Apoptosis during brain development
Estimated that ~1/3 of the neurons that differentiate during development ultimately die before adulthood
* Progenitor cells in the ventricular zone show high levels of apoptosis during late development
How do we assess cell death?
TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling
- Detects apoptotic DNA fragmentation
- Relies on terminal deoxynucleotidyl transferase
- An enzyme that attaches deoxynucleotides tagged with a marker to DNA double strand breaks
- DNA double helix breaks during apoptosis
Development of the frog hindlimb
- Peak motor neuron population is ~4000 during
development of the limb - By adulthood, ~1200 motor neurons exist
- 60% are eliminated
~50% of rat retinal ganglion cells also die during development
Stages of mouse development when apoptosis is blocked
- A/B: embryonic day 10.5
- C/D: embryonic day 13.5
- E/F: embryonic day 16.5
Defects and too much apoptosis is also lethal
Trophic Factors
Survival factors
Necessary for maintenance of neuronal connections and neuronal survival
Provided in limited quantities
Trophic Factors Sources
- Neurons receive trophic factors from:
- Target tissues that they innervate
- Synaptic inputs
- Neighboring neurons that do not synapse directly on them - Paracrine
- Distant cells through the circulatory system
- Glial cells
Nerve growth factor (NGF)
First trophic factor identified
Produced by target tissues of sympathetic neurons
* Sympathetic nervous system
Homeostasis regulation, pupil dilation, gut motility, fight or flight
Experiments implicating target tissue in ganglion size
Manipulation of limb bud number directly dictates
size of dorsal root ganglia sensory neurons
Hamburger’s prediction
Discovery of NGF
- The hypothesis: The target tissue (in this case limb) secretes a factor that stimulates neuron proliferation - Hamburger’s prediction
- The experiment: Remove the limb and count neurons
- The results: No change in number of differentiated neurons. Proliferation not changed! But neurons die when their axons reach the amputated stump
Target tissue secretes a factor that is essential for neuronal survival
New goal: Identify that factor using tumors promoted neuronal survival and snake venom also promoted neuronal survival
Discovery of NGF
Inhibit function and assess neuron survival
Tumors and snake venom also promoted neuronal survival
Raised an antibody against the unknown protein and
inject it into rabbits
* Antibodies bind to proteins – block protein from normal functions
* E = experimental animal
* C = control animal
Ganglia are reduced with antibody injection
Sympathetic neurons undergo programmed cell death within 24- 48hrs after NGF withdrawal
The internal cell death program
- After NGF withdrawal, mitochondrial cell death program is activated
- Release of Cytochrome C from mitochondria activates caspases that initiate cell death
Neurotrophins
NGF is one
Subset of neurotrophic factors that have similar structures
Neurotrophin Structure
- Growth factors such as NGF are produced as propeptides. ~250 amino acid long protein
- Processed post-translationally and cleaved. ~120 amino acid peptide is the final product
- Peptides homodimerize to create a biologically
active molecule
NGF, BDNF, NT-3, and NT-4 also share conserved protein sequences
- Share 50% protein homology
- Concentrated in six hydrophobic regions
Also have a unique domain necessary to bind specific receptors
Neurotrophin Receptors
Neurotrophin receptors bind to 1 or 2 neurotrophins
* High homology between receptors
* ~50% homology in the extracellular domain
* Each receptor is also highly spliced leading to a wide array of variability in receptor sequence.
* NGF bind to TrkA
Neurotrophin signaling is critical to the survival of disparate neurons
Neurotrophin Signaling
- Signals received from target tissue can function locally in the axon terminal or distantly in the cell body.
- Supported by work in vitro
- Neurons can be cultured in a device that isolates their cell bodies from their processes
- Addition of neurotrophin to axons allows and supports target dependent neuronal survival and allows cell body survival but axon degeneration
- Supports a role for neurotrophin signaling in the cell body for neuronal survival AND a role for local neurotrophin activity in the axon terminal for axon maintenance
Neurotrophins from target tissues are essential for:
- Local processes in the axon terminal that support axon maintenance
- Distant processes in the cell body that support neuron survival
- This can be circumvented by direct application of the neurotrophin to the cell body in cultured neurons
Retrograde transport of neurotrophin signaling endosomes
Changes in transcriptional programs
* Example: NGF signaling activates CREB and NFAT, two powerful transcription factors
Produces proteins essential for axon maintenance and cell survival
* Prevents mitochondrial induced apoptosis
Retrograde transport of signaling endosomes
For this, ligands (eg NGF) and receptors (eg TrkA) must be internalized into a signaling endosome
