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Neurotrophic Factors and Programmed Cell Death. 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.

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Neurotrophic Factors

and

Programmed Cell Death


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.


Effect of removing or augmenting neural targets on the survival of related neurons
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.


Effect of removing or augmenting neural targets on the survival of related neurons1
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.


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.


1954: neurite outgrowth assay that the targets of innervating neurons provide signals that recruit undifferentiated cells to develop into sensory or motor neurons.

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


Neurotrophins in the cns
Neurotrophins in the CNS that the targets of innervating neurons provide signals that recruit undifferentiated cells to develop into sensory or motor neurons.

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.


  • 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.


Regulation of neurotrophin synthesis by physiological activity
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)


- seizure activity (generalized activation) Activity

- stimulation of LTP

- normal physiological stimuli, such as light  visual cortex; general physical activity, sensory stimulation, enriched environments.


Regulation of synaptic transmission by neurotrophins
Regulation of Synaptic Transmission by Neurotrophins Activity

  • 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).


Some other growth factor families cytokines
Some Other Growth Factor Families: Cytokines Activity

  • 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.


  • Include: Activity

    - 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:


  • Neuropoietic Cytokines: Activity

    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.


  • TGF Superfamily: Activity

    - 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).


FGF: Activity

- 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.






Tyrosine kinase Activity

Receptor activation:


Tyrosine kinase Activity

Receptor activation:



NGF: Activity 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


Neurotrophin Evolution Activity

Neurotrophins have only been isolated from chordates

Hallbook (1999) Curr Opin Neurobiol9: 616-21



The trk family of receptor tyrosine kinases for the neurotrophins
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


Alternative splicing generates many Trk receptor isoforms Neurotrophins

NGF KD for TrkA=10-11M

Roux and Barker (2002) Prog Neurobiol 67:203-233


p75 NeurotrophinsNTR structure

NGF KD=10-9M

(all neurotrophins

can bind p75NTR)

Roux and Barker (2002) Prog Neurobiol 67:203-233


Models for Trk and p75 NeurotrophinsNTR interaction

Chao and Bothwell (2002) Neuron 33:9-12


p75 NeurotrophinsNTR is required for developmental myelination

Cosgaya et al. (2002) Science298:1245-1248


(Found only in fish) Neurotrophins


The effect of NT/NTR knockouts on neurons in the DRG Neurotrophins

Fariñas et al. (2002) Brain Res Bull 57:809-816


Trk receptor signaling Neurotrophins

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


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


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


Ngf signal can be transduced at the tips of growing neuronal processes
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.


Does internalized ngf contribute to sympathetic neuron survival
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.


Activated Trk can signal locally and retrogradely using different signalling pathways

Slow (2-20 mm/hr)

Miller and Kaplan (2001) Neuron 32:767-770


Differential control of different signalling pathwaysTrkA 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


In vitro different signalling pathways 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.


Neurotrophins’ roles in neuronal development different signalling pathways

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.


Neurotrophins in the CNS different signalling pathways

The highest levels of neurotrophins are found in the hippocampus

Lindsay et al. (1995) Trends Neurosci 17:182-190.



  • 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.



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


Transfer of GFP-BDNF requires synaptic activity neuron

Kohara et al. (2001) Science 291: 2419-2423


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


Result: of neurotrophins in dendritic remodeling: local release of BDNF alters nearby dendritic structure

Horch and Katz (2002) Nature Neuroscience 5: 1177-1184


Selection of CNS synapses via BDNF of neurotrophins in dendritic remodeling:

Strong presynaptic activity results in release of more BDNF. The postsynaptic site responds by elevating the amount of AMPA receptors and nNOS. This mechanism could contribute to selective facilitation (e.g. maintenance of LTP).


BDNF is also involved in regulating energy balance/feeding behavior

BDNF/TrkB signaling is downstream of the melanocortin-4 receptor

Xu et al. (2003) Nature Neuroscience6: 736-742


Something to contemplate: behavior

In rodents, a diet that is high in saturated fat and refined sugar results in reduced hippocampal BDNF expression

Molteni et al. (2002) Neuroscience112: 803-814


Cytokines and Growth Factors in the Nervous System behavior

CNTF, LIF (oncostatin M, cardiotrophin-1): neuropoietic cytokines. These factors may be important in neuronal response to injury.

GDNF, neurturin, artemin, persephin: exhibit distant homology with the TGF-b family. They signal through a receptor complex composed of the Ret tyrosine kinase and a GPI-linked binding subunit (GFRa family; GFRa1, a2, a3, and a4). These factors are potent axon-promoting growth factors in vivo for developing sympathetic and parasympathetic neurons.


Signaling behavior

Pathways:







Neurotrophins can protect striatal neurons from excitotoxic injury

Albrech et al. (2002) Brain Res Bull 57:817-822.

In the striatum, TrkB receptors are the most abundant, followed by TrkC



Sensitive Period injury

Anatomy and physiology are especially sensitive to modulation by experience.

Critical Period

An extreme form of Sensitive Period.

Appropriate expression is essential for the normal development of a pathway or set of connections (and after this period, it cannot be repaired).

e.g., There was a critical period for the formation of ocular dominance columns, based on neuronal activity/input from both spontaneous firing and visual inputs from the eyes.


If appropriate information is not received during the critical period (from experience), this pathway never attains the ability to process information in a normal fashion, and as a result, perception or behavior can be permanently impaired.

E.g., development of appropriate social and emotional responses to others.

E.g., development of language skills in humans.


More on learning and early experiences later
More on Learning and Early Experiences Later critical period (from experience), this pathway never attains the ability to process information in a normal fashion, and as a result, perception or behavior can be permanently impaired.


What happens to neurons in the absence of neurotrophic factors?

Apoptosis is a mechanism contributing to programmed cell death (PCD)


Morphological types of cell death factors?

Apoptosis: originally defined according to a set of characteristic ultrastructural features that include nuclear and cytoplasmic condensation, cell fragmentation and phagocytosis.

Necrosis: cell death as the result of injury, disease, or pathological state (usually involves large numbers of cells and is associated with inflammation). Chromatin condenses in multiple small clumps and at later stages, cell membranes and organelles disintegrate.

Autophagy: (from the Greek, self-eating) Cytoplasm is destroyed by lysosomal enzymes before any nuclear changes become visible. A characteristic feature is the appearance of large autophagic vacuoles in the cytoplasm. At later stages, chromatin condenses, DNA laddering is evident and phagocytosis occurs.


Necrosis factors?

PCD

apoptotic

autophagic

Morphology of cell death


Ultrastructure of cell death factors?

apoptosis

necrosis




Regulation of PCD in mitotic neural progenitors depends, in part, on asymmetric distribution of PAR-4 and elevated ceramide

Ceramide (a lipid that can act as an intracellular 2nd messenger)

Bieberich et al. (2003) J. Cell Biol. 162: 469-579.


Genes involved in PCD are highly conserved part, on asymmetric distribution of PAR-4 and elevated ceramide

C. elegans

Mammalian homologs of the C. elegans PCD genes

Ced-3 = caspases (cysteine aspartate proteases)

Ced-4 = Apaf-1

Pro-death: Bax, Bak [Bim, Brd, Dp5/Hrk, Bad]

Pro-life: Bcl-2, Bcl-XL, IAPs

Ced-9 = Bcl-2 family


pro-casp3 part, on asymmetric distribution of PAR-4 and elevated ceramide

Death signaling pathways

Extrinsic

Intrinsic

FasL

Bim

Bcl-2

FasR

Bax

PTP

FADD

Bid

Cyt C

pro-

casp9

APAF

pro-casp8

casp8

casp9

“Apoptosome”

APOPTOSIS

casp3


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