Neurotrophic Factors and Programmed Cell Death

1. Neurotrophic Factors and Programmed Cell Death

2. The Neurotrophic Hypothesis: Targets of innervation secrete limiting amounts of survival factors to generate a balance between the size of the target organ and the number of innervating neurons.

3. Effect of Removing or Augmenting Neural Targets on the Survival of Related Neurons • PN23091.JPG 1934: Victor Hamburger discovered that removal of a limb bud resulted in reduced numbers of sensory and motor neurons in the spinal cord.

4. Effect of Removing or Augmenting Neural Targets on the Survival of Related Neurons • PN23092.JPG 1939: Victor Hamburger showed that transplantation of a supernumerary limb resulted in increased numbers of sensory and motor neurons in the spinal cord.

5. Based on his limb-bud experiments, V. Hamburger hypothesized that the targets of innervating neurons provide signals that recruit undifferentiated cells to develop into sensory or motor neurons. (he was wrong) In 1942, Levi-Montalcini and Levi proposed that target derived signals maintain survival of differentiating neurons. In 1949, Hamburger and Levi-Montalcini repeated the limb bud experiments and found that their results supported the neurotrophic hypothesis. Hamburger, V. and Levi-Montalcini, R. (1949) J. Exp. Zool. 111: 457-502.

6. 1954: neurite outgrowth assay 1960: NGF purified 1969: NGF purified to homogeneity – extract Stanley Cohen Rita Levi-Montalcini 1986: Levi-Montalcini and Cohen split the Nobel prize for Physiology or Medicine “for their discovery of growth factors” + extract

7. Neurotrophins in the CNS 1. In the CNS, neurotrophins have important roles in neuron and glial survival, as well as differentiation and growth (as they do in the PNS). • In fact, the functions stretch beyond the time of peak synapse formation (both before and after); e.g., BDNF mRNA increases to maximal levels in postnatal animals.

8. The many possible sources of trophic support are illustrated in the CNS – many types of neurotrophic factors with compensatory/synergistic effects exist. * this may be why transgenics missing one of them often have no blatant developmental abnormalities – or at least, can survive. Note: autocrine, paracrine, afferants (anterograde transport). 3. BDNF, NT3, NT4, and their receptors are most widespread in the brain (NGF less so - mostly periphery), particularly in the cortex and hippocampus.

9. Regulation of Neurotrophin Synthesis by Physiological Activity • The transcription of genes for CNS neurotrophins is regulated by various forms of neuronal activity. • It has been observed that levels of BDNF mRNA in hippocampus, cortex, and cerebellum can be changed by: - depolarization and Ca2+ influx - excitatory neurotransmission (glu, kainate increase; GABA decrease)

10. - seizure activity (generalized activation) - stimulation of LTP - normal physiological stimuli, such as light  visual cortex; general physical activity, sensory stimulation, enriched environments.

11. Regulation of Synaptic Transmission by Neurotrophins • One important postnatal function of neurotrophins (after synaptogenesis and normal cell death): - from the anterograde transport (afferent sources) - including transient modulation of synaptic transmission (e.g., increased efficacy of inputs to CA1 pyramidal neurons (Schaffer collaterals). • Maintenance of LTP. • Alterations in morphology of synaptic elements. • Endocrine control of cell survival. • Maintenance of neuron size and arbourization. • Facilitation of activity-dependent enhancements (i.e., complexity of dendriticarbours or spine formation and remodeling).

12. Some Other Growth Factor Families: Cytokines • Several other families of signaling molecules with actions both inside and outside the nervous system exist: • Like neurotrophins, these diffusable factors regulated growth and maintenance: Cytokines = “cell movement factors”. • So named because they were first known to regulated chemotaxis and migration.

13. Include: - Interleukins (central changes in immune system). - TNF – proimflammatory. - interferons – inhibit viral replication and growth. • Several cytokines have activities in the developing and adult nervous system. • The following are several families:

14. Neuropoietic Cytokines: e.g., ciliary neurotrophic factor – promote survival of developing motor neurons, hipp., sensory neurons, parasympathetic ciliary ganglionic neurons. - induction of neural cell precursors to differentiate  astrocytes. e.g., leukemia inhibitory factor – induces changes in gene expression that occurs in neurons after injury.

15. TGF Superfamily: - recall role in early development and induction processes. - may have distinct functions later in development. - TGF and a close relative, GDNF (glial-derived neurotrophic factor) protect the survival and function of dopaminergic neurons (note the enhanced survival in animal models of Parkinson’s Disease). - Also, - survival of motor neurons. - peripheral sensory autonomic neurons. - other systems (kidneys, enteric, nervous).

16. FGF: - Mitogenesis during early embryonic development (stim proliferation of many embryonic tissues). - in brain, FGF1 and FGF2 expression remain high in nervous system throughout life. - Signal through tyr kinase receptors that are similar to the trks for neurotrophins. - Have important roles promoting survival after injury and can also signal differentiation.

17. Bioassay with cultured ganglia

18. Transport of NGF

19. Structure of neurotrophins

20. Structure of neurotrophin receptors

21. Tyrosine kinase Receptor activation:

22. Tyrosine kinase Receptor activation:

23. Distribution of brain neurotrophins

24. NGF: sympathetic neurons and some sensory neurons (CNS neurons do not require NGF for survival) BDNF: NGF-related factor purified in 1982 from pig brain (shares ~50% homolog with NGF) NT-3 and NT-4/5: were obtained by PCR cloning All these factors are synthesized as ~250 aa precursors that are processed into 120 aa proteins

25. Neurotrophin Evolution Neurotrophins have only been isolated from chordates Hallbook (1999) Curr Opin Neurobiol9: 616-21

26. Structure of NGF bound to its receptor

27. The Trk Family of Receptor Tyrosine Kinases for the Neurotrophins • PN23160.JPG p75NTR: purified and cloned 1st, homology to TNFR Trk: tropomyosin-related kinase, originally known as orphan receptors

28. Alternative splicing generates many Trk receptor isoforms NGF KD for TrkA=10-11M Roux and Barker (2002) Prog Neurobiol 67:203-233

29. p75NTR structure NGF KD=10-9M (all neurotrophins can bind p75NTR) Roux and Barker (2002) Prog Neurobiol 67:203-233

30. Models for Trk and p75NTR interaction Chao and Bothwell (2002) Neuron 33:9-12

31. p75NTR is required for developmental myelination Cosgaya et al. (2002) Science298:1245-1248

32. (Found only in fish)

33. The effect of NT/NTR knockouts on neurons in the DRG Fariñas et al. (2002) Brain Res Bull 57:809-816

34. Trk receptor signaling When a neurotrophin binds to a trk receptor, the kinase domain is activated resulting in autophosphorylation. Autophoshorylation results in further activation of the kinase domain, leading to activation of three potential signaling cascades: MAPK PI3K PLC-g

35. Our axons can be >1 m in length---how does the neurotrophin/receptor complex signal to the neuronal cell body? Miller and Kaplan (2001) Neuron 32:767-770

36. Campenot, RB (1977) Local control of neurite development by nerve growth factor. Proc Natl Acad Sci U S A. 74(10):4516-9. (A method that can be used to study how NTs added to distal axons signal retrogradely) NGF +K252a Miller and Kaplan (2001) Neuron 32:767-770

37. NGF signal can be transduced at the tips of growing neuronal processes Sympathetic neurons were placed in a TC system that allowed the somas and neurites to be bathed in different media. L: Most neurons die when grown without NGF for 30 hr. R: Neurons can be kept alive by adding NGF only to the compartments with growing neurites. In both cases, anti-NGF prevented TrkA activation in the central compartment.

38. Does internalized NGF contribute to sympathetic neuron survival? L: NGF covalently bound to beads – preventing internalization, but allowing local activation of the TrkA receptor. R: sympathetic neuron soma are exposed to prot-delivery system (Pep-1-antiGF complex) that allows anti-NGF to enter cells  with anti-NGF now inside the cells, 40% of sympathetic neurons die.

39. Activated Trk can signal locally and retrogradely using different signalling pathways Slow (2-20 mm/hr) Miller and Kaplan (2001) Neuron 32:767-770

40. Differential control of TrkA trafficking and signaling may also be the basis for the different functions of NGF and NT-3 Kuruvilla et al. (2004) Cell 118: 243-255

41. In vitro assays have shown that neurotrophins enhance both axonal and dendritic growth In vivo, the situation is more difficult to study Why? In standard knockouts, it is difficult to separate the survival effects of neurotrophins from their effects on the morphology of neurons. This problem has begun to be addressed by using conditional knockouts, or by crossing neurotrophin knockouts with mouse mutants lacking pro-apoptotic genes. Recent evidence from these kinds of experiments suggests that long distance peripheral sensory axon growth in vivo is neurotrophin-dependent.

42. Neurotrophins’ roles in neuronal development and function • NT’s are expressed in regions of the developing embryo that are traversed by sensory axons en route to their targets. • NT’s affect the proliferation and differentiation of CNS neuroepithelial progenitors, neural crest cells, and progenitors of enteric neurons in vitro (and in some cases also confirmed in vivo). • In the CNS, BDNF/TrkB signaling is implicated in the development and maintenance of cortical circuits.

43. Neurotrophins in the CNS The highest levels of neurotrophins are found in the hippocampus Lindsay et al. (1995) Trends Neurosci 17:182-190.

44. Trophic support for peripheral and central nervous system neurons

45. BDNF can be secreted by the presynaptic neuron in an activity-dependent fashion • Indirect Evidence: • BDNF is found in synaptic vesicle preparations • TrkB receptors are found in dendrites • Axotomy of axons from BDNF-expressing neurons results in a depletion of BDNF in their target area • Direct Evidence: • Kohara, K. et al. (2001) “Activity-dependent transfer of brain-derived neurotrophic factor to postsynaptic neurons” Science 291: 2419-2423.

46. Experimental Strategy employed by Kohara et al.

47. Transfer of GFP-BDNF from a presynaptic to postsynaptic neuron axon DsRed GFP-BDNF DsRed+GFP-BDNF Kohara et al. (2001) Science 291: 2419-2423

48. Transfer of GFP-BDNF requires synaptic activity Kohara et al. (2001) Science 291: 2419-2423

49. Recently, a similar technique was used to examine the role of neurotrophins in dendritic remodeling: Horch and Katz (2002) Nature Neuroscience 5: 1177-1184 eGFP Effect on dendrites? RFP + BDNF-myc Visual cortex pyramidal neurons in a slice culture

50. Result: local release of BDNF alters nearby dendritic structure Horch and Katz (2002) Nature Neuroscience 5: 1177-1184