Cellular NeurobiologyBIPN140

1st Midterm Exam Ready for PickupBy the elevator on the 3rd Floor of Pacific Hall (waiver)

Exam Depot Window at the north entrance to Pacific Hall (no waiver)Mon-Fri, 10:00 AM to 4:00 PM

1st midterm regrade: contact the IA who graded the question directly before Nov 14, 2016.

PS4 Q&A will be posted on October 27th

Nick’s Office Hour this Wednesday (Oct 26) is canceled.

Chih-Ying’s Office Hour: Monday, 1:00-2:00 PM, Bonner Hall 4146

BIPN140 Lecture 10: Intracellular Signaling

Su (FA16)

1. Overview of Chemical Signaling

2. G protein signaling & Phosphorylation

3. Nuclear Signaling

4. Receptor Tyrosine Kinase Signaling

Chemical Signaling in Neurons (Fig. 7.1)

Amplification in Signal Transduction Pathways (Fig. 7.2)

Enzyme Enzyme

Depends on the half life of the activated receptor

Depends on the half life of the GTP-bound G

Three Classes of Cell Signaling Molecules (Fig. 7.3)

Lipophilic (e.g. steroidhormone)

Categories of Cellular Receptors for Secreted Signals (Fig. 7.4)

e.g. membrane bound guanylyl cyclase

Types of GTP-binding Proteins: Large G Proteins (Fig. 7.5)

GEFs: Guanine nucleotide exchange factors

GAPs: GTPase activating proteins (the endogenous GTPase activity is usually too slow to terminate the signal)

Effector Pathways Associated with GPCRs (Fig. 7.6)

Types of GTP-binding Proteins: Small G Proteins (Fig. 7.5)

GEFs: Guanine nucleotide exchange factors

GAPs: GTPase activating proteins (the endogenous GTPase activity is usually too slow to terminate the signal)

Neuronal Second Messengers (Fig. 7.7B-D)

Ca2+-induced Ca2+ release (CICR)

Membrane phospholipid

Neuronal Second Messengers (Fig. 7.7A)

Gs

Gi/o

Gq

Extracellular Ca2+

Intracellular Ca2+

Different PLC isoforms are differentially regulated by Gq, Ca2+, and receptor tyrosine kinase

Requires ATP

Powered by Na+ gradient

Regulation of Cellular Proteins by Phosphorylation (Fig. 7.8)

Phosphorylation Sites

(Mendez et al., Neuron 28, 153-164, 2000)

Mechanism of Protein Kinase Activation (Fig. 7.9)(cAMP-dependent protein kinase)

(calcium/calmodulin-dependent protein kinase II )

(protein kinase C)

Regulation of Tyrosine Hydroxylase by Phosphorylation (Fig. 7.14)

Receptor Tyrosine Kinases

Kandel et al., Principles of Neural Science, 5th Edition, Figure 11-9

Ligand bindingDimerizationPhosphorylation of its partner

Receptor auto-phosphorylationRecruitment of adaptor proteinsDifferent downstream signaling events

Mitogen-activated protein kinase (MAPK): activate other kinases by direct phosphorylation (MAPKK or MAP2K, MAP3K, MAP4K)

GRB2

GEF

Tyrosine Kinases (Fig. 7.12)

Nuclear Signaling/Transcription (Fig. 7.10)

Upstream activator site

Transcriptional Regulation by CREB (Fig. 7.11)

Background: Target tissues of sympathetic and sensory neurons secret NGF to promote nerve growth/differentiation and survival. The neurotrophic effect of NGF requires activation of CREB transcription factor at the cell body. However, it is unclear how NGF signaling at the axon terminal is propagated back to the soma which can be over 1 meter away. Is NGF internalized by itself with activated TrkA back to the cell body for the transmission of the signal? Alternatively, is some downstream signaling proteins (ras, MAPK?) or 2nd

messengers generated at the axon terminal upon NGF-TrkA interaction transported back to the cell body?

Experiments: Use compartmentalized cultures of sympathetic neurons (axon terminals and cell bodies are cultured in different chambers). Generate phospho-specific antibodies that label site specific-phosphorylated and unphosphorylated CREB (anti-P-CREB) and TrkA (anti-P-TrkA). This system enables exposure of terminals or cell bodies to NGF and then to assess by immunocytochemistry the phosphorylation (activation) state of CREB and TrkAin cell bodies.

1. Maintain the culture in low NGF (2 ng/ml).2. Stimulate the culture with high NGF (200 ng/ml) at

either axon terminals or cell bodies.3. Immuno-stain with anti-P-CREB and/ot anti-P-TrkA4. Speed: 2-4 mm/hour

Whole-cell lysates (controls)

Cell bodies

Axon terminals and distal processes

Cell bodies

Axon terminals and distal processes (center-plated)

Side-plated

Fig. 1. Phosphorylation of CREB after application of NGF to sympathetic neurons

Campenot Chamber

anti-P-CREB

Fig. 2. Internalization of NGF is required for CREB activation at cell bodies

NGP-coupled microbeads: can activate TrkA but can not be internalized

anti-P-TrkA

Results: Internalization of NGF and its receptor TrkA, and their transport to the cell body, were required for transmission of the transmission of target-derived NGF signaling. The tyrosine kinase activity of TrkA was required to maintain it in an auto-phosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus the NGF-TrkAcomplex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons.