Neurotrophins.

Transcription

1 Neurotrophins Nature seems unaware of our intellectual need for convenience and unity, and very often takes delight in complication and diversity. Santiago Ramón y Cajal, Nobel Prize Lecture,

2 History: Ross G. Harrison Early 1900s Harrison isolated neural tissues from amphibian embryos and grew them in vitro. He is credited with developing the method of tissue culture. In 1910 Harrison observed processes projecting from neural explants which were grown in the presence of a preparation derived from clotted lymph. Harrison thought that the projections (axons) might be directed by a physical property of the substrate or by electrical fields. Harrsion also showed that the amphibian nervous system could innervate limbs and organs grafted from other species.

3 Viktor Hamburger: 1930s and 1940s 1934: Hamburger developed the chick embryo model for studying innervation of peripheral tissues; this model came to replace the amphibian model used by Harrison and other investigators. He found that removal of the wing bud led to hypoplasia of the innervating motor and sensory neurons. Subsequent studies revealed that the hypoplasia resulted from death of differentiated neurons, rather than a failure to recruit neurons from a pool of precursor neurons. Hamburger also used the chick embryo model to study the innervation of tissue grafts.

4 Rita Levi-Montalcini She graduated summa cum laude from the University of Turin Medical School in 1936, and then completed a degree for specialization in neurology and psychiatry in Fascist laws prevented Italian Jews from practicing medicine or working in universities at that time, so Levi- Montalcini set up laboratory equipment in her bedroom to continue her research on neurogenesis. When the family was forced to leave Turin in 1941due to the heavy Allied bombing of the city, Levi-Montalcini rebuilt her laboratory in the family's country cottage. When the Germans invaded Italy in the fall of 1943, the family moved to Florence where they lived underground until the end of the war. After Allied armies forced the Germans out of Florence in August 1944, Levi-Montalcini worked as a medical doctor in an Italian refugee camp, treating epidemics of infectious diseases and abdominal typhus. After the war, her family returned to Turin and Levi- Montalcini resumed her position as an assistant at the University of Turin Institute of Anatomy. Two articles that Levi-Montalcini had published in foreign scientific journals interested Viktor Hamburger, head of the Zoology Department of Washington University in St. Louis. In September 1947 Rita Levi-Montalcini accepted Hamburger's invitation to collaborate with him as a research associate. Though she initially planned to stay at Washington University for less than one year, Levi- Montalcini stayed for thirty years. Text from:

5 Levi-Montalcini and Hamburger: 1948; sarcoma : Elmer D. Bueker, a former student of Hamburger, reported that the chick embryo nervous system could innervate a grafted mouse tumor ( sarcoma 180 ) isolated from connective tissue. Bueker observed that sensory nerve fibers from the dorsal root ganglia adjacent to the grafted tumor had innervated the tumor, while the tumor was devoid of innervation by motor neurons. Levi-Montalcini and Hamburger decided to use this experimental system, rather than studying grafted limbs and organs.

6 Levi-Montalcini and Hamburger: They reproduced the studies of Bueker but also noted that, in addition to sensory neurons, sympathetic neurons innervated the tumor. They noted that the sympathetic ganglia innervating the tumor were greatly (~6X) increased in size compared to control ganglia. They proposed that the sarcoma 180 tumor was releasing a soluble, diffusible, growth-promoting factor that altered the differentiative and growth properties of the target cells.

7 Levi-Montalcini and Hamburger: In order to test their proposal, they transplanted a mouse sarcoma onto the chorioallantonic membrane (an extraembryonic membrane) of the chick embryo to prevent direct contact of the tumor and chick tissues. The circulatory system connects the two tissues. It was found that the extra-embryonic transplants elicited the same effects as the intraembryonic grafts, supporting the proposed diffusible nature of the nerve-growth promoting agent being produced by the tumor. Their attempts to replicate their findings using dried tumors and by injecting tumor extracts into the embryos were unsuccessful. They reasoned that the use of cell culture techniques would greatly facilitate the identification of the factor.

8 Levi-Montalcini goes to Rio de Janeiro Dr. Levi-Montalcini developed a collaboration with Professors Carlos Chagas and Hertha Meyer at the University of Brasil in Rio de Janerio. She reports boarding an airplane (presumably in St. Louis) carrying in my handbag two mice bearing transplants of mouse sarcomas 180 and 37. (Levi-Montalcini 1986 Nobel lecture). The tumor had given a first hint of its existence in St. Louis, but it was in Rio de Janeiro that it revealed itself, and it did so in a theatrical and grand way, as if spurred by the bright atmosphere of that explosive and exuberant manifestation of life that is the Carnival in Rio [ref. 14 in 1986 Nobel lecture].

9 Halo effect identified by Rita Levi-Montalcini (1954) Sensory and sympathetic ganglia were removed from chick embryos and cultured in a semi-soft medium in proximity to, but not in contact with, fragments of mouse sarcoma tumor 180 or 37. Within 24-hours, Levi-Montalcini and colleagues noted a halo of nerve fibers growing out from the ganglia, with the highest density of fibers being on the side facing the tumor. Figure 3. Eight-day-old sensory ganglia from chick embryos. (a) The ganglion, which faces a fragment of chick embryonic tissue (ct), shows fibroblasts but few nerve fibers. (b) Ganglion cultured in the presence of fragments of mouse sarcoma for 24 h. (c) Ganglion cultured in the presence of fragments of mouse sarcoma for 48 h. In (b) and (c), the ganglia, facing fragments of sarcoma (s), show the typical halo effect elicited by the growth factor released from the sarcoma. In (c), note the first evidence of a neurotropic effect of the growth factor. Reproduced, with permission, from Ref. [7]. (1986) The Nobel Foundation. Aloe 2004

10 Effect of NGF Figure 4. Photomicrographs of sensory ganglia removed from an eight-day-old chick embryo and cultured for 24 h at 37 8C. Ganglia were cultured (a) in a medium containing no nerve growth factor (NGF) and (b) in a medium containing 10 ngml of NGF. Note that only the ganglion that was exposed to NGF displays a dense halo of nerve fibers. Reprinted, with permission, from Ref. [14]. q (1964) American Association for the Advancement of Science ( Aloe 2004

11 1954: Stanley Cohen Levi-Montalcini returned to St. Louis ( ?). Shortly before her return, Stanley Cohen, a biochemist, had begun working with Viktor Hamburger. Cohen isolated a fraction from the sarcomas that promoted neuronal growth in vitro. Cohen, Hamburger, and Levi-Montalcini proposed the name Nerve Growth Stimulating Factor, which was later shortened to Nerve Growth Factor (NGF).

12 : Cohen purified and characterized NGF, first from snake venom, then from mouse submandibular salivary glands. 1960: Cohen, while purifying NGF from submandibular glands, began to test for other growthpromoting activities. He noted that, when fractions that did not contain NGF-like activity, were injected into newborn mice, there was a precocious opening of the eyelids, an early eruption of the incisors, and a stunting of growth. 1962: Cohen isolated the second factor and called it tooth-lid factor 1964: Cohen renamed the second factor epidermal growth factor (EGF), based on his observation that in vitro it had a direct effect on epidermal cell growth.

13 Identification of other neurotrophins (post 1964) 1982: brain-derived neurotrophic factor (BDNF) was purified 1990: neurotrophin-3 (NT3) was identified by cloning techniques : the gene for neurotrophin 4 (NT4) was cloned from Xenopus laevis; subsequently, the mammalian homolog of NT4 was cloned but was given the name neurotrophin 5 (NT5) because its sequence is more divergent from the Xenopus counterpart than are the sequences of other neurotrophin homologs. Generally, the term NT4/5 is used to refer to this gene and its product.

14 Summary 4 neurotrophin genes: NGF BDNF NT3 NT4/5

15 Neurotrophins are synthesized as proneurotrophins The protein product of each neurotrophin gene includes a signal sequence, which is cleaved in the ER, a prodomain, and the mature neurotrophin sequence. Proneurotrophins are cleaved intracellularly by furin and other prohormone convertases in the trans-golgi network. Extracellular probdnf is cleaved by several matrix metalloproteinases (MMPs) and by plasmin following its activation (cleavage) by tissue plasminogen activator (tpa).

16 Pro- and mature neurotrophins Both pro- and mature forms of neurotrophins are released by cells. Both processed (mature) and unprocessed (proform) neurotrophin proteins form homodimers in solution. NGF is primarily secreted in its mature form, whereas BDNF is primarily secreted as probdnf. NGF is released through the constitutive secretory pathway, while BDNF (probdnf) is predominantly secreted through the regulatory (activity-dependent) pathway.

17 Pro- and mature neurotrophins Pro-domains play a role in protein folding and dimerization Pro domain appears to play a role in apoptosis In general, the proneurotrophins and the neurotrophins exert opposing biological actions

18 BDNF gene Each neurotrophin is translated from a single coding exon. In the case of BDNF, multiple mrna transcripts are present within a cell. From Aid et al J Neurosci Res. 85:

19 Why so many transcripts? May allow for cell-type or subcellular localization differences May (probably) have different mechanisms for controlling expression from differing promoters. Different signals may generate different transcripts. Transcripts may have differing stabilitymay be able to regulate duration of local BDNF production

20 Functions of proneurotrophins prongf: apoptosis probdnf: apoptosis; dendritic remodeling probdnf: may regulate synaptic efficacy during development probdnf: induction of LTD

21 Functions of neurotrophins Target-derived survival factor for developing neurons (original identification) Growth cone guidance Synaptic modulation Roles in learning and memory Neurogenesis

22 Neurotrophin-receptor interactions Two types of neurotrophin receptors have been characterized: 1. Trk (tropomyosin receptor kinase) family 2. p75 NTR (p75 neurotrophin receptor), which is a member of the tumor necrosis factor receptor (TNFR) superfamily. A third neurotrophin receptor has been identified but not well characterized: sortilin. Reichardt 2006

23 Complexity of the TrkB gene

24 Complexity of the TrkB gene

25 Complexity of TrkB proteins

26 Neurotrophin-receptor interactions Trk receptors display selectivity for neurotrophins. p75 NTR binds all neurotrophins with similar affinity. Trk and p75 NTR bind neurotrophins with an equilibrium binding constant of K d ~ 10-9 M when expressed alone. When they are co-expressed, the K d is increased to ~10-11 M. The ligand specificity of the Trk-p75 NTR is determined by the Trk moiety. Neurotrophins have been proposed to physically interact solely with the Trk constituent of the Trkp75 NTR complex. p75 NTR is thought to alter the conformation of the Trk receptor, increasing its affinity for ligand. Sortilin binds NGF with relatively low affinity (K d ~10-8 M) and prongf and probdnf with realtively high affinity: 10-9 M and K d ~10-10 M, respectively). Sortilin associates with p75 NTR further increasing it affinity for prongf (~10-10 M). Schweigreiter (2006) BioEssays

27 p75 NTR complexes with sortilin and with TrkA Nykjaer et al (2004) Nature 427:

28 Functions of prongf binding to the p75 NTR /sortilin complex cell death occurring during development injury- e.g., acute neuronal loss following spinal cord injury and in seizure models disease progression- e.g., neuronal loss during aging and in Alzheimer s Disease patients Antagonists of prongf binding to the p75ntr/sortilin complex may be useful in the treatment/prevention of disorders resulting from injury and in the treatment of Alzheimer s Disease and aging

29 Functional duality of neurotrophins and proneurotrophins in the nervous system Mature neurotrophins and/or Trk receptor Proneurotrophins and/or p75 NTR /sortilin Cell fate survival apoptosis Glial migration & differentiation Stimulatory & inhibitory, respectively Inhibitory & stimulatory, respectively Transmitter phenotype noradrenergic cholinergic Activity-dependent synaptic plasticity LTP LTD Schweigreiter 2006

30 Neurotrophin signalling. Reichardt L F Phil. Trans. R. Soc. B 2006;361:

31 BDNF and synaptic plasticity BDNF produces fast effects on synaptic transmission by posttranslational modification of synaptic proteins in both the presynaptic and postsynaptic compartments. BDNF increases depolarization-dependent glutamate release (note: a presynaptic event), possibly as the result of the phosphorylation of components of the exocytotic machinery (e.g., synapsin I and II). BDNF also exerts effects on postsynaptic events via mechanisms dependent on protein phosphorylation: BDNF increases NMDA receptor single channel open probability as the result of NMDA receptor phosphorylation; BDNF stimulates delivery of GluA1- containing AMPA receptors as the result of protein phosphorylation. BDNF also regulates synaptic plasticity via effects on gene trascription and mrna translation.

32 BDNF-dependent regulation of synaptic transmission Fig. 2. BDNF regulates glutamatergic synaptic transmission by acting at the pre- and post-synaptic level. BDNF sequestered in secretory vesicles present in the post-synaptic region is released by a Ca2+-dependent mechanism, following activation of glutamate receptors. BDNF acts on pre-synaptic TrkB receptors, potentiating glutamate release, and exerts shortand long-term effects in the post-synaptic cell. BDNF induces the translocation of AMPA receptors to the synapse and increases the activity of NMDA receptors by phosphorylation-dependent mechanisms. Furthermore, BDNF induces local protein synthesis at the synapse from mrnas transported along dendrites in RNA granules, by promoting the disassembly of the granules (1) and activating the translation machinery (2). Additional effects of BDNF include the regulation of RNA transport along dendrites, which is mediated by kinesin motor proteins, and activation of gene expression (3).

33 Local protein synthesis The majority of mrna translation in neurons occurs in the cell body Translation of some mrnas can occur independent of the soma at synaptic sites in dendrites and growing axons Various stimuli induce local protein synthesis at synapses Local protein synthesis has been shown to play a role in synaptic plasticity, neurite growth and development. BDNF regulates translation initiation and elongation through mechanisms dependent on the activation of mtor, PI3-K and ERK signaling pathways.

34 BDNF, like netrin-1, induces synthesis of β- actin in neuronal growth cones under attractive conditions Figure 1. The differential translation model for local translation in growth cones. (a) A gradient of attractive guidance cue, such as netrin-1, induces asymmetrical activation of translation and transport of mrnas, causing asymmetrical translation of proteins that build up the cytoskeleton, which leads to attractive turning. (b) A gradient of repulsive guidance cue, such as Slit-2, induces similar asymmetrical activation of translation but induces transport and translation of different mrnas, causing asymmetrical translation of proteins that disassemble the cytoskeleton, which leads to repulsive turning. Lin & Holt 2008

35 Figure 2. Local translation and communication between the axon and cell body. (1) Stimulation of axons leads to transcription-independent differential localization of mrnas to the axon through transport on microtubules, changing the population of mrnas available for local axonal translation. (2) Newly synthesized transcription factors can be retrogradely transported on microtubules to the cell body where they influence transcription. Lin & Holt 2008

36 Why synthesize proteins locally rather than transport proteins (either mature or immature ) that were synthesized in the soma? increased flexibility for the regulation of localization and activation: regulatory elements in the 5 UTR and 3 UTR do not affect function of the protein; in contrast, regulatory elements in the protein do affect localization and function increased speed of response more efficient: possible macromolecular crowding in axons/synapses may preclude storage of multiple copies of each of the proteins that may be needed; it is more efficient to store mrna and synthesize and degrade proteins, as needed.

37 Mitogen-activated protein kinases (MAP kinases; MAPKs)

38 Mitogen-activated protein kinases (MAPKs) proline-directed, serine/threonine kinases- i.e., MAPKs phosphorylate serine or threonine residues that precede proline residues: PX(S/T)P Three families: 1. extracellular signal-regulated protein kinase (ERK) 2. C-Jun amino-terminal kinase (JNK, also called stressactivated protein kinases, SAPK) 3. p38 stress-activated protein kinase (p38; also called RK/CSBP) Activated by dual phosphorylation on both tyrosine and threonine residues in the activation lip. Regulate several MAPKAPKs (MAPK-activated protein kinases), transcription factors, and post-translational processes (e.g., mrna stability).

39 MAPK activation/inactivation MEK 2ATP 2ADP MAPK T-X-Y MAPK T-X-Y P P 2P i MKP

40 MAPK phosphorylation sites minimal consensus substrate sequence of (S/T)-P optimal consensus substrate sequence of P-X-(S/T)-P ~90% of all proteins contain an (S/T)-P sequence, yet, not all of these are MAPK substrates

41 Mechanisms of MAPK interaction with a substrate 2 site model: phospho-acceptor site and docking site 2 types of docking domains: 1. D-domain; Related motifs have been identified in a number of proteins and have been given various names, including DEJL (docking sites for ERK and JNK, LXL) domain, kinase interaction motif (KIM), MAPK-docking site, D box, D-site and D-domain. 2. FXFP motif; also called the DEF (docking site for ERK, FXFP) motif

42 D-domains Although these domains were identified based on the ability to bind one or more MAPK, there are differences in the consensus sequences used to identify each of them. Consensus sequences D-domain (Kornfeld et al.): (K/R)-X-(X/K/R)-(K/R)-X (1-4) -(L/I)-X-(L/I) (K/R)-(K/R)-(K/R)-X (1-5) -(L/I)-X-(L/I) KIM sequence (McKenzie et al.): (V/L)X2(R/K)(R/K)X (3-6) L MAPK-binding site (Bardwell et al.): (R/K) 2 X (2-6) (L/I)X(L/I)

43 D-domains Model of Sharrocks and colleagues Basic LXL Φ

44 The CD domain and docking groove on MAPKs Proteins bind to a region on MAPKs termed the common docking (CD) domain. This region is enriched in negatively charged amino acids. Docking of proteins to the CD domain is mutually exclusive. Regions adjacent to the CD domain are also important for docking; thus, the concept of a docking groove

45 MAPK pathways MAPKKK MAPKK MAPK

46 MAPK pathway inhibitor

47 References Aloe (2004) Trends Cell Biol. 14: Keshishian (2004) J. Exp. Zoo. 301A: Reichardt (2006) Phil. Trans. R. Soc. B. 361: Schweigreiter (2006) BioEssays 28: