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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
Cha
pter
15
Fun
ctio
n an
d A
nato
my
of t
he M
amm
alia
n R
etin
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!
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