RhoA is one of the members of the Ras Superfamily, which has been shown to modulate the actin cytoskeleton. RhoA through its downstream effectors such as RhoA-associated Kinase (ROCK) and Myosin Light Chin Kinase (MLCK) modulates actin stress fiber formation (Tapon and Hall, 1997).

Although modulation of stress fibers is one of the most characteristic functions of RhoA GTPase, numerous studies highlight the role of RhoA in neurophysiological processes. For instance, in cerebellar granular neurons, RhoA was found to bidirectionally control axonal elongation either promoting it via the Rho-mDia pathway or repressing it via the Rho-ROCK pathway, depending on the concentration of stromal cell-derived factor-1 (SDF-1 Alpha) chemokine used for stimulation (Arrazola Sastre et al., 2020). Moreover, RhoA was shown to regulate the cellular and molecular processes underlying synaptic plasticity. Through its downstream effectors, ROCK or p160^ROCK, RhoA promotes actin polymerization and long-term potentiation (LTP) in rat hippocampal neurons (Rex et al., 2009). Dominant negative form RhoA was found to promote dendritic arborization in Xenopus retinal ganglion cells (Newey et al., 2005). RhoA is also involved in neuronal survival, as shown by studies in which combined suppression of RhoA and Rac promoted neural stem/progenitor cell (NSPCs) survival in the injured spinal cord (Numano et al., 2009).

RhoA signaling has been directly implicated in neurodegenerative diseases, including Alzheimer’s disease (AD), Picks disease (PiD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS; Huesa et al., 2010; Aguilar et al., 2017; Schmidt et al., 2022). Dendritic spine loss, a hallmark of early neurodegeneration, is regulated by RhoA signaling. Treatment of cultured neurons with oligomeric beta amyloid (Aβo) activates the intracellular tyrosine kinase Pyk2 (PTK2B) which then phosphorylates and reduces the activity of the RhoGAP protein called GTPase regulator associated with focal adhesion kinase-1 (Graf1). Reduced RhoGAP activity results in increased RhoA activity which leads to dendritic spine loss (Lee et al., 2019).

In the human brain with AD or PiD, RhoA co-localizes with neurofibrillary tangles (NFTs), which are enriched with hyperphosphorylated Tau proteins (Huesa et al., 2010). Similarly, in the brain of Tg2576 AD model mice, RhoA level is reduced in the synaptic terminals, while dystrophic neurites show elevated RhoA expression (Huesa et al., 2010). Several studies evaluated neurodegeneration-associated changes in the level of RhoA effectors, such as ROCK1 and ROCK2. In human brains with mild cognitive impairment (MCI) and AD, ROCK1 levels are increased compared to non-AD brain matter (Henderson et al., 2016). This finding is further supported by studies using cellular models of AD: exposure of neurons to oligomeric Aβ42 increases ROCK1 and ROCK2 expression and induces phosphorylation of Lim Kinase 1 (LIMK) downstream of RhoA, while depletion of ROCK1 reduces toxic Aβ42 production (Henderson et al., 2016). In a study that looked at the effect of Aβ42 on BV2 microglial cells, Aβ42 treatment was shown to stimulate the production of reactive oxygen species (ROS), that was inhibited by blocking RhoA activity (Moon et al., 2013). ROCK involvement in PD progression was demonstrated by a study in which treatment with Fasudil, a small molecule ROCK inhibitor, promoted dopaminergic neuron survival and improved motor performance in a PD mouse model (Tonges et al., 2012). In an ALS mouse model, constitutive activation of Rho GTPase leads to the death of motor neurons via a mechanism that depends on Rho kinase or p160^ROCK activity (Stankiewicz et al., 2020).

Activation of Rac1 is required for the formation of membrane ruffles and lamellipodia and involves the WAVE complex (Parri and Chiarugi, 2010). Via its downstream effectors such as PAK, p67phox, and MLK 2/3, Rac1 regulates the dynamics of actin cytoskeletal proteins, as well as transcription and nuclear signaling processes (Bosco et al., 2009).

Rac1 protein belongs to the Rac GTPases subfamily which also includes Rac2, Rac3, and RhoG (Narumiya and Thumkeo, 2018). Rac1 is involved in establishing neuronal polarity and in neuronal survival. For example, the Rac/Cdc42 effector IQGAP3 is known to promote axonal outgrowth in hippocampal neurons (Wang et al., 2007). Rac1’s interaction with p21-activated kinase (PAK) promotes neuronal survival via inhibition of the Bcl-2-associated death promoter (Bad; Stankiewicz and Linseman, 2014). Relevant to ALS pathogenesis, NSC23766, a selective inhibitor of the Rac-specific GEFs Tiam1 and Trio, is sufficient to induce the death of embryonic stem cell (ESC)-derived motor neurons (Stankiewicz et al., 2020).

In the human AD cortex, Rac1 expression decreases in parallel with increased Rac1 level in the plasma (Borin et al., 2018). Similarly, in the hippocampus of 3xTg-AD mice, Rac1 was reported to be active, but by 7 months of age, the total Rac1 level decreases (Borin et al., 2018). In mouse hippocampal neurons, pharmacological inhibition of Rac1 with NSC23766 reduced the expression of Amyloid Precursor Protein (APP) via decreasing the level of APP mRNA (Wang et al., 2009). Paradoxically, Manterola and colleagues demonstrated that in neuron-like cell lines (SN4741) and rodent primary neuron cultures, Rac1 activation by oligomeric Aβ42 involves Protein Kinase C (PKC), Phosphatidylinositol 3-Kinase (PI3K) and Phosphoinositol-Dependent Kinase (PDK1), causing loss of neurons. Pharmacological inhibition of PDK1 by OSU03012 resulted in Rac1 inactivation and subsequent inhibition of neuronal death (Manterola et al., 2013). Fibrillary Aβ was shown to interact with integrin receptors on microglial cells causing activation of a Rac1 GEF, Vav, which leads to enhanced Rac1 activity. Moreover, Vav^−/− microglial cells showed reduced production of ROS (reactive oxygen species) compared to Vav^+/+ microglial cells (Wilkinson et al., 2006). Rac1 signaling is also involved in Tau pathology, which is another neuropathological hallmark of AD. In human neuroblastoma cells, Rac1 was found to indirectly increase Tau phosphorylation via translocation of SET, a protein phosphatase 2A (PP2A) inhibitor (Borin et al., 2018).

Cdc42 induces actin polymerization by interacting with the Wiskott–Aldrich syndrome protein (WASp) and neuronal-WASp (N-WASp). Cdc42 can also modulate the organization of microtubules by interacting with Cdc42-Interacting protein (CIP4). As a modulator of both actin and microtubule assembly, Cdc42 can induce both filopodia and membrane protrusions (Gerasimcik et al., 2015).

There are three small GTPases belonging to the Cdc42 subfamily: Cdc42, RhoQ (TC10), and RhoJ (TCL; Narumiya and Thumkeo, 2018). In hippocampal neurons, Cdc42 was shown to promote actin polymerization via its effector N-WASP and the Arp2/3 complex. Through Cdc42, N-WASP induces filopodia and therefore modulates axon formation (Kitamura et al., 2003). By regulating cofilin, Cdc42 also plays an important role in establishing neuronal polarity. This is supported by findings that in Cdc42^−/− neurons, phosphorylation of cofilin increases, leading to abnormal axonal morphology (Garvalov et al., 2007; Hall and Lalli, 2010) Involvement of Cdc42 in synaptic plasticity, dendritic spine structural plasticity, and learning and memory was demonstrated by studies that show impaired synaptic plasticity and hippocampus-dependent memory in conditional Cdc42 KO mice (Kim et al., 2014).

Several studies reported Cdc42 involvement in the progression of neurodegeneration. One study found that both AD and FTLD (frontotemporal lobar degeneration) are associated with elevated expression of Cdc42 in the frontal cortex, compared to age-matched controls (Saraceno et al., 2018). Several downstream target molecules have been proposed to mediate the Cdc42-dependent changes associated with neurodegeneration, including N-WASP. In cultured rat hippocampal neurons, Aβ exposure activated Cdc42 and Rac1, leading to increased F-actin formation (Mendoza-Naranjo et al., 2007).

As a functional regulator of Wnt signaling, one of the Cdc42 effectors is GSK3β (Etienne-Manneville and Hall, 2003), a kinase highly expressed in the brain with a critical role in neuropsychiatric and neurodegenerative disorders including AD (Llorens-Martin et al., 2014). Via phosphorylation of Tau, GSK3β contributes to NFT formation (Pelleieux et al., 2018). Interestingly, compounds identified from the Enamine’s screening collection can inhibit GSK3β-dependent Tau phosphorylation and reduce Tau aggregation in human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregation mutation ΔK280 (Chiang et al., 2021). Based on these studies, it is postulated that pharmacological modulation of Cdc42 can be a potential avenue to better understand the role of Cdc42 signaling in neurodegeneration (Aguilar et al., 2017; Guiler et al., 2021).

Although the main function of Rab1 is to regulate the ER-to-Golgi transport, it is also implicated in signaling pathways associated with other cellular processes such as nutrient signaling, integrin-dependent cell migration, and autophagy. Rab1 signaling is unique among small GTPases, as they do not activate their effectors; instead, they regulate their targeting and formation of active signaling complexes on their respective membranes (Yang et al., 2016).

Rab1 is involved in neuronal development, particularly in the formation of neurites (Villarroel-Campos et al., 2014) Rab1 tubules are able to move bidirectionally between the central body of the neuron and neurites that connect to the central Golgi and ER exit sites (Saraste, 2016).

Rab1 dysfunction has been implicated in the pathogenesis of PD. In human neuroblastoma cells, α-synuclein overexpression disrupts Rab1A activity resulting in a hindered ability of autophagosome synthesis. Moreover, in a rodent model of PD, increasing Rab1 expression reduces α-synuclein-mediated Golgi fragmentation, prevents the death of dopaminergic neurons, and ameliorates PD-like motor deficits (Winslow et al., 2010).

Members of the Rab3 subfamily (Rab3A, Rab3B, Rab3C, and Rab3D) are primarily restricted to neurons, but the A, B, and C isoforms can be found in endocrine tissue as well. The D isoform is expressed in adipose tissue, exocrine glands, and the endocrine pituitary. The GDP/GTP exchange cycle of Rab3A is required for the Ca²⁺-regulated exocytosis to occur (Schluter et al., 2004).

Rab3 GTPases are predominantly localized to the presynaptic active zone where they regulate the priming and docking of synaptic vesicles (Vieira, 2018). Interestingly, deleting one or two of these genes in mice had no phenotype; however, triple or quadruple knockouts resulted in lethality if Rab3A was part of the deletion (Schluter et al., 2006).

Several findings directly implicate Rab3 in synaptic dysfunction associated with Parkinson’s disease (Chen et al., 2013). Similar to Rab1, Rab3 overexpression partially rescues the α-synuclein-induced dysfunction of dopaminergic neurons (Gitler et al., 2008). In another study, Rab3 was shown to directly interact with α-synuclein. It was reported that Rab3A recycling machinery regulates the membrane binding of α-synuclein (Chen et al., 2013).

Rab4 is involved in cell secretion and metabolism via regulating the vesicular trafficking between the recycling and degradation pathways (Conti et al., 2009). Transfer of the vesicular cargo from early endosomes to the recycling endosomes requires Rab4 recruitment to the intermediate sorting endosomes (Grant and Donaldson, 2009).

In neurons, Rab4 is involved in axonal outgrowth via endosome recycling. In a study using Xenopus neurons, Rab4 downregulation resulted in axonal elongation (Falk et al., 2014).

Expression levels of Rab4 were found to be increased in human brains with MCI as well as in late onset AD brain matters (Cataldo et al., 2000; Ginsberg et al., 2011). These findings are supported by studies using cellular and murine models of AD. For example, the level of Rab4 significantly increases in neurons that carry AD-relevant mutations in the PSEN1 gene and in the brain of 3xTg-AD model mice as the disease progresses (Soejima et al., 2013). CD2AP, a scaffolding protein for the actin cytoskeleton and a Rab4a effector, is another potential link between Rab4a and AD (Monzo et al., 2005). Loss of function mutation of CD2AP leads to enhanced Aβ production (Guimas Almeida et al., 2018; Van Acker et al., 2019) and increased Tau-induced neurotoxicity (Shulman et al., 2014).

Rab5 GTPase plays a critical role in clathrin-mediated endocytosis and endosomal maturation (Semerdjieva et al., 2008). Rab5 also helps mediate the kinetics of nuclear membrane disassembly and plays an important role in the morphology of the peripheral ER (Audhya et al., 2007).

In the Drosophila neuromuscular junction, Rab5 promotes synaptic endosomal integrity (Wucherpfennig et al., 2003). In mice, Rab-5-dependent endosomal sorting regulates the size uniformity of synaptic vesicles (Shimizu et al., 2003).

Rab5 has been implicated in neurodegeneration, particularly in AD and HD. In the human brain with late onset AD, the size of Rab5 positive early endosomes is significantly larger than in the healthy non-AD brain (Cataldo et al., 2000). In murine models of AD and in the brain of mice overexpressing Rab5, it has been shown that overactivation of Rab5 is sufficient to drive AD-like neurodegeneration and cognitive decline (Pensalfini et al., 2020). Mechanistically, Rab5 involvement in AD might be due to its association with early endosomes (EE) that are upregulated and enlarged in the aging neurons, partly due to increased APP endocytosis (Burrinha et al., 2021). Consequently, Rab5 upregulation in mature neurons significantly increases intracellular Aβ42 production and induces synaptic dysfunction (Burrinha and Guimas Almeida, 2022). Studies using cellular models of HD found that Rab5 interacts with the Huntingtin-HAP40 complex that regulates the activity of early endosomes (Pal et al., 2006; Ravikumar et al., 2008). Disruption of the Rab5-Huntingtin-HAP40 complex leads to a reduction in endosomal motility (Pal et al., 2006).

Rab6 plays a role in the control of retrograde vesicle transport from the Golgi apparatus toward the endoplasmic reticulum (ER; Opdam et al., 2000). Rab6 is also critical for basement membrane formation (Homma et al., 2019).

Rab6 is highly expressed in the brain where it has predominantly pre-synaptic distribution and plays a role in the control of retrograde vesicle transport from the Golgi apparatus toward the ER (Opdam et al., 2000). Studies in Drosophila demonstrated that Rab6 plays a role in synaptic specificity via activation of Ric1 homologue-Rab6-N-Cadherin pathway (Tong et al., 2011). In a more recent study, Rab6 was identified in cargo delivery to synapses, a function that requires ELKS1 (Nyitrai et al., 2020).

Rab6 might be involved in neurodegenerative disorders, particularly AD (Elfrink et al., 2012). Elevated Rab6 expression has been found in the AD brain, as well as in cells carrying PSEN1 gene mutations (Scheper et al., 2004, 2007). Early studies of the role of Rab6 in AD pathogenesis suggest that Rab6 can influence whether APP is sorted into the α-secretase pathway or the amyloidogenic β-secretase pathway. In HEK 293 cells co-expressing a defective GTP-binding Rab6 mutant and APP, secretion of the soluble APP alpha increased, whereas production of the peptides derived from the amyloidogenic processing pathway was either slightly inhibited or unaffected (Mcconlogue et al., 1996).

Members of the Rab8 subfamily regulate cell shape and protrusion formation (Hattula et al., 2006; Peranen, 2011). Activation of Rab8 positively regulates the formation of lamellipodia and filopodia, whereas its inhibition negatively impacts these structures (Peranen et al., 1996).

Rab8 proteins are involved in the transport of GluA1-AMPA receptors from the ER-Golgi network to the postsynaptic membrane (Gerges et al., 2005; Hausser and Schlett, 2019). Inhibition of neuronal Rab8 compromises membrane transport, resulting in impaired neurite outgrowth. Mechanistically, Rabin8, a shared GEF between RAB8, RAB10, and RAB11 is central to control of neurite outgrowth (Homma and Fukuda, 2016).

Rab8 involvement in AD neurodegeneration is supported by a study showing a significant reduction in PSEN1 mutated cells compared to cells that expressed normal PSEN1 (Kametani et al., 2004). Rab8 has also been implicated in PD. In a cellular PD model, gain-of-function mutations in LRRK2 caused lysosomal sequestration of endogenous Rab8a, which was reversed by pharmacological inhibition of LRRK2 (Mamais et al., 2021).

Rab8 also has HD implications. In a Drosophila model of the disease, increased Rab8 level provided neuroprotection against mutant Huntingtin (HTT) via reducing the accumulation of downstream soluble toxic species (Delfino et al., 2020). Another member of the Rab8 GTPase subfamily, Rab10, has been strongly implicated in AD pathogenesis (Yan et al., 2018; Tavana et al., 2018). In cellular models of AD, Rab10 silencing was shown to significantly decrease the production of toxic Aβ42 (Ridge et al., 2017). Genetic studies of elderly individuals at high-risk for AD (APOE ε4 carriers) who escaped the disease, identified a rare Rab10 variant as a potential provider of molecular resilience against AD (Ridge et al., 2017).

Proteins belonging to the Rab11 subfamily regulate the trafficking of the recycling endosomes and early endosomes to the trans-Golgi network (Wilcke et al., 2000).

Rab11 plays a major role in dendrite outgrowth and is also involved in receptor trafficking and recycling (Dong et al., 2010). It is critical for the activity-dependent delivery of GluA-containing AMPA receptors to the synapses (Park et al., 2004).

Several studies implicate Rab11 in neurodegeneration (Bhuin and Roy, 2015). Rab11 was shown to be neuroprotective in a Drosophila model of PD by restoring the size of synaptic vesicles and rescuing α-synuclein-induced locomotor impairment (Breda et al., 2015) as well as by protecting against mitochondrial dysfunction induced by parkin mutations (Rai and Roy, 2022). In a Drosophila model of HD, overexpression of Rab11 rescued synaptic dysfunction and behavioral deficits (Steinert et al., 2012). Similarly, in mouse models of HD, elevating Rab11 activity was sufficient to reduce oxidative stress and neuronal loss (Li et al., 2009, 2010).

RNAi screening of all human Rab proteins that regulate membrane trafficking using cells with robust Aβ and sAPPβ production (primary neuron cultures derived from AD model mice and cell lines with stable expression of APP with Swedish mutation) identified Rab11 as a key regulator of Aβ production. The molecular mechanism involves Rab11-mediated recycling of BACE-1 to EE, followed by enhanced Aβ production (Udayar et al., 2013).