1. Rho GTPases as regulators of
morphological neuroplasticity
Ahmet GÜNER
60051105

2. 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.

3. • 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.
The Ras superfamily of GTPases
Ras Rho Rab Arf Ran
particularly relevant for cell biology
by regulating morphogenesis,
polarity, migration and division
• At the molecular level Ras and Rho family are involved in cytoskeletal
dynamics, vesicular transport and gene expression.

4. 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.

5. Rho Family
Rho GTPases
Rho Rac Cdc42 Rnd RhoD TTF
RhoA Rac1 Cds42Hs RhoE/Rnd3
RhoB Rac2 G25K Rnd1/Rho6
RhoC Rac3 TC10 Rnd2/Rho7
RhoG

6. Structure of the
Rho protein family.
Structure
of
the
Rho
protein
family.
Auer et al. 2011

7. 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.

8. 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.

9. 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.

10. Regulation
• 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.

11. 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.

12. Regulation
Table1 Summary of the cellular activities which involve Rho, Rac and Cdc42
NADPH G1 cell-cycle Cell-cell Cell Transform-
Actin SRF JNK/p38 NF-ĸβ Secretion
oxidase progression contacts polarity ation
Rho + + - + - + + + - +
Rac + + + + + + + + - +
Cdc42 + + + + - + + ? + +
SRF and NF-ĸβ are both MAP kinase pathways only been shown to
JNK and p38 are Secretion has
These activitiesfactors. to biological in professional phagocytic cells
transcription refer
The NADPH oxidase complexis present onlypathways GTPasescanmast cells.
involve Rho which in be
induced by the activated Rho GTPases indicated and / or which
can be inhibited by dominant negative constructs of the
appropriate Rho GTPases.

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

14. 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.

15. 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.

16. 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.

17. The effector remainsInactivation of the effector occurs though
active until
GTP hydrolysis takes place.of modification Y (e.g. dephosphoryl- (Y,
Alternatively a modification of the effector
removal
Binding of Rho GTPase to ellipses) (e.g. autophosphorylation, as is the
orange effector relieves an
antoinhibitory intramolecularorPAK, phosphorylationactivating protein),
ation removal of a bound
case for interaction (this is by a separate kinase
clearly the case for the kinase PAK the fordifferent activating its inactive
allowing effector to reenter
or bindingand a the scaffold
to protein) may
conformation. also for other
proteins Dia and WASP, and mayactivity even after dissociation of the
maintain be
effectors which contain auto inhibitory domains.
GTPase.

18. 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.

19. 2. Axon elongation, sprouting and
collateralization
• 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.

20. • 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.

21. • 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.

22. 2. Axon elongation, sprouting and
collateralization
• 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.

23. 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.

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

25. 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.

26. • 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).
substrate for integrin receptors to
stimulate the axonal growth
machinery.
• 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.

27. 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.