Alpha-synuclein, autophagy-lysosomal pathway, and Lewy bodies: Mutations, propagation, aggregation, and the formation of inclusions

Abstract

Research into the pathophysiology of Parkinson's disease (PD) is a fast-paced pursuit, with new findings about PD and other synucleinopathies being made each year. The involvement of various lysosomal proteins, such as TFEB, TMEM175, GBA, and LAMP1/2, marks the rising awareness about the importance of lysosomes in PD and other neurodegenerative disorders. This, along with recent developments regarding the involvement of microglia and the immune system in neurodegenerative diseases, has brought about a new era in neurodegeneration: the role of proinflammatory cytokines on the nervous system, and their downstream effects on mitochondria, lysosomal degradation, and autophagy. More effort is needed to understand the interplay between neuroimmunology and disease mechanisms, as many of the mechanisms remain enigmatic. α-synuclein, a key protein in PD and the main component of Lewy bodies, sits at the nexus between lysosomal degradation, autophagy, cellular stress, neuroimmunology, PD pathophysiology, and disease progression. This review revisits some fundamental knowledge about PD while capturing some of the latest trends in PD research, specifically as it relates to α-synuclein.

Keywords: Lewy bodies; Lysosomes; autophagy-lysosomal pathway; neurodegeneration; neuroimmunology; synucleinopathies; α-synuclein.

Figure 1

The pathological hallmark of PD and the downstream effects of the SNc’s dopaminergic output into the basal ganglia.A, postmortem samples show the loss of pigmented DA neurons in the SNc. Healthy patients show a high density of the melanin-filled DA neurons in the substantia nigra, while PD patients exhibit the loss of cells in this region. Upon closer inspection, many of the remaining DA neurons in PD patients show cytoplasmic inclusions, referred to as Lewy bodies. B, DA neurons of the substantia nigra release dopamine to stimulate and inhibit nuclei located in the lateral striatum, which include the caudate and the putamen. Both pathways require DA input from the SNc to modulate the planning and selection of motor actions appropriately. The dopaminergic input into the basal ganglia is shown in black. Blue lines represent the direct pathway, and the red lines represent the indirect pathway. C, consequences of Parkinson’s disease on the basal ganglia pathways. With adequate dopaminergic stimulation of the lateral striatum via DA projections, both the direct and indirect pathways can modulate the planning and selection of movement. However, the loss of dopaminergic input results in decreased activity of the direct pathway and increased activity of the indirect pathway, which culminates in the lack of thalamic input to the motor cortex. DA, dopaminergic; PD, Parkinson’s disease; SNc, substantia nigra pars compacta.

Figure 2

α-syn protein structure and important regions, SNCA multiplication, and aggregation of α-syn.A, α-syn, a 14 kDa protein with 140 amino acids, contains three domains: The membrane-binding domain, the NAC (nonamyloid component) region, and the protein-binding domain. The membrane binding domain, which is on the N-terminal side of the protein, contains most of the KTKEGV (imperfect) repeats that aid in membrane binding. The NAC domain is the hydrophobic region that is critical for the aggregation of α-syn monomers. The C terminal contains the protein-binding domain, which includes the most widely known phosphorylation site: serine 129. Familial PD, caused by mutations in SNCA, translates to the variants detailed in the membrane-binding region of the protein. These mutations include the following: A53T, A53E, A53V, A30P, H50Q, G51D, and E46K. B, patients with familial cases of PD, due to multiplication of the SNCA locus (located on chromosome number 4), have one additional copy of the SNCA gene compared to WT when an extra copy is inherited from one parent (i.e., heterozygous duplication). In cases where additional copies of the gene are inherited from both parents, patients have two additional copies of the SNCA gene. Such cases are referred to as homozygous duplication. Finally, patients known to have a heterozygous triplication of the SNCA gene inherited three copies of the SNCA gene from one parent and only one copy of the SNCA gene from another parent. Theoretically, homozygous duplication and heterozygous triplication should express similar amounts of α-syn, but epigenetic factors and allele preferences create a lot of variety in protein expression. Both homozygous duplication and heterozygous triplication are associated with earlier onset of PD. C, α-syn monomers have been shown to aggregate into oligomers and protofibrils before their fibrillization. Protocols have been established for the generation of α-syn monomers, along with protocols to produce full-length fibrils (larger than 100 μm) or preformed fibrils (smaller than 100 μm), usually made from the sonication of full-length fibrils into smaller chunks (250, 251, 252). PD, Parkinson’s disease; SNc, substantia nigra pars compacta.

Figure 3

α-syn internalization pathways. Multiple pathways have been proposed for the internalization of α-syn monomers, oligomers, and fibrils. Here, we illustrate five of the most reported pathways: receptor-mediated (CME), lipid raft, caveolae, macropinocytic, and phagocytic internalization pathways. Cargo internalized through CME, lipid raft, and caveolae go through the endosomal system, undergo endosomal maturation, and are then transported to lysosomes. Although traditional macropinocytosis also colocalizes with markers of early and late endosomes, the type of macropinocytic pathway we discovered did not undertake this maturation, allowing cargo within macropinosomes to be quickly transported to lysosomes. Finally, phagocytosis, an internalization mechanism specific to macrophages, has been shown to take up α-syn, be transported into phagosomes, and eventually be transported to lysosomes. Conventional phagocytic internalization also colocalizes with early endosome markers and undergoes maturation before reaching lysosomes. CME, clathrin-mediated endocytosis.

Figure 4

α-syn aggregates in different synucleinopathies. Structures of α-syn fibrils and filaments, seeded in vitro or isolated from brains, were resolved using Cryo-EM. The protein data bank (PDB) number for each structure is listed. The structure in 7NCA was assembled using an MSA seed and incubated with recombinant α-syn. This structure was resolved by Lovestam et al. (156). 8CYV shows the resolved structure of α-syn fibrils amplified using CSF from a (Dementia with Lewy bodies) DLB case, resolved by Sokratian et al. (253). 7V49, shows an α-syn fibril seeded by CSF of a PD patient (254). 8BQV, shows the structure of α-syn filament from juvenile-onset synucleinopathy (255). Although α-syn is the building block of all these aggregates, different diseases and physiological conditions lead to structurally distinct 3D structures. CSF, cerebrospinal fluid; MSA, multiple system atrophy; PD, Parkinson’s disease.

Figure 5

Models for the formation of LBs through fibrillization of α-syn and autophagosomal dysfunction.A, internalization of PFFs and their accumulation in lysosomes (1) is followed by the introduction of cytosolic α-syn into lysosomes through CMA (2), initiating the first steps of endogenous α-syn aggregation. Lysosomal stress caused by α-syn accumulation and aggregation, both from the cytosol and PFFs, and potentially external factors, leads to lysosomal membrane permeabilization (3). Aggregated α-syn oligomers/protofibrils then gain access to the cytosol and seeds the aggregation of endogenous α-syn (4 and 5). Fibrillization of α-syn continues, and α-syn aggregates begin to associate with different organelles through membrane and protein interactions (6–8). α-syn fibrils then act as tethers and form a cytoplasmic inclusion, eventually maturing into LBs (9). B, formation of membrane-bound LBs can occur through the internalization of aggregate forms of α-syn, potentially coupled with an external factor (1). Endogenous α-syn, whose concentration is controlled by CMA (47), meets internalized PFFs in lysosomes, leading to α-syn aggregation occurring within lysosomes (256) (2). Eventually, mounting lysosomal stress caused by PFFs (and potentially an external factor) leads to lysosomal membrane permeabilization (lysosomal leakage), leading to the leaking of PFFs into the cytosol (3). PFFs then seed the aggregation of endogenous α-syn (4 and 5). Impaired lysosomal activity is then exacerbated by aggregated forms of α-syn blocking the CMA pore, composed mainly of LAMP2A, leading to further accumulation of monomeric α-syn (6). Autophagosomes attempt to clear aggregation and damaged/dysfunctional organelles through autophagic uptake (7). Due to impaired lysosomal activity, contents within autophagosomes are not degraded, and autophagosomes begin expanding in size as the sequestration of aggregates and organelles continues (8 and 9). Eventually, this leads to the formation of membrane-bound LB inclusions (10). CMA, chaperone-mediated autophagy; LB, Lewy body; PFF, preformed fibril.

Figure 6

Model of LB-like inclusion formation through a dual hit treatment regime. Internalization of an aggregate form of α-syn, coupled with exposure to proinflammatory cytokines (released by microglia or T-cells), results in two different pathways that work to stress the cell. PFF internalization (a PD insult) results in lysosomal stress, and their occupation of lysosomes results in impaired cell degradative activity. The introduction of proinflammatory cytokines activates immune-related pathways in the cell that alter gene expression. Downregulation of lysosomal proteins results in exacerbating lysosomal dysfunction. Proinflammatory cytokines also lead to the rise of oxidative stress and elevated expression of endogenous α-syn in the cell. The combination of PFF accumulation in lysosomes, altered gene expression, and oxidative stress leads to lysosomal membrane permeabilization and the leaking of PFFs into the cytosol. PFFs gain access to the cytosol and seed the aggregation of endogenous α-syn. Autophagosomes attempt to clear aggregation and dysfunctional organelles, but they cannot due to impaired lysosomal activity. This leads to the formation of membrane-bound LB-like inclusions. LB, Lewy body; PD, Parkinson’s disease; PFF, preformed fibril.