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

2. Rab GTPases in Neurodegeneration 

Small GTPases of the Rab family are responsible for controlling vesicular transport and membrane trafficking. They regulate all the steps of this transport; the biogenesis of carriers, their movement across the cytoskeleton, and their tethering in the target membranes [38,39]. As with the rest of the members of the Ras superfamily, the activity of Rab GTPases is regulated by GEFs, GAPs, and GDIs. Two main families of RabGEFs have been described. The first is the DENN domain-containing family of GEFs, which can activate different Rab GTPases [40]. DENN is the catalytic domain that interacts directly with Rab GTPases [40]. The second is the Vps9 domain-containing family of GEFs, which are specific for Rab5 GTPases [41]. 

    click to jade cistanche for Alzheimer's disease and Parkinson's disease

Apart from these two families, other proteins have been shown to act as GEFs for Rab GTPases, such as the TRAPP I and Mon1/Ccz1 complexes, which are GEFs for Rab1 and Rab7, respectively [41]. On the other hand, whereas GEFs share low sequence homology amongst them, Rab GAPs are classified into a unique family, the Tre-2/Bub2/Cdc16 (TBC)-domain GAPs. In humans, there is a single GAP that does not contain this TBC domain, the Rab3GAP complex [41]. Unfortunately, GEFs and GAPs for several of the Rab GTPases have not been described yet [41,42]. Apart from being regulated by their activation state (GDP-bound/GTP-bound), Rab GTPases can be found both in their active and inactive state in the cytosol or membranes. 

This localization is controlled by the prenylation of the C-terminal cysteine residues. Once the vesicular transport is complete, Rab GTPases must be recycled and transported from membranes back to the cytosol. GDIs bind to prenylated and inactive (GDP-bound) Rab GTPases and then, the GTPases are removed from the membrane. Thus, the recycling of Rab GTPases is only accomplished once the vesicular transport is complete and the GTPase is inactivated by a GAP [41]. Nevertheless, prenylation is not the unique post-translational modification that regulates Rab GTPases. Some Rabs can be phosphorylated by kinases such as p34cdc2 or the PD-related kinase LRRK2 [41,43]. The pathogenic variants of LRRK2 associated with PD result in an increase in such phosphorylation. This post-translational modification occurs in the switch II domain, which is crucial for the GTPase interaction with its regulators. Specifically, phosphorylation reduces the interaction of the GTPase with its regulators [43,44]. 

As previously mentioned, Rab GTPases control all of the key steps of vesicular transport and membrane trafficking, due to their ability to interact with different effector molecules [45]. For cargo selection, budding, and coat formation, Rab GTPases interact with proteins such as TIP47 or retromer. For instance, Rab9-GTP interacts with TIP47 in late endosomes, increasing TIP47's affinity towards the cargo that must be transported [46]. TIP47 recognizes the cytoplasmic domains of mannose 6-phosphate receptors (MPR), activating the transport from endosomes to the Golgi complex [46]. Another example is the interaction of Rab7 with the retromer complex to mediate the endosome-to-Golgi complex transport [47]. Regarding the regulation of vesicular transport, Rab GTPases interact with motor proteins such as kinesins and dyneins. Kinesins and dyneins are ATPases that use ATP hydrolysis to induce conformational changes that generate the motive force to move the cargo towards the plus-end and the minus-end of microtubules, respectively [48]. 

The Rab GTPases such as Rab3A, 6, 8A, 10, 11A, 14, 27A, and 39B interact with myosin type V to transport organelles and vesicles through actin filaments [49]. For instance, Rab27A interacts with myosin type V and melanophilin, forming a ternary complex to transport melanosomes toward actin filaments [50]. For the control of the uncoating and tethering of vesicles, Rab GTPases associate with proteins such as TRAPP, Exocyst, or p115/Golgins. One example is the interaction of Rab1 with p115, which is a tethering protein that induces the formation of the SNARE complex and stimulates the docking of COP I-coated vesicles in Golgi membranes [51]. Moreover, Rab1 also interacts with other tethering factors such as GM130 and GRASP65 to facilitate the fusion of the Golgi membranes vesicles [52]. 

GM130 is then responsible for the maintenance of the Golgi structure [52]. It is known that Rab GTPases interact with proteins such as Sro7 and Rabenosyn-5 [45]. For example, Rab8 interacts with Sro7, regulating SNARE protein functions in the fusion of vesicles to the cell membranes while Rabenosyn-5 serves as a nexus between Rab and hVPS45 [53,54] by bringing together Rab4 and/or Rab5 and hVPS45-associated Rabenosyn-5, which then binds SNAREs. In conclusion, Rab GTPases are the master regulators of cargo selection, formation, transport, docking, and the fusion of vesicles with target membranes. Taking into account the importance of membrane trafficking in the nervous system, neurons have developed specific mechanisms to control the transport of proteins, organelles, and receptors through long distances in axons and dendrites. Rab GTPases regulate the recycling, exocytosis, and endocytosis of synaptic vesicles; the liberation of neurotransmitters; the traffic of receptors; and the anterograde and retrograde axonal transports [15].

 What is more, they are also involved in the branching and morphogenesis of dendrites, neurite growth, and neuronal migration during development. Considering the importance of such processes, the dysregulation of Rab GTPases has been related to various neurodegenerative diseases such as AD, PD, amyotrophic lateral sclerosis (ALS), and Charcot–Marie–Tooth (CMT) [8,15]. In AD, various Rab GTPases are involved in the transport of proteins related to the pathology, such as Tau, APP, BACE1, α-secretase, γ-secretase, and Aβ peptides. Furthermore, the expression of these GTPases is altered in the post-mortem AD brain [55]. Regarding PD, these GTPases control the transport of α-syn [56]. Additionally, Rab GTPases could be mediating the toxicity caused by the LRRK2 kinase in PD [57]. As mentioned above, some Rab GTPases are substrates of LRRK2 and the dysregulation in this phosphorylation has been described to induce neurotoxicity and the degeneration of dopaminergic neurons in vivo [57,58]. Hereunder, we describe the specific roles of the main Rab GTPases in the onset and progression of AD and PD (Figure 2).

2.1. Rab1 

Rab1 GTPases control the bidirectional transport between the endoplasmic reticulum (ER) and the GA, as well as the formation, integrity, and recycling of Golgi membranes [38,59]. The Rab1 family is composed of two isoforms: Rab1A and Rab1B. The GEF for both isoforms is TRAPP I. TRAPP I is a complex of proteins that activates Rab1 and is involved in the ER–Golgi transport [41,60]. On the other hand, the molecule responsible for the inactivation of Rab1 is TBC1D20 GAP [41,61]. Many studies highlight the importance of Rab1, as well as its regulators, in the maintenance of the integrity of Golgi membranes. 

The overexpression of dominant-negative forms of Rab1A and Rab1B, the depletion of both GTPases and the overexpression of TBC1D20 GAP induce the fragmentation of the GA [38]. 2.1.1. Rab1 and the ER–Golgi Traffic Rab1 controls the transport between the ER and the GA, as it can interact with p115 and GM130-GRASP65 to favor the fusion of ER-vesicles in the GA [62–64]. Through its interaction with these effector molecules, Rab1 governs the formation, integrity, and recycling of GA membranes. On the one hand, Rab1 interacts with p115 protein, which is a vesicle tethering factor, to control this ER–GA traffic [65]. On the other hand, when Rab1 associates with the GM130-GRASP65 complex in the GA, it regulates the stacking of the GA and vesicle binding [66,67]. 

GM130 is responsible for the integrity of Golgi membranes [52]. Moreover, it is believed that p115 can interact with GM130-GRASP65 for ER-vesicle fusion in the GA [62,64]. Furthermore, Rab1 also controls the retrograde transport between GA and the ER. To do so, the GTPase interacts with GBF1, a GEF for the Arf1 GTPase that is involved in the biogenesis of COP I vesicles [68,69]. Although the role of Rab1 in the ER–GA traffic in AD pathogenesis is not yet clear, it has been described that this GTPase could prevent the loss of dopaminergic neurons in PD [19]. In PD, one of the possible mechanisms by which α-syn could be inducing neurodegeneration is by inhibiting the ER–GA traffic [19]. 

It has been described that wild-type (WT) α-syn, as well as the mutant α-synA53T that causes early-onset PD, block the ER–GA traffic, although α-synA53T initiates this blockage more rapidly than the WT. Cooper and collaborators have demonstrated that this α-syn-induced toxicity is prevented in the presence of Rab1 [19]. In fact, in Drosophila melanogaster (D. melanogaster), Caenorhabditis elegans (C. elegans), and primary cultures of rat neurons that express WT α-syn or α-synA53T, the expression of Rab1 rescued the loss of dopaminergic neurons [19]. These data suggest that Rab1 could play a protective role in the control of ER–GA traffic and, therefore, could prevent neurodegeneration in PD. Rab1 and its function in the control of ER–GA traffic are also related to ALS. The mutations in SOD1, TDP-43, or FUS proteins that cause this neurodegenerative disease result in a mislocalization of Rab1, as well as in impaired ER–GA transport and increased ER stress [8]. Rab1 overexpression, on the contrary, exerts a protective role against this stress [8,21].

2.1.2. Rab1 and the Integrity of the GA 

Apart from the classical hallmarks of AD and PD pathologies, it has been described that neurons present a fragmented GA in both cases [70]. This fragmentation has been attributed to various causes, such as the presence of protein aggregates in the cytoplasm, alterations in the cytoskeleton, or the malfunction of intracellular trafficking. In this regard, Martínez-Menárguez et al. state that the main reason for GA fragmentation in neurodegenerative diseases is the alterations in the intracellular transport [70]. Several studies have demonstrated that in neurodegenerative pathologies, Rab1- mediated traffic dysregulation induces GA fragmentation [16,17,70]. In the case of AD, those GA alterations have been ligated to pTau levels [71,72]. 

In 2014, the study of Jiang and collaborators revealed that GA fragmentation preceded Tau hyperphosphorylation [71]. According to them, GA fragmentation promotes Tau phosphorylation through the activation of cyclin-dependent kinase-5 (cdk5) and ERK. Moreover, in AD patients, neurons exposed to NFTs present bigger defects at the Golgi in comparison to neurons without NFT [72]. Neurons that accumulated intermediate levels of play before NFT formation showed intermediate defects in the GA [72]. This supports that the progressive accumulation of play is associated with structural alterations in the GA. According to Antón-Fernández and collaborators, these alterations could affect the processing and trafficking of proteins, and therefore, they could contribute to neuronal dysfunction in AD [72]. Furthermore, the overexpression of Rab1A in HeLa cells expressing human Tau and primary neurons of rat cortex prevented GA fragmentation, whereas the silencing of the GTPase by siRNA induced its fragmentation [16,17]. 

They observed that Rab1A was co-localizing with GM130 in primary cultures of neurons from the rat cortex [16]. Another effect of Rab1A silencing was the up-regulation of Tau secretion. Thus, the authors proposed that Rab1 could be a therapeutic target to modulate Golgi dynamics and Tau secretion in AD [16]. In summary, GA fractioning is associated with Tau phosphorylation [71], pTau accumulation in NFT [72], and Tau secretion [16]. Hence, Rab1 GTPase regulation could modulate such neurodegenerative processes. Regarding PD, dopaminergic neurons also display GA fragmentation. Specifically, dopaminergic neurons from the substantia nigra par compacta that overexpress human α-syn exhibit GA fragmentation, which is reduced when overexpressing Rab1A [17]. 

Additionally, apart from rescuing GA fragmentation, Rab1A overexpression in dopaminergic neurons induced improvements in motor functions. Conversely, overexpression of the non-printable Rab1A (Rab1A-∆CC) was not able to rescue GA from fragmentation. This demonstrated the importance of Rab1A in the maintenance of the GA integrity, and consequently, in the control of motor functions [17]. These data suggest that Rab1A GTPase overexpression could be a therapeutic approach for this pathology. A recent study has analyzed dopaminergic neurons from the substantia nigra of human patients with PD, and they have demonstrated that GA is fragmented and that the surviving neurons show a high overexpression of Rab1 GTPase [18].

 The authors suggest that this overexpression of Rab1 could induce the GA fragmentation by two theoretical mechanisms proposed: (1) overexpressed Rab1 could alter ER–Golgi transport, therefore causing an imbalance in the GA; (2) Rab1 could be interacting with Golgin-84, which would be inducing the fragmentation [18]. Overall, there are discrepancies regarding the role of Rab1 in either inducing or preventing GA fragmentation in PD. Apart from AD and PD, ALS is another neurodegenerative disease that presents GA fragmentation. The major cause for this seems to be the disturbances in the secretory pathway dependent on Rab1 [70]. Thus, Rab1 and its role in maintaining GA integrity are involved in different neurodegenerative diseases.

2.1.3. Rab1 and the Control of the Autophagosome 

Rab1 GTPase, along with other Rab GTPases such as Rab5, Rab7, Rab9A, Rab11, Rab23, Rab32, and Rab33B, participates in the formation of the autophagosome [73] at its beginning by recruiting the autophagy-related protein 9 (Atg9), a transmembrane protein responsible for transporting membranes to the phagophore, which is the structure preceding the formation of the autophagosome [74,75]. As previously mentioned, α-syn overexpression induces GA fragmentation. This leads to the dysregulation of autophagy in the SKNSH human neuroblastoma cell line, HeLa, HEK293, and M7-α-syn mice [20]. Winslow and colleagues described that α-syn alters the activity of the Rab1A/Atg9 axis. When silencing Rab1A and overexpressing α-syn, the Atg9 protein stopped localizing at a perinuclear position and passed to a diffuse distribution, resulting in a reduction in the autophagosome formation [20]. Thus, an increase in Rab1A activity could favor autophagy and therefore reduce the severity of the disease, as this mechanism could be used to recycle and eliminate protein aggregates.

2.2. Rab5 

Rab5 plays an important role in endocytosis, being responsible for the fusion of endocytic vesicles coming from the plasma membrane to form early endosomes. By this mechanism, Rab5 regulates the internalization and the trafficking of membrane receptors [76]. The two GEFs described for Rab5 are Ras/Rab Interactor 3 (RIN3) and Rabex5. RIN3 is a member of the RIN family of GEFs, together with RIN1 and RIN2. All three have a Vps9 domain, which is the Rab5-specific GEF catalytic domain [77]. Regarding Rabex5, it is the best-understood member of the Vps9 domain-containing GEFs. Apart from its catalytic domain, Rabex5 contains a Rabaptin5 binding site, which is a Rab5 effector molecule. Thus, Rabex5 binds tightly to Rab5-regulated Rabaptin5, which in turn regulates Rabex5 GEF activity, forming a feedback loop [78]. Rab5 recruits Rabaptin5 in early endosomes, the latter being responsible for the docking and fusion of membranes [79]. 

Once activated, the Rabex5/Rab5/Rabaptin5 complex is localized in endocytic vesicles and early endosomes [79–81]. The three molecules work to stabilize active Rab5 once it reaches its targeted localization, forming a positive feedback loop that potentiates this pathway [38]. As the Rab5 signaling to Rabaptin5 [79] above described, Rab5 can signal through the PI3K hVPS34-p150 complex, which increases the levels of PI3P in early endosomes [25,82,83]. This PI3P permits the recruitment of the EEA1, another Rab5 effector molecule that regulates the docking of endocytic vesicles before their fusion with the early endosomes [84]. Moreover, hVPS34-p150 can activate a negative feedback loop by activating TBC1D2 GAPs, resulting in Rab5 GTPase inactivation [85]. 

The TBC domain-containing GAPs TBC1D3, RUTBC3, and USP6NL have been described as Rab5 GAPs [12,41]. The role of Rab5 in neurodegenerative diseases has been circumscribed to endosomal trafficking. In this regard, various studies have detected an increase in Rab5 activity in AD [12,22,86–91], as well as in murine models of PD [12,92,93]. In Huntington’s disease (HD), Rab5 also controls the motility of early endosomes. HD is caused by mutations in the huntingtin (Htt) protein, which is located on the GA and vesicles. Htt forms a complex with Htt-associated protein 40 (HAP40) and serves as an effector molecule of Rab5 [94]. In HD, HAP40 is upregulated and the Htt-HAP40 complex is disrupted. Consequently, the motility of the early endosomes is reduced [94]. Thus, Rab5 could be a therapeutical target to improve endosomal motility in HD.

2.2.1. Rab5 and APP Processing 

The anomalies in endocytic trafficking are one of the main characteristics of AD, and according to Cataldo and collaborators, they precede the Aβ deposits [95]. A later study demonstrated that Rab5 overexpression can reproduce such endocytic anomalies by increasing the highly active processing of APP in endosomes [22]. The overexpression of Rab5 in murine cells induced endocytic changes related to AD, such as the presence of big endosomes similar to those observed in neurons from AD brains [22]. Furthermore, Rab5 overexpression increased by 2.5 times the levels of Aβ1-40 and Aβ1-42 secretion [22]. 

The authors also observed an increase in the βCTF levels. These βCTFs colocalize with early endosomes, suggesting a direct relationship between the endosomal pathway, βCTF generation, and Aβ production. Therefore, the endosomal anomalies observed in AD could be associated with defects in APP proteolysis [22]. This suggests that Rab5 could be a therapeutic target due to its relevance in the control of APP processing and consequently, in Aβ1-40 and Aβ1-42 generation. The role of βCTF in the recruitment of pleckstrin homology and phosphotyrosine binding domain- and leucine zipper motif-containing adaptor protein (APPL1) has also been described [91]. In endosomes, APPL1 stabilizes active Rab5-GTP, leading to a pathologic dysregulated endocytosis [91]. 

Taking into account the role of Rab5 in the endosomal pathway, Grbovic and collaborators defend that the dysregulations in the endosomes give rise to an increase in βCTF [22], and Kim and collaborators defend that βCTFs induce those endosomal dysregulations [91]. Additionally, shRNA silencing of BACE1 reverted the endocytic defects, suggesting that APP proteolysis could be the cause of the endocytic defects [96]. In conclusion, these studies point out a positive feedback loop in which the APP processing could lead to dysregulation of the endosomal pathway, and the defects in the endocytic pathway could in turn increase APP processing

2.2.2. Rab5 and Axonal 

Transport In normal basal forebrain cholinergic neurons (BFCNs), the nerve growth factor (NGF) binds and activates the TrkA receptor at axonal ends. The NGF-TrkA complex is then internalized by endocytosis mediated by Rab5. The endosomes are transported in a retrograde direction through microtubules to the cell body, where the growth and differentiation signals are propagated to the nucleus [12]. In pathological conditions, there is an overactivation of Rab5 in BFCN neurons, which results in bigger early endosomes. These endosomes interfere in the retrograde axonal transport of NGF signals. Additionally, an increase in Rab5 activity can also affect motor proteins, altering axonal transport, and defects in the transport of trophic signals to the cell body lead to neuronal atrophy [12]. 

In this regard, the GEF RIN3 has been related to the overactivation of Rab5 in the transport of trophic signals [77,97]. Moreover, genome-wide association studies (GWAS) have linked RIN3 with the risk of developing AD [12,98–100]. However, it still needs to be clarified whether RIN3 function and expression are altered in AD and if other Rab5 GEFs underlie Rab5 over-activation in AD [12]. Nevertheless, there is another possible mechanism that could explain Rab5 overactivation. As previously mentioned, βCTF recruits APPL1 to the endosomes, which stabilizes Rab5-GTP. This complex leads to dysregulated endocytic pathways, as well as altered axonal transport [12,91]. Regarding PD, murine models constitutively expressing human α-syn have demonstrated the α-syn-dependent activation of Rab5 leading to dysregulation in Rab5 and dynein complex resulting in endosomal dysfunction. This could be the underlying mechanism that would explain the dysregulation in retrograde axonal transport and the consequent neuronal atrophy in PD [12,93].

2.3. Rab7 

Rab7 GTPase regulates vesicular transport, specifically the late endocytic pathway [101]. It presents a fundamental role in the maturation of endosomes, in the transport of endosomes and lysosomes, in the fusion of late endosomes and lysosomes, and in the lysosomal biogenesis [26,101,102]. Rab7 also participates in the traffic of autophagosomes [103]. Considering the importance of all these processes, Rab7 has been proposed as a therapeutic target for cancer [26] and neurodegeneration [104]. Rab7 activation is mediated by the GEF Mon1-Ccz1 [27,105,106]. The mechanism by which Mon1-Ccz1 mediates Rab7 activation consists of its ability to be an effector molecule of Rab5 and interact with PI3P in early endosomes [102,107]. 

This way, there is an exchange between Rab5 and Rab7 and the endosome passes from an early endosome to a late endosome [105,107]. On the other hand, the GAPs described for Rab7 are TBC1D2A, TBC1D5, TBC1D15, and EVI5-L [41]. Rab7-GTP in late endosomes and lysosomes can signal through its effector molecule the Rab-interacting lysosomal protein (RILP) [108]. RILP recruits dynein–dynactin motor complexes and consequently, the endosomes are transported toward the minus end of the microtubules [109]. The FYVE and coiled-coil domain-containing protein 1 (FYCO1) is another effector molecule of Rab7 that mediates the vesicular transport towards the plus end of microtubules [110]. Moreover, FYCO1 forms a complex with Rab7 and the LC3 protein, which is in charge of the maturation of the autophagosome [111]. 

Once this complex is formed, autophagic vesicles are transported toward the plus end of the microtubule [110]. Regarding the nervous system, both autophagy and the endolysosomal traffic governed by Rab7 have been associated with pathologies such as AD, PD, HD, or Charcot– Marie–Tooth type 2B (CMT2B) [104,112]. Rab7 is involved in the traffic of toxic peptides such as Aβ vesicles [23] or Tau secretion in AD [29] and α-syn clearance in PD [30].

2.3.1. Rab7 and Trafficking of Toxic Peptides 

In AD, Aβ accumulation can be the consequence of a dysregulation in the APP processing, as well as a defect in the elimination of the toxic oligomers [113]. Therefore, the Rab5 and Rab7-controlled endolysosomal traffic are important for the clearance of toxic peptides such as Aβ. In this regard, studies in the N2a neuroblastoma mouse cell line, as well as in primary neuronal cultures from mice, have demonstrated that Aβ1-42 is internalized in Rab5-positive early endosomes at initial states and later, in Rab7-positive late endosomes [23]. These data suggest that the endocytic pathway is actively involved in the clearance and/or elimination of Aβ. 

The overexpression of the dominant-negative forms of Rab5 and Rab7, unable to bind and transmit the signal through their effector molecules, inhibited the colocalization of these GTPases with Aβ1-42 monomers and oligomers in the endosomes [23]. This supports the involvement of these GTPases and endocytosis in Aβ clearance. Some studies suggest that the Rab5- and Rab7-mediated dysregulated endolysosomal pathway has toxic effects [24,87,88]. Post-mortem AD brains have shown increased Rab5 and Rab7 protein levels [87,88]. Moreover, a study in primary neurons from the rat cortex has demonstrated that a Rab5- and Rab7-mediated active internalization of Aβ1-42 leads to neuronal death [24], and adding that the endocytosis general inhibitor phenyl arsine oxide (PAO) attenuated the toxicity. 

These results suggest that blocking Rab5- and Rab7-mediated endocytosis could be a therapeutic strategy to prevent neuronal death in AD [24]. As for Tau, the brains of patients with rapid progressive AD and 5XFAD mice brains exhibited increased Rab7A protein levels colocalized with pTau [28]. Moreover, Rab7A overexpression in primary cortical neurons and HeLa cells induced Tau secretion [29]. Conversely, Rab7A silencing, as well as the overexpression of its dominant-negative form, partially blocked Tau secretion [29]. All these data could mean that Rab7 dysregulation could contribute to Tau accumulation, as well as to the propagation of its toxic effects in AD [114].

2.3.2. Rab7 and Endolysosomal Trafficking of Membrane Receptors 

Endolysosomal pathway dysfunction has been related to PD, and genes that participate in this pathway have been related to this pathogenesis [115]. Lrrk, the homolog of the LRRK2 kinase in D. melanogaster, interacts with Rab7 in the membranes of late endosomes and lysosomes and has been shown to inhibit the Rab7-dependent perinuclear localization of lysosomes [116]. Conversely, the mutant form of Lrrk, the analog to the pathogenic LRRK2G2019S, promotes the perinuclear clustering of lysosomes. Thus, Rab7 and the LRRK2G2019S could underlie the dysfunctional endolysosomal pathway in PD [116]. 

It has been described that LRRK2 regulates the Rab7-dependent endocytic traffic of the epidermal growth factor receptor (EGFR) [31]. The expression of the mutant LRRK2G2019S caused a delay in early-to-late endosomal EGFR trafficking and a consequent delay in EGFR degradation. These defects were reverted by overexpressing the constitutively active form of Rab7 [31]. The ability of Rab7 to regulate the trafficking of receptors has already been used in therapeutic approaches for multiple sclerosis (MS) [33]. The overexpression of Rab7 can regulate the presence of Toll-like receptors (TLRs) and therefore control the inflammatory response [33]. However, Rab7 is not the only Rab GTPase that regulates the trafficking of receptors. 

Rab11, for instance, controls TLR trafficking via the endosomes [117]. In this regard, the presence of specific single nucleotide polymorphisms (SNPs) in Evi5, a Rab11GAP, has been correlated to higher susceptibility for developing MS [118]. This suggests that Rab11 could be recycling TLR receptors, affecting innate immunity. More recently, Evi5 has been associated with MS [119] and it has been used as a marker for the disease [120]. These data invite one to explore Rab GTPases signaling regulation as an approach to promote the recycling of receptors in neurodegenerative diseases.

2.3.3. Parkin/Rab7/RILP 

Parkin is a ubiquitin E3 ligase associated with PD, as mutations in this enzyme are the second most common genetic risk factor for the development of the disease [121]. Rab7 K38 residue ubiquitination maintains Rab7 in an active form and consequently affects the endocytic traffic [32]. Experiments with primary fibroblast cultures from PD patients deficient of functional Parkin and in cells overexpressing the Rab7K38R mutant that cannot be ubiquitinated demonstrated that in these situations, the Rab7 capacity of binding to its effector molecule Rab7-Interacting Lysosomal Protein (RILP) is diminished [32]. RILP is a Rab7 effector molecule involved in transducing the Parkin/Rab7 axis signaling. 

Specifically, RILP recruits dynein–dynactin motor complexes so that vesicles can be transported toward the minus end of the microtubules [108,109]. According to Song and collaborators, Rab7 dysregulation could be the main cause of endocytic alterations in Parkin-/- cells. Moreover, these dysregulations of the Parkin/Rab7/endocytosis axis could contribute to the progression of PD pathology [32].

2.3.4. Rab7 and Autophagy 

Rab7 in its active form can regulate the formation of the autophagosome, as well as its maturation and transport toward the microtubules [104]. The study of Rab7 and its role in autophagy could facilitate the development of strategies for the treatment of neurodegenerative diseases [104]. Rab7 is related to autophagy in CMT2B neurodegenerative disease. This pathology is caused by different missense mutations in Rab7 that lead to the reduced localization of Rab7 to autophagic compartments and decreased autophagy [8,34]. It is described that CMT2B is a direct consequence of Rab7 dysfunction, although it still needs to be clarified whether the pathology is a consequence of a reduction in the autophagic pathway due to Rab7 loss of function [8]. 

Regarding PD, studies with HEK293 and D. melanogaster α-synA53T demonstrated that Rab7 overexpression favors the clearance of α-syn aggregates [30]. Moreover, the authors identified that Rab7 localized in the neuromelanin granules in the human substantia nigra [30]. The Rab7/neuromelanin granules are autophagosome-like protective organelles. Rab7 participates in the biogenesis of these granules and the clearance of α-syn aggregates [30]. In addition, Rab7 overexpression in D. melanogaster rescued the phenotype and improved the locomotor deficits [30]. Nevertheless, Rab7 is not the only Rab GTPase described to control the α-syn clearance through autophagy. Recently, Rab27b has been shown to control the endolysosomal traffic and thereby the secretion and clearance of α-syn through autophagy [122]. 

Accordingly, the silencing of Rab27b by shRNA increased the intracellular levels of insoluble α-syn. Additionally, the post-mortem brains of PD patients have shown increased protein levels of Rab27b [122]. Although they are not related to autophagic processes, other Rab GTPases also participate in the homeostasis of α-syn; whereas some of them favor the clearance of the aggregates, others favor their formation. For instance, Rab39B classically regulates the transport between the GA and the post-synaptic membrane. In PD, mutations in Rab39B have resulted in the loss of function of the GTPase and, consequently, in the dysregulation of α-syn homeostasis [123,124]. 

Conversely, PD patients have shown increased levels of Rab35, which promotes an augmented aggregation and secretion of α-synA53T [125]. Besides, primary cell cultures and in vivo experiments demonstrated that LRRK2-mediated Rab5 dysregulation induced severe neurotoxicity and the loss of dopaminergic neurons [57,58].

why eating cistanche can prevent Alzheimer's disease and Parkinson's disease

Cistanche contains several active compounds that have been shown to have neuroprotective effects, which may help prevent or slow the progression of Alzheimer's disease and Parkinson's disease. These compounds include echinacoside, acteoside, and verbascoside, which have been found to have anti-inflammatory and antioxidant properties that can protect neurons from damage and reduce inflammation in the brain. Additionally, cistanche has been shown to increase levels of acetylcholine, a neurotransmitter that is important for learning and memory, which can be decreased in Alzheimer's disease. While more research is needed to fully understand the potential benefits of cistanche for preventing these diseases, these initial findings are promising.

Reference

19. Cooper, A.A.; Gitler, A.D.; Cashikar, A.; Haynes, C.M.; Hill, K.J.; Bhullar, B.; Liu, K.; Xu, K.; Strathearn, K.E.; Liu, F.; et al. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science 2006, 313, 324–328. [CrossRef] 

20. Winslow, A.R.; Chen, C.-W.; Corrochano, S.; Acevedo-Arozena, A.; Gordon, D.E.; Peden, A.A.; Lichtenberg, M.; Menzies, F.M.; Ravikumar, B.; Imarisio, S.; et al. α-Synuclein impairs macroautophagy: Implications for Parkinson’s disease. J. Cell Biol. 2010, 190, 1023–1037. [CrossRef] [PubMed] 21. Soo, K.Y.; Halloran, M.; Sundaramoorthy, V.; Parakh, S.; Toth, R.P.; Southam, K.A.; McLean, C.A.; Lock, P.; King, A.; Farg, M.A.; et al. Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43, and FUS-associated ALS. Acta Neuropathol. 2015, 130, 679–697. [CrossRef] [PubMed] 

22. Grbovic, O.M.; Mathews, P.M.; Jiang, Y.; Schmidt, S.D.; Dinakar, R.; Summers-Terio, N.B.; Ceresa, B.P.; Nixon, R.A.; Cataldo, A.M. Rab5-stimulated up-regulation of the endocytic pathway increases intracellular beta-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Abeta production. J. Biol. Chem. 2003, 278, 31261–31268. [CrossRef] [PubMed] 

23. Li, J.; Kanekiyo, T.; Shinohara, M.; Zhang, Y.; Liu, M.J.; Xu, H.; Bu, G. Differential Regulation of Amyloid-β Endocytic Trafficking and Lysosomal Degradation by Apolipoprotein E Isoforms. J. Biol. Chem. 2012, 287, 44593–44601. [CrossRef] 

24. Song, M.S.; Baker, G.B.; Todd, K.G.; Kar, S. Inhibition of β-amyloid1-42 internalization attenuates neuronal death by stabilizing the endosomal-lysosomal system in rat cortical cultured neurons. Neuroscience 2011, 178, 181–188. [CrossRef] [PubMed] 

25. Gillooly, D.J.; Raiborg, C.; Stenmark, H. Phosphatidylinositol 3-phosphate is found in microdomains of early endosomes. Histochem. Cell Biol. 2003, 120, 445–453. [CrossRef] [PubMed] 

26. Guerra, F.; Bucci, C. Role of the RAB7 Protein in Tumor Progression and Cisplatin Chemoresistance. Cancers 2019, 11, 1096. [CrossRef] 27. Nordmann, M.; Cabrera, M.; Perz, A.; Bröcker, C.; Ostrowicz, C.; Engelbrecht-Vandré, S.; Ungermann, C. The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7. Curr. Biol. 2010, 20, 1654–1659. [CrossRef] [PubMed] 

28. Zafar, S.; Younas, N.; Correia, S.; Shafiq, M.; Tahir, W.; Schmitz, M.; Ferrer, I.; Andréoletti, O.; Zerr, I. Strain-Specific Altered Regulatory Response of Rab7a and Tau in Creutzfeldt-Jakob Disease and Alzheimer’s Disease. Mol. Neurobiol. 2017, 54, 697–709. [CrossRef] 29. Rodriguez, L.; Mohamed, N.; Desjardins, A.; Lippé, R.; Fon, E.A.; Leclerc, N. Rab7A regulates tau secretion. J. Neurochem. 2017, 141, 592–605. [CrossRef] 

30. Dinter, E.; Saridaki, T.; Nippold, M.; Plum, S.; Diederichs, L.; Komnig, D.; Fensky, L.; May, C.; Marcus, K.; Voigt, A.; et al. Rab7 induces clearance of α-synuclein aggregates. J. Neurochem. 2016, 138, 758–774. [CrossRef] 

31. Gómez-Suaga, P.; Rivero-Ríos, P.; Fdez, E.; Blanca Ramírez, M.; Ferrer, I.; Aiastui, A.; López De Munain, A.; Hilfiker, S. LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity. Hum. Mol. Genet. 2014, 23, 6779–6796. [CrossRef] [PubMed] 

32. Song, P.; Trajkovic, K.; Tsunemi, T.; Krainc, D. Parkin Modulates Endosomal Organization and Function of the Endo-Lysosomal Pathway. J. Neurosci. 2016, 36, 2425–37. [CrossRef] [PubMed] 

33. Klaver, E.J.; van der Pouw Kraan, T.C.T.M.; Laan, L.C.; Kringel, H.; Cummings, R.D.; Bouma, G.; Kraal, G.; van Die, I. Trichuris suis soluble products induce Rab7b expression and limit TLR4 responses in human dendritic cells. Genes Immun. 2015, 16, 378–387. [CrossRef] [PubMed] 

34. Colecchia, D.; Stasi, M.; Leonardi, M.; Manganelli, F.; Nolano, M.; Veneziani, B.M.; Santoro, L.; Eskelinen, E.-L.; Chiariello, M.; Bucci, C. Alterations of autophagy in the peripheral neuropathy Charcot-Marie-Tooth type 2B. Autophagy 2018, 14, 930–941. [CrossRef] [PubMed] 

35. Hill, K.; Li, Y.; Bennett, M.; McKay, M.; Zhu, X.; Shern, J.; Torre, E.; Lah, J.J.; Levey, A.I.; Kahn, R.A. Munc18 Interacting Proteins: ADP-ribosylation factor-dependent coat proteins that regulate the traffic of β-Alzheimer’s precursor protein. J. Biol. Chem. 2003, 278, 36032–36040. [CrossRef] 36. Bansal, A.; Kirschner, M.; Zu, L.; Cai, D.; Zhang, L. Coconut oil decreases expression of amyloid precursor protein (APP) and secretion of amyloid peptides through inhibition of ADP-ribosylation factor 1 (ARF1). Brain Res. 2019, 1704, 78–84. [CrossRef] 

37. Griffin, E.F.; Yan, X.; Caldwell, K.A.; Caldwell, G.A. Distinct functional roles of Vps41-mediated neuroprotection in Alzheimer’s and Parkinson’s disease models of neurodegeneration. Hum. Mol. Genet. 2018, 27, 4176–4193. [CrossRef] [PubMed] 

38. Goud, B.; Liu, S.; Storrie, B. Rab proteins as major determinants of the Golgi complex structure. Small GTPases 2018, 9, 66–75. [CrossRef] [PubMed] 

39. Homma, Y.; Hiragi, S.; Fukuda, M. Rab family of small GTPases: An updated view on their regulation and functions. FEBS J. 2021, 288, 36–55. [CrossRef] 

40. Marat, A.L.; Dokainish, H.; McPherson, P.S. DENN Domain Proteins: Regulators of Rab GTPases. J. Biol. Chem. 2011, 286, 13791–13800. [CrossRef] 

41. Müller, M.P.; Goody, R.S. Molecular control of Rab activity by GEFs, GAPs and GDI. Small GTPases 2018, 9, 5–21. [CrossRef] [PubMed] 

42. Koch, D.; Rai, A.; Ali, I.; Bleimling, N.; Friese, T.; Brockmeyer, A.; Janning, P.; Goud, B.; Itzen, A.; Müller, M.P.; et al. A pull-down procedure for the identification of unknown GEFs for small GTPases. Small GTPases 2016, 7, 93–106. [CrossRef] 

43. Steger, M.; Tonelli, F.; Ito, G.; Davies, P.; Trost, M.; Vetter, M.; Wachter, S.; Lorentzen, E.; Duddy, G.; Wilson, S.; et al. Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases. Elife 2016, 5, e12813. [CrossRef] [PubMed] 

44. Madero-Pérez, J.; Fdez, E.; Fernández, B.; Ordóñez, A.J.L.; Ramírez, M.B.; Gómez-Suaga, P.; Waschbüsch, D.; Lobbestael, E.; Baekelandt, V.; Nairn, A.C.; et al. Parkinson's disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation. Mol. Neurodegener. 2018, 13, 3. [CrossRef] 45. Hutagalung, A.H.; Novick, P.J. Role of Rab GTPases in membrane traffic and cell physiology. Physiol. Rev. 2011, 91, 119–149. [CrossRef] 

46. Carroll, K.S.; Hanna, J.; Simon, I.; Krise, J.; Barbero, P.; Pfeffer, S.R. Role of Rab9 GTPase in facilitating receptor recruitment by TIP47. Science 2001, 292, 1373–1376. [CrossRef] [PubMed] 

47. Liu, T.-T.; Gomez, T.S.; Sackey, B.K.; Billadeau, D.D.; Burd, C.G. Rab GTPase regulation of retromer-mediated cargo export during endosome maturation. Mol. Biol. Cell 2012, 23, 2505–2515. [CrossRef] 

48. Horgan, C.P.; Mccaffrey, M.W. Rab GTPases, and microtubule motors. Biochem. Soc. Trans. 2011, 39, 1202–1206. [CrossRef] 

49. Lindsay, A.J.; Jollivet, F.; Horgan, C.P.; Khan, A.R.; Raposo, G.; McCaffrey, M.W.; Goud, B. Identification and characterization of multiple novel Rab-myosin Va interactions. Mol. Biol. Cell 2013, 24, 3420–3434. [CrossRef] [PubMed] 

50. Nagashima, K.; Torii, S.; Yi, Z.; Igarashi, M.; Okamoto, K.; Takeuchi, T.; Izumi, T. Melanophilin directly links Rab27a and myosin Va through its distinct coiled-coil regions. FEBS Lett. 2002, 517, 233–238. [CrossRef] 

51. Guo, Y.; Linstedt, A.D. Binding of the vesicle docking protein p115 to the GTPase Rab1b regulates membrane recruitment of the COPI vesicle coat. Cell. Logist. 2013, 3, e27687. [CrossRef] [PubMed] 

52. Nakamura, N. Emerging new roles of GM130, a cis-Golgi matrix protein, in higher order cell functions. J. Pharmacol. Sci. 2010, 112, 255–264. [CrossRef] 

53. Nielsen, E.; Christoforidis, S.; Uttenweiler-Joseph, S.; Miaczynska, M.; Dewitte, F.; Wilm, M.; Hoflack, B.; Zerial, M. Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through an FYVE finger domain. J. Cell Biol. 2000, 151, 601–612. [CrossRef] 

54. Rahajeng, J.; Caplan, S.; Naslavsky, N. Common and distinct roles for the binding partners Rabenosyn-5 and Vps45 in the regulation of endocytic trafficking in mammalian cells. Exp. Cell Res. 2010, 316, 859–874. [CrossRef] 

55. Zhang, X.; Huang, T.Y.; Yancey, J.; Luo, H.; Zhang, Y.-W. Role of Rab GTPases in Alzheimer’s Disease. ACS Chem. Neurosci. 2019, 10, 828–838. [CrossRef] 

56. Shi, M.; Shi, C.; Xu, Y. Rab GTPases: The Key Players in the Molecular Pathway of Parkinson’s Disease. Front. Cell. Neurosci. 2017, 11, 81. [CrossRef] [PubMed] 

57. Jeong, G.R.; Jang, E.-H.; Bae, J.R.; Jun, S.; Kang, H.C.; Park, C.-H.; Shin, J.-H.; Yamamoto, Y.; Tanaka-Yamamoto, K.; Dawson, V.L.; et al. Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration. Mol. Neurodegener. 2018, 13, 8. [CrossRef] 

58. Steger, M.; Diez, F.; Dhekne, H.S.; Lis, P.; Nirujogi, R.S.; Karayel, O.; Tonelli, F.; Martinez, T.N.; Lorentzen, E.; Pfeffer, S.R.; et al. Systematic proteomic analysis of LRRK2-mediated rab GTPase phosphorylation establishes a connection to ciliogenesis. Elife 2017, 6, e31012. [CrossRef][PubMed] 

59. Liu, S.; Storrie, B. How Rab proteins determine Golgi structure. Int. Rev. Cell Mol. Biol. 2015, 315, 1–22. [PubMed] 

60. Ishida, M.; Oguchi, M.E.; Fukuda, M. Multiple Types of Guanine Nucleotide Exchange Factors (GEFs) for Rab Small GTPases. Cell Struct. Funct. 2016, 41, 61–79. [CrossRef] [PubMed] 

61. Fukuda, M. TBC proteins: GAPs for mammalian small GTPase Rab? Biosci. Rep. 2011, 31, 159–168. [CrossRef] [PubMed] 

62. Sztul, E.; Lupashin, V. Role of tethering factors in secretory membrane traffic. Am. J. Physiol. Cell Physiol. 2006, 290, C11–C26. [CrossRef] 

63. Sztul, E.; Lupashin, V. Role of vesicle tethering factors in the ER–Golgi membrane traffic. FEBS Lett. 2009, 583, 3770–3783. [CrossRef] 

64. Grosshans, B.L.; Ortiz, D.; Novick, P. Rabs and their effectors: Achieving specificity in membrane traffic. Proc. Natl. Acad. Sci. USA 2006, 103, 11821–11827. [CrossRef] [PubMed] 

65. Grabski, R.; Hay, J.; Sztul, E. Tethering factor P115: A new model for tether-SNARE interactions. Bioarchitecture 2012, 2, 175–180. [CrossRef] [PubMed] 

66. Hu, F.; Shi, X.; Li, B.; Huang, X.; Morelli, X.; Shi, N. Structural basis for the interaction between the Golgi reassembly-stacking protein GRASP65 and the Golgi matrix protein GM130. J. Biol. Chem. 2015, 290, 26373–26382. [CrossRef] [PubMed] 

67. Zhang, X.; Wang, Y. GRASPs in Golgi Structure and Function. Front. Cell Dev. Biol. 2015, 3, 84. [CrossRef] 68. Alvarez, C.; Garcia-Mata, R.; Brandon, E.; Sztul, E. COPI Recruitment Is Modulated by a Rab1b-dependent Mechanism. Mol. Biol. Cell 2003, 14, 2116–2127. [CrossRef] [PubMed] 

69. Monetta, P.; Slavin, I.; Romero, N.; Alvarez, C. Rab1b interacts with GBF1 and modulates both ARF1 dynamics and COPI association. Mol. Biol. Cell 2007, 18, 2400–2410. [CrossRef] [PubMed] 

70. Martínez-Menárguez, J.Á.; Tomás, M.; Martínez-Martínez, N.; Martínez-Alonso, E. Golgi Fragmentation in Neurodegenerative Diseases: Is There a Common Cause? Cells 2019, 8, 748. [CrossRef]

to be continued

Alazne Arrazola Sastre 1,2, Miriam Luque Montoro 1 , Hadriano M. Lacerda 3 , Francisco Llavero 1,4,* and José L. Zugaza 1,2,5,

1 Achucarro Basque Center for Neuroscience, Science Park of the UPV/EHU, 48940 Leioa, Spain; alazne.arrazola@ehu.eus (A.A.S.); miriamluquem@gmail.com (M.L.M.) 

2 Department of Genetics, Physical Anthropology and Animal Physiology, University of Basque Country UPV/EHU, 48940 Leioa, Spain 

3 Three R Labs, Science Park of the UPV/EHU, 48940 Leioa, Spain; hadrilac@gmail.com 

4 Hospital 12 de Octubre Research Institute (i+12), 28041 Madrid, Spain 5 IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain * Correspondence: fcollavero.imas12@h12o.es (F.L.); joseluis.zugaza@ehu.es (J.L.Z.)