Brain—Target interactions Genes Hormones-Thyroid Proliferation MZ Migration IZ Differentiation VZ IZ Maturation Interactions Limb LMC Neuron Death Trophic Factors • Victor Hamburger The Nature of Science • The Dogma: • Observation; Hypothesis; Experimental tests; Conclusions. The Most important factors. Curiosity • Preparation • Free your mind • And the rest will Follow • Serendipity Indirect Factors Timing! • Appropriate Questions. • Money • Technical skills and the availability of appropriate scientific equipment • The Old Boy/Old Girl Network • Mentoring and Sponsorship Neurotrophins • Trophic factors (survival signals). First discovered in 1950’s • Gene family. In mammals includes NGF, BDNF, NT3 and NT4/5. • Individual neurotrophins activate different different receptor tyrosine kinases • Have a primary role in promoting the survival of motor neurons and peripheral sensory neurons. It is known that survival of neurons that innervate the periphery depend on the size of target tissue (Neurotrophic Factor Hypothesis ). • Also have a role in regulating proliferation and differentiation of neuronal and glial precursors. Rita-Levi Montalcini (1909-?) Turin Italy Nobel Prize-1986 102 yrs old 2011 Life is Good, But Parental Rebellion Rita Levi-Montalcini describes being raised in a cultured, intellectually stimulating environment, even though her father believed that “a professional career would interfere with the duties of a wife and mother. At twenty (1929), I realized that I could not possibly adjust to a feminine role as conceived by my father, and asked him permission to engage in a professional career. In eight months I filled my gaps in Latin, Greek and mathematics, graduated from high school, and entered medical school in Turin. Invasions; Decisions Trying years Nerve Growth & Neoplasms (crazy experiments) In a 1948 article, a former student of Viktor Hamburger, Elmer Bueker, reported the results of a bold and imaginative experiment consisting in grafting fragments of mouse sarcoma 180 into the body wall of three-day chick embryos. The histological study of the embryos fixed 3-5 days later, showed that sensory nerve fibers emerging form adjacent dorsal root ganglia had gained access into the neoplastic tissue while no motor nerve fibers entered into the tumor. Figure 3. Drawings illustrating the in vitro "halo" effect on 8-day chick embryo sensory ganglia cultured in the presence of fragments of mouse sarcoma 180 for 24 hours (b) or 48 hours (c). In (a), the ganglion, which faces a fragment of chick embryonic tissue, shows fibroblasts but few nerve fibers. In (b) and (c), the ganglia, facing fragments of sarcoma 180, show the typical "halo" effect elicited by the growth factor released from the sarcoma. Note in (c) the first evidence of a neurotropic effect of the growth factor. Stanley Cohen Fractionated tumor homogenate Got an active nucleoprotein Nerve growth factor Serendipity:snake venom Snake venom was known to contain phosphodiesterases that should break down the nucleic acid part of the nucleo protein NGF-snake venom 2 • Either the snake venom destroyed an inhibitor factor in the tumoral fraction—that is the tumoral fraction might contain a “brake” on its ability to stimulate nerve outgrowth; or the snake venom itself could stimulate outgrowth. • In 1957 Cohen purified the NGF from snake venom. •Mouse salivary glands—even better Cohen made an NGF antiserum Caused immunosympathectomy • Neurotrophin Receptors TRK receptors • A digression: Viruses and transforming viruses • A second digression, Oncogenes and Protooncogenes. • A third digression, skeletal muscle physiology—tropomyosin Contraction and myosin heads – A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences was called trk for tropomyosin receptor kinase. The name trk was then taken for the neurotrophin receptors. – Martin-Zanca D, Hughes SH, Barbacid M. – A biologically active complementary DNA clone of a transforming gene present in a human colon carcinoma contains gene sequences of both tropomyosin and a previously unknown protein tyrosine kinase. The predicted protein (641 amino acids) encoded by this oncogene seems to have been formed by a somatic rearrangement that replaced the extracellular domain of a putative transmembrane receptor by the first 221 amino acids of a non-muscle tropomyosin molecule. Parada: Trk tyrosine kinase is a receptor for NGF; antibody to phosphotyrosine on precipitated trk protein with and without NGF NGF-induced phosphorylation of trk protein in DRG cells Neurotrophins bind to a family of receptor tyrosine kinases called Trks NOTE: Like BDNF, NT4/5 binds to TrkB Different neurotrophins are required for the survival of distinct populations of neurons in the nervous system Nerve growth factor (NGF) mediated signaling P75NTR binds all NT’s with low affinity. Its intracellular domain has no catalytic activity but it interacts with adapters Thoenen and Sendtner, 2002. Nature Neuroscience 5:1046-1050 22.13 The influence of neurotrophins. (Part 1) 22.13 The influence of neurotrophins. (Part 2) Cerebral Cortex neurons: Left-transfected with green fluorescent protein(GFP) right— transfected with GFP + BDNF 22.14 Evidence that NGF can influence neurite growth by local action. 22.15 Neurotrophin receptors and their specificity for the neurotrophins. 22.16 Signaling through the neurotrophins and their receptors.