NP Postsynaptic Processes

Learning Objectives:

• Identify the major elements of post-synaptic densities and their roles in synaptic transmission

• Discuss mechanisms that organize post-synaptic densities and key proteins involved

• Discuss trans-synaptic mechanisms by which postsynaptic density proteins help organize presynaptic active zones.

What you should know:

• What is a postsynaptic density

• Postsynaptic densities differ according the type of synapse

• Neuroligins and other CAMs form key parts of the density and signal via different pathways

• PSD95 is a core organising molecule due to its 3 PDZ domains and other signalling/binding domains

• Cytoskeletal proteins like Shank can have important signalling functions

The Postsynaptic Density

Post-synaptic specialization

• Membrane and cytoplasmic proteins clustered immediately opposite release sites or active zones at synapses

• These proteins include:

  • Ligand gated ion channels

  • Anchoring proteins

  • Cytoskeleton

  • Regulatory proteins

• Known as density because appears dense in electron microscopy

EM of a glutamatergic synapse.

PSD core: high concentration of PSD-95.

• PSD-95 is orientated with N-terminus near the plane of postsynaptic membrane and C-terminus deep in the spine.

• This close apposition suggests PSD-95 in a position to bind neurotransmitter receptors.

PSD pallium: deeper layer, containing a scaffold of Shank and Homer proteins.

• The pallium becomes denser and more prominent during intense synaptic activity due to reversible addition of calcium calmodulin Kinase II and other proteins.

There are a large variety of proteins in the postsynaptic density

• In the brain, excitatory synapses probably have more than 1000 different proteins

• Inhibitory synapses have at least 250 different proteins

• Protein names are often uninformative about function

  • e.g. Homer, Shank, PSD95, PSD93, gephryn, collybistin

Some proteins we know

• MAGUKs (membrane-associated guanylate kinases).

  • The most abundant MAGUK at the mammalian post synaptic density is PSD-95 (DLG4, SAP90);

    • others include SAP97(DLG1), PSD-93 (DLG2,Chapsyn-110), and SAP102 (DLG3).

• GKAP (guanylate kinase-associated protein)

  • also called PSD- 95- associated protein, or disks large-associated protein (DAP-1).

  • Binds to Shank and PSD-95.

• Shank (SH3 and multiple ankyrin repeat domains protein)

  • also called ProSAP (Proline-rich synapse-associated protein), Synamon and CortBP (Cortactin binding protein).

• Homer also called Vesl, Cupidin, and PSD-Zip45

  • Major function is to crosslink different proteins

Many PSD proteins are cell adhesion molecules

• Need cell adhesion molecules to keep structures intact

• Many rare mutations in synaptic function genes underly autism (red)

• Central role for neuroligin-neurexin is maintaining synapse function

Summary

• Membrane and cytoplasmic proteins clustered immediately opposite release sites or active zones at synapses

  • Needed to anchor receptors, signalling molecules etc.

• PSD core (superficial) and pallium (deep)

• Difference in protein composition of excitatory vs inhibitory synapses

  • More in excitatory synapses

• Key proteins such as PSD-95, GKAP, Shank and Homer

• Many PSD proteins are CAMs

Neuroligins and Neurexins

Neuroligins

Neuroligins organize the post synaptic density

• Neuroligin (NLGN), a type I membrane protein, is a cell adhesion protein on the postsynaptic membrane that mediates the formation and maintenance of synapses between neurons.

  • Neuroligins act as ligands for β-Neurexins, which are cell adhesion proteins located presynaptically.

• Neurexins (NRXN) are a family of presynaptic cell adhesion proteins that have roles in connecting neurons at the synapse.

  • They are located mostly on the presynaptic membrane and contain a single transmembrane domain.

Neuroligins

• Postsynaptic cell adhesion molecules that bind across synaptic cleft to neurexins

• Neuroligin 1 characteristically found in all glutamatergic synapses

  • Some nicotinic synapses in the peripheral nervous system?

• Neuroligin 2 found preferentially in some inhibitory synapses (GABA)

  • And in some cholinergic (acetylcholine) synapses

• Neuroligin 3 found in excitatory and inhibitory synapses

  • Forms heterodimers with neuroligin 1

• Neuroligin 4 found preferentially at glycinergic synapses in retina

Neuroligins bind to PSD95

• Postsynaptic density protein 95 (or synapseassociated protein 90 (SAP-90) or Disks large homolog 4 (DLG4))

• PSD95 has 3 PDZ binding domains

  • PDZ for Postsynaptic density protein 95, Drosophila disc large tumor suppressor (Dig 1) and zona occludens protein (ZO-1)

  • PDZ motif 80-90 amino acids

• Neuroligins bind to PSD95 via 3rd PDZ domain

• PSD95 binds AMPA glutamate receptors via 1st PDZ domain

• Neuroligin/neurexin interaction holds AMPA receptors in place

PDZ binding domains

• Grip has 7 PDZ binding domains and stands for glutamate receptor interactive protein

  • Hold glutamate receptors close to their effector binding domains

PSD95 localisation

• Cortical neuron of a mouse injected with CAG-PSD95 WPRE plasmids into lateral cerebral ventricles (E16 embryos in utero)

• RED: CAG-DSRedExpress-WPRE showing dendrite

• GREEN: CAG-PSD-95-WPRE showing presence of PSD-95 localization.

PSD95 has other domains

• Have 3 PDZ domains binding to PSD95

PSD95 Binding

PSD95 binds to many things:

• NMDA receptors

• Calcium-calmodulin protein kinase II (CaMKII)

  • Regulates NMDA and AMPA cycling

  • Implicated in memory formation

• Neuronal nitric oxide synthase

  • Implicated in synaptic plasticity and neuroprotection

• Shank proteins, indirectly via GKAP

  • Shanks can also bind directly to neuroligins

Summary

• Neuroligins expressed on postsynaptic cell membrane and Neurexins expressed on presynaptic membranes, these come together to form that synapse and form the presynaptic an postsynaptic structures together.

• There are 4 types of neuroligins and these are expressed on different types of synapses.

• Neuroligins bind to PSD-95

• PSD-95 binds to a number of partners via the PDZ domains including NMDA receptors, CaMKII, nNOS

Regulation of presynaptic specialisations

Post-synaptic specializations regulate pre-synaptic specializations via cell adhesion molecules (CAMs) and vice versa

• Major example is the neurexin-neuroligin interaction

• Conformational change occurs when this interaction occurs

• But deletion of neuroligin does not prevent synapse formation

• Other cell adhesion molecules can substitute

Postsynaptic density

Postsynaptic densities include dozens of signal transduction molecules including:

• glutamate receptors (NMDA-R; mGluR),

• tyrosine kinase receptors (RTK),

• many intracellular signal transduction molecules,

• most notably the protein kinase CaMKII

There are many receptors and protein scaffolding proteins that bind the receptors to their effective proteins

Shanks

• Important scaffolding proteins that tether other immediate scaffolding proteins

• Cytoskeletal proteins coupling via contact into the actin cytoskeleton

• Bind via Homer to metabotropic glutamate receptors

• Also via Homer to IP3 receptors on the smooth endoplasmic reticulum

  • Regulate Ca release from the SER

• Mutations in Shank proteins can cause presynaptic changes signalling via neuroligin to presynaptic neurexins

Inhibitory synapses

• Key organising molecule specific for GABA and glycinergic synapses is gephyrin

• Gephyrin self-assembles into a hexagonal lattice and interacts with various inhibitory synaptic proteins

• Anchors the receptors and important proteins to the post synaptic membrane

• Both GABA and glycine receptors are from the same superfamily as the nicotinic acetylcholine receptor

  • Pentameric transmembrane proteins

  • Contain at least 2 α-subunits

Summary

• Post-synaptic specializations regulate pre-synaptic specializations via CAMs and vice versa

• Postsynaptic density includes signal transduction proteins

• Key organising molecule specific for GABA and glycinergic synapses is gephyrin