1Life and Death for NeuronsWe lose 20-80% of our neurons during developmentMost neurons die right around the time that axons are invading the target1Expt: Look at the effect of removing or adding a limb on neuron survival in the spinal cord of the Xenopus frogResult: Adding a limb increases neuronal survival, while removing one reduces it.Model: The target is making something in limited quantities that promotes cell survival. Viktor Hamburger, 1920sWhy do neurons live or die?2Experiment : Transplant mouse sarcoma tumors near limb bud.Result: More neurons in the dorsal root ganglia survive.What makes neurons survive?ÎThey isolated the factor and called it nerve growth factor.Nobel 1986: Rita Levi-Montalcini and Stanley CohenIdentification of nerve growth factor32How Nerve Growth Factor was isolated1. Develop an assay: cultured Dorsal Root Ganglia in a dishAdded tumor extract, neuron outgrowth is dramatically enhanced2. Identify a rich source of NGF: purified NGF from snake venom4Î NGF is the trophic factor that promotes cell survivalÎ NGF is made by the target neuronDRG + Sympathetic NeuronsThey DIEThey SURVIVE+ NGFThey DIEvenom + NGF AbDoes NGF really promote cell survival?5The Neurotrophic Hypothesis• The target cells release a factor that promotes cell survival• This factor is found in limiting quantities• Cells compete to get enough factor to survive63There are many different trophic factors•Neurotrophins•NGFÆ TrkA Receptor•BDNF Æ TrkB Receptor•NT3Æ TrkC Receptor•NT4/5Æ TrkB Receptor•TGF-Beta Family•Interleukin – 6 related cytokins•FGFs•SHHThey all also bind the p75 receptor7Neurotrophins act throughreceptor tyrosine kinase signaling cascades8Signal transduction for Neurotrophins94Other actions of neurotrophins• Neuronal survival• Nerve growth• Nerve sprouting• Differentiation• Modulation of synaptic transmission• Electrical properties10ANeurons approach targetDegenerating ne uronTarget source of neurotrophic fac torLimited supply of neurotrophic fac torBThe Neurotrophic Hypothesis: neurons compete for limiting amounts of a neurotrophinsome neurons survive, other neurons die11How do neurons die?Two kinds of cell death1) NecrosisDeath by accident, ie trauma to tissue2) Programmed Cell Death (Apoptosis)Death by designControlled cellular self-destruction125Normal CellCell shrinks away from neighboursPlasma membrane blebbingCytoplasmic and nuclear condensationChromatin condensesNuclear and cellular fragmentationApoptotic BodiesPhagocytosisThe process of Programmed Cell Death (PCD)13PCD NecrosisNucleiChromatincondensation, fragmentationIrregular chromatin clumpingCytoplasmicOrganellesMembranes intact DisruptedvsMorphological Differences betweenProgrammed Cell Death and NecrosisDNA Cleaved into fragments No damage14AAAAMorphological Appearance of PCDNucleus is fragmented, blebby15normalPCD6Morphological appearance of PCDDNA is fragmented into 180 bp pieces16normal PCDC. elegans cell death ProgramComplete lineage description of all 1090 cells- 131 cells die during developmentMutagenIncreased survivalDecreased survivalIsolate geneMutated Gene Phenotypeced3 blocked all 131 embryonic cell deathsced4 blocked all 131 embryonic cell deathsced9(g.o.f) absence of cell death, embryonic lethalityWhat molecules cause Programmed Cell Death?17The apoptotic pathway is conserved in worms and humans187Most of the molecules involved in PCD are novelCED3 caspase is a cysteine protease….cleaves proteinsCED4 adaptor activates CED3CED9 inhibits CED4EGL-1 inhibits CED919ANeurons approach targetDege nerating neuronTarget source of neurotrophic factorLimited supply of neurotrophic factorBDo trophic factors inhibit Programmed Cell Death?20Neurotrophins increase Bcl-2 expression to inhibit PCD218Overexpression (OE) of Bcl-2 gives bigger brains12% volume increase in Bcl-2 OE brainsNormalBcl-2 OEbcl-2 OE normal22Why is PCD used during development?1. sculpting structures2. deleting unneeded structures3. controlling cell numbers4. eliminating non-functional or harmful cells23Diseases Associated with Deregulated ApoptosisIncreased ApoptosisAIDSNeurodegernative disordersAlzeheimer’s disease, Parkinson’s disease,Amyotrophic lateral sclerosisRetinitis pigmentosaMyelodysplastic syndromesAplastic anaemiaIschaemic InjuryMyocardial infarction, Stroke, Reperfusion injuryToxin-Induced liver diseaseAlcoholCancerFollicular lymphomascarinomas with p53 mutationshormone dependent tumours:breast cancer, prostate cancer,ovarian cancerAutoimmune DisordersSystemic lupus erythematosusImmune-mediated glomerulonephritusViral InfectionsHerpesvirus, poxvirus, adenovirusInhibition of Apoptosis249Why does a neuron live or die?1. Neurotrophins promote cell survivalby inhibiting Programmed Cell Death2. Programmed Cell Death allows death to occur without harming nearby