Perspectives of RAS and RHEB GTPase Signaling Pathways in Regenerating Brain Neurons

Abstract

Cellular activation of RAS GTPases into the GTP-binding "ON" state is a key switch for regulating brain functions. Molecular protein structural elements of rat sarcoma (RAS) and RAS homolog protein enriched in brain (RHEB) GTPases involved in this switch are discussed including their subcellular membrane localization for triggering specific signaling pathways resulting in regulation of synaptic connectivity, axonal growth, differentiation, migration, cytoskeletal dynamics, neural protection, and apoptosis. A beneficial role of neuronal H-RAS activity is suggested from cellular and animal models of neurodegenerative diseases. Recent experiments on optogenetic regulation offer insights into the spatiotemporal aspects controlling RAS/mitogen activated protein kinase (MAPK) or phosphoinositide-3 kinase (PI3K) pathways. As optogenetic manipulation of cellular signaling in deep brain regions critically requires penetration of light through large distances of absorbing tissue, we discuss magnetic guidance of re-growing axons as a complementary approach. In Parkinson's disease, dopaminergic neuronal cell bodies degenerate in the substantia nigra. Current human trials of stem cell-derived dopaminergic neurons must take into account the inability of neuronal axons navigating over a large distance from the grafted site into striatal target regions. Grafting dopaminergic precursor neurons directly into the degenerating substantia nigra is discussed as a novel concept aiming to guide axonal growth by activating GTPase signaling through protein-functionalized intracellular magnetic nanoparticles responding to external magnets.

Keywords: Magneto Protein Therapy; Parkinson’s disease; RAS GTPase; RHEB GTPase; axonal guidance; brain regeneration; magnetogenetics; nanoparticle; optogenetics; survival.

Figure 1

GTPase cycling between GTP-bound “ON” state and GDP-bound “OFF” state. Upon an external signal, a guanine nucleotide exchange factor (GEF) is activating small GTPases (here H-RAS) by exchanging GDP to GTP and thereby modulating cell signaling cascades. To return into the “OFF” state, GTPase-activating proteins (GAPs) accelerate GTP hydrolysis to GDP.

Figure 2

Signaling pathways of RAS GTPase. Receptor tyrosine kinases (RTKs)-mediated activation of RAS in the presence (“+”) and absence (“-“) of ligand is shown as an example. However, multiple upstream regulators of RAS exist. The SHC-GRB2-SOS complex promotes the exchange of GDP to GTP and thereby switching RAS GTPase to the active conformation. Hence, signaling is propagated through the diverse pathways of RAS such as the most prominent RAF/MEK/ERK cascade. RAS-mediated PI3-kinase (PI3K) activation results in protein kinase B (AKT) activation, which is inhibited by phosphatase PTEN (illustrated by T-bar). TIAM1/RAC and RAL-GEF/RAL are involved in cytoskeletal dynamics and cell migration. PLCε/DAG/PKC is associated with Ca²⁺-dependent cellular functions. AF6 forms electrical synapses by localizing at gap junctions. RIN controls axonal pathfinding and via RAB endomembrane signaling. RASSF promotes apoptosis and Canoe regulates cellular polarity.

Figure 3

Schematic overview of RAS and PI3K pathway and their interconnection. Binding of growth factors to receptor tyrosine kinases results in activation of RAS and PI3K signaling pathway. Complex-formation of NORE1, RASSF1A, and MST1, together with activated RAS leads to JNK-mediated senescence, cell-cycle arrest or apoptosis [16,84]. RAS-mediated activation of RAF/MEK/ERK signaling plays a role in survival, proliferation, neurite growth, and neuronal regeneration, but the effect differs between various types of neurons [85]. PI3K-mediated phosphorylation of AKT promotes TSC dissociation from lysosomes and RHEB activation. The activation of RHEB by GEF is still under discussion (dashed arrow). Downstream of RHEB, mTORC1 and S6K suppress autophagy and promote survival, neurite growth, and neuronal differentiation [86]. Furthermore, PI3K-mediated mTORC2 activation plays a role in PKC-dependent synaptic plasticity, actin cytoskeleton dynamics, and neuron morphology [87,88]. Strong activation of AKT directly inhibits RAF (illustrated by T-bar) and thereby leading to downregulation of ERK1/2 signaling [89].

Figure 4

Concept of Magneto Protein Therapy. (A) Somatic cells obtained from Parkinson’s patient by biopsy are reprogrammed [287] or directly converted [288] to dopaminergic (DA) precursor neurons in vitro. After cell sorting and loading with protein-functionalized magnetic nanoparticles (pMNPs), these cells are grafted to the striatal target regions or substantia nigra, where neurons degenerate. (B) In the patient’s brain, the grafted DA precursor cells convert into mature DA neurons. By the application of an external magnetic field, pMNPs are driven towards the inner membrane of the growing axon thereby activating small GTPases allowing spatially controlled fiber growth.