Synaptic Plasticity

Question 1

Apoptosis during brain development

Answer 1

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

Question 2

How do we assess cell death?

TUNEL assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling

Answer 2

• 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

Question 3

Development of the frog hindlimb

Answer 3

• 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

Question 4

Stages of mouse development when apoptosis is blocked

Answer 4

• 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

Question 5

Trophic Factors

Survival factors

Answer 5

Necessary for maintenance of neuronal connections and neuronal survival

Provided in limited quantities

Question 6

Trophic Factors Sources

Answer 6

• 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

Question 7

Nerve growth factor (NGF)

First trophic factor identified

Answer 7

Produced by target tissues of sympathetic neurons
* Sympathetic nervous system

Homeostasis regulation, pupil dilation, gut motility, fight or flight

Question 8

Experiments implicating target tissue in ganglion size

Answer 8

Manipulation of limb bud number directly dictates
size of dorsal root ganglia sensory neurons

Question 9

Hamburger’s prediction

Discovery of NGF

Answer 9

• 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

Question 10

Discovery of NGF

Inhibit function and assess neuron survival

Tumors and snake venom also promoted neuronal survival

Answer 10

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

Question 11

The internal cell death program

Answer 11

• After NGF withdrawal, mitochondrial cell death program is activated
• Release of Cytochrome C from mitochondria activates caspases that initiate cell death

Question 12

Neurotrophins

NGF is one

Answer 12

Subset of neurotrophic factors that have similar structures

Question 13

Neurotrophin Structure

Answer 13

• 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

Question 14

Neurotrophin Receptors

Answer 14

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

Question 15

Neurotrophin Signaling

Answer 15

• 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

Question 16

Neurotrophins from target tissues are essential for:

Answer 16

• 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

Question 17

Retrograde transport of neurotrophin signaling endosomes

Answer 17

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

Question 18

Retrograde transport of signaling endosomes

Answer 18

For this, ligands (eg NGF) and receptors (eg TrkA) must be internalized into a signaling endosome