α-Synuclein is a low-molecular weight protein that is composed of 140 amino acids and is divided into three distinct regions—a positively charged N-terminal region (1–60), a central hydrophobic region (61–95), which is known as the non-amyloid-beta component (NAC) and is involved in α-synuclein aggregation and a highly acidic C-terminal domain (96–140) [19,20]. α-Synuclein is an intrinsically disordered protein abundantly expressed in the central nervous system (CNS) [21] and was first purified in vitro in soluble and monomeric forms [22]. Usually, disordered proteins possess primary sequences enriched in charged residues and prolines [23] that avoid misfolding and aggregation. In the case of α-synuclein, the NAC domain is partially protected by the positive and negative charges of the N- and C-terminal regions. However, mutations change their environmental conditions and disturb the native compactness of α-synuclein, thereby triggering aggregation. To date, six point-mutations (A53T, A30P, A53E, E46K, G51D, and H50Q) of α-synuclein were reported to be associated with synucleinopathies (Figure 1) [24,25,26,27,28,29]. Their rates of aggregation and oligomerization were studied widely in vitro [30,31,32,33]. Among them, α-synuclein in E46K, H50Q, and A53T mutations are more prone to aggregation, while α-synuclein in the A30P mutation appeared to be more prone to oligomerization [34]. All mutations identified to date are located in the N-terminal domain but do not promote changes in the major structure to the α-synuclein monomer. Only the A30P mutation suggested a reduced propensity to adopt alpha-helical content [35]. The membrane-binding properties of mutation types are likely affected by their localization in the N-terminal domain. For example, A53T and H50Q suggest a higher propensity to bind with membranes [36]; however, A30P, A53E, and G51D suggest a lower propensity to bind with membranes [37,38,39]. Additionally, these mutations alter long-range electrostatic interactions between the N- and C-termini, thereby, affecting the stability of the native α-synuclein [40]. Therefore, aberrant internal contacts of mutated α-synuclein might cause a partially folded intermediate that increases the propensity for self-assembly [41].

The definition of α-synuclein oligomers is rather ambiguous, owing to their heterogeneity, instability, transient existence, and variability. Oligomers are regarded as intermediates of the aggregation process but suggest higher toxicity than the other forms [42,43,44] and cause a wide range of damaging effects, such as inhibition of exocytosis and dopamine release in PC12 cells [45], complex I-dependent, Ca²⁺-induced mitochondrial dysfunction in an in vitro system comprising isolated mitochondria [46], and altering synaptic transmission in primary hippocampal and dissociated nigral neurons [47]. Additionally, α-synuclein oligomers can permeabilize lipid bilayers [48,49] and alert plasma and intracellular membrane structures [50], by rearranging the lipid microenvironment and accelerating the flux of hydrophilic molecules in the surroundings [49,51]. On the other hand, α-synuclein oligomers also induce pore formation and act as pathological membrane channels, while tubular amyloid or ring-shaped oligomers are integrated into the membrane [48,52]. Although α-synuclein oligomers seem like they connect with plasma membrane in neurons, the structural characterization of the putative pores remains unclear. Furthermore, it was reported that α-synuclein oligomers inhibit tubulin polymerization, affect the cytoskeleton of cells, and damage the integrity of the neurite network in a human dopaminergic cell line [28,53]. Similarly, α-synuclein overexpression also causes microtubule destabilization and neuritic degeneration in cultured cells [54].

Misfolded and damaged polypeptides such as α-synuclein oligomers and aggregates in PD are mainly degraded by two major protein degradation systems—the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) [55]. However, in the brains of patients with PD, lysosomal activity [56] and various lysosomal markers (LAMP1, heat-shock protein 73, and cathepsin D) were significantly decreased, especially in nigral neurons containing α-synuclein inclusions [57]. Consistently, dementia with Lewy body (DLB) brains also exhibits ALP depletion [58]. Additionally, α-synuclein aggregates inhibit macroautophagy by binding with essential ALP components, such as p62 and LC3 [58]. In cultured cells, chaperone-mediated autophagy was inhibited by mutated α-synuclein expression [59]. This inhibition results from α-synuclein binding with the specific receptor on the lysosome LAMP2, which might induce α-synuclein aggregation and injure the entire homeostasis of cells [60]. UPS dysfunction is also present in PD brains, such as decreased proteasome enzymatic activities and structural defects of proteasomes [61,62], and proteasome dysfunction is considered to be of primary or secondary consequence in a wide array of chronic neurodegenerative diseases [63,64,65,66,67]. As reported previously, both wild-type α-synuclein and mutant αSyn (A53T) expression resulted in a time-dependent and significant decrease in proteasome dysfunction in cellular models, such as SH-SY5Y cells [68] and PC12 cells [69]. A possible hypothesis about UPS inhibition by α-synuclein is the steric blocking of the proteasome machinery. Seventy percent of Lowy bodies, extracted from post-mortem DLB brains, are positive for 20S proteasome components [70]; more importantly, the 20S component was found in α-synuclein aggregates extracted from pre-formed α-synuclein fibrils-treated and α-synuclein-expressing HEK293 cells [71] and the 26S proteasome was isolated together with α-synuclein oligomers [72]. Additionally, aggregated proteins such as amyloid-β [70], tau [73], and α-synuclein [72] interact with and impair proteasome functions, and aggregated proteins such as α-synuclein and tau oligomers impair 20s proteasome function through allosteric impairment of the substrate gate in the 20S core particle and prevents the 19S regulatory particle from injecting substrates into the degradation chamber [73,74]. Altogether, these findings suggest that α-synuclein, especially α-synuclein oligomers, can block the UPS, thereby, damaging the clearance of other substrates and unbalancing proteostasis.

The inherent ability of α-synuclein to bind to lipids and membranes, especially membranes with negatively charged surfaces, increases the potential for interaction with the mitochondrial membrane [75]. Thus, the properties of α-synuclein in lipid binding were extensively investigated [76]. Several studies indicated that the ability for α-synuclein docking to the lipid surface is regulated by the deletion or insertion of charged amino groups in the first 25 residues of the N-terminal domain, adopting an α-helix conformation [77,78,79]. Thus, some mutations of α-synuclein, such as A30P, E46K, and A53T, in this domain might strongly affect this ability. Regarding the mitochondrial outer membrane (MOM) proteins, which α-synuclein are associated with, the in vitro pulldown experiments of extracts of the mitochondria with α-synuclein peptide composed of the last 40 residues of C-terminal retrieved TOM22, TOM40, voltage-dependent anion channels (VDAC)-1, 2, 3, and Samm50 as binding partners (Figure 2). However, the S129 phosphorylation distinctly inhibited α-synuclein association with TOM40 and Samm50 [80]. VDAC is a vital and recurrent binding partner of α-synuclein, and co-immunoprecipitation analysis revealed an association between the two proteins in the brainstem, striatum, and cerebral cortex in A53T human α-synuclein transgenic mice [81]. Moreover, recombinant α-synuclein monomers in vitro suggested marketable inhibition ability in the VDAC-1 channel activity in planar lipid bilayers [82].

Therefore, although α-synuclein is the able to bind membranes directly, especially the proteins that mirror the composition of mitochondria, and the protein–protein interactions with the MOM were consistently reported, the mechanism for the mitochondrial protein import machinery, particularly TOM20, remains unclear.

The association between PD and mitochondria was reported in 1976; 1-Methyl-1,2,3,6-tetrahydropiridine (MPTP) was discovered as a mitochondrial complex I inhibitor, resulting in PD-like motor symptoms. Furthermore, complex I activity decreased by approximately 30% to 40% in the substantia nigra pars compacta (SNpc) of patients with PD. This further supports the vital role of mitochondria in PD development [83]. In contrast, experimental rodent models and patients with PD indicated strong evidence of damage to mitochondrial dynamics and increased reactive oxygen species (ROS) [84,85]. The main PD-connected genes are involved in mitochondrial homeostasis. Mutations in these genes are associated with mitochondrial dysfunction and might cause familial PD, as reported elsewhere [84]. New findings also described the role of α-synuclein in mitochondrial injury [86,87]. It is possible that there is a salient relationship between α-synuclein and mitochondria under physiological or pathological conditions.

Recently, the toxicity of α-synuclein in the mitochondria was extensively studied. Constitutive low α-synuclein levels in the mitochondria might play an essential role in maintaining normal functions of mitochondrial complexes. However, high levels of α-synuclein accumulation in the mitochondria contribute to the impairment of complex I function [12]. Furthermore, overexpression of wild-type or A53T mutant α-synuclein in SHSY-5Y cells or treating aggregated α-synuclein in an isolated mitochondrion, induces cytochrome c release, thereby increasing mitochondrial oxidative stress and causing mitochondrial dysfunction and fragmentation [11,87,88]. Although the mechanism through which α-synuclein translocates to the mitochondria remains unclear, the N-terminal domain of α-synuclein mimics mitochondrial-targeting sequence properties and contributes to the anchoring of α-synuclein to the mitochondrial membrane [88]. Additionally, α-synuclein suggested a high-affinity binding to the TOM20 pre-sequence receptor of the mitochondrial protein import machinery. When α-synuclein translocates to the mitochondrial membrane, it disrupts the translocase of the inner and MOM complex assembly process and impairs mitochondrial protein import from the cytosol [87,89,90,91]. Interestingly, α-synuclein also functions in the mitochondrial matrix, indicating that physiological α-synuclein accelerates ATP synthase activity by directly binding to its subunit, thereby ensuring mitochondrial health and proper ATP fueling for synaptic function [87]. Ludtmann et al. reported that α-synuclein monomers interact with and enhance ATP synthase under normal conditions [91]. Under pathological conditions, α-synuclein aggregation is stimulated and generates accumulated α-synuclein oligomers with increasing enrichment of β-sheet structures that are in close proximity to the inner mitochondrial membrane. They inhibit complex I function, thereby triggering oxidation events and ultimately leading to mega-channel opening and mitochondrial permeability transition events [91]. Therefore, α-synuclein itself does not interfere with the normal functions of mitochondria, and the toxicity of α-synuclein in the mitochondria is regulated by its oligomerization and aggregation events, which are associated with other pathological factors.

In cells, fatty acids with a small number of carbon atoms are water-soluble; however, long-chain fatty acids with 12 or more carbon atoms are insoluble. Therefore, to move long-chain fatty acids inside the cells, an FABP that binds to and solubilizes long-chain fatty acids is required. The FABP family is a low-molecular-weight intracellular protein, with a molecular weight of 14–15 kDa, and up to 12 types of molecular species were identified in mammals [92]. They serve intracellular lipid chaperones and regulate the uptake and transportation of long-chain FAs [93,94], such as eicosanoids and bile acids. Their functions are thought to include intracellular uptake of ligands, transport to lipid metabolism pathways, and regulation of lipid signal transduction [95]. FABP is named after the first isolated tissue or cell, heart-type (H-), brain-type (B-), epidermis-type (E-), adipocyte-type (A-), liver-type (L-), intestinal-type (I-) FABP, etc. However, due to the diversity of tissue and cell expression, they were recently called L-FABP (FABP1), I-FABP (FABP2), and H-FABP (FABP3), in the order of isolation.

Among them, three FABPs, the heart-type (FABP3), brain-type (FABP7), and epidermal-type (FABP5), are expressed in the brain. FABP7 localizes to the neural stem cells of astrocytes and the hippocampal granule cell layer. This suggests high expression levels in the embryonic brain after birth; however, the expression levels decrease in the mature brain, astrocytes, and OPCs [96,97]. This expression is localized in astrocytes and the hippocampal neural stem cells [98]. Analysis using FABP7 gene knock-out mice revealed that FABP7 is involved in n-3 long-chain unsaturated fatty acid uptake into astrocytes.

Interestingly, no abnormalities were observed in spatial memory and learning in FABP7-deficient mice, but aversive memory, fear, and anxiety were enhanced [96]. On the other hand, FABP5 is most widely expressed in the epidermis, liver, and adipocytes and is similar to FABP7. It is highly expressed in the nerve and glial cells in embryonic brains but is decreased in mature brains [97,99,100]. The expression of mRNA peaks from neurogenesis (E17) to the process of nerve differentiation (E19), and its expression decreases during the synaptogenesis period (P5-P10), suggesting that it is involved in the migration and differentiation of nerve cells [101]. On the other hand, FABP3 is not expressed in an embryonic brain but is expressed in nerve cells in a mature brain [99]. FABP3 was first isolated from the heart, although it is widely expressed in the tissues and cells in the body, it was reported to be expressed in the brain, skeletal muscle, mammary gland, and ovary, in addition to the heart. In contrast to FABP7, FABP3 has a high affinity for n-6 unsaturated fatty acids, such as arachidonic acid [102]. It was also shown to bind to epoxyeicosatrienoic acid, an arachidonic acid metabolite, and to prolong its half-life [103]. Furthermore, it was reported that FABP3 expression is increased in the serum and cerebrospinal fluid (CSF) of patients with neurodegenerative disorders, including PD, AD, and other neurological disorders [104,105], suggesting that it might not only be related to normal brain function but also to neurological disease.