Blindess Studyguide PDF

Blindness Module Notes: Unit 1) The retinal Anatomy Retina: visual processing center where the photoreceptor cells and neurons are locations (fovea and macula) Fovea: part of retina/macula with highest acuity vision, finer details, center vision (no rods, but many cones) F= Focus...

Blindness Module Notes: Unit 1) The retinal Anatomy Retina: visual processing center where the photoreceptor cells and neurons are locations (fovea and macula) Fovea: part of retina/macula with highest acuity vision, finer details, center vision (no rods, but many cones) F= Focus Macula: pigmented area at center of retina containing the fovea (dense in photoreceptors) Macula Dracula is Dense in photoreceptors Photoreceptors (Rods and Cones): at the back of the retina, detect the light Bipolar Cells: connect photoreceptors to ganglion cells - relay signal to brain Ganglion Cells: leave eyes in large cluster at the optic disk (detect light, not process) Optic Disk: does not contain any photoreceptors, “natural blind spot” Optic Nerve: Collection of Ganglion cell axons that relay information from the retina to the brain Horizontal Cells: receive input from photoreceptor cells, integrate signaling adjust signaling, and regulate activity in photoreceptor cells Amacrine cells: receives signals from bipolar cells, regulate/integrate activity in bipolar and ganglion cells. Retinal Pigment epithelium (RPE): recycling of phototransduction molecules (retinol), which protrudes around the outer segments. The RPE phagocytosis shed photoreceptor membranes and recycles their components Bruch's Membrane: thick layer to separate RPE and choroid Muller Glia: provides nutrients to neurons and is a light guide, phagocytes debri. (NOT A NEURON) Notes: -phototransduction occurs in the outer segments (long cell of the photoreceptors) -plexiform layers of retina are synaptic layers Unit 2- Retinal Physiology Notes: -All trans retinol travels through the membrane to the RPE where it is transferred back into 11-cis retinol -rhodopsin: where retinol is, important protein in the light cascade -outersegments of photoreceptors have lots of disks (membranes) where this all occurs Unit 3 Genetics Background: Dominant: AA or Aa: one allele to see phenotype Recessive aa : two alleles to see phenotype Autosomal Recessive - loss of function mutations - skips generations (two unaffected individuals have affected children) - Two affected parents will have affective children Autosomal Dominant Diseases - gain of function - equally affected male and female - Two affected parents can have an unaffected child - Do not skip generations X-Linked Recessive - There is a greater chance of males being affected - All the sons of an affected female will be affected - The trait can skip more than one generation - An affected male yields daughter who are carriers X-linked Dominant - If the father has it, the daughters will - Affected males must come from affected mothers - Does not skip generations To Calculate Probability: - If child is unaffected but parents are carriers, use a punnett square and denote that if they do not have the disease they have a ⅔ chance of being affected - Assume that those married in do NOT have the disease because it is “rare” Notes: -deleterious: bad genes -Deletion: loss of DNA sequence -Frameshift mutation: removing a base, switched all amino acid codons and leads to a nonfunctional protein -Insertions: causes a frameshift mutation by introducing stop codons earlier than necessary -RNA is transcribed in a 5 to 3 direction, which is a process of generating RNA from DNA. Then translation is the process of generating a protein from mRNA. Unit 3) Types of Eye Diseases 1) Age related Macular Degeneration -Onset: happens in older people -Progression: lose your central vision first -retinal image shows spots around the fovea -blurred or distorted central vision -there is a defect in RPE, blood vessels, and photoreceptors -Not hereditary How to diagnose arMD? - Amsler Grid Test: if you see curvatures - Fundus image: discoloration of macula, larger yellow spots of drusen buildup Types 1. Dry AMD - Why? Due to impairment of cells in the macula - What? RPE and Cones photoreceptors are impaired - How? A buildup of Drusen (protein and lipid bunch) between the RPE and Bruch's Membrane causes the RPE and cones to be starved for nutrients, then the RPE and Cones start to die, then you have a GRADUAL loss of vision - 80-90% of MD is a dry form, but 20% of those affected will develop Wet MD - Treatment? Vitamin supplement: ARED or ARED 2 which slows the progression 2. Wet AMD - How? Drusen is much more severe/larger and so RPE cells start to make VEGF (vascular endothelial growth factor). VEGF makes capillaries defective and then blood starts leaking into the retina which breaks down Bruch's Membrane and kills the cells. This leads to a rapid loss of vision - Treatment? (1) Anti-angiogenic drugs: binds and inactivate VEGF - Have to continuously get injections subretinal - Not getting rid of the problem but stabilizing growth (2) Laser Therapy: infusion of fluorescent compound - Laser zaps and kills capillaries - RPE is still not getting nutrients - Only works for a short time (3) Photodynamic Laser therapy: combination of laser destruction and a drug Risk factors of arMD - Genetic Predispositions: certain defects increase your chances of having it but it's not a genetic defect in itself - More likelihood: - Age-older - Caucasian - Female - Fair skin - Smoker - Obese 2) Retinitis Pigmentosa (Retinitis= Rods die first) -early onset (early teens to 20s) -rods die first, and cones die later -Fundus/retinal image shows black pigment (cells dying) -midline scan will show a thinning retina (loss of photoreceptor cells) -lose peripheral vision first (tunnel vision) -Defect in the photoreceptor cells -Can be autosomal recessive, dominant, or x-linked recessive -can be from a large number of gene mutations (ex. Mutation in RPE65), at least 35 different genes but all result in rods dying -most common mutation is in rhodopsin for 20-30% 3) Leber's Congenital Amaurosis -affects people from birth or in first year of life -rods/cones do not die -mutation in the RPE 65 gene -Fundus image looks normal/retina in tact (can’t tell) -Some genes that cause this: (Cep 290, Guanylyl cyclase, RPE 65) Briard Dog w/ LCA - increased number and size of vacuoles in the RPE cells. The all-trans retinal come in through vacuoles and with a nonfunctional RPE, the vacuoles start to build up with the retinol - Dog becomes blind - outer nuclear level/rods are normal (no loss of cells) - normal fundus image - abnormal ERG (electroretinogram) - DNA analysis showed 4 base pair deletion/ frameshift mutation Mouse w/ LCA - disrupted packaging of the rod disks (abnormal outer segments) - rhodopsin is still in the disks, but they have less rhodopsin protein (reduced rhodopsin rat) - rods are not dying and are not absent - normal fundus image - abnormal ERG - Used immunoblot Notes for diseases: -both LCA and RP are commonly recessive How to determine mutations of a disease: - DNA sequencing of the wild type and mutant DNA (GENOME SEQUENCE) - Restriction digestion of a PCR amplified DNA fragment - Pedigree analysis Notes: -RPE65: converts all trans retinal back into 11-cis (issues with this protein can be bad) -Arrestin: common protein to turn off Rhodopsin 7-trans protein -if you lose photoreceptor cells, the retina becomes thinner Unit 4) Gene Therapy for LCA -gene therapy: for humans -gene rescue: animals Necessary Components 1) Wild Type Gene Did not know function of gene when testing in animals DNA is so large that mRNA is selected so introns are spliced out to make it smaller into CDNA 2) Virus/Vector: deliver it to the correct cells (RPE) “Vehicle” Ex: AAV (adeno associated Virus)- infects RPE cells (good at targeting specific cell types) 3) Promoter: A regulatory DNA sequences that allows the transgene to be transcribed at the right time and allows the polymerase to bond (directs transcription of the transgene) 4) Non Dysfunctional cells have to be alive Ex: can use for LCA but not RP 5) Must be a recessive disease (loss-of-function): insert a dominant gene in 6) Where you inject is important! Notes: - Experimental Timeline: (1)2005 Briard Dog experiments at UPENN (2)2013 Spark therapeutics: identified promoter and DNA that would work for LCA (3) 2017 Clinical results published (4)2017 FDA Approves Luxterna - Transgene= gene(usually CDNA) +promoter(usually smaller) - In dogs, you do not need a wild type human RPE65 protein (you need a promoter, wildtype copy or cDNA, and a virus) How is it tested? -trials on dogs or other animals -must be certain it works before clinical trials -tests a variety of different viruses and genes Unit 5) Stem Cells What are stem cells: - undifferentiated: not committed to specific cell type (not reached “final status”) - self renewing: divides while maintaining undifferentiated state (asymmetric or symmetric) - potency: capable of producing many types of sells - Present throughout the life of an organism Types of Cell Division: - asymmetric: produces two cells of different types - symmetric: produces two cells of the same type - Extrinsic (Environmental) and Intrinsic (intercellular) signals determine the type of cell division and identity of new cells Potency Amongst Cells: - Morulas= totipotent because they can become any part of the new organism and its extra-embryonic tissue. - Blastocyst(inner cell mass cells)= pluripotent because can become any cell in the new organism - Ectoderm/Mesoderm/Endoderm= Multipotent because they can become anything in that sublevel - Progenitor Cells= Multipotent because they commit to being a number of different but similar cell types (can’t self renew) - Precursor Cells= Unipotent because can only commit to being one type of cell  - Differentiated cells= no potency because now this cell has become a single type As they lose potency, there is less expression of the pluripotency genes. If you want to see if genes are potent, you can do multiple tests to see if they are expressing the potency genes. Pluripotent cells - Pluripotent cells: capable of becoming any type of cell in the organism Knockout Mouse experiment: - (1) Embryonic stem cells were isolated from a mouse containing an altered gene - (2) They injected these cells into a female black mouse's blastocyst - (3) The female black mouse gives birth to mice with these altered gene - (4) These mice mated and produced fully gene-manipulated mice How to Determine Pluripotency: 1) Teratoma assay - Inject pluripotent cells into the abdomen of a mouse or rat and a tumor should form - If you isolate that tumor it should contain endoderm, ectoderm, and mesoderm cells 2) RNAseq or Microarray analysis (Gene Expression) - Figure out of cells express pluripotency marker genes at a high level - Common markers: Oct4, Sox2, C-Myc, SSEA-4 - Tested using gene expression analysis 3) In vitro embryoid bodies - Isolate human stem cells after in vitro fertilization from the inner cell mass - Put human ES cells in a liquid culture and allow them to grow and divide - Add specific growth factors - Label with antibodies and analyze cell results Adult (somatic Stem cells) - Multipotent stem cells - Where: found in bone marrow - Already present in the body (not rejected) - Cannot divide indefinitely - Less likely to have immune response Vs Embryonic stem cells - Pluripotent stem cells - Found in inner cell mass of the blastocyst (5-7 days after fertilization) - Embryo is destroyed (ethical issues) - Can divide indefinitely - Can self renew - Possibility for rejection by patient - Isolated from the blastula/blastocyst - Can form a teratoma when injected into a mouse - Easier to culture Notes:  - Commitment: Cell is going down a stage in potency and cannot get back up. They are choosing to be something else - Differentiation: final commitment where the cell now is in its final form (specialized) Induced Pluripotent stem cells iPSCs: pluripotent cells produced by treating differentiated adult cells with different factors to make them express pluripotency again (close to embryonic cells but have cellular memory) - Why important/ How can we use - Study development of cells (watch how they aggregate) - Study disease progression/development - Cell Therapy (transplantation therapy to replace lost or dead cells in tissues or organs) - Drug Development History: 1st step: John Gordan’s Somatic Cell Nuclear Transfer (FROG CLONING) 1. Isolated the nucleus from a frog’s epithelial cell 2. Remove the nucleus from a frog’s egg cell 3. Put the nucleus of epithelial into the egg cell 4. Egg cell developed into a full frog Why important? - The nucleus of differentiated cells can be pluripotent in the right place with the right factors. Led to the creation of iPS cells Why is it not useful? - Very low success with more complex animals - Not ethical Neurons?? -if you wanted to differentiate neurons in a culture dish, you could use embryonic stem cells, neural stem cells, or induced pluripotent stem cells. Yamanaka and iPSCs  - Took mice fibroblast cells and took a transgene with pluripotency genes (about 24 options) and added a virus - How did it work: if we could make cells pluripotent, the promoter would be turned on that was on the transgene. - Discoveries: pluripotency causes cells to change morphology and shape, they expressed genes of pluripotency. You do not have to use egg cells Unit 6 Cell Therapy Cell Therapy: a technique that can be used on certain cell types in order to replace old/dead cells. - Cannot use neurons because they have to make certain synaptic connections and have a neural framework that would not work great. - But RPE cells would work because you don't have to make neural connections (replacing the dying RPE cells) - Tested: grow cells in a dish and program them to be how you want them to be. You need to first test on an animal that would have a similar organ to that of a human Cell therapy for AMD - You can use hESCs, iPSC, or RPE cells, make them all into RPE cells by growing on a dish, then inject them into eye Cell therapy for RP - Does not work because photoreceptors are neurons - Not successful yet How to Determine Success - Immunostaining: shows cells express targeted cell’s proteins - Percent protein expression levels: make sure it is constant - Electron microscopy: check for similarities in cell - Ex: RPE cells are polarized, check for apical side (microvilli) and basolateral side - Functional Tests: make sure the cells are doing what they are supposed to do (RPE phagocytosis) - Ensure there are no other pluripotent cells present - Examine retinal thickness-> thicker with new RPE layer - BCVA- test confirms improvement of vision and reading Concerns - May be rejection, especially from hESCs Notes: -In vitro: culture -in vivo: animal De cruz studied fixing mouse retina: -to demonstrate that they produced stable RPE cells -to demonstrate that the RPE cells were not pluripotent or multipotent -to demonstrate that the RPE cells generated from stem cells did not proliferated Unit 7 Interpreting data from figures 1. Western Blot (immunoblot) - detects and quantifies protein of choice from a biological sample - Band size and intensity tells you about protein expression - Ex: found that mice had less rhodopsin in their rods 2. Polymerase Chain reaction (PCR) - Amplifies a targeted DNA sequence, allowing us to inspect a fraction of DNA - We can assess the presence or absence of certain DNA fragments or a deduction in size - Ex: in the PCR we find their is missing codons 3. Reverse Transcriptase PCR - Detect and quantify RNA by converting RNA into cDNA - Used in Yamanaka to analyze ES cell marker genes 4. Tissue immunostaining - Visualize the presence of specific proteins or cells in tissue sections - Provides information about whether studies work, the formation and set of certain things - Technique to stain thin section of retina with antibody to highlight microglia 5. Fundus image - Detailed image of the back of the eye with retina, blood vessels, optic nerve, macula and fovea - You can identify RP or Macular Degeneration along with any abnormalities like hemorrhaging or lesions or dark spots. -Northern Blot: RNA -Southern Blot: DNA -Western Blot: Protein -(see above) Nat1 is a positive control (confirm that expression is present) -RT minus is a negative control (confirm no DNA contamination)