Rho GTPases as regulators

of morphological

neuroplasticityAhmet GÜNER

60051105

1. Introduction

• The cell, in its tissue environment, receives many signals that act on membrane receptors.

• This leads to the activation of multiple signal transduction pathways and

a particular cellular response is generated after integration of these via intracellular effectors.

• Over the recent years, several small GTPases have been identified as key mediators of the interactions between cell adhesion molecules and cytoskeleton constituting axonal and dendritic morphology.

• Rho GTPases are now regarded as major regulators of axonal and dendritic growth.

• At the molecular level Ras and Rho family are involved in cytoskeletal dynamics, vesicular transport and gene expression.

The Ras superfamily of GTPases

Ras Rho Rab Arf Ran

particularly relevant for cell biology by regulating morphogenesis,

polarity, migration and division

1. Introduction

• Small GTPases of the Rho family are pivotal regulators of several signaling networks that are activated by a wide variety of receptor types.

• When activated, Rho GTPases affect many aspects of cell behavior, including – actin cytoskeleton dynamics, – transcriptional regulation, – cell cycle progression, and – membrane trafficking.

Rho Family

Rho GTPases

Rho

RhoA

RhoB

RhoC

Rac

Rac1

Rac2

Rac3

RhoG

Cdc42

Cds42Hs

G25K

TC10

Rnd

RhoE/Rnd3

Rnd1/Rho6

Rnd2/Rho7

RhoD TTF

Structure of

the Rho

protein family.

Structure of the Rho protein family.

Auer et al. 2011

Rho Family

• When bound to GDP they are inactive; – upstream events lead to

• the exchange of GDP for GTP and • the protein switches into an active conformation.

• It is this form of the protein that can interact with downstream targets or effector molecules to produce a biological response. An intrinsic GTPase activity completes the cycle.

Rho Family

• At least three classes of molecules are capable of interacting with Rho GTPases and regulating their activation state:

– (i) Guanine nucleotide exchange factors (GEFs) catalyze the exchange

of GDP for GTP,

– (ii) GTPase activating proteins (GAPs) stimulate the intrinsic GTPase

activity, and

– (iii) guanine nucleotide dissociation inhibitors (GDIs) inhibit the

exchange of GDP for GTP and might also serve to regulate their

association with membranes.

Rho Family

• The most extensively characterized members are Rho, Rac and Cdc42.

• Each of these GTPases act as a molecular switch, cycling between an active GTP-bound, and an inactive GDP-bound, state.

• Activation of Rho GTPases is mediated predominantly through cell surface receptors (cytokine-dependent, tyrosine kinase or G-protein coupled).

• Receptor tyrosine kinases (RTKs) are activated by their respective ligands, which lead to the

» dimerization and » autophosphorylation of the receptor and » to the stimulation of various signaling pathways including

small Rho GTPases.

Regulation

Regulation

• Cycling between the GTP-and GDP-bound states is regulated by numerous cellular proteins.

• Although still poorly characterized, over 30 guanosine nucleotide exchange factors (GEFs) have been identified that facilitate the exchange of GDP for GTP.

Regulation

Actin SRF JNK/p38 NF-ĸβ NADPH oxidase

G1 cell-cycle progression

Cell-cell contacts Secretion Cell

polarityTransform-ation

Rho + + - + - + + + - +Rac + + + + + + + + - +

Cdc42 + + + + - + + ? + +

Table1 Summary of the cellular activities which involve Rho, Rac and Cdc42

These activities refer to biological pathways which can be induced by the activated Rho GTPases indicated and / or which can be inhibited by dominant negative constructs of the appropriate Rho GTPases.

SRF and NF-ĸβ are both transcription factors.

JNK and p38 are MAP kinase pathways

The NADPH oxidase complexis present only in professional phagocytic cellsSecretion has only been shown to involve Rho GTPases in mast cells.

Effectors

• Rho proteins act on several downstream effectors involved in the – stabilization, – contraction, – polymerization and – capture of cytoskeletal building blocks.

Regulation and downstream effectors of Rho GTPases RhoA, Cdc42 and Rac1 involved in shaping neuronal morphology.

Effector proteins downstream of small GTPases are involved in restructuring the cytoskeleton.

Effectors

• Microtubule stabilization is regulated by • RhoA, • Rac1 and • Cdc42 through the actions of

» mDia, » PAK (p21-activated kinase) or » PAR6 (partitioning defective-6)

• Moreover, RhoA activates several other effector proteins, among them;

ROCKI and ROCKII

which in turn phosphorylate myosin light chain (MLC) and its phosphatase resulting in enhanced actomyosin-based contractility.

Effectors

• Inhibition of ROCK in semaphorin-treated embryonic hippocampal neurons reverses the stimulatory effect on axonal branching and increases axonal length.

• In contrast, dendritic branching is not markedly altered by ROCK inhibition.

• Other downstream signaling molecules of Rho proteins are not directly related to the cytoskeleton, such as p38α, which is required for calcium-dependent excitotoxic cell death.

Binding of Rho GTPase to effector relieves an antoinhibitory intramolecular interaction (this is clearly the case for the kinase PAK and for the scaffold proteins Dia and WASP, and may be also for other effectors which contain auto inhibitory domains.

The effector remains active until GTP hydrolysis takes place.Alternatively a modification of the effector (Y,

orange ellipses) (e.g. autophosphorylation, as is the case for PAK, phosphorylation by a separate kinase or binding to a different activating protein) may maintain activity even after dissociation of the GTPase.

Inactivation of the effector occurs though removal of modification Y (e.g. dephosphoryl-ation or removal of a bound activating protein), allowing the effector to reenter its inactive conformation.

2. Axon elongation, sprouting and collateralization• Axon branches are formed by two different ways. Terminal branching

is characterized by the bifurcation of the growth cone that gives rise to two or more separate axons shafts.

• Alternatively, interstitial branching occurs by the de novo initiation of axon branches from previously quiescent regions of the axon.

• Terminal branching occur in the process of axonal outgrowth.

• Interstitial branching takes place after axonal development and target contact.

• During transition from a dynamic filopodium to a stable branch, – unbundling of axonal microtubules is necessary

• to enable microtubule ends • to interact with actin bundles • to form the emanating branch.

• Microtubules then splay apart and invade actin-rich filopodial-like structures on the axon shaft.

• Subsequently, microtubules become bundled again, and the generation of new actin protrusions stops in the plasma membrane lateral to the bundled microtubules, thereby stabilizing the newly formed branch.

2. Axon elongation, sprouting and collateralization

• The Ras/Raf/ERK and PI3K/Akt signaling pathways are both required for axon outgrowth, and each pathway induces distinct axonal morphologies.

Activation of PI3K is necessary for branch formation induced

by neurotrophic growth factors (NGFs).

Inactivation of GSK-3β leads to enhanced axon growth by

adult DRG neurons and by hippocampal neurons.

• Over expression of Ras and Raf stimulates elongative axon growth by embryonic DRG neurons,

whereas • over expression of Akt or PI3K enhances axon calibre and branching.

• Furthermore, neurotrophin-3 (NT3) induces more highly branched and thicker axons than NGF (nerve growth factor) in different types of embryonic neurons.

• In addition, the ERK pathway is involved in the regulation of gene expression underlying axon maintenance.

• However, ERK is required for local axon assembly and regulates axonal microtubules and actin filaments as well.

• Inhibition of ERK results in depolymerization of actin and growth cone collapse, a phenomenon induced by the activation of RhoA, too.

2. Axon elongation, sprouting and collateralization

3. Specific functions of Rho GTPases in neuronal and glial cells

• In general, Rho proteins inhibit neurite extension, whereas

• Cdc42 and Rac act as positive regulators of neurite outgrowth and dendritic spine formation by promoting membrane protrusion through actin filament assembly.

• Rho GTPases in glial cells enfold multiple layers of plasma membrane around the axon to form myelin.

• Ablation of Cdc42 in cells of the oligodendrocyte lineage results in a stage-specific myelination phenotype characterized by an enlargement of the inner tongue of the oligodendrocyte process.

• Similarly, knockout of Rac1 results in abnormal accumulation of cytoplasm in oligodendrocytes as well.

• In Schwann cells, the lack of Rac1 produces a delay in the process of radial sorting of axons and arrests myelination.

• Cdc42 deficient Schwann cells are defective in axon myelination, and • perturbation of RhoA activity inhibits

» glial migration and » defasciculation of sensory axons.

3. Specific functions of Rho GTPases in neuronal and glial cells

4. The role of RhoA in myelin-dependent inhibition of axon regeneration

• In contrast to axons in the adult central nervous system (CNS), peripheral axons are capable of regrowth after axotomy.

• Whereas CNS axon tracts contain myelin-associated neurite growth inhibitors, the micro-environment of the peripheral nerve is regarded to – lack these inhibitors and – provides sufficient support to stimulate and – maintain axon regeneration into the enervated muscle or skin.

• It is generally accepted that peripheral glia (Schwann cells) secretes neurotrophic factors required for survival and regeneration as well as appropriate extracellular matrix molecules (laminins).

• Moreover, the formation of scars or cysts, which act as major barriers for regeneration in the injured CNS, is greatly reduced in the lesioned peripheral nervous system.

substrate for integrin receptors to stimulate the axonal growth

machinery.

5. Conclusions and outlook

• Rho family GTPases act as molecular switches that couple changes in the extracellular environment to various intracellular signal transduction pathways.

• They are influenced by cell surface receptors and regulate distinct aspects of the cytoskeletal protein machinery, such as – actin polymerization and depolymerization, – anchoring and cross-linking, – myosin motor activities and – microtubule stabilization.