1. Life and Death for Neurons We lose 20-80% of our neurons during development Most neurons die right around the time that axons are invading the target 1

2. Expt: Look at the effect of removing or adding a limb on neuron survival in the spinal cord of the Xenopus frog Result: 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, 1920s Why do neurons live or die? 2

3. Experiment : Transplant mouse sarcoma tumors near limb bud. Result: More neurons in the dorsal root ganglia survive. What makes neurons survive?  They isolated the factor and called it nerve growth factor. Nobel 1986: Rita Levi-Montalcini and Stanley Cohen Identification of nerve growth factor 3

4. How Nerve Growth Factor was isolated 1.Develop an assay: cultured Dorsal Root Ganglia in a dish Added tumor extract, neuron outgrowth is dramatically enhanced 2. Identify a rich source of NGF: purified NGF from snake venom 4

5.  NGF is the trophic factor that promotes cell survival  NGF is made by the target neuron DRG + Sympathetic Neurons They DIE They SURVIVE + NGF They DIE venom + NGF Ab Does NGF really promote cell survival? 5

6. The 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 survive 6

7. There are many different trophic factors Neurotrophins NGF  TrkA Receptor BDNF  TrkB Receptor NT3  TrkC Receptor NT4/5  TrkB Receptor TGF-Beta Family Interleukin – 6 related cytokins FGFs SHH They all also bind the p75 receptor 7

8. Neurotrophins act through receptor tyrosine kinase signaling cascades 8

9. Signal transduction for Neurotrophins 9

10. Other actions of neurotrophins Neuronal survival Nerve growth Nerve sprouting Differentiation Modulation of synaptic transmission Electrical properties 10

11. The Neurotrophic Hypothesis: neurons compete for limiting amounts of a neurotrophin some neurons survive, other neurons die 11

12. How do neurons die? Two kinds of cell death 1)Necrosis Death by accident, ie trauma to tissue 2)Programmed Cell Death (Apoptosis) Death by design Controlled cellular self-destruction 12

13. Normal Cell Cell shrinks away from neighbours Plasma membrane blebbing Cytoplasmic and nuclear condensation Chromatin condenses Nuclear and cellular fragmentation Apoptotic Bodies Phagocytosis The process of Programmed Cell Death (PCD) 13

14. PCDNecrosis Nuclei Chromatin condensation, fragmentation Irregular chromatin clumping Cytoplasmic Organelles Membranes intactDisrupted vs Morphological Differences between Programmed Cell Death and Necrosis DNACleaved into fragmentsNo damage 14

15. A A A A Morphological Appearance of PCD Nucleus is fragmented, blebby 15 normal PCD

16. Morphological appearance of PCD DNA is fragmented into 180 bp pieces 16 normal PCD

17. C. elegans cell death Program Complete lineage description of all 1090 cells - 131 cells die during development Mutagen Increased survival Decreased survival Isolate gene Mutated GenePhenotype ced3 blocked all 131 embryonic cell deaths ced4 blocked all 131 embryonic cell deaths ced9(g.o.f) absence of cell death, embryonic lethality What molecules cause Programmed Cell Death? 17

18. The apoptotic pathway is conserved in worms and humans 18

19. Most of the molecules involved in PCD are novel CED3 caspase is a cysteine protease….cleaves proteins CED4 adaptor activates CED3 CED9 inhibits CED4 EGL-1 inhibits CED9 19

20. Do trophic factors inhibit Programmed Cell Death? 20

21. Neurotrophins increase Bcl-2 expression to inhibit PCD 21

22. Overexpression (OE) of Bcl-2 gives bigger brains 12% volume increase in Bcl-2 OE brains Normal Bcl-2 OE bcl-2 OEnormal 22

23. Why is PCD used during development? 1.sculpting structures 2. deleting unneeded structures 3. controlling cell numbers 4. eliminating non-functional or harmful cells 23

24. Diseases Associated with Deregulated Apoptosis Increased Apoptosis AIDS Neurodegernative disorders Alzeheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis Retinitis pigmentosa Myelodysplastic syndromes Aplastic anaemia Ischaemic Injury Myocardial infarction, Stroke, Reperfusion injury Toxin-Induced liver disease Alcohol Cancer Follicular lymphomas carinomas with p53 mutations hormone dependent tumours: breast cancer, prostate cancer, ovarian cancer Autoimmune Disorders Systemic lupus erythematosus Immune-mediated glomerulonephritus Viral Infections Herpesvirus, poxvirus, adenovirus Inhibition of Apoptosis 24

25. Why does a neuron live or die? 1.Neurotrophins promote cell survival by inhibiting Programmed Cell Death 2.Programmed Cell Death allows death to occur without harming nearby cells 25