α-Synuclein (αsyn) is an abundant brain neuronal protein that can misfold and polymerize to form toxic fibrils coalescing into pathologic inclusions in neurodegenerative diseases, including Parkinson's disease, Lewy body dementia, and multiple system atrophy. These fibrils may induce further αsyn misfolding and propagation of pathologic fibrils in a prion-like process. It is unclear why αsyn initially misfolds, but a growing body of literature suggests a critical role of partial proteolytic processing resulting in various truncations of the highly charged and flexible carboxyl-terminal region. This review aims to 1) summarize recent evidence that disease-specific proteolytic truncations of αsyn occur in Parkinson's disease, Lewy body dementia, and multiple system atrophy and animal disease models; 2) provide mechanistic insights on how truncation of the amino and carboxyl regions of αsyn may modulate the propensity of αsyn to pathologically misfold; 3) compare experiments evaluating the prion-like properties of truncated forms of αsyn in various models with implications for disease progression; 4) assess uniquely toxic properties imparted to αsyn upon truncation; and 5) discuss pathways through which truncated αsyn forms and therapies targeted to interrupt them. Cumulatively, it is evident that truncation of αsyn, particularly carboxyl truncation that can be augmented by dysfunctional proteostasis, dramatically potentiates the propensity of αsyn to pathologically misfold into uniquely toxic fibrils with modulated prion-like seeding activity. Therapeutic strategies and experimental paradigms should operate under the assumption that truncation of αsyn is likely occurring in both initial and progressive disease stages, and preventing truncation may be an effective preventative strategy against pathologic inclusion formation.
Neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and Lewy body dementia (LBD), are collectively the leading cause of dementia worldwide with devastating human and economic costs (
1
, 2
). There currently exist no disease modifying therapies that slow or prevent the onset of dementia in these diseases (2
), and further research is needed to understand underlying pathophysiologic processes with the goal of identifying targets for therapeutic strategies. Common to most neurodegenerative diseases is the presence of amyloidogenic aggregates comprised predominantly of misfolded proteins of neuronal origins (3
, 4
); these diseases are typically classified based on the clinical presentations and identity of the misfolded proteins (3
).α-Synuclein (αsyn) is a 140-residue, 14.46-kDa protein that is predominantly found within the presynaptic region of neurons in the central nervous system, where it has important functions in vesicle trafficking (
5
, 6
). Misfolding of this protein and consequent intracellular inclusion formation is a hallmark of the class of neurodegenerative diseases termed synucleinopathies, which includes PD, LBD, and multiple system atrophy (MSA) (7
). αsyn isolated from these inclusions is misfolded into pathologic, β-sheet–rich polymers assembled into various oligomers or larger amyloidogenic fibrils (8
, 9
, 10
, 11
, 12
, 13
), which collectively can contribute to neuronal toxicity and dysfunction along with prion-like progression of disease (11
, 14
, 15
). In addition to polymerization, pathologic αsyn harbors extensive post-translational modifications (PTMs), including phosphorylation, truncation, ubiquination, nitration, sumoylation, and multiple others (16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
17
, 18
).The consequences of misfolded oligomeric and fibrillar forms of αsyn in preclinical models have been extensively studied and reviewed elsewhere (
11
, 19
, 20
), and current evidence suggests that disease progression may be difficult to thwart once these forms of αsyn are widespread due to prion-like recruitment of endogenous αsyn into further pathologic forms that can spread within and between neuronal and glial cells in a vicious cycle (20
, 21
). A more attractive therapeutic target may be the dysfunctional processes resulting in initial αsyn misfolding and fibril formation through mechanisms such as aberrant αsyn PTMs if these occur early or are essential for disease progression. Some PTMs have been shown in vitro to alter the propensity of αsyn to form pathologic fibrils similar to those isolated from disease inclusions (18
, 22
), and corollary in vivo processes likely responsible for the appearance of these PTMs such as oxidative stress and impaired protein clearance pathways are demonstrable in animal models of synucleinopathy and tissue from diseased patients (23
, 24
, 25
). Indeed, PTMs of αsyn are abundant in disease, with 90% or more of αsyn being phosphorylated at Ser-129 and 15–20% being C-terminally truncated within pathologic inclusion extracts (16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
17
, 26
, 27
, 28
). C-terminal truncation of αsyn may be particularly detrimental among PTMs, as it has been consistently demonstrated in vitro that C-truncated αsyn self-assembles into fibrils far more readily than full-length (FL) αsyn or even familial disease causing missense mutant forms of αsyn (17
, 29
, 30
, 31
, 32
, 33
, 34
, 35
, 36
). Proteolytic formation of truncated αsyn (32
, 37
, 38
, 39
, 40
, 41
, 42
), possibly promoted by impairment of proteostasis as in aging (43
), may initiate inclusion pathology formation and progression. This review aims to summarize the evidence for the accumulation of diverse types of truncated αsyn in disease, survey the physiological processes involved in their formation, and discuss the functional implications in the context of pathologic fibril formation, toxicity, and prion-like disease progression.αsyn background
αsyn has structurally distinct regions important to function and disease
αsyn was first isolated from synaptic vesicles in the Torpedo fish and was termed “synuclein” when a homologous protein was detected in rat neuronal synaptic vesicles and nuclear envelopes (
44
). The majority of αsyn is in a cytoplasmic, soluble form as an intrinsically disordered protein (6
); in association with synaptic vesicles, the repeat-rich N terminus (residues 1–60) and “nonamyloid component” (NAC) domain (residues 61–95) adopt a helical structure necessary for the regulatory role of αsyn in vesicle trafficking that is thought to be a main function of αsyn in the neuron (45
, 46
). However, αsyn also functions in synaptic maintenance and SNARE protein assembly, as evidenced by its ability to mitigate phenotypes in CSPα null mice (47
), and αsyn can function as a chaperone, which is made possible by labile intermolecular interactions inherent to unstructured proteins (48
, 49
). These biological functions of αsyn are attributed to its three overarching structural domains: the amphipathic N terminus, the hydrophobic NAC, and the acidic C terminus (residues 96–140). The N terminus harbors most of the seven imperfect KTKEGV 11-mer repeats important in helix formation in association with phospholipid surfaces (45
, 46
). The NAC region is hydrophobic and acts as a lipid “sensor,” mediating specificity for synaptic vesicle binding (5
, 45
). Last, the C terminus remains unstructured in nearly all conformations due to its highly charged 15 acidic residues; the charged state and ever-changing structure of this region permits most of the chaperone activity of αsyn along with its ability to bind metals, polyamines, and positively charged proteins such as tau (5
, 45
, 48
, 50
, 51
).Due largely to the hydrophobic nature of the NAC region (
52
), a confluence of pathologic processes can result in the disease-associated misfolding of αsyn into dense fibrils that are highly insoluble in detergents (8
). Using proteolytic digestion of fibrils, antibody accessibility, and structural techniques such as cryo-EM, it was determined that the typical αsyn amyloid fibril core is composed roughly of residues ∼31–102 stacked as in-register β-sheets (31
, 51
, 53
, 54
, 55
, 56
, 57
, 58
, 59
). The extreme N terminus and most of the C terminus are not within the amyloid core, but the N terminus is still relatively structured compared with the fully disordered C terminus (53
, 55
, 60
). The C terminus may also govern higher-order assembly of fibrils, as its highly negative charge governs lateral packing of fibrils and possibly association of protofilaments into more mature amyloid species (33
, 56
, 60
).αsyn-laden inclusions are hallmarks in multiple neurodegenerative diseases
αsyn was first associated to neurodegenerative diseases when the NAC region of the protein was detected in association with amyloid plaques in AD (
61
, 62
). A more direct involvement in neurodegenerative diseases was supported by the seminal discovery that the A53T missense mutation in the SNCA gene encoding αsyn was responsible for autosomal dominant PD in the Contursi kindred (63
). Immediately thereafter, αsyn was identified as the major component of Lewy bodies (LBs) and Lewy neurites (LNs) that have been pathologic hallmarks in PD and LBD for 100 years prior (64
, 65
).αsyn pathological inclusions are known to be present in a number of heterogeneous neurodegenerative diseases, including in about ∼50% of AD patients (
65
, 66
, 67
). Symptomatically and pathologically, various diseases afflicted by αsyn inclusions can be very different. PD and LBD are pathologically akin with LB and LN pathology (Lewy-related pathology; LRP) prevalent in the degenerating dopaminergic neurons of the substantia nigra pars compacta (SNpc) that underlies the parkinsonism movement disorder (66
, 68
). In stark contrast to the aforementioned diseases where αsyn aggregates are mainly found in the neuronal cytoplasm as LBs and LNs, MSA is a movement disorder that may present with cerebellar signs or parkinsonism and is characterized by αsyn-containing glial cytoplasmic inclusions (GCIs) within oligodendrocytes (7
, 66
, 69
). GCIs are predominantly formed in white matter tracts, but morphologically unique nuclear neuronal αsyn inclusions and neuronal cytoplasmic inclusions can also be found in various brain regions in MSA.In the stereotyped pathologic progression of PD, αsyn inclusions are typically observed early in the peripheral nervous system (particularly in the gastrointestinal tract) and caudal brainstem coinciding with minor autonomic symptoms, followed by the onset of parkinsonism with aggregate formation and neurodegeneration in the SNpc, with telencephalic pathology and cortical symptoms such as dementia and psychosis occurring in late stage (
65
, 66
). In LBD, progression of disease is more varied than PD in that LRP may appear in limbic and cortical regions earlier than in PD, resulting in more rapid onset of dementia that can even precede motor symptoms or SNpc pathology (66
, 67
). MSA has its own unique progression patterns, further demonstrating the variance in disease states induced by misfolding of the same protein (7
, 66
, 69
). Additionally, ∼40–60% of AD cases present LRP, often restricted to limbic regions, that bears similarities to that of PD and LBD, albeit with some differences (66
, 67
). There exist many other less common neurodegenerative diseases exhibiting diverse αsyn aggregates either as primary or secondary lesions with their own unique symptoms and progression patterns (65
). In addition to the diversity in progression and morphology of αsyn inclusions between diseases and even across brain regions in the same disease, there is diversity in the cell types affected, as astrocytic and oligodendroglial αsyn pathologies are present in addition to neuronal αsyn aggregates in varying abundance, depending on the disease (21
, 66
, 67
).Variations among the synucleinopathies in terms of pathology, symptomatology, and rate of progression suggest that the form of pathologic αsyn present may differ between these diseases at the molecular level similar to the concept of strains in prion disease (
20
, 66
, 70
). Additionally, the pathologic and symptomatic progression of PD in particular seem to be linked to the “spread” of pathologic αsyn aggregates through interconnected neurons in the autonomic nervous system, which again bears similarities to prion disease (71
, 72
). Indeed, much of the recent research on the molecular mechanisms of synucleinopathies is now based on the assumption that misfolded αsyn harbors prion-like activity.Molecular mechanisms of αsyn aggregation, prion-like spread, and toxicity are key to understanding synucleinopathies
Aggregates comprised of misfolded αsyn are directly implicated in mechanisms of cytotoxicity and disease progression. Evidence for the toxic role of αsyn is supported to date by the identification of seven missense mutations in the SNCA gene resulting in autosomal dominant synucleinopathies with associated symptoms (
73
, 74
, 75
). These mutant forms of αsyn are thought to cause disease by either accelerating polymerization into oligomers/fibrils, altering the physiologic function of αsyn, or a mixture of these mechanisms (74
, 76
). Additionally, increased gene dosage through duplication or triplication of the SNCA gene similarly results in familial synucleinopathy (73
, 74
). In animal models, overexpression of αsyn and usage of aggregation-prone familial mutant forms such as A53T αsyn results in robust Lewy-like neuronal αsyn inclusions, neuronal death, and motor symptoms (77
, 78
). Increasing the tendency of αsyn to polymerize through mutation or increased gene dosage leads to disease in these familial cases, and in sporadic synucleinopathies, aggregation-promoting PTMs may play a similar role (22
, 67
).The assembly of αsyn monomers into oligomeric species and eventually β-sheet–rich fibrils is important to the disease process; the formation and continued presence of αsyn oligomers and fibrils in inclusion-bearing cells has been directly implicated in neuronal and glial toxicity through a number of pathways, including impairment of axonal transport, blockage of lysosomal autophagy and other proteolytic pathways, mitochondrial toxicity and oxidative stress, synaptic dysfunction, and functional insufficiency of monomeric αsyn from its typical presynaptic location (reviewed in Refs.
19
, 79
, and 80
). In addition to the direct toxic mechanisms of intracellular aggregated αsyn, neuroinflammation is induced by glial detection of the αsyn aggregates and cytotoxicity, which may result in further damage (reviewed in Refs. 21
, 81
, and 82
). Although there is debate as to which form of misfolded αsyn (oligomers, protofibrils, fibrils, etc.) is the most toxic, it is likely that all of these species are present in diseased cells (80
).In addition to their purported toxicity, αsyn aggregates demonstrate prion-like induction of further αsyn pathology and progression of disease (
83
). It was suggested that αsyn could potentially act similar to a prion when it was found that fetal stem cell grafts placed into the brains of PD patients developed αsyn inclusions within the grafts, demonstrating that a “spread” of pathology had occurred (84
, 85
). Stemming from the in-register β-sheet structure of αsyn fibrils (58
) and the intrinsic lability of monomeric αsyn in adopting diverse conformations necessary for its physiologic function, it became apparent from early in vitro observations that the addition of preformed αsyn fibril “seeds” to a solution of αsyn monomers could induce fibril elongation as monomers assume a pathologic β-sheet conformation induced from the exposed “templates” at the ends of the fibrils in a process termed “conformational templating” (13
, 83
, 86
, 87
, 88
). This seeded templating process bears similarities to the protein misfolding cyclic amplification technique common to the prion field, and further prion-like properties of αsyn were quickly explored (89
). The introduction of preformed αsyn fibrils (PFFs) to cultured cells expressing monomeric αsyn induced a polymerization phenomenon similar to that seen in vitro (90
, 91
, 92
, 93
, 94
). In animal models, intracerebral or even peripheral injection of PFFs or human synucleinopathy brain lysate into rodents can robustly trigger the formation of LB-like pathology and motor symptoms, further corroborating the importance of prion-like conformational templating in synucleinopathies (20
, 95
, 96
, 97
, 98
). The prion-like theory of αsyn conformational templating may explain propagation of pathology in synucleinopathies, where for PD in particular it has been proposed that spread of fibrils through anatomic connections sufficiently predicts which regions of the brain will be stereotypically affected (the Braak staging schema) and subsequently when various symptoms will arise based on regions affected (71
, 72
, 99
, 100
). Indeed, experimentation in various preclinical models has demonstrated that even nanomolar quantities of αsyn fibrils are able to spread between neurons and even glial cells through multiple extracellular and intercellular uptake mechanisms (reviewed in Refs. 20
and 21
), suggesting that the appearance of misfolded αsyn aggregates may be sufficient to kick start a vicious cycle of further αsyn aggregation and prion-like spread.Pathologic αsyn adopts different strains that may underlie heterogeneity in disease
Like prions, misfolded αsyn within fibrils can also adopt varied conformations, which bear similarities to “strains” in prion disease whereby the unique conformations of misfolded prion protein can be propagated, resulting in characteristic pathologies and symptoms particular to the initiating strain (reviewed in Ref.
70
). Strains may explain why aggregation of the same protein, αsyn, is able to cause a spectrum of diseases, each with overlapping but separate pathologies, symptoms, regional and cellular distribution of inclusions, and rates of progression (66
).Monomers of recombinant αsyn can be induced to form into PFFs similar to fibrils found in LBs when incubated with agitation for several days (
31
); it was noted that upon microscopic examination, extensive heterogeneity exists in the ultrastructural appearance of these PFFs, with some displaying helical periodicity of paired protofibrils, which others lack, hinting at the existence of differing strains of αsyn fibrils (31
, 54
, 57
). Further experimentation in vitro demonstrated that modifying the buffer parameters used (pH, ionic strength, etc.) for PFF production results in PFFs with unique structural and biochemical characteristics stemming from the modified conformations of the misfolded αsyn monomers comprising the aggregates (58
, 70
, 101
, 102
, 103
, 104
, 105
). In cellular and animal models, these variant PFFs can be functionally different in terms of their potency in seeding αsyn pathology, rate of disease progression, the cell types they affect, and even the morphology of inclusions that result in mice injected with the variants PFFs, which is strongly reminiscent of differing diseases resulting from prion strains (14
, 70
, 103
, 105
).Extending the strain concept to human disease, αsyn fibrils from MSA brain lysate have been compared with lysate from LBD and PD brains to investigate whether there are unique structural and functional differences in fibrils between these diseases (reviewed in Ref.
70
). Structurally, there are purported differences in the biochemical and ultrastructural characteristics between αsyn fibrils derived from MSA and LBD/PD brains; MSA fibrils appear to have more “twisted” fibril variants characterized by undulating fibril width along their length, which may not be as prevalent in LBD/PD αsyn fibrils (10
, 31
). In addition, MSA αsyn fibrils can be more stable to protease digestion (106
) and detergent extraction (107
) and less stable in denaturation with specific solvents (105
) and interact with protein aggregate binding dyes differently from LBD/PD αsyn fibrils (108
), which cumulatively demonstrates significant biochemical, and possibly conformational, differences in the fibrils between these diseases. Additionally, αsyn inclusions between MSA and LBD/PD are not only different in their morphologies and cellular locations, but also in detection, as evidenced by monoclonal antibodies that selectively label αsyn in MSA inclusions over LBD/PD (106
) and the Gallyas–Braak silver staining technique that detects MSA inclusions and not typically those of LBD/PD (109
).Experimentally, MSA αsyn fibrils appear to be up to 1000 times more potent in their prion-like activity compared with LBD/PD αsyn fibrils or any PFF variant as measured by the time to inclusion formation and disease onset, amount of pathology, and amount of insoluble αsyn formed in animal and cellular models injected with various disease lysates and PFFs (
106
, 110
, 111
, 112
, 113
). In addition to the increased potency, MSA αsyn fibrils can impart their unique structural conformation in induced pathology measured in cell culture assays and passaging studies in mice (105
, 106
, 114
). It is increasingly evident that different strains of αsyn fibrils likely contribute to the different synucleinopathies or even rates of progression within the same synucleinopathy; however, it is still not understood why specifically these different strains arise.Mechanisms by which the differing strains of misfolded αsyn and subsequent fibrils could arise include modification of αsyn through PTMs or altered subcellular environment promoting certain conformations. It is likely that PTMs could induce unique αsyn strains, as even single residue mutations in αsyn are able to structurally alter resulting αsyn fibrils and functionally impact prion-like seeding and biochemical properties in cellular and animal models (
115
, 116
, 117
, 118
). Likewise, there exists a “species barrier” between mouse and human αsyn, where due to their 7-amino acid difference, fibrils comprised of the αsyn of one species do not seed monomers of the other species as efficiently due to presumed structural incompatibility (119
, 120
, 121
, 122
). The majority of misfolded pathologic αsyn harbors PTMs such as phosphorylation at Ser-129 (pSer-129), and recent work is establishing the role of these PTMs in modifying prion-like seeding of αsyn fibrils. For pSer-129 in particular, two papers have observed that αsyn inclusions in MSA appear to be less reactive than LBD for common antibodies targeted against pSer-129, and biochemical analysis suggests less pSer-129 in the insoluble αsyn fibrils extracted from MSA brains, suggesting that differing PTMs may indeed result in different fibril strains (106
, 123
).- Mavroeidi P.
- Arvanitaki F.
- Karakitsou A.-K.
- Vetsi M.
- Kloukina I.
- Zweckstetter M.
- Giller K.
- Becker S.
- Sorrentino Z.A.
- Giasson B.I.
- Jensen P.H.
- Stefanis L.
- Xilouri M.
Endogenous oligodendroglial α-synuclein and TPPP/p25α orchestrate α-synuclein pathology in experimental multiple system atrophy models.
Experimentation using animal models to explore the effect of PTMs on aggregation is extensive, but far fewer studies have been conducted to determine how the presence of PTMs within αsyn fibrils impacts prion-like seeding (
18
). Compared with a single point mutation or phosphorylation site, removal of multiple residues through truncation of αsyn may have a large impact on prion-like seeding activity and strain-like alterations in structural and biochemical properties, which will be discussed herein to determine how the presence of this PTM in disease could impact progression.Evidence for truncated αsyn in health and disease
Detection in human neurodegenerative diseases
Although truncation of αsyn may theoretically increase the propensity of the protein to aggregate into pathologic forms, it is important to first confirm that truncated αsyn is actually present in human disease and, if so, which specific ones. Note that post-mortem interval does not affect the observation of truncated αsyn, showing that truncation is not an artifact of post-mortem decay (
16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
26
, 124
). Studies aiming to detect truncated forms of αsyn typically rely on immunoblotting, immunohistochemistry (IHC) with truncation-specific antibodies, or MS. Immunoblotting in the form of Western blotting (WB) is the most common method utilized; however, this analysis is not always straightforward, as human αsyn has an aberrant apparent mobility of ∼19 kDa on SDS-PAGE compared with the predicted 14.4 kDa (6
); thus, some truncations can result in unexpected shifts in mobility. In addition, the C-terminal truncation of αsyn removes acidic residues, resulting in an increased protein pI, and thus typical WB procedures may not detect the full extent of C-truncated forms of αsyn, as the pH levels of common blotting buffers are not ideal for the electrophoretic transfer onto membranes (22
, 26
, 125
). The IHC approach that relies on antibodies specific for modified forms of the protein presents its own challenges, as it is difficult to produce antibodies specific for a truncated form of a protein without any cross-reactivity for the full-length form. MS avoids many of the shortcomings inherent to other techniques but is also the least common approach for detecting truncated αsyn. Despite these challenges, robust evidence exists that truncation of αsyn is occurring in a way that is largely disease-specific, and the estimated or exact truncated forms of αsyn identified are summarized in Table 1 (16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
17
, 26
, 27
, 28
, 62
, 67
, 124
, 126
, 127
, 128
, 129
, 130
, 131
, 132
, 133
, 134
, 135
). Overall, studies relating to truncated αsyn forms noted their presence on WB from LBD, PD, or MSA brain lysate; however, a few were particularly comprehensive in detecting their presence and attempting to identify exact truncation sites through the generation and IHC application of truncation-specific antibodies (16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
128
, 129
, 130
, 131
, 132
, 133
) and MS (16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
27
, 132
, 134
) to study diseased brain lysate.Table 1Summary of observational studies of truncated αsyn in human synucleinopathies
| Reference | Design | Identified truncations | Remarks |
|---|---|---|---|
126 | WB of MSA brains | ∼12-13 kDa | Strong 12 kDa band in MSA and not controls. |
62 | WB of LBD, PD brains | 12 kDa 6 kDa | Both bands detected only with NAC antibody. 6 kDa more disease-specific. |
28 | WB of purified LBs from LBD, PD | 14 kDa 16 kDa | 14 kDa more abundant than FL (∼19 kDa) in purified LBs. |
127 | WB of LBD, PD brains | 15 kDa | 15 kDa band C-truncated at indeterminate location. Most abundant in disease-associated detergent-insoluble fraction. |
26 | WB and MS of PD brains | 12 kDa (1–119/125), 10 kDa, 8 kDa | 12 kDa suggested to be truncated somewhere between 119-125; likely 1–119 and 1–122 from MS. 8 kDa band is both N- and C-truncated. 8 kDa most disease-specific. Up to 50% of αsyn truncated in PD-insoluble fraction. |
17 | WB of PD, LBD brains | ∼12 kDa (1–110/124), ∼9 kDa(1–100/110), ∼6 kDa (15/60-100/110) | 6 kDa is N- and C-truncated. 6 kDa most disease-specific. ∼15–30% of αsyn truncated in PD/LBD insoluble fraction. |
16
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease. | WB and MS of LBD and PD brains; C truncation–specific antibody cleavage at residue 119 for LBD, PD, and MSA. | 15 kDa (1–133/135), 13.5 kDa (1–126/129), 12.5 kDa (1–122), 12 kDa (1–119), 11.5 kDa (1–115), 10 kDa (1–96/105) | C truncation at 119 likely most common, but least specific for disease. C-truncated 119 αsyn found in LBs and GCIs. Other C truncations found only in insoluble fraction and disease-specific. All truncated αsyn examined had intact N terminus. 1–115, 1–119, 1–122, 1–133, and 1–135 αsyn confirmed with MS. |
128 | WB and protein sequencing of LBD and PD brains; IHC with truncation-specific/selective antibodies for cleavage at residue 9 or at 122 | ∼10 kDa (10–122), ∼12 kDa (1–122) | N-terminal truncation at residue 10 mainly detected LBs and not neuritic pathology; mainly central portion of LBs, suggesting role in early formation of LB. Truncated αsyn present in 70–90% of LBs. C-terminal truncation antibody (residue 122) had similar pattern and results to N-terminal truncation antibody staining. |
129 | WB and IHC of LBD and AD brains using truncation-specific antibodies for cleavage at residues 110 or 119 | 110 C-truncated αsyn, 119 C-truncated αsyn | 119 C-truncated αsyn more common in LBs and LNs in LBD and AD brains than 110 C-truncated αsyn. |
| Most LBs contained both FL and 119 C-truncated αsyn, and rarely only 119 αsyn. | |||
| 110 C-truncated αsyn more specific for disease on WB than 119 αsyn. | |||
124 | WB of LBD brain extracts | ∼15 kDa (1–115/120), ∼10 kDa, ∼12 kDa | 15 kDa band only truncation evident in soluble fraction of controls and LBD; epitope mapping suggests it is not N-truncated and is C-truncated between residues 115–122. 15 kDa band (likely 1–119) is enriched in the lysosomal fraction. 10 kDa and 12 kDa bands detected upon further subcellular purification of the cytosol. C-truncated αsyn enriched in center of LBs. |
130 | IHC of PD brains using truncation-specific antibodies for cleavage after residue 119 or 122 | 119 C-truncated αsyn, 122 C-truncated αsyn | C-truncated αsyn at residue 119 or 122 concentrated in the core of LBs and LNs; also found in pale bodies. |
131 | IHC and WB of LBD brains using truncation-specific antibody after residue 122 | 122 C-truncated αsyn | C-truncated αsyn at 122 increased in LBD compared with control brain lysate even in soluble fraction. |
| IHC detected extensive 122 αsyn in dystrophic neurites and cortical LBs in the hippocampus and putamen | |||
27 | MS of PD brain lysate (frontal cortex) | In order of abundance: 5-140 > 1–122 > 1–119 > 1–135 > 39–140 > 68–140 | 1–122, 1–135, and 1–119 truncated forms of αsyn had abundances of 0.23-0.26 compared with 1–140 αsyn in the SDS-insoluble fraction. Significant amounts of purely N-truncated αsyn detected. |
| Found in S.D.S insoluble fraction: 1–119, 1–135, 1–122, 5-140, 68–140 | |||
132 | MS, WB, and IHC with truncation-specific antibody at residue 103 for PD and LBD brains | 1–103 C-truncated αsyn | 1–103 αsyn present in both soluble and insoluble fractions of PD and LBD but not controls. Inclusions labeled in PD and LBD but not controls with the 103-specific antibody. |
| Ratio of 1–103 αsyn to FL αsyn increased in SNpc relative to cortex in PD. | |||
133 | WB of PD brain lysate and IHC with antibody specific for pSer-129 only if also C-truncated | 12.5 kDa (suggested to be residues 15–130), 25 kDa (dimer of truncated αsyn) | Particularly localized to mitochondria, where it exerts toxicity as small SDS-resistant aggregates. The 12.5 kDa band not detected by N- or C- terminal αsyn antibodies. |
134 | WB and MS of PD intestinal appendix lysate | 1–125, 18–125, 19–125, 1–124, 18–124, 19–124, 1–114, 19–114, 19–115, 19–113 truncated αsyn | 1–125, 1–124, and 19-125 appear to be particularly abundant. Significant increase in truncated αsyn in appendix compared with SNpc for the same brain and ratio of truncated to FL αsyn in appendix greatly increased for PD compared with controls. |
67 | WB of LBD lysate | ∼15 kDa C-truncated ∼16 kDa C-truncated | Truncation bands mainly apparent in LBD insoluble fraction; some minor truncation bands in soluble fractions. More C-truncated αsyn in the amygdala/medial temporal lobe compared with cingulate. |
135 | IHC of PD SNpc and colon with truncation-specific antibody at residue 103 | 103 C-truncated αsyn | Usually co-located in inclusions with truncated tau. |
| C-truncated 103 αsyn in SNpc and colonic neurons in PD and not controls. |
In almost all studies utilizing WB of LBD and PD lysate, a common trend is observed where in the insoluble fraction containing aggregated αsyn there is a major ∼17–19 kDa band representing FL αsyn; a ∼12–15 kDa band only reactive to N-terminal αsyn antibodies, suggesting C-truncated forms of αsyn; and 1–2 additional minor bands of ∼9–10 and ∼6–8 kDa, which may be both N- and C-truncated αsyn (Table 1). Each truncation band seems to contain a mix of 2–3 truncated αsyn species that can be resolved using MS or specific antibodies. Confirmed C-truncated forms of αsyn found in detergent-insoluble disease fractions studied by combinations of epitope mapping, MS, and/or truncation-specific antibodies end at residues 103, 110, 113, 114, 115, 119, 122, 124, 125, 133, and 135 (Table 1). Confirmed N-truncated forms of αsyn studied with similar techniques are those starting at residues 5, 10, 18, 19, and 68 (Table 1). In particular, 1–119 and 1–122 appear to be some of the most common forms of truncated αsyn and comprise the major portion of the ∼12–15 kDa band; these have each been detected with multiple specific antibodies and MS experiments where their relative abundances were as high as 20–25% that of FL αsyn in the insoluble brain fraction (Table 1). The forms of αsyn truncated at only the extreme N or C terminus, such as 1–135 and 5–140, have also been determined to be highly abundant in two separate MS experiments (
16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
27
); however, they may not be distinguishable from FL αsyn on a typical WB due to size similarities and both becoming pSer-129–positive (16
) (Table 1). A caveat to determining the most common truncated forms of αsyn is that each study often examined tissue from only one region, and the few studies that compared truncation in differing regions often demonstrated variation in the amount of truncated αsyn from one region to the next (- Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
67
, 132
, 134
). It is difficult to arrive at even a rough number for the percentage of αsyn that is truncated in disease compared with controls due to the variety of truncated forms, regional variation, and detection issues; however, based only on the 2–3 major truncation bands visible on Western blots from multiple studies, it seems that ∼15–30% of insoluble αsyn in PD/LBD is truncated (Table 1). Notably, almost all studies examining truncation have focused on LBD and PD and not MSA, although at least two studies have observed a ∼12–15 kDa band in MSA and 119 C-truncated αsyn in GCIs with a specific antibody (16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
126
).In the detergent-insoluble fraction of LBD/PD lysate (containing aggregated forms of αsyn), multiple studies have noted an increase in the truncated αsyn/FL αsyn ratio compared with the soluble fraction and control fractions, suggesting that the presence of certain truncations may be enriched in pathologic inclusions with disease implications (
16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
17
, 26
, 28
, 67
, 127
, 128
, 129
, 131
, 132
, 134
) (Table 1). IHC of pathologic inclusions from synucleinopathy samples using truncation-specific antibodies corroborates the suggested importance of these species. Antibodies specific for αsyn truncated at residues 9, 103, 110, 119, and 122 have been used to detect truncated αsyn within LBs, LNs, and GCIs (16
, - Anderson J.P.
- Walker D.E.
- Goldstein J.M.
- de Laat R.
- Banducci K.
- Caccavello R.J.
- Barbour R.
- Huang J.
- Kling K.
- Lee M.
- Diep L.
- Keim P.S.
- Shen X.
- Chataway T.
- Schlossmacher M.G.
- et al.
Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body disease.
128
, 129
, 130
, 131
, 132