Lecture 10 - Neurotrophic Factors I Flashcards
Developmental process— Neurulation
(Folding process in vertebrate embryos -transformation of the neural plate into
the neural tube.)
What determines differentiation/formation
of neural tissues?
Phylogenetically similar
Neural tube — forms the central nervous system
Induction & patterning are guided by:
* Intrinsic factor: Molecules induced in cells upon exposure to an inducing factor.
* Extrinsic factors: signaling molecules provided by other cells.
* Local events: contact and diffusible factors.
Thus, cells must be “competent’ and precisely timed to provide and receive signals. A complex multistep process.
How is phenotype controlled by signals from the neuronal target, using sympathetic neurons as a model?
Sympathetic neurons
* Most produce norepinephrine as transmitter.
* But those innervating sweat glands in foot pads uses acetylcholine.
How can this arise? - switching of biosynthetic machinery
Early observations (1920s-40s)
Transplantation of target organ
* Interpretation: factor/s produced by the target tissue increase the number of neuron or prevent the number of neurons from dying naturally.
* Target tissue influence the number of neuron in mature animal
Neurotrophic factor hypothesis
* Developing neurons compete with each other for a limited supply of neurotrophic factor/s (NTF) provided by the target tissue.
* Successful competitors survive; unsuccessful ones die.
What was the evidence that NGF affects sympathetic neuron development?
Administration of anti-NGF to new born mice
Loss of neuron in ganglia and
Complete disappearance of sympathetic chain of ganglia.
When deprived of NGF for 3-5 days.
Neuroactive factors: affecting survival of neurons
What are examples of some factors?
Neurotrophins
NERVE GROWTH FACTOR
NEUROTROPHIN-3
BRAIN-DERIVED NEUROTROPHIC FACTOR
NEUROTROPHIN-4/5
GDNF family of ligands (GFL)
GLIAL CELL-LINE DERIVED NEUROTROPHIC FACTOR (GDNF)
NEURTURIN (NTN)
PERSEPHIN (PSP)
ARTERMIN (ART)
Neurotrophic factors (NGF, BDNF, NT3, NT4/5)
What are their effects?
Classical effects — neuronal cell survival
Others:
Regulate axonal/dendritic growth and guidance.
Synaptic structures/connections
Neurotransmitter release
LTP
Synaptic plasticity.
Development of other non-neuronal organs.
Involvements:
Glial biology.
Vascular formation.
Tumor progression.
Dorsal root ganglia — sensory ganglion derived from neural crest
What is their function?
Cells involved in sensing pain, touch, pressure.
Cells express peptides, small molecules as neurotransmitters.
Some lessons from knockout studies
* Homozygous knock-out of neurotrophins (NGF and NT 1-4) — lethal mutations.
* Haploinsufficiency — (reduced amount in +/- mice) — many other deficiencies…
Neurotrophic factors requirement may be switched in the lifetime of the organ
What is an example?
- At birth (day1-P1) - 80% neuron express TrkA
- As DRG develops into adult, the number of neuron expressing TrkA receptor decreases.
- Dependency of NGF is decreased with development in small diameter neuron (IB4+ cells).
- Remove NGF(<2 d) — decrease nociceptive neurons and hypoalgesia
GDNF is the dependent factor when DRG cells develop to maturity
How was this found?
- A subpopulation of DRG cells (IB4+) switch growth factor dependence from NGF in early development to GDNF.
- Most IB4 cells express Ret (GDNF receptor system) in late development.
Phenotypic changes when NGF deprived 4-11 days — - No. of DRG cells - unchanged.
- Increasing Ret-expressing population of nociceptors differentiates into 3 subclasses of neurons expressing specific Mas-related G-protein-coupled receptors (Mrgpr) - MrgprA3; MrgprB4; MrgprD
What is the first neurotrophin discovered?
NGF is the first Neurotrophin discovered
4 others
Brain derived neurotrophic factor (BDNF), Neurotrophin 3 (NT 3), Neurotrophin 4/5 (NT4).
Phylogenetics: ligand and receptor found in lower animals- snail, frogs, avian, fish, zebra fish, fugu. snake But not in flies or c. elegans.
What are the features of neurotrophins?
Neurotrophins associate into non-covalent dimers
Neurotrophin-Receptor interactions
* Neurotrophins bind to TrK (tropomyosin-related kinase)
* Ratio of binding: 2:2 (neurotrophins: Trk)
Distinct contact sites of Trk binding with neurotrophins
* Shows high identities between different Trk — involve in direct binding of ligands.
What are the features of TrK?
TrK biogenesis and trafficking
* TrkA-containing endosomes in neurons are traffic in anterograde & retrograde
* NGF-TrkA binding results in internalization of ligand-receptor complex
Internalized NGF-TrkA have activities — Endosomal signalling
* Signalling is prolonged.
* Retrograde traffic is critical.
Trk can interact with other membrane components
Early studies
* TrK show specificity of ligand interactions and affinity to receptor is increased in the presence of a co-receptor, p75
No evidence of TrkA-p75 directly binding to each other
Free flow in membrane to partner with more
What are some interesting and puzzling observations regarding Trk and NGF?
How can they happen?
- p75 can enhance neurotrophin binding to Trk — increase affinity and specificity of ligand-Trk interactions.
- High doses of mature neurotrophin elicit cell death through p75NTR independent of Trk.
- Activation of p75NTR can also induce apoptosis in oligodendrocytes, neurons, and vascular smooth muscle cells when Trk activation is reduced or absent.
How can this happen?
* Tissue specific expression of multiple NGF forms
*Multiple isoforms of NGF with Mw 13.5 — 30 Kd —due to different processing of the proprotein.
NGF can bind to p75 directly too but not high affinity. Why?
- p75NTR also known as Low-Affinity Nerve Growth Factor Receptor, LNGFR (nerve growth factor receptor (TNFR superfamily, member 16)
- Note- contact sites are similar with all NT — thus, p75 binds all.
- However, proNGF (proNT) can bind to p75 with high affinity in the presence of yet another co-receptor, Sortilin.
What is Sortolin?
Sortilin: prototype of the Vps10p domain receptor family
Involve in both
* Cell Signalling
* Intracellular protein sorting
Sortolin plays important roles in TrkA neuronal trafficking
Proneurotrophins can bind to co-receptors directly
What is the outcome of the different partnerships?
Sortilin > proneurotrophins > p75NTR > death
Sortilin > neurotrophins > Trk > survival
p75NTR > neurotrophins > Trk > survival
Multicomponent receptor system with multiple interacting partners
What are the factors contributing to signalling specificity: to live or die?
- Type of heterodimeric neurotrophin receptor pairing
- Activation by mature versus pro-neurotrophins
- Post-activation proteolytic processing & trafficking
- Transactivation
What is GDNF?
Member of GDNF family of ligands (GFL)
Glial Cell Line-Derived Neurotrophic Factor
Produced by glia cells and promotes neuron survival
What is neuturin?
Member of GDNF family of ligands (GFL)
Factor with sympathetic survival-promoting activity
What is perserphin?
Another member of the GDNF family of ligands (GFL)
What is artermin?
Another member of GDNF family of ligands (GFL)
What is the mechanism of GDNF ligands (GFLs) signalling?
Multicomponent system - GFLs bind to GFRα1-4 then pair with p75, c-RET, N-CAM etc.
As they are not transmembrane, can be cleaved to soluble GFRα1-4 and bind to other cells
How does p75 neurotrophin receptor directly
interact with Ret and GFRα?
p75 enhances GFL signaling by modulating Ret level on the cell surface
Loss of p75 leads to defects in GFL-mediated survival of Ret expressing neurons in vitro
p75 is required for the postnatal diversification of nonpeptidergic nociceptors in vivo
What can GDNF and other GFL do?
Neuronal & Extraneuronal activities
- Neuronal survival (dopaminergic/ cholinergic/ sympathetic)
- Motor neuron protection
- Retinal photoreceptor/retinal ganglion survival.
- Cochlear nerve development.
- Intestinal ganglion cell development.
- Testis — spermatogonial stem cell development.
- Promotes primordial follicle development
- Neural stem cell differentiation — “maturation” to DA neurons
- Kidney absence — GFRα1 knockout —die. Similar to RET KO.
- Memory deficiency/enteric neurons — GFRα2 knockout.
- Adenoma (MEN2A/2B)/GIioma/Hirshprung disease
- Disorders of CNS — Parkinson’s disease/Stroke/epilepsy/Alzheimer’s disease/ Neuropsychiatric disorders
What is the EVIDENCE that GDNF may be a useful therapeutics?
Survival of midbrain dopaminergic neuron in vitro.
Rescue of dopaminergic neuron (100%) in post MPTP (10 days treatment)
treated mouse
Rescue of dopaminergic neuron in post MPTP treated mouse using gene therapeutics (Adenoviral in the striatum/ AAV or Lentiviral in SN-striatal)
Cell-based therapy
(GDNF producing fibroblast/P19/HEK/astrocytes/C2C12 cells preclinical study)
Monkey (Rh) MPTP model: Single i.c.v. dose of GDNF
- Reverse MPTP induced behavioural changes (last for 2-3 wk).
- Neurochemical parameters: SN/ globus pallidus showed increase in DA (106% & 86% increase respectively).
- Protective and restorative properties of GDNF
No good animal model that completely recapitulate Parkinson’s disease
Genetic Knockout studies
How come NO changes in catecholaminergic neuron number but yet GDNF seem to help?
Limitation of knockout assays - lethal at birth/compensation. Cannot decipher late developmental stages and switch of dependence.
Absolute requirement of GDNF for adult catecholaminergic neuron survival
o Significant selective/extensive catecholaminergic neuronal death, (locus coeruleus, substantia nigra and Ventral tegmental area).
Other neuronal systems, e.g, GABAergic and cholinergic pathways, appeared unaffected.
o Mutants -progressive behavioural motor disturbances, consistent with the parallel neurochemical/histological losses.
Conflicting results
Various genetic models including same approach did not show same results
o Possibly due to mice genetic difference, method of KO not reproducible
o Compensation by gdnf production from other cells
Resolution?
These genetic models are not perfect
o KO still have GDNF expressed but low levels
o Reducing levels have corresponding behavioural deficits
o Likely GDNF is required for maintenance of DA neuron
What is the EVIDENCE that Neurturin (NTN) may be a more useful therapeutic than GDNF?
Survival of midbrain dopaminergic neuron in vitro.
Rescue of dopaminergic neuron in post MP TP treated mouse
Important differences:
* GDNF: allodynia
* NTN: no observable side effects
