416 Lecture 12 by 8lhHc75x


									ANAT 416                                                                            February 16, 2012
Dr. Cayouette                                                                       Lecture 12

ANAT 416
Lecture 12 – Eye Disease and Regeneration
Dr. Michel Cayouette – February 16, 2012

NOTE: This NTC is meant to be used as a study aid to supplement your own class notes. Hence,
not all of the text contained in the lecture slides will be reproduced here.

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Class Outline
Part I: Major Diseases of the Eye
Part II: Regeneration of the Lens
Part III: Regeneration of the Retina
Part IV: Retinal Stem Cells

I. Major Diseases of the Eye
1. Cataracts
       A cataract is characterized by clouding, caused by denaturation of lens proteins that develops in
        the lens of the eye or in its envelope, obstructing the passage of
       Risk factors include long-term exposure to UV light (plays a big
        role), exposure to radiation, secondary effects of diseases such as
        diabetes, hypertension, and advanced age, or trauma. There are
        also congenital (genetic) forms of cataracts.
       Age-related cataract is responsible for 48% of world blindness
       Surgery is the main treatment for cataracts, but in developing
        countries, sometimes don’t always have this chance – people end
        up with cataracts and become blind.
                                                                             Figure 1: Cataracts

2. Glaucoma
       Glaucoma is a heterogenous group of disorders (genetic or
        spontaneous) which constitute the leading cause of
        irreversible blindness worldwile. This is unlike cataracts,
        which is reversible.
       Glaucoma is characterized by a progressive loss of retinal
        ganglion cells (RGCs – transmit nervous impulse to the
        brain, not the neurons that see light), specific visual field
        deficit, and a characteristic atrophy of the optic nerve due to
        pressure that builds up in the posterior chamber of the eye.
       Progressive open angle glaucoma (POAG) is the most
        common type of glaucoma.
       Abnormally elevated intraocular pressure (IOP) is frequently
        associated with glaucoma and is a major risk factor for this             Figure 2: Glaucoma

ANAT 416                                                                            February 16, 2012
Dr. Cayouette                                                                       Lecture 12

Increases intraocular pressure is caused by obstruction of the trabecular meshwork
       There are many risk factors, but most often the cause is that the cells that are located along
        Schlemm’s canal and the trabecular meshwork, which is the draining system for the anterior
        chamber of the eye. This fluid needs to exit with constant change via osmolarity. It is drained
        into the lymph system and from there enters the mesh system.
       When this draining system is obstructed by a growth of cells, the fluid can't escape the eye
        anymore and begins to accumulate – he gives the example of blowing a balloon too much – the
        fluid stays inside and intraocular pressure increases  pressure on RGC axons  degeneration.
       The primary event is a growth of cells – similar to cancer. The cells lose control over their
        normal proliferation rate, which can be due to certain genes, as well as spontaneous mutation.

        Figure 3: Glaucoma

Mouse models of glaucoma: Induced model
       There are two “induced” models used in mice to study glaucoma.
                    Mechanically crush the optic nerve slightly; this causes damage to the RGC
                       axons, similar to what you would see when there is increased intraocular pressure.
                       See degeneration of optic nerve over time.
                    NMDA injections activate NMDA receptors that will open a channel that allows
                       the influx of calcium causing cell death, in this case, the progressive degeneration
                       of RGCs.
           o These two models are not perfect – those that have glaucoma don’t really have a crushed
               nerve or have NMDA injections. This is the issue with studying glaucoma as right now
               there are really no good models; however, people are working hard to develop better

Mouse models of glaucoma: Inherited model
       Recently, mutations which cause glaucoma in humans have been discovered. The DBA/2J mouse
        model was found by chance. It contains homozygous mutations in two separate genes: Tyrp1b
        (melanosomal protein) and Gpnmp (transmembrane glycoprotein). If we can knock out these
        genes, we can simulate what happens in glaucoma.
       This mouse model was found by chance, people noticed ganglion cells missing in the animals
        they were studying. When these genetic studies started to come out, perhaps the mouse was a
        natural mutant for genes that led to glaucoma. They discovered that when they did knock these
        genes out there was retinal degeneration.
       In the experiment, they labeled optic nerves with a red dye. In the mutant, there is a decrease in
        the number of retinal ganglion cells.
       Use of gene therapy – if could correct these mutations, maybe you could prevent the
        degeneration of these stem cells.

ANAT 416                                                                            February 16, 2012
Dr. Cayouette                                                                       Lecture 12

       Mutations:
            o Gain of Function (GOF) – protein acquires function not usually there
            o Loss of Function (LOF) – there is a truncation in the transcription of the gene
       If could re-express that gene in a cell, could correct that defect and prevent the degeneration of
        the RGCs.
       Or, could introduce survival factors, or trophic support. These are proteins that help slow down
        degeneration (ex: BDNF, CNF, NGF, FGF). These proteins are able to prevent the start of a
        degeneration pathway.
       People tried to increase the concentration of these trophic factors in the eye. This is actually
        possible in clinic, in which a doctor can insert a mini pump into someone’s sclera to slowly
        deliver a drug over many months. Actually, this is used in some AIDs patients who have CMV.
        This provides viral protection to the eye.
       People trying to develop this approach – instead of putting antibacterial drug, they can put
        trophic factor to protect RGCs from cell death. This would not cure the disease, it would simply
        slow down the degeneration of these cells.

3. Retinitis Pigmentosa (RP)
       A group of genetic eye diseases that affect the retina, causing gradual, permanent loss of vision
        due to the degeneration of rod photoreceptors first (tunnel vision), often followed by cones
        (no env factors in developing this)
       Prevalence of RP is approx. 1 in 5000 worldwide.
       There are many forms: autosomal dominant, autosomal recessive, and X-linked forms. But all of
        these forms are characterized by the degeneration of the rod photoreceptors first, which leads to
        the loss of peripheral vision (tunnel vision). In humans, there are cones that are in the fovea of
        the eye. That’s where the light is focused on the retina. Use cones in daylight. Rods at night,
        which are mostly located in the peripheral of the fovea?
       A “fundus photo” is a picture taken of the back of eye. You can see a healthy retina in the normal
        patient, versus an infected periphery in the RP patient, in which pigmented cells behind retina are
        seen at back of eye in RP.
       You are okay if you lose 70% of rods – you wouldn’t do as well in dim light, but you could drive
        well. You are perfectly fine in daylight with cones. The issue is when this disease advances, the
        loss of rod photoreceptors causes cone pr’s, as they provide trophic factors to the cones. When
        there is a decrease in the number of rods, there is an indirect decrease in the number of cones
        receptors  loss of daylight vision  blindness.
       No cure for this disease.

Figure 4: Retinitis Pigmentosa

ANAT 416                                                                            February 16, 2012
Dr. Cayouette                                                                       Lecture 12

Mouse models of RP
       You can use natural mutants, or:
       Engineered mouse models (knockouts and transgenics)
           o 97% of the photoreceptors in mice are Rods, so they are a great model for studying RP.
           o Rd1 mouse has a mutatation in phosphodiesterase gene, and is a major contributor to RP.
              If this gene is mutated in mice, after 21 days only, the retina is completely missing all of
              the photoreceptors – there is only an interneuron layer.
           o rd1/rd1 mouse has a mutation in rhodopsin, the main protein that is hit by late and
              changes conformation and intiaties the photoreduction cascade. Mutations in rhodopsin
              are a major contributor to RP. There are up to 50 mutations identities in rhodopsin genes.

4. Age-related macular degeneration (AMD)
       AMD is characterized by chronic, age-related, degenerative disease of the macula aka cone
        photoreceptor degeneration. Prevalent in older individuals. A person with AMD cannot see
        anything in the middle of their visual field, only the periphery. They will often turn their head
        from side to side.
       There are genetic and environmental components. For example, it could be due to exposure to
        bright light.
       It is hard (and pretty much unethical) to design studies to test AMD in human subjects. However,
        it is important to carry out environmental studies.
       There are two major forms of AMD:
             o Dry AMD – more common, 90% of AMD cases. Caused by accumulation of “drusen” –
                 yellowish deposit of cell debris secreted be cell, which accumulates in cone cell death.
             o Wet AMD – must more severe form, but less common. Growth of abnormal blood
                 vessels under the macula causing blisters that lead to cone photoreceptor death. Will
                 become blind in a few years. Due to growth of abnormal blood vessels under the macula
                 causing blisters that lead to cone photoreceptor death.

Animal models of AMD
       Transgenic mice expressing a mutant form of human ELOVL4 (enzyme involved in the
        elongation of long-chain fatty acids that causes dry AMD.
       There is some evidence that perhaps overactivity of this enzyme is responsible for extra
        production of fatty acids  drusen in the extracellular space.
       Issue with this model: you are using mice which have 97% rods to model a disease of cone
        photoreceptor degeneration. In the retina, there is clearly photoreceptor degeneration, therefore
        there is probably some modeling of the disease artificially in rods that are not normally exposed
        to this kind of degeneration.
       Chicks and dogs can also be used as an animal model.
       Note that there are various ways to introduce DNA into cell, such as transfection techniques.

II. Regeneration of the Lens
The Newt – An Expert in Regeneration
       The newt (salamander), is an expert in regeneration. However, there has been a shift in the field
        to study fish and frogs.
       Regeneration involves transdifferentiation, which is the transformation of one differentiated
        cell type into another. There are three types of transdifferentiation:
ANAT 416                                                                                 February 16, 2012
Dr. Cayouette                                                                            Lecture 12

             o Direct – rare; take pancreatic duct cells which can be directly transformed into liver cells,
               for example, without any intermediate cell. Simply by removing one gene and adding
               another one was sufficient to transform these into liver cells.
             o Dedifferentiation model – any cell type which would stop expressing certain genes that
               would characterize them as a liver or kidney cell, for example. These cells are
               dedifferentiated, then go to an intermediate stage where they start amplifying and then
               finally undergo redifferentiation into a different cell type.
             o Stem cell intermediate – have stem cells that remain in certain tissues for all the life of
               the animal that are self-proliferative, like HSCs and can regenerate these.

Regeneration of lens in salamander via dedifferentiation
       Through the process of dedifferentiation of the pigmented epithelial cells of the iris, in which
        these cells stop expressing iris cell genes and this leads to a proliferation of stem cells. After
        some time, they start expression of lens genes and regenerate the lens.
       Remove the lens only, then wait for these cells to proliferate and then differentiate. This is in
        vivo regeneration of a tissue.
       Note, the dorsal part of the iris has a special area that secretes molecules, such as wnt, which
        affect differentiation of normal development.
       This experiment raises a number of questions:
            o How does wnt control the proliferation of these cells?
                     Wnt triggers the expression of many transcription factors that lead to
            o How do these cells not overproliferate? What controls their decision to differentiate?
            o How does the regenerated lens grow back to the exact right size?
                     Are there intrinsic triggers or is there some feedback mechanism with various

Figure 5: Regeneration of the Lens via dedifferentiation

III. Regeneration of the Retina
Different Source of Retinal Regeneration
       The retinal pigment epithelium (RPE) is the primary source of RPCs or
        stem cells in amphibians.
           o The RPE has a neural origin as it comes from the neural tube. It is
               an epithelial cell with microvilli and neural membranes, but it is
               not a neuron. Recently, a paper came out showing you can take
               RPE cells from humans, even 99 year old humans and put them in
               culture and they will start proliferating through a special technique
               and you can redifferentiate them into many different cell types.

                                                                   Figure 6: different sources of retinal regeneration
ANAT 416                                                                               February 16, 2012
Dr. Cayouette                                                                          Lecture 12

       The ciliary margin zone (CMZ) is the primary source of neurogenesis in response to damange
        of the anterior retina in amphibian, fish, and birds to a lesser extent.
       In fish, the primary source of retinal regeneration is intra-retinal; either the rod progenitors or an
        intrinsic stem cell
       In fish and birds, there is evidence for Muller cell-mediated retinal regeneration.

Regeneration from the ciliary margin zone (CMZ)
       The CMZ varies from animal to animal.
       In fish and amphibians, it allows retinal growth to keep pace with the overall growth of the eye
        and animal.
            o Fish continue to grow their eyes throughout their life, so there must be a system to
                continuously produce retinal cells.
       In warm-blooded vertebrates, there is a limitation in both the amount of neurogenesis at the
        retinal margin and the regeneration potential (local regeneration, restricted to a few hundred
        microns from the marginal zone)
       In mammals, there is no regeneration in vivo.

Regeneration from the Retinal Pigment Epithelium (RPE)
       This regeneration is observed in amphibians (adult and
        embryonic) and embryonic chick and mammals.
       RPE cells lose pigmentation, proliferate, and generate
        two new epithelial layers: a pigmented and a non-
       The non-pigmented layer begins to express genes of
        retinal progenitors that generate new retinal neurons,
        much like normal retinal development
       The RPE generates normally oriented retinas in
        amphibians, but generates retina of inverted polarity in
        embryonic chicks and mammals
       In the mouse embryo, up to a certain stage of
        development, you can remove the RPE and stimulate it
        to regenerate some RPE cells, but the retina generated
                                                                             Figure 7: Regeneration from RPE
        is upside down.
            o Normally, you would have the photoreceptor cells on top of the RPE and the interneuron
                layer. In chicks, and to a certain extent in mammals, you can get some regeneration of the
                RPE cells, they proliferate, but the retina generated is upside down. You have
                photoreceptors facing the inside of the eye and the interneurons facing the outside of the
                eye, as opposed to having a normal oriented retina in which these epitheliums
                dedifferentiate further and have photoreceptors facing down (or facing the outside/apical
                surface of the eye) and the RPE cells are in the right place. The key is the detachment
                from the membrane and dedifferentiation and reproliferation.
            o The PE cells are pigmented epithelial cells, which means they have apical-basal polarity.
                This detachment from membrane, and this rounding up from cells in which they lose
                polarity. RPE cells have apical membrane, but apical membrane of RPE faces apical
                membrane of photoreceptors. There are two apical membranes are facing each other.
                When there is a detachment from Broch’s membrane, you lose orientation and you can
                regenerate retina in right orientation. If maintain attachment, you don’t lose polarity and
ANAT 416                                                                                February 16, 2012
Dr. Cayouette                                                                           Lecture 12

                these RPE cells maintain orientation, their apical membrane is on top and normally facing
                the apical membrane of photoreceptors, if these cells proliferate and you regenerate a
                retina, the apical surface is on top and interneuron underneath. This light grey line
                denotes the apical membrane. The front part becomes retina and the back part becomes
                the RPE. Because of this fold, you can get two apical membranes facing each other. That
                causes an orientation of the RPE that is basically opposite the orientation of the retina,
                therefore if you regenerate without losing polarity you generate a retina that is upside
       Thus, it is critical to understand the reasons of these important differences in retinal regeneration
        between amphibians and mammals, if the process is to be stimulated in mammalian retina.

Regeneration from intrinsic retinal sources
       In fish, the RPE does not contribute to retinal regeneration.
       There are different sources possible for the regeneration in fish:
            o The intrinsic rod precursor
            o A quiescent stem cell located in the INL
            o A subpopulation of Muller glial cells – these can stop proliferation and generate some
                 RPE cells.
       Population of neural glial cells
       Also quiescent stem cell located in eye of fish
       Intrinsic rod precursor stem cell – generates rod and nothing else
       What is so special about these cells?

The rod precursor and intrinsic stem cells
       When there is an insult to the retina of the fish, these cells can start to proliferate to generate a
        blastema. This is followed by migration of these cells into the retina and the generation of new

Muller glial cells
       In the chick, Muller cells can re-enter the cell cycle after damage to the retina and start
        expressing markers of RPCs.
            o You can visualize this proliferation with BrdU staining.
       A small proportion of these Muller glial cells regenerate some neuronal cell types of the retina
        such as amacrine, ganglion, and bipolar cells (no photoreceptors or other cell types)
       In mammals, an even smaller proportion of Muller glial cells can re-enter the cell cycle and
        generate new neurons.
            o This is rare, in one retina, can only get a few cells. The results of these experiments are
                not convincing, but there is potential.

IV. Adult retinal stem cells
       The CMZ is a specialized region for genesis of new retinal
        neurons in frogs, fish, and birds, but this zone is not found
        in the mammalian retina.
       Nevertheless, there are reports demonstrating that the
        anterior eye of mammals contains cell that can express
        progenitor cell markers and            Figure 8: Adult retinal stem
ANAT 416                                                                             February 16, 2012
Dr. Cayouette                                                                        Lecture 12

        differentiate into retinal neurons under certain conditions.
       This source of retinal stem cells appears to include the iris and the ciliary epithelium of the
        ciliary body (muscle which controls movement of the lens).
       Experiment: you could take these cells out, expand them in a dish and re-implant in patient that
        has retinal degeneration.

Retinal stem cells from the ciliary body grow neurospheres and can generate some retinal
       When you want to isolate a stem cell, the classic approach is take a tissue, then dissociate it into
        a single cell suspension and put these cells in low density in dish and let them float in culture.
        The cell culture only contains what these cells need to survive. These cells will begin to
        proliferate and form a ball of cells called an embryonic body (if they do not adhere) or a
        neurosphere if in the nervous system, in the absence of cell contact and adherence to any
        substrates. Main criteria to select stem cells.
            o Stem cells have the capacity to self-renew and in the absence of environmental signals.
                These cells, because can self-renew indefinitely, they will expand and form sphere,
                whereas the cells that divide a few times would not form this sphere.
            o The stem cells would keep dividing forever and sphere will keep growing. When the
                sphere reaches 100 microns, it is known as a stem cell sphere and you can isolate these.
            o The ciliary body is right next to the retina, it’s the closest we have to a CMZ in humans –
                perhaps it is evolutionary conserved to the RPE.
       Over time, the spheres get bigger but start to lose pigment. So first there is growth and then
        constant growth.
       Also, you need to be able to passage the sphere to demonstrate indefinite self renewal. So in the
        sphere, is there is even one stem cell, you should be able to dissociate the embryonic body if you
        plate each cell in a separate dish. If another embryonic body develops, this demonstrates self-
        renewal capacity.
       You also need to demonstrate that there is multi-lineage stem cell capacity ie it can generate
        neurons of different types. Might be able to take cells from patients with RP and graft them in,
        but there is poor regeneration of these cells. These cells are very immature stem cells, when graft
        them in there could be issues with survival. This is an adult retina now, so they may be missing
        some survival factors that were available earlier in development.

Retinal regeneration by cell grafting: The developmental timing is critical for use of stem cells
       Stem cell transplantation in the retina did not produce satisfactory results (very few grafted cells
        integrate the retina and generate new neurons).
       Grafting of committed photoreceptor precursor cells isolated at a developmental stage that
        correspond to the peak of photoreceptor cell production dramatically improved the results

Grafted cells generate photoreceptor cells and integrate the right retinal layer in wildtype mice
       So you would isolate the stem cells at the time they would generate specific cell types.
       Example: Isolate rod precursors from P3-4 mice, which is the time there is peak rod production.
        If you express GFP in rod precursors cells that you isolated and graft them into adults, can watch
        and see that they are retained in vivo in the photoreceptor level. They have all the correct
        markers, which suggest these were correctly differentiated.
       If isolate cells from the embryonic retina, this experiment would not work.

ANAT 416                                                                               February 16, 2012
Dr. Cayouette                                                                          Lecture 12

Grafted cells generate photoreceptor cells and improve light response in retinal degeneration
mutant mice
       Now the question is, do these P3-4 progentor-induced photoreceptors work? You could graft
        these photoreceptors into the RP model mouse:
            o Look at their response to light: the RP response curve is shifted to the left and you are
               restoring their response.
            o Look at their iris reflex: this reflex is triggered by exposure to light in which the iris is
               open in the dark and shuts in the light. In blind animals, there is no reflex and it stays
               shut. There is a shift from a blind animal that has no grafts, and there is a shift in the
               curve with a higher reflex in animals that received the graft.

       A better understanding of the normal developmental processes regulating cell fate specification
        is needed in order to stimulate retinal regeneration, either trough intrinsic sources or cell grafting
       Take cells at right stage, can regenerate photoreceptor progenitors
       Need to take adult retinal stem cells bring to right stage for grafting so that they can introduce.


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