Small GTPases Of The Rab And Arf Families: Key Regulators Of Intracellular Trafficking in NeurodegenerationⅢ

3. Arf GTPases in Neurodegeneration 

Arf GTPases belong to a family of 29 members classified into different subfamilies: Arf1- 6, Arf-like proteins (Arl), SARs, and Trim23 [9,126,127]. Arf GTPases are differentiated from Ras, Rho, and Rab families as they possess an N-terminal extension of about 14 amino acids that can be covalently modified. In this regard, Arf GTPases can be N-myristoylated whereas Arl GTPases can be myristoylated, palmitoylated, or acetylated [9]. Arf GTPases control cellular processes such as the bidirectional trafficking of membranes (secretion and endocytosis), metabolism of lipids, motility, division, apoptosis, and gene transcription [9,127]. 

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However, their main role is the recruitment of coat proteins and complexes during vesicle formation in membrane trafficking, particularly in the Golgi [9]. Thus, Arf GTPases, as well as their GEFs and GAPs, are localized in the plasma membrane, endosomes, lipid droplets, mitochondria, and lysosomes [9]. 

Like all GTPases of the Ras superfamily, the activity of Arf GTPases is regulated by GEFs, GAPs, and GDIs. In humans, 15 Arf GEFs have been described, and are classified into six families depending on their domains: GBF, BIGs, Cytohesins, EFA6/Psd, BRAG/IQSec, and FBX [9]. All of them share in common the Sec7 catalytic domain [9,128,129]. Regarding the Arf GAPs, they are classified into 10 subtypes: ArfGAP1, ArfGAP2/3, ADAP1/2, SMAP1/2, AGFG1/2, GIT1/2, ASAP1-3, ACAP1-3, ARAP1-3 and AGAP1-11 [130–132]. They are characterized by their Arf GAP catalytic domain, although a family of proteins known as ELMOD has been demonstrated to possess GAP activity towards some Arf GTPases without having the Arf GAP domain [133–135]. 

Additionally, Arf GTPases can be regulated by post-translational modifications such as phosphorylation or ubiquitination [9]. Various Arf GEFs and GAPs have been described to play an important role in the nervous system. For instance, the Arf6 GAP, also known as ACAP3, has been shown to regulate neurite outgrowth in hippocampal neurons in mice [136]. Arf6 EFA6 GEF is involved in the arborization of dendrites and the formation of dendritic spines [137]. 

Moreover, mutations in the GEF BRAG1/IQSec2 have been associated with nonsyndromic X-linked intellectual disability [138]. Another example is that mice with Schwann cell-specific GEF BIG1 knockout display reduced myelin thickness [139]. All of these studies demonstrate the fundamental importance of Arf GTPases, as well as their regulators in the nervous system. About Arf GTPase's main effector molecules, they are components of vesicle coating, such as COP I, adaptor proteins (AP), GGA, and MINT, which are the most studied [140]. COP I is a vesicle-coating protein complex [141]. AP-1, AP-3, and AP-4 are clathrin adaptor proteins [9,140]. The GGAs participate in the TGN. Finally, MINTs interact with Munc18, a neuronal protein required for the exocytosis of synaptic vesicles [142]. 

Arf GTPases have been correlated to pathologies of the nervous system, such as ALS, retinal disease, and Creutzfeldt–Jakob disease [143]. Moreover, Arf GTPases are associated with AD, as MINTs regulate APP trafficking [35] and the GGAs interact with BACE1 to control APP processing [144] (Figure 3)

3.1. Arf/MINT and APP Trafficking and Processing 

MINTs are a family of three proteins that are specifically expressed in neuronal tissue. They are essential components for the fusion of neuronal synaptic vesicles [142,145]. MINT proteins directly bind to Arf-GTP and colocalize with APP in TGN regions [35]. MINT overexpression in HEK293 cells increased Arf GTPase-mediated APP protein levels [35]. Conversely, the siRNA-specific silencing of MINT3 in HeLa cells reduced APP protein levels. This demonstrated the Arf/MINT axis controls APP trafficking [35]. Another study demonstrated that MINT3 colocalized with APP in purified APPcontaining vesicles in the SH-SY5Y neuroblastoma cell line [146]. The siRNA silencing of MINT3 impacted APP trafficking, as well as its processing, inducing an increase in Aβ1-40 secretion [146]. 

Recently, a study in the N2a/APP695 mouse neuroblastoma cell line demonstrated that treatment with coconut oil reduced mRNA and protein levels of Arf1. Additionally, their results showed that Aβ1-40 y Aβ1-42 secretion levels were decreased [36]. All these studies suggest that Arf GTPase, along with its effector molecule MINT3, could be a therapeutic target to help regulate pathognomonic Aβ secretion in AD.

3.2. Arf/GGA/BACE1 

The GGAs participate in the transport and sorting of proteins in the TGN [147]. One of the best-studied functions of the GGAs is to direct the transport of ubiquitinated proteins to the endolysosomal pathway, as they possess ubiquitin-binding sites [148–150]. It has been described that GGA3 binds to the ubiquitinated BACE1 secretase to regulate its proteasomal degradation [151]. In this regard, the ectopic expression of GGA3 in GGA3-knock-out H4 neuroglioma cells blocked BACE1 accumulation, as well as Aβ1-40 secretion [151]. Therefore, the potentiation of the Arf/GGA3 axis activity could reduce BACE1 levels and consequently, Aβ secretion. Nevertheless, a study demonstrated in HEK293 cells cotransfected with APP695 and BACE, that GGA1 sequesters APP together with BACE1 into the Golgi [152]. 

Consequently, BACE1 processes APP increasing βCTF levels. Curiously, neither the intracellular levels nor the secretion of Aβ1-40 was increased. The authors conjectured that GGA1 sequesters the APP into the Golgi together with BACE1, where the β-cleavage takes place, and GGA1 also prevents these βCTF fragments from being transported and processed by the γ-secretase. In this way, although βCTF is increased, GGA1 would be negatively regulating its processing by the γ-secretase and, consequently, the generation of Aβ1-40 [152]. In the same line, another study in HEK293 and N2a cell lines showed that GGA overexpression resulted in a reduction in the secretion of the soluble APP alpha (sAPPα), sAPPβ, and Aβ. siRNA silencing of GGA reversed this effect [144].

3.3. Arl8 and Neuroprotection Against Aβ 

Arl8 GTPase promotes pre-synaptic vesicular and endocytic macromolecule traffic toward the lysosomes [153,154]. Various effector molecules are recruited to lysosomal membranes by Arl8-GTP [155]. For instance, the HOPS complex is responsible for the fusion of compartments in the late endocytic pathway [153,156,157]. Another effector molecule described for Arl8 in mammals is the SKIP/PLEKHM2, which is a linker protein that recruits kinesin-1 to lysosomal membranes [155,158]. It has been described that silencing the expression of Arl8 in C. elegans neurons provokes Aβ-mediated neurodegeneration [37]. On the contrary, the overexpression of Arl8 partially blocked this neurodegeneration. 

The authors demonstrated that the neuroprotective role of Arl8 depended on its state of activation. Constitutively active ArlQ75L partially reduced the neurodegeneration, whereas the dominant-negative ArlT34N did not have any protective effect [37]. The authors suggest that Arl8 could inhibit neurodegenerative processes through the activation of autophagy [37,159].

3.4. ArfGAP1/LRRK2 

LRRK2 is a multidomain protein that presents kinase activity as well as GTPase activity [160], described to be controlled by ArfGAP1 in HEK293 and brain extracts from mice [161]. The ArfGAP1/LRRK2 regulation is reciprocal, as LRRK2 can phosphorylate ArfGAP1 and increase its activity. Moreover, ArfGAP1, apart from activating GTP hydrolysis, also increased LRRK2 kinase activity, suggesting that ArfGAP1 activity could be implicated in this kinase activation [161]. It is known that primary neurons from LRRK2G2019S mice display neurite retraction. Stafa and collaborators rescued this phenotype by silencing ArfGAP1 [161]. Therefore, this GAP could be a possible therapeutic target for PD [161].

4. Future Perspectives 

The purpose of the drugs used for the treatment of AD and PD is to enhance cognition and ameliorate the symptoms. Thus, the FDA-approved drugs for both diseases include cholinesterase inhibitors and N-Methyl D-Aspartate (NMDA) receptor antagonists [162]. However, the symptomatic treatments do not strike the origin and the progression of the disease. In this regard, some approaches are currently being studied to decrease Aβ production, reduce the aggregation or enhance its clearance on the one hand, and inhibit Tau phosphorylation on the other [162], and new dopaminergic drugs are currently being tested for PD [162]. 

Both diseases, AD and PD, share common features such as the generation and accumulation of toxic peptides. Nevertheless, a few studies are focusing on the role of membrane and vesicular trafficking in the generation, accumulation, and clearance of those peptides. Specifically, the position of the Rab and Arf GTPases in these processes should be given more attention, and targeting them could be a promising therapeutic approach. One strategy could be modulating Rab and Arf GTPase activity depending on their state in the pathology. The expression of constitutively active or dominant-negative forms of these GTPases may be another alternative. 

For instance, constitutively active ArlQ75L reduced Aβ-induced neurodegeneration in C. elegans [37]. Another example is the dominant-negative form of Rab7A, which partially blocked Tau secretion [29]. Moreover, targeting the expression by siRNA silencing techniques could serve as a therapeutic strategy. Rab7A silencing by siRNA has been proven to be effective in the reduction of Tau secretion too [29]. A recent study has hinted that modifying Arf GTPases could be a feasible approach. Coconut oil treatment in N2a/APP695 cells reduced Arf1 mRNA and protein levels, which resulted in a decrease in Aβ1-40 and Aβ1-42 secretion levels [36]. 

Additionally, targeting the PTMs that allow GTPases to be anchored to cellular membranes could be a promising approach in the case of the Rab and Arf families, as they are key regulators of membrane and vesicle trafficking. This strategy has already been proven to be effective in other families of the Ras superfamily [5,163]. For instance, the inhibition of Rho family PTMs by lovastatin promotes myelin repair [163]. The regulation of GEFs, GAPs, and GDIs that control specific Rab and Arf GTPases could be an additional approach to treating neurodegenerative diseases. Lastly, peptides that interfere with protein–protein interactions between GTPases and their respective effector molecule is a promising alternative, as specific interactions can be inhibited without affecting the GTPase interaction with other effector molecules [5]. The problem lies in the fact that some Rab and Arf GTPases can sometimes have a neuroprotective role, whereas at other times they can be neurotoxic.

For instance, Rab5 overexpression has been shown to increase Aβ1-40 and Aβ1-42 secretion [22] and Rab7 seems to contribute to Tau secretion [29]. However, endolysosomal traffic controlled by Rab5 and Rab7 appears to favor the clearance of Aβ [23,24]. Another example is Rab1. 

Whereas Rab1 has been described to prevent GA fragmentation [16,17], it has recently been reported to possibly induce this fragmentation in neurons from human PD patients [18]. The presence of GTPases with opposite roles in neurodegenerative diseases further complicates the development of therapeutic approaches. The pathological state of activation in each specific case should be taken into consideration when focusing on small GTPases as therapeutical targets. In addition, it is important to describe the whole signaling cascade controlled by each GTPase in each specific pathological condition before considering a GTPase as a therapeutic target. 

While many signaling cascades are described in neurodegeneration in the Ras and Rho families [5], pathways controlled by Rab and Arf families need deeper study. Thus, the description of the exact axis controlling each toxic response would help us to identify therapeutic objectives. Considering this, the field should advance in describing the precise signaling cascades controlled by Rab and Arf GTPases in neurodegeneration to detect potential targets. However, not only is it important to describe the signaling cascades, but identifying the location of the specific pool that is being activated in a cell is relevant too [5]. Furthermore, the implication of glial cells should not be disregarded. 

Most studies in Rab and Arf GTPases have been carried out in neuronal cells, ignoring the involvement of glial cells in the pathogenesis of neurodegenerative diseases. For instance, microglial cells present a high volume of membrane trafficking as they actively participate in the clearance of protein aggregates. Therefore, methods specifically targeting glial cells could be a promising therapeutic option. Apart from emphasizing the search for therapies, studies should focus on the search for early detection of neurodegenerative diseases. For instance, a liquid biopsy-based early diagnosis would improve the outcome of neurodegenerative diseases and researchers are currently trying to find biomarkers for early detection [164]. 

However, if the biomarker needs to be present in the blood, it has to be able to cross the blood–brain barrier [165]. Another problem is that the concentration of the biomarker in blood could be lower than in the cerebrospinal fluid; for instance, Aβ concentrations are 10-fold lower in plasma [165]. Thus, highly sensitive techniques would be required for the detection of biomarkers in blood. Despite these challenges, the liquid biopsy-based diagnosis will soon be performed in the field of neurodegenerative diseases [164].

5. Conclusions 

The Ras superfamily of GTPases has long been disregarded as potential players in neurodegenerative diseases. Recently, we reviewed the role of Ras and Rho families, as well as their regulatory and effector molecules, as potential participants in the pathogenesis of neurodegeneration [5]. When it comes to Rab and Arf GTPases, the field is less explored, and very few studies have associated these molecular switches with AD and PD. However, these studies are a clear indication that the Rab and Arf families are involved in the pathogenesis of neurodegenerative diseases. In a broad concept, Rab GTPases in physiological conditions are responsible for vesicular transport and membrane trafficking [38]. 

They control the integrity of the GA, the processing and trafficking of toxic peptides such as the APP, and the axonal transport of proteins such as membrane receptors, and autophagy. Regarding Arf GTPases, their main function is to control vesicle formation, although they are also regulators of the membrane's bidirectional trafficking [9]. In this way, they manage the trafficking of proteins such as APP and BACE1. We expect that future research will allow us to characterize the whole signaling cascades controlled by Rab and Arf GTPases in neurodegeneration, and this will hopefully facilitate the development of therapeutic strategies. However, it must be highlighted that most studies have been done on neuronal cells, ignoring the involvement of glial cells in the pathogenesis of neurodegeneration. Thus, the role of these GTPases in AD and PD should be studied not only in neurons but in the nervous system as a whole.

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