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Biochemistry & Molecular Biology
Mitochondrial dysfunction and vesicular trafficking alterations have been implicated in the pathogenesis of several neurodegenerative diseases. It has become clear that pathogenetic pathways leading to neurodegeneration are often interconnected. Indeed, growing evidence suggests a concerted contribution of impaired mitophagy and vesicles formation in the dysregulation of neuronal homeostasis, contributing to neuronal cell death. Among the molecular factors involved in the trafficking of vesicles, Ras analog in brain (Rab) proteins seem to play a central role in mitochondrial quality checking and disposal through both canonical PINK1/Parkin-mediated mitophagy and novel alternative pathways. In turn, the lack of proper elimination of dysfunctional mitochondria has emerged as a possible causative/early event in some neurodegenerative diseases.
- vesicular trafficking
- neurodegeneration
- Ras analog in brain (Rab)
- mitophagy
1. Introduction
Vesicular trafficking alterations have been implicated in the pathogenesis of several neurodegenerative diseases (NDs) as likely early events [1,2]. Two main branches of vesicle trafficking exist in neuronal cells: the canonical shuttling of cellular components and the mobilization of synaptic vesicles. Both processes are tightly regulated and orchestrated by multiprotein complexes. Their malfunction causes profound alterations in cellular homeostasis.
As for canonical vesicle trafficking, the three central pathways are: (i) the secretory pathway, (ii) the endocytic pathway, and (iii) specialized/organelle-specific vesicular transports [3]. The secretory pathway, through which proteins and lipids move from the ER through the Golgi to the extracellular environment, involves two main processes, namely, the anterograde (from ER to Golgi) and the retrograde (from Golgi to ER) transports, which are mediated by COPII and COPI coatomers, respectively. The endocytic pathway, in which several cargos can be internalized by cells, involves the formation of early and late endosomes, which eventually fuse with lysosomes; in this process, several proteins assist vesicle formation, e.g., clathrin, caveolins, adaptors, GTPase dynamins, Ras analog in brain (Rab) proteins, and vacuolar protein sorting (VPS) proteins. Eventually, several specialized vesicle transports have been recently discovered, which mirror the high dynamism and social behavior of cellular organelles [4]. The endoplasmic reticulum (ER), Golgi apparatus, lysosomes, peroxisomes, and mitochondria are known to physically interact and exchange components thanks to both contact sites and vesicles formation; this allows organelles to modulate specific cellular functions, pathways, and metabolism. As an example, mitochondria are known to communicate with the ER, Golgi apparatus, lysosomes, lipid droplets, peroxisomes, and melanosomes [5]. The contact sites between mitochondria and the ER (i.e., mitochondria-associated membranes, MAMs) are important hubs for lipid trafficking, Ca2+ homeostasis, ER stress, apoptosis, and macroautophagy. On the other hand, the mitochondria-lysosome axis leads to the formation of vesicles of mitochondrial origin (i.e., mitochondria-derived vesicles, MDVs), whose generation and release are central to the disposal of defective components without triggering the mitophagic process. However, the content of MDVs is still mostly unknown; some components seem to play a role in the activation of inflammatory processes and are being investigated as candidate plasma biomarkers for NDs [6,7].
As for synaptic vesicles, neuronal synapses are specialized subcellular compartments, where the cycles of exocytosis and endocytosis are regulated by a complex molecular machinery, including several proteins, e.g., SNAREs, synaptotagmin, synaptophysin, and some small GTPases belonging to the Rab family (mainly Rab3, Rab5, and Rab27) [8]. Synaptic vesicles are classified in pools based on their function: they can either be part of the reserve pool or be involved in active exo- and endocytotic cycles. The mobilization of vesicles from one pool to another is an ATP-dependent process, fueled by the mitochondria that are resident in the synapses. Thus, synapses which lack mitochondria display marked defects in vesicular trafficking that are mirrored by deficits in neuronal functionality [9]. This further supports a clear link between mitochondrial dysfunction, which is a crucial pathogenetic process in many NDs, and defects in vesicular trafficking.
Mitochondria are central organelles in all cell types, being responsible for energy production in the form of ATP, the biosynthesis of amino acids and steroids, β-oxidation of fatty acids, the maintenance of cytosolic calcium homeostasis, the production and modulation of reactive oxygen species (ROS), and the triggering of the apoptotic cell death program. Moreover, they are dynamic organelles that are transported on cytoskeletal proteins and organized in networks, which are continuously remodeled by fusion and fission events [10]. This allows for both the exchange of mitochondrial components and the elimination of damaged parts to maintain a functional pool of healthy mitochondria. Damaged mitochondria must be disposed of through a macroautophagic pathway called mitophagy. Canonical mitophagy involves PTEN-induced kinase 1 (PINK1) and Parkin proteins, which are responsible for the recruitment of the molecular machinery that guides the formation of the autophagosome and the subsequent fusion with lysosomes [11]. When mitophagy is hampered, dysfunctional mitochondria accumulate within cells, thus increasing ROS concentration and oxidative load in general. Considering the huge energy demand and the terminally differentiated state of neuronal cells, it appears evident that any alterations to physiological mitochondrial homeostasis can have devastating effects on neuronal survival.
Nowadays, there is convincing evidence of impaired mitochondrial function as a cause rather than a consequence of the neurodegenerative process in several NDs, such as Alzheimer’s disease (AD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and Parkinson’s disease (PD). For instance, in the frame of PD etiopathology the vast majority of genetic PD-related loci are associated with mitochondria, and aberrant mitochondrial function has emerged as central also in sporadic PD [12]. Recently, vesicular trafficking defects have also been highlighted as important pathogenetic factors [13]. Alpha-synuclein, the major component of toxic protein aggregates in PD, has been shown to directly bind the SNARE protein VAMP2 and to be critical for the proper assembly of the SNARE complex [14]. LRRK2 protein, whose gene mutation is linked to autosomal dominant inherited familial PD, has been demonstrated to co-localize with Rab5 on endocytic vesicles [15] and to be the kinase of several Rab proteins [16,17]. The substrate repertoire of Parkin, which acts as an E3 ubiquitin ligase and is found to be mutated in autosomal recessive juvenile PD, includes several proteins involved in vesicular trafficking, such as Synphilin1 and Synaptotagmin XI [18]. In addition, recent genome-wide association studies (GWASs) have proposed new loci associated with an increased risk of PD, which encode proteins involved in vesicular trafficking, such as VPS35, Synaptojanin1, VPS13C, Rab39B, Rab7L1/Rab29, and Synaptotagmin XI [13,19,20].
2. Mitochondrial Dynamics Altered in Neurodegeneration
Mitochondria are double-membraned organelles strictly involved in the regulation of cellular homeostasis and metabolism. Among the most common known functions, such as ATP production, ion homeostasis, biosynthesis of precursors for macromolecules, and management of toxic metabolic by-products, mitochondria are emerging as being more and more relevant to regulating signaling pathways and cellular stress responses [21]. They are interconnected entities that constitute branched tubular networks, which are not fixed structures; rather, they are highly dynamic and their shape is deeply affected by perturbations to cellular functions [22].
Mitochondrial dynamics collectively refer to all the processes involved in the morphological changes of the mitochondrial network, and they include fusion, fission, mitophagy, and mitochondrial transport [23]. These processes are deeply interrelated and part of the quality control of mitochondrial organelles. The fine tuning of fusion and fission dynamic transitions is orchestrated mainly by the dynamin-related family of large GTPases, and the resulting balance between the two processes is responsible for the number and size of mitochondria [10]. Mitophagy is the process that allows dysfunctional mitochondria to be degraded. It can be triggered by different stimuli and completed by distinct pathways [24]. The altered balance of the abovementioned processes often results in the intracellular accumulation of dysfunctional organelles, thus contributing to the onset and progression of several NDs.
3. Rab Proteins at the Crossroads of Mitochondrial Dysfunction and Neurodegeneration
Rab proteins represent the largest family of Ras-like GTPases. They are largely distributed in eukaryotic cells with more than 70 members identified in mammals and 11 homologs in budding yeast [75,76]. Rab proteins exist within cells as molecular switches and shift between an active GTP-bound form, which associates with intracellular membranes, and an inactive GDP-bound conformation, which is present in the cytosol. These small GTPases require prenylation at their C-terminal cysteine residues to associate with membranes. This process is aided by geranylgeranyl transferases (GGTs) and Rab escort proteins (REPs). When membrane-bound, Rab proteins are activated by the exchange of GDP with GTP, and this is catalyzed by guanine nucleotide exchange factors (GEFs). In their active state, Rab proteins perform a myriad of functions, including vesicular budding, trafficking, and fusion with target membranes. On delivering cargo, Rabs undergo hydrolysis of the bound GTP to GDP with the help of GTPase-activating proteins (GAPs). In the inactive state, Rab proteins are retrieved and sequestered in the cytosol by GDP dissociation-inhibitor (GDI) proteins [77,78].
Rab proteins have been linked to multiple diseases, including NDs, cancer, and diabetes [79]. Among NDs, Rab proteins have been linked to dementia, AD, PD, HD, and ALS. In humans, 24 Rab proteins were found to be involved in the central nervous system [80], where many of them perform specialized neuronal functions [81]. More recently, the involvement of altered vesicle trafficking, particularly that which is Rab protein-mediated, has been highlighted in ND pathobiology. This finding has also proven to be a challenge, as studies are endeavoring to determine whether Rab proteins are causes or effects in the pathogenesis of NDs.
As Rab proteins are master regulators of vesicle trafficking, it is not surprising that they are emerging as key players in the molecular pathways of PD through a complex interplay with several PD-related genes. The question to be addressed is which Rab proteins are relevant to the mechanisms involved in PD, and to what extent. The most striking evidence of Rab proteins’ involvement came from the discovery of genetic alterations in familial forms of PD. Rab39B has a high brain-specific and exclusively neuron-specific expression [82]. Mutations in Rab39B resulted in a complete loss of protein expression and function and X-linked intellectual disability with early-onset PD [83]. Moreover, in Rab39B knockout mice, deficiency in Rab39B led to impaired learning and memory and impaired basal autophagic flux [84]. Another Rab gene, Rab29 (Rab7L1), has emerged as a leading candidate for sporadic PD, being one of the five genes that belongs to the PARK16 locus. Its role in PD pathogenesis is not completely understood; however, several studies have pointed to its upstream regulatory role in relation to LRRK2, whereby Rab29 increases LRRK2 activity and its localization to the trans-Golgi network [16,85,86]. LRRK2 mutations are the most common causes of familial PD and are also implicated in sporadic PD. The link between LRRK2 and Rab proteins came from the discovery that LRRK2 phosphorylates a wide subset of them, including Rab1A, Rab1B, Rab3, Rab5, Rab8A, Rab10, Rab12, Rab29, Rab35, and Rab43 [86]. All pathological LRRK2 mutations lead to hyperactivation of the protein, followed by abnormal Rab phosphorylation, which is abrogated upon pharmacological inhibition of LRRK2. Indeed, clinical trials are currently ongoing using small molecules as inhibitors of LRRK2 kinase activity, with potential beneficial effects in both familial and sporadic PD cases. As a downstream effect of LRRK2 inhibition, the restoration of physiological activity of Rab proteins is crucial to explanations of the therapeutic effects of these drugs [87]. An integrated omics analysis revealed dysregulation of the endocytic pathway in iPSC-derived dopaminergic neurons carrying the G2019S mutation in LRRK2. This dysregulation could be due to changes in the activity of several Rab proteins, including Rab5B, Rab7, and Rab10 [88].
Several studies have also established the role of Rab proteins in modulating α-synuclein biology in different model systems. Rab5, an early endosomal protein, is integral to the biogenesis of early endosomes [89]. It has been reported in the endocytosis of α-synuclein leading to neuronal death [90]. Another endosomal Rab, Rab7, was shown to clear α-synuclein aggregates. In HEK293 cells and Drosophila melanogaster, overexpression of Rab7 led to the clearance of α-synuclein aggregates and reduced cell death, thereby showing its protective role. This protective effect was specific to Rab7, as Rab5, Rab9, and Rab23 failed to rescue the phenotype [91]. Moreover, over-expressed α-synuclein was found to interact with dynein and induced an increase in the levels of GTP-bound Rab5 and Rab7 [92]. Eventually, overexpression of Rab11, physiologically involved in endosomal recycling, has also been shown to regulate α-synuclein dynamics in Drosophila models of α-synuclein toxicity [93].
As mitochondrial dysfunction makes a clear contribution to NDs, particularly PD, it is imperative to understand the pathways that regulate mitochondrial health. It is quite evident that mitochondrial quality-control pathways involve an intricate network of proteins and pathways, many of which have not been clearly understood and identified yet. For quite some time, macroautophagy was believed to be the only mechanism for degrading organelles, including mitochondria. Many studies are building up evidence that brings Rab proteins to the forefront in the clearing of damaged mitochondria via different pathways. Studies have also reported that alterations to Rab proteins related to their phosphorylation states, expression levels, and localization can lead to defects in mitophagy.
This entry is adapted from the peer-reviewed paper 10.3390/ijms24076268
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