Growth factors and cell death. Urmas Arumäe Institute of Biotechnology

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1 Growth factors and cell death Urmas Arumäe Institute of Biotechnology

2 Growth factors stimulate cell survival (among other activities). Or conversely: absence of the growth factors causes cell death.

3 Survival-promoting activity = trophic activity. Survival-promoting factors = trophic factors. Two meanings: - Anabolic, nutritional, stimulating metabolism, cell growth etc. Greek trophikos [τροϕικός] = of food, nourishment, nutrition. - Antagonizing cell death program (mainly apoptosis). Anti-apoptotic activity. Trophic factors stimulate both. Topic here only the anti-apoptotic activity.

4 Generally, the healthy cells in vivo do not require survival stimulation. Life a default, normal status. No special efforts for survival required. During development, many (if not all?) cells have special periods (stages) where their life must be actively maintained by trophic factors from other cells. Programmed (ontogenetic) cell death periods Many cells die due to lack of survival stimulation by trophic factors. Healthy cells die (just not required for the organism). Developmental checkpoints. Best studied in neurons (neurotrophic factors) and lymphocytes (interleukines). Coherent models. Other cell types less studied. Increasing number of factors that regulate survival of embryonic cells (sonic hedgehog, netrin, BMPs, ephrins etc). No coherent models yet.

absence of a neurotrophic factor NGF (all other conditions are the same).

5 Sympathetic neurons at the programmed death stage (first neonatal week) cultured with nerve growth factor (NGF) The same culture deprived of NGF Healthy neurons die only due to absence of a neurotrophic factor NGF (all other conditions are the same). After programmed death period, the mature neurons do not die any more due to NGF deprivation. Arumäe, unpublished

6 Same occurs in the sympathetic ganglia in vivo during programmed death period (about first neonatal week) Before programmed death During programmed death Counting the dying (pyknotic) neurons from the sections of embryonic ganglia. The number of dying neurons increases in the wild-type animals at the time of programmed death (E18, P1). No dying neurons after this period (adult animals) (not shown). NGF knockout (NGF -/- ) ganglia (= NGF deprivation) almost all neurons die. Middleton & Davies, Development Dec;128(23):

7 Growth factors critically control embryonic development at two principal levels: - when bound to the receptors, they trigger proliferation, differentiation, survival etc. - their absence (at some ontogenetic stages) causes cell death Both activities are absolutely required for the proper embryonic development.

8 Apoptosis: overview of the basic principles

9 Modes of cell death Programmed cell death: intentional removal of unwanted cells. Active, controlled. Cell participates in its own death. - Apoptosis: major morphologically, biochemically and genetically defined cell death program. - Other poorly studied non-apoptotic death programs (death with increased autophagy, necroptosis). - Pathological cell death often mixed (both apoptotic and non-apoptotic features). Non-programmed cell death: unintentional killing by overwhelming injury. Traumatic. Accidental. Passive. Uncontrolled. - Necrosis: major morphological appearance of accidental death.

10 Cells are dying by death programs: Physiologically: normal, healthy cells that are not required any more. This death is as much normal and essential aspect of development and physiology as any other (proliferation, differentiation, adaptation etc.). Pathologically: sick, stressed cells that are requied but become harmful or dysfunctional.

11 Programmed death of healthy cells in development Sculpting the shape of developing organs, e.g.: cavity of epiblast lumenae of ducts interdigital tissue etc. Removing temporary organs not any more necessary, e.g.: Müllerian ducts in males and Wolffian duct in females pronephric kidneys in mammals tadpole tails in frogs larval muscles and neurons in insects etc. Establishing proper organ size (opposite to proliferation). Cell overproduction apoptotic removal of surplus. Removal of self-reactive or non-reactive lymphocytes.

Early foetus: both Müllerian and Wolffian ducts Müllerian duct removal Wolffian duct removal The lumenae of gut (and other tissues) are sculpted by programmed cell death.

12 Early foetus: both Müllerian and Wolffian ducts Müllerian duct removal Wolffian duct removal The lumenae of gut (and other tissues) are sculpted by programmed cell death. Programmed cell death eliminates temporary ducts during development of the male and female inner reproductive organs. The interdigital mesoderm, initially formed between fingers and toes, is removed by programmed cell death.

13 Morphogenetic cell death in brain development (brain shape formation) e.g. flexures are generated by cell death; the biology of this death almost not studied Massive collective apoptosis in some brain areas vitally important for morphogenesis.

14 Programmed cell death of healthy cells in adult organism Removal of unnecessary cells (e.g. activated T or B cells after immune response, intestinal epithelial cells etc.). Regulation of cell number in tissues (opposite to proliferation): tissue homeostasis, tissue turnover. Especially in highly proliferative tissues. In adult human several million cells are produced every second by mitosis and a similar number die by apoptosis. During life span, most of our cells die. During a year, we lose about one body weight by programmed death.

15 Physiological cell death in development In most cases the actual triggers are not yet known. Often absence of survival factors. Sometimes actively killing factors/receptors.

Martin Raff (University College London): theory of social control in the tissues. Cells secrete survival-promoting factors for each other that keep the neighbours alive (block apoptosis).

16 Martin Raff (University College London): theory of social control in the tissues. Cells secrete survival-promoting factors for each other that keep the neighbours alive (block apoptosis). In development, there are periods where cell death seems to occur by default if not suppressed by signals from other cells. Programmed (ontogenetic) cell death periods. The only thing our cells can do on their own is to kill themselves, and the only reason they normally remain alive is that other cells are constantly stimulating them to live. (Raff M. Social controls on cell survival and cell death. Nature, 1992, 356: ).

17 Necrosis Healthy cell Pyknosis, chromatin condensation Apoptosis Necrotic swelling Fragmentation (apoptotic bodies) Cell rupture From: Kerr et al., Methods Cell Biol., 1995, 46:1 Inflammation Immune response Phagocyte Removal of apoptotic bodies (often whole cells)

18 Biochemical-cellular pathways of apoptosis 1. Mitochondrial (intrinsic) pathway: death program is released inside the cell. Mitochondria involved. 2. Death receptor (extrinsic) pathway: death program is activated at cell surface by other cells (ligation of death receptors). The non-apoptotic pathways (autophagic death, necroptosis etc.) are poorly studied. Mostly in pathological situations. KEGG (Kyoto Encyclopedia of Genes and Genomes) database: genes coordinately working in apoptosis

19 Mitochondrial pathway of apoptosis: main characteristics Triggered by mild stress, absence of survival (trophic) factors, or still undiscovered stimuli. Bcl-2 family proteins are activated/inactivated. Apoptotic molecules (cytochrome c, Smac, AIF, Omi etc) released from the mitochondria to the cytosol. Cytochrome c triggers activation of caspases. Activated caspases destroy the cell.

20 Bcl-2 family proteins Proteins that control the survival/death decisions of the cells. Divided by activity: - pro-apoptotic (Bax, Bak, Bok etc.) - anti-apoptotic (Bcl-xL, Bcl-2, Bcl-w etc.) - regulatory BH3-only (Bid, Bad, Bim etc.) Death stimulus activates first the BH3-only proteins. Activated BH3-only proteins inactivate anti-apoptotic members. This leads to activation of pro-apoptotic members (details not clear). Activated pro-apoptotic Bax, Bak etc. generate pores (channels) into mitochondrial membranes (and endoplasmic reticulum).

Interactions between the classes of Bcl-2 family proteins in the (mitochondrial) apoptosis: a current model Willis et al., Science, 2007, Vol. 315. no. 5813, pp.

21 Interactions between the classes of Bcl-2 family proteins in the (mitochondrial) apoptosis: a current model Willis et al., Science, 2007, Vol no. 5813, pp BH3-only proteins are activated first by apoptotic stimulus. Each have its own mechanism of activation (proteolysis, dephosphorylation, transcription etc.). BH3-only proteins bind to and inactivate antiapoptotic proteins. Inactivation of anti-apoptotic proteins releases block from pro-apoptotic effector proteins (indirect activation). How - is not fully clear.

22 Core machinery of the mitochondrial apoptotic pathway inactive Healthy cell active Apoptotic cell inactive inactive active active inactive caspase-9 Apoptotic stimulus activates BH3-only Bcl-2 family proteins (e.g. Bim, Bad, Bid). These in turn inactivate anti-apoptotic members (e.g. Bcl-xL, Bcl-2) that leads to activation of pro-apoptotic members (Bax, Bak). Activated Bax and/or Bak generate pores (channels) to the mitochondrial membranes. Mitochondrial proteins (cytochrome c, Smac, Omi etc.) are released to the cytosol. In the cytosol, these proteins activate caspases apoptotic proteases. Active caspases cleave downstream substrates, that in turn cause apoptotic death.

Mol Interv. 2003 Feb;3(1):19-26 Caspases Family of cysteine proteases specifically activated in apoptosis. Present in the cells as inactive zymogens. Activated proteolytically by caspases themselves.

23 Mol Interv Feb;3(1):19-26 Caspases Family of cysteine proteases specifically activated in apoptosis. Present in the cells as inactive zymogens. Activated proteolytically by caspases themselves. Initiator caspases are activated at specific platforms (DISC, apoptosome); they in turn activate executional caspases (cascade). Cut specifically selected proteins that are thereby activated or inactivated. Cell death is irreversible (point of no return) when caspases are activated above a certain threshold. Mitochondrial apoptotic pathway is specifically initiated by caspase-9, that activates caspase-3.

(e.g. absence of trophic (survival) factors) Activation of caspase cascade at the apoptosome (mitochondrial pathway) Pro-caspases and Apaf-1 (a scaffold for procaspase accumulation) are inactive in

24 (e.g. absence of trophic (survival) factors) Activation of caspase cascade at the apoptosome (mitochondrial pathway) Pro-caspases and Apaf-1 (a scaffold for procaspase accumulation) are inactive in the cytosol of healthy cells Release of cytochrome c to the cytosol triggers assembly of the apoptosome (Apaf-1 and pro-caspase-9, an initiator caspase) Pro-caspase-9 is activated at the apoptosome active caspase-9 Activated caspase-9 cleaves and activates an effector caspase (caspase-3) that in turn activates caspases -6 and -7 From Biochimie (2-3) apoptosis

25 Caspases make one or two cuts to the selected substrates, that either inactivates (loss-of-function) or activates (gain of apoptotic function) them. Cleave and inactivate proteins (e.g. nuclear lamins nuclear shrinking; cytoskeletal fodrin loss of cell shape; polyadp-ribose polymerase (PARP) reduced genome surveillance etc.) Cleave and inactivate inhibitors of death proteins (e.g. ICAD cleaved CAD released DNA cleavage) Separate regulatory and effector domains of proteins activation or inactivation (e.g. other caspases, PAK2 kinase and gelsolin increase in cytoskeletal tension membrane blebbing apoptotic bodies) No wholesale protein degradation. Caspases are among the most specific proteases. Casbah: The CAspase Substrate database

26 Trophic factors actively suppress apoptosis at programmed death period Factor bound survival signaling? Survival (with factor): BH3-only inactive Bax inactive Bcl-xL active Caspases inactive Death (no factor): BH3-only active Bax active Bcl-xL inactive Caspases active Factor bound - receptor signaling actively suppresses death program. PI3K-Akt pathway essential. Link between Akt and death machinery poorly known. Factor removal - death program passively released.

27 Trophic factor signaling prevents activation of BH3-only proteins.? Other possible checkpoints to suppress apoptotic machinery. Poorly known. PI3K - Akt kinase pathway essential (but not the only one). Bad phosphorylation (inactivation) shown only in few cases. Not general.

28 PI3K-Akt pathway potently blocks apoptosis PI3K: phosphoinositide 3-kinase Akt1 = protein kinase B (PKB): serine-threonine kinase Overexpression of Akt or constitutively active Akt increased survival of the cells in apoptotic conditions. Blockage of Akt (by e.g. dominant-negative constructs) increased sensitivity to apoptosis (including the cancer cells). Virtually every hormone and growth factor that has ever been investigated has been shown to have some effect on PI3K-Akt pathway activity.

29 Liao Y, Hung MC. Am J Transl Res Jan 1;2(1):19-42 Consensus Akt phosphorylation motif analysis: there are potentially thousands of cellular substrates for Akt; about 50 of them have been characterized so far. Among them: Apoptotic proteins, activated/inactivated by phosphorylation: Bad, caspase-9, ASK1, apoptosis signal-regulating kinase 1 (ASK1), forkhead box O transcription factors (FoxOs), Bim, FasL, inhibitor of nuclear factor-κb kinase (IKK-NFκB), p53. Regulators of protein synthesis or cell growth: tuberous sclerosis complexes 1 and 2 (TSC1/2), mtor, elongation-initiation factor 4E binding protein-1 (4E-BP1), S6K.

Activation of PI3K-Akt pathway by trophic factors. A generic (consensus) pathway Target substrates of PI3K/PKB whose proapoptotic activities are suppressed by phosphorylation.

30 Activation of PI3K-Akt pathway by trophic factors. A generic (consensus) pathway Target substrates of PI3K/PKB whose proapoptotic activities are suppressed by phosphorylation. Role of these substrates in cell survival controversial. These substrates shown in some situations/cells only. No common, main survival-promoting substrate. Not a common pathway (yet?). Akt is just one, best-studied survival-promoting mechanism of growth factors.

31 An example of physiological apoptosis caused by lack of survival (trophic) factors): regulation of neuronal number by neurotrophic factors. A best studied model.

Adjusting the number of postmitotic neurons during programmed (ontogenetic) cell death periods Almost every neuronal population has a developmental period where 20-80% of initially

32 Adjusting the number of postmitotic neurons during programmed (ontogenetic) cell death periods Almost every neuronal population has a developmental period where 20-80% of initially generated neurons die. Oppenheim RW (1991) Cell death during development of the nervous system. Annu Rev Neurosci 14: Ronald W. Oppenheim, Wake Forest University, Winston-Salem, NC, USA

33 Oppenheim RW (1991) Cell death during development of the nervous system. Annu Rev Neurosci 14: Table 1. A partial list of instances of normal neuronal death in vertebrates. Neuronal population Animal Motoneuron Cochlear nuclei chicken, mouse spinal fish, frog, turtle, opossum, wallaby, mouse, Inferior olive chicken, rat rat, chicken, quail, monkey, human Inferior colliculus rat trochlear salamander, frog, chick, quail, duck, human Isthmo-optic nucleus chicken, duck abducens duck Optic tectum hamster, chicken, rat, lizard oculomotor duck, mouse, chicken Ectomammillary nucleus chicken facial mouse Various visual nuclei tree shrew trigeminal chicken Para bigeminal nucleus rat electro-motor electric fish Lateral geniculate nucleus mouse Spinal ganglion Pineal ganglion Ciliary ganglion frog, chicken, rat chicken chicken Retina mouse, rat, rabbit, guinea pig, cat, marsupial cat, quokka (marsupial), wallaby, frog, chicken, hamster, monkey, human Sympathetic ganglion chicken, rat Cerebellum chicken, li7.ard, mouse, rat Cochlear ganglion chicken Habenulae nucleus mouse Vestibular ganglion chicken Thalamus lizard Nodose ganglion chicken, quail Hippocampus mouse, rat Trigeminal ganglion chicken, mouse, rat Corpus striatum lizard, rat Otocyst rat Cerebral hemispheres (forebrain) chicken, zebrafinch Enteric neurons Lateral line Sympathetic preganglionic cells guinea pig frog chicken Olfactory cortex Cerebral cortex rat rat, cat, mouse, hamster Dorsal motor nucleus of vagus chicken Mesencephalic nucleus of trigeminal chicken, hamster, frog Rohon-Beard (sensory) neurons frog Neuronal precursor cells of the adrenal rat

34 Programmed death period of postmitotic neurons occurs during synaptogenesis with the targets. Neurons are initially overproduced, then their number is reduced to normal by programmed death. At the programmed death period, all neurons are intrinsically apoptotic. Model: their apoptosis is suppressed by neurotrophic factors from the innervated tissues (targets). Targets produce limited amounts of neurotrophic factors. Neurons that do not get enough neurotrophic (survival) factors, die due to non-suppressed apoptosis.

35 Target-derived neurotrophic model for neuronal survival/death during programmed death period All neurons of this population, just innervating the target, are intrinsically apoptotic. Target cells produce neurotrophic factors that block neuronal apoptosis. Neurotrophic factors are transported from nerve terminals to the cell bodies. Neurons not receiving neurotrophic factors die (not rescued from apoptosis). Neurotrophic factors: not enough for all neurons. Only for required number of neurons. Tested rigorously for peripheral (sensory and sympathetic) neurons and spinal motoneurons. From: Barde, Neuron, 1989, 2:1525

36 Programmed death periods of peripheral and spinal motoneurons mouse sensory neurons (third embryonic week): NGF, BDNF, NT-3 mouse sympathetic neurons (first postnatal week): NGF chick ciliary parasympathetic neurons (second embryonic week): CNTF mouse spinal motoneurons (third embryonic week): NGF, BDNF, NT-3, CNTF, GDNF, NRTN, ARTN, others

Survival/death of the neurons at programmed death stage is controlled by neurotrophic factors (limited amounts) No target Two targets Less neurons More neurons, overinnervation Knockout mice of

37 Survival/death of the neurons at programmed death stage is controlled by neurotrophic factors (limited amounts) No target Two targets Less neurons More neurons, overinnervation Knockout mice of neurotrophic factors or their receptors: respective (peripheral) neurons die at programmed death stage. Excess NGF at the time of programmed death more NGF-dependent neurons survived. Function-blocking NGF antibodies less neurons survived (immunosympathectomy). Main biological result: systems matching. Quantitative optimization of connectivity between neurons and their targets.

Neurotrophic factors control the apoptotic machinery of the neurons During programmed death periods, neurotrophic factors actively suppress apoptosis. In their absence, apoptosis acts freely.

38 Neurotrophic factors control the apoptotic machinery of the neurons During programmed death periods, neurotrophic factors actively suppress apoptosis. In their absence, apoptosis acts freely. Neurotrophic factors activate survival kinase Akt (protein kinase B), that somehow keeps the core apoptotic machinery suppressed. In the absence of neurotrophic factors, BH3-only proteins are activated. Bim, Hrk/DP5 and Noxa are induced transcriptionally. BH3-only proteins activate Bax mitochondrial apoptosis. Link(s) between Akt, BH3-only proteins and Bax poorly known. Eugene M. Johnson Washington University School of Medicine, St. Louis When programmed death period is over, neurons survive without neurotrophic factors (apoptosis suppressed by default).

39 Gilley J, Coffer PJ, Ham J (2003) FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. J Cell Biol 162: An example how a trophic factor (NGF) controls transcriptional activation of BH3-only protein Bim in the sympathetic neurons during programmed death period. FOXO3a (Forkhead Box O transcription factor) transcriptional activity is inhibited by NGF via PI3-K signaling and Akt/SGK-mediated phosphorylation. (SGK: Serum- and Glucocorticoid-inducible Kinase, related to Akt) NGF deprivation activates FOXO3a (dephosphorylation) that binds to and activates Bim promoter. Bim activates Bax mitochondrial apoptosis. This is just one link between trophic factors, Akt kinase and Bax activation. Generic?

40 How does the programmed death period end?

41 Easton RM, Deckwerth TL, Parsadanian AS, Johnson EM, Jr. (1997) Analysis of the Mechanism of Loss of Trophic Factor Dependence Associated with Neuronal Maturation: A Phenotype Indistinguishable from Bax Deletion. J Neurosci 17: Putcha GV, Deshmukh M, Johnson EM, Jr. (2000) Inhibition of apoptotic signaling cascades causes loss of trophic factor dependence during neuronal maturation. J Cell Biol 149: In mouse sympathetic neurons, programmed death period occurs during first postnatal week. At that time, the neurons die in culture without NGF. After this period, the neurons do not die by NGF deprivation any more. A. Mature neurons (23 days with NGF) + 16 days with NGF C. Immature neurons (5 days with NGF) + 2 days with NGF B. Mature neurons (23 days with NGF) deprived of NGF for 16 days Living neurons with reduced size (atrophic) D. Immature neurons (5 days with NGF) deprived of NGF for 2 days All neurons dead Reduction in size of NGF-deprived neurons loss of anabolic stimulation.

Mature neurons: Bax levels were not changed. Also the levels of Bcl-xL, Bcl-2, caspases. NGF deprivation did not activate Bax, cytochrome c not released, caspases not activated.

42 Mature neurons: Bax levels were not changed. Also the levels of Bcl-xL, Bcl-2, caspases. NGF deprivation did not activate Bax, cytochrome c not released, caspases not activated. BAX and cytochrome c undergo subcellular redistribution during trophic factor withdrawal in immature, but not mature, sympathetic neurons. Neurons were maintained in NGF for 5 (immature; DIV 5) or 25 d (mature; DIV 25) with caspase inhibitor BAF and then deprived of NGF for 24 or 96 h respectively, and immunostained for Bax. Punctate localization in b shows apoptotic activation of Bax in the NGF-deprived immature neurons. Not in the mature NGF-deprived neurons. Bax-activating mechanisms are free during programmed death period, but under control (repressed) after it.

43 Kole AJ, Swahari V, Hammond SM, Deshmukh M (2011) mir-29b is activated during neuronal maturation and targets BH3-only genes to restrict apoptosis. Genes & Development 25: Micro-RNA mir-29b represses BH3-only protein mrnas in the mature but not young neurons. No BH3-only proteins no Bax activation. Binding of micro-rnas to the untranslated regions of mrna translational arrest or degradation of the mrna mir-29b binding sites on the untranslated regions of BH3-only proteins Bim, Bmf, Hrk, Puma and N-Bak in the sympathetic neurons

44 mir-29b is upregulated during neuronal maturation. Overexpressed mir-29b prevented death of young apoptotic neurons. mir-29b repressed mrnas of BH3-only proteins in the luciferase assay. BH3-only proteins were induced in the young but not mature neurons by apoptotic stimuli. Proposed model showing that high mir-29b levels in mature neurons prevent induction of BH3- only proteins after apoptotic stimuli. Apoptotic stimuli cause cytochrome c release and death in young neurons, while mature neurons remain resistant.

45 Death of non-reactive immature thymocytes during lymphopoiesis another example of programmed cell death period.

46 90% of all thymocytes fail to express functional T cell receptor die by mitochondrial apoptosis (death by neglect, lack of positive selection). A checkpoint in T cell development, where nonreactive thymocytes are removed. T-cell receptor activation expression or function of cytokine (mainly interleukine-7, IL-7) receptors IL-7, secreted by the thymic stromal cells, promotes survival. Conversely: absence of T-cell receptors absence/non-functionality of IL-7 receptors IL-7 cannot promote survival. Apoptosis is free in the developing (but not mature) thymocytes. Must be suppressed by cytokines (IL-7). Only in the cells that generate functional T Cell Receptor complex. For review: Opferman Apoptosis in the development of the immune system. Cell Death and Differentiation (2008) 15,

47 T cell receptor (TCR) engagement with self-peptide MHC complexes promotes expression or function of IL-7R. Mechanism not fully clear. Binding of IL-7 to IL-7R suppresses intrinsic apoptotic machinery in the T-cell progenitors. Akt active, BH3-only proteins (in particular Bim) suppressed, Bax not activated, caspases not activated. Absence of IL-7R no trophic stimulation mitochondrial apoptotic machinery kills the cells. Dramatic loss of thymocytes and mature T cells in mice that lack IL-7 or IL-7Rs.

48 Ontogenetic death of thymocytes: Occurs during specific stage of T-cell development (selection of functional T-cells). Lymphopoiesis occurs throughout the lifetime (neurogenesis once in the lifetime). Healthy cells die (not required for the organism). Developmental checkpoint. Only those thymocytes that bind the MHC/antigen complex with adequate affinity will receive a vital survival signal. Intrinsic apoptotic machinery is temporarily free, trophic factors (interleukin-7) suppress it. Trophic factor receptor is the bottleneck (neurons trophic factor itself).

49 Main points At programmed death periods, cells are competent to die by lack of survival signaling (death program free). Other cells produce survival/trophic factors that suppress death program in the wanted cells, but let it be activated in the unwanted cells (social control). Ligand-bound receptors (generally) signal survival (among other activities), deligation of the receptors (sometimes) allowes cell death to occur. De-ligation of the receptors during programmed death periods leads to activation of the core mitochondrial apoptotic machinery. The details are yet poorly known. In addition to neurotrophic factors and interleukines, many more growth factors may control the survival/death of the developing cells. Yet poorly studied. Apoptosis can be also triggered by various activated death receptors. Topic of next lecture.

Cell Death & Trophic Factors II. Steven McLoon Department of Neuroscience University of Minnesota

Cell Death & Trophic Factors II Steven McLoon Department of Neuroscience University of Minnesota 1 Remember? Neurotrophins are cell survival factors that neurons get from their target cells! There is a