L5: Second Messengers and Intracellular Signalling

What is the difference between primary and secondary messengers?

Primary

- carries signals between cells (e.g NT, hormones )

Secondary

- carries signals within cells , following primary messenger

Why are second messengers important?

They are involve in Synaptogenesis, Nociception, NT release, Neurite outgrowth, Proliferation, Gene expression, Synaptic plasticity, Inflammation, Enzyme regulation and Cell viability

Describe the association of 3 major R classes with 2nd messenger function ?

1) GPCRs

- G alpha s => increase cAMP by adenylate cyclase

- G alpha i => decrease cAMP promo by adenylate cyclase

- G alpha q/11 => increase IP3 and DAG production via PLC

2) Ligand-gated ion channels => Ca 2+ entry (VGCCs)

3) Tyrosine Kinase-linked R => phosphorylation cascade activate effector enz PLC b and protein K

What is signal amplification

Activating one receptor activates multiple second messenger molecules and each has an effect on cellular process

Give examples of signal amplification ?

Pain sensitisation, NGF and protein kinase cascade

What is the role of NGF ?

Neuronal proliferation and survival during nervous system development (bind to TrkA on peripheral neurons )

When is NGF release ?

During tissue damage and inflammation => increased excitability of sensory nerves

How does NGF alter pain processing ?

Altered gene expression (e,g CGRP, sub P)

Reducing Nav activation threshold => increasing the activity of TRPV1

Describe what happens after NGF binds to trkA ?

1) 2 monomers that are kept apart => dimerise and phosphorylate

2) Conformational change => GRB2 adhere to it => SOS binds to GBR2 => recruit RAS => forming a complex

3) Exchange of GDP for GTP => RAS activated => recruits RAF => phosphorylates MEK

4) Phosphate gc attached to MAP kinase => activates MAP => cytosolic effects and can translocate to nucleus so affects transcription

How is activation of trkA an example of signal amplification ?

Many molecules of MEK can be phosphorylates by one RAF

What is the role of AKAP ?

Scaffolding protein binds signalling molecules in close proximity so ensures signals are created, terminated and signalling is restricted to micro domain not entire cytoplasm

What modulates memory formation ?

cAMP via activation of PKA or CREB phosphorylation

How is cAMP synthesised and metabolised

ATP to cAMP via Adenylate Cyclase

cAMP to AMP via Phosphodiesterase

How many isoforms of adenylate cyclase their in the CNS ?

8

- Isoform 1,3,8 activated by calcium/calmodulin (CaM)

- Isoform 5&6 inhibited by calcium (CaM-independent)

What activates and inhibits all isoforms of AC ?

G alpha s = activates

G alpha i = inhibits

What processes does Ca2+ regulate ?

Memory, function, vesicular trafficking and neurogenesis

State the short term and longer term effects of cAMP ?

Short term effects = strengthening synapses by phosphorylation and increase trafficking of GluR, Kv channels and GABA R

Long term effects = transcriptional regulation via CREB

What are the characteristics of CREB knockout mice ?

- Memory deficits

- Impaired hippocampal LTP

(CREB suggested to be important survival factor for dopaminergic neurons in the substantia nigra)

As most proteins do not bind Ca2+ ions directly what is the alternative ?

Effects via calmodulin (Calcium modulated protein - CaM)

What are the two type of Ca2+/Calmodulin-dependent protein kinases (CaMKs) that many effects rely on ?

1) Specialised (e.g myosin light chain kinase)

2) Multifunctional (e.g CaMK II, primarily expressed in neurons) - one target of CaMKII is CREB

What does sustained increase in Ca2+ cause ?

Apoptotic/necrotic cell death = excitotoxicity

Describe how increase in calcium leads to chopping up of DNA ?

Increase Ca 2+ => activation of proteases, endonucleases, phospholipases => damaging cytoskeleton, cell membrane and DNA

What pathologies are high levels of Ca 2+ associated with ?

Stroke

Traumatic brain injury

Neurodegenerative disease = PD and AD

How does the cell regulate Ca2+ conc ?

Primary and Secondary AT

- conc kept low via SERCA ATPase, PMCA ATPASE, NCKX, NCX

What happens when Ca2+ is needed ?

- Enters via Ca2+ permanent channels

- Released from Ca2+ intracellular stores by activation of GPCR => activate phospholipid so generate IP3 and DAG => IP3 binds to R on endoplasmic reticulum => Ca2+ release