MCDB 423 Lecture 28: Lecture 28

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University of Michigan - Ann Arbor
Molecular, Cellular and Developmental Biology
MCDB 423
John Kuwada

Lecture 28 Slide 1 All of our cells have this apoptosis machinery -Can lose neurons in an unwanted fashion by activating this mechanism or by losing control of metabolic pathways Apoptosis is a program which has signaling molecules that happens in the cell Necrosis happens as a default mechanism Slide 2 Neurotrophin signaling by binding to a neurotrophin receptor is triggering downstream signaling including -Transcriptional responses that can stimulate transcription of anti-apoptic factor such as Bcl2 Not having neurotrophin signaling would be a bad thing for the maintaince of your neurons in the brain Slide 3 We also saw that target derived neurotrophins is important for inhibiting apoptosis In the chick, there are target derived neurotrophins secreted from the developing muscles from the limp buds Slide 5 One thing one can do to study signaling within axons specifically from cell bodies is to culture neurons in compartmentalized chambers which will allow you to expose axons and cell bodies to different manipulative environments -The classic name for these chambers is called a campenot chamber In the top-down view (left), there’s a chamber where you put the cell bodies and there’s another chamber where the axons are growing -In the side view (right), the axons are growing out in a tiny space there where only axons can go through -There’s vasline coating so that fluid can’t exchange between the chambers -It’s a way to manipulate the environment of different parts of the neuron slide 6 This uses nano-fabrication There are tiny groves where the neurons can grow their axons in the yellow part You can have fluid in the yellow and black part -Due to differences in pressure, the fluids won’t mix -Another way to keep the fluid separate in the chambers The red dye is just a waterphilic so it doesn’t bind to membranes But the green can bind to membranes and fill the membranes of anything it touches -The green dye is labeling neurons whose axons are going through the grove This can be used to look at neurotrophin signaling or axon guiding signaling specifically in axons Slide 7 Answer = c A classic experiment that was done was to grow your DRG neurons in culture and they require serums which has growth factors to keep them alive -You can inhibit nerve growth factor signaling by adding antibodies that will bind to the nerve growth factor and prevent the factor from binding to the receptor -This is a quick way to inhibit NGF If you add NGF to this chamber, within a day all the neurons will die But you can prevent that death by adding recombinant NGF just to the axon terminals -You can also see phosphorylated TrkA, NGF, etc. of these neurons slide 8 Receptors are present at synapses and axons Receptors when bound to ligand are physically trafficked into endosomes all the way to the cell body -Then we can see downstream signaling occurring in the cell body slide 9 Receptor signaling works more effectively once the receptor has been endocytosed The endosome can be trafficked or recycled Slide 10 Answer = b Adding the NGF to the distal axons triggers formation of endosomes that can be trafficked back to the cell body Old experiment that suggested that this trafficking was important: -Instead of providing soluble NGF, provide NGF only in the association of a huge bead that is too big to be endocytosed by the neuron -These NGF beads were then added to the container If this retrograde hypothesis is correct, what will happen? -The neurons with the NGF bead will die slide 11 You could take labeled NGF -Quantum dot label is a very bright label Within 30 secs, the labeled NGF is physically moving in the axon to the cell body in a very direct and rapid manner Slide 12 This is requiring then endocytosis and requiring retrograde transport vi
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