinTranscription Translation

X No\not functional protein funktional protein+X

Adeno$Associated$Virus$(AAV)

✓ Naturally replication-deficient and nonpathogenic.

✓ Ability to transduce both mitotic and post-mitotic cells.

✓ Efficient long-term gene transfer in a number of cells types, including eye, CNS and muscle.

✓ Low immunogenicity – no inflamation when administered to brain.

How$to$get$the$gene$into$re'nal$cells?

AAV$as$a$gene$shuLle

..... .

..... ...... .

..... . ..... .

corr. DNA AAV

+

..... .

..... ...... .

..... . ..... .

AAV + corr. DNA

Miika

The Lancet, Volume 374, Issue 9701, Pages 1597 - 1605,

:'Injec#on'of'AAV'in'worse'eye

:'No'side'effects

:'12'pa#ents''in'the'range'of'8':'44'years

:'Improvement'in'all'pa#ents

:'8'year'old'showed'almost'normal'visionCorey'Haas

http://www.youtube.com/watch?v=FyR99anGBqEhttp://> 10 clinical trials currently under way

- several patients now received injection in second eye

Gene therapy clinical trials for retinal diseases

Summary$Gene$Therapy$

Gene:therapy'for'recessive'forms'of'IRD'are'in'clinical'trials'(for'RPE)'

Gene:therapy'for'dominant'forms'of'IRD'are'in'pre:clinical'models

Ques'ons:

Long:term'effects

Gene'defect'(muta#on)'has'to'be'iden#fied'

Cells's#ll'have'to'exist':'no'cells,'no'gene'therapy!

61

Replacement$of$photoreceptors$Implants

Regenera'onTransplanta'on

Make$other$cells$light$sensi've Optogene'cs

Other$Op'ons$for$Regaining$Light$Sensi'vity

62

Optogene'cs

Microbial'channelrhodopsin

directly'light'gated'ca#on'channel

Chlamydomonas reinhardtii

channelrhodopsinbacteriorhodopsin

halorhodopsin

63

64Activation of 25% residual cones sufficient for visual response

Volker Busskamp will visit Dresden on the 12.July!!!!

Photoreceptor replacement?

66

Re'nal$ImplantsTransplanta#on'of'ar#ficial'light'sensing'devices'that'provide'electrical's#muli'to'remaining're#nal'neurons

Photoreceptor$Replacement$

Cell$Transplanta'onTransplanta#on'of'cells'that'generate'func#onal'photoreceptors'and'replace'lost'cells

Regenera'onAc#va#on'of'intrinsic'progenitors'to'differen#ate'into'photoreceptors

Artificial vision - retinal implants

68

RPE

rodcone

Müller glia

horizontal cellbipolar cells

amacrine cell

RGC

ONL

INL

RGLKarl & Reh, 2010

‘Intrinsic’$Regenera'on

Karl & Reh, 2010

Retinal Regeneration

Thummel et al., 2008 Karl et al., 2008

Transplanted Retinal Cells Differentiate into Photoreceptors

OS

IS

OP

N

IP

ST

retina

ONL

INL

GCL

OSactin-EGFP

EGFP/Bassoon/dapi

ONL

OPL

EGFP/PKCα

ONL

INL

OPL

Bartsch et al., 2008

Eberle et al., 2012

Correlative Light and Electron Microscopy (CLEM)

Outer Segment Formation in a Heavily Degenerated Mouse Model (P347S)

Eberle et al., 2012

unsorted A CD73- B

CD73+ C D

ONL

OS

IS

OPL

ONL

OS

IS

OPL

ONL

OS

IS

OPL

AT

OS

IS

CB

E

INL

INL

INL

n = 6

n = 7

n = 7

Enrichment of photoreceptors by CD73-based MACS

Eberle & Schubert et al., 2011

MACS: magnetic associated cell sorting

unsorted CD73- CD73+

rhoGFP

Func'onality?

Nature, 2012

Transplanta#on'of'young'photorecptors'into'a'mouse'model'of'slow're#nal'degenera#on'(Gnat1:/:)

Pearson et al., 2012

Primary'photoreceptor'precursors'are'sub1op2mal'for'therapies

How'to'get'efficient'amounts'of'donor'cells?

Stem/Progenitor'Cells!

'Highly'expandable

'Mul2potent

supplyProblems'may'arise:

logis2c

ethics

Photoreceptor$genera'on$from$pluripotent$stem$cell$lines?

embryonic stem cells induced pluripotent stem cells

79

mouse ESCs

embryoid bodies differentiation

80

Eiraku et al., 2011

Sandra Grahl

phase Rx-GFP

ESC-derived photoreceptors

Sandra Grahl

Eiraku et al., 2011

82

Gene$Therapy

Re'nal$Implants

‘Intrinsic’$Regenera'on

Cell$Transplanta'on

Approaches*to*Cure*Blindness Problems

cells's#ll'have'to'existmuta#on'must'be'knownmonogenic'

proper'func#onalitysignal'processingresolu#on

photoreceptorconnec#onfunc#onality

integra#on'numbersconesconnec#oncell'source

Thank You For Your Attention!