α-synuclein phosphorylation and truncation are normal events in the adult human brain

doi:10.1016/j.neuroscience.2011.10.042

Neuroscience

Volume 200, 3 January 2012, Pages 106-119

https://doi.org/10.1016/j.neuroscience.2011.10.042 Get rights and content

Abstract

α-synuclein is a key protein in Lewy body diseases (LBDs) and a major component of Lewy bodies and related aberrant cytoplasmic and neuritic inclusions. Regional differences in α-synuclein have been associated with selective neuronal vulnerability to Lewy pathology. Furthermore, phosphorylation at serine 129 (Ser129) and α-synuclein truncation have been considered crucial in the pathogenesis of Lewy inclusions. The present study shows consistent reduction in α-synuclein protein expression levels in the human substantia nigra and nucleus basalis of Meynert compared with other brain regions independently of age and pathology. Phosphorylated α-synuclein at Ser129 is naturally increased in these same regions, thus inversely related with the total amount of α-synuclein. In contrast, truncated α-synuclein is naturally observed in control and diseased brains and correlating with the total amount of α-synuclein. Several truncated variants have been identified where some of these variants are truncated at the C-terminal domain, whereas others are truncated at the N-terminal domain, and all are present in cases with and without Lewy pathology. Although accumulation of truncated α-synuclein variants and phosphorylated α-synuclein occurs in Lewy bodies, α-synuclein phosphorylation and truncation can be considered constitutive in control and diseased brains.

Highlights

▶αsynuclein protein levels are lower in the human substantia nigra and nucleus basalis of Meynert compared with other brain regions. ▶The reduction in α-synuclein protein levels in these two human brain regions is independent of age and pathology. ▶Phosphorylated α-synuclein is increased in substantia nigra and nucleus basalis of Meynert, inversely correlating with the total amount of α-synuclein. ▶Truncated α-synuclein is a general event present in normal and Lewy body disease brains and is not related to pathology or age.

Section snippets

Human brain samples

The brains of 13 patients were obtained at autopsy following informed consent of the patients or their relatives and the approval of the local ethics committee. Eight different areas were dissected, frozen on dry ice, and stored at −80 °C until use. The areas were frontal cortex (area 8), temporal cortex (anterior part of the superior gyrus), caudate (head), putamen (medial), nucleus basalis of Meynert, amygdala, substantia nigra, and cerebellum (upper vermis). Both genders were equally

α-synuclein levels in human samples

Total levels of α-synuclein were studied by Western blotting in eight regions of 13 human cases classified according to their clinical and pathological identification in three groups: control (C), tauopathies (T; including AD-related and AGD cases), and Lewy body diseases (LBD); were analyzed using four different antibodies directed against different α-synuclein amino acid sequences: AB1, AB2, AB3, and AB4 (Table 2, Fig. 1). Fig. 2A shows immunodetection with all four antibodies of a

Discussion

The present results have shown that α-synuclein protein levels are lower in the substantia nigra and nucleus basalis of Meynert, which are more susceptible than other more resistant regions to Lewy body diseases. This is in agreement with previous α-synuclein mRNA studies (Rockenstein et al., 2001), but differs from other α-synuclein protein reports (Neystat et al., 1999, Kingsbury et al., 2004, Chiba-Falek et al., 2006, Tong et al., 2010). Such differences may be explained by different

Acknowledgments

This work was supported by project PETRI 2007-0397, Ministry of Science and Innovation. We are grateful to B. Wolozyn and A. Pujol for their help in providing the pcDNA 3.1 vector and reagents, respectively; and to the Proteomics Service at the Science Park of Barcelona for characterization α-synuclein by mass spectrometry. We thank T. Yohannan for editorial assistance.

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Posttranslational modifications of α-synuclein, such as truncation or abnormal proteolysis, are implicated in Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). A key focus of this article includes the proteases responsible for inducing truncation, the specific sites susceptible to truncation, and the resultant influence of these truncated species on the seeding and aggregation of endogenous α-synuclein. We also shed light on the unique structural attributes of these truncated species, and how these modifications can lead to distinctive forms of synucleinopathies. In addition, we explore the comparative toxic potentials of various α-synuclein species. An extensive analysis of available evidence of truncated α-synuclein species in human-synucleinopathy brains is also provided. Lastly, we delve into the detrimental impact of truncated species on key cellular structures such as the mitochondria and endoplasmic reticulum. Our article discusses enzymes involved in α-synuclein truncation, including 20 S proteasome, cathepsins, asparagine endopeptidase, caspase-1, calpain-1, neurosin/kallikrein-6, matrix metalloproteinase‐1/‐3, and plasmin. Truncation patterns impact α-synuclein aggregation — C-terminal truncation accelerates aggregation with larger truncations correlated with shortened aggregation lag times. N-terminal truncation affects aggregation differently based on the truncation location. C-terminally truncated α-synuclein forms compact, shorter fibrils compared to the full-length (FL) protein. N-terminally truncated monomers form fibrils similar in length to FL α-synuclein. Truncated forms show distinct fibril morphologies, increased β-sheet structures, and greater protease resistance. Misfolded α-synuclein can adopt various conformations, leading to unique aggregates and distinct synucleinopathies. Fibrils, with prion-like transmission, are potentially more toxic than oligomers, though this is still debated. Different α-synuclein variants with N- and C-terminal truncations, namely 5–140, 39–140, 65–140, 66–140, 68–140, 71–140, 1–139, 1–135, 1–133, 1–122, 1–119, 1–115, 1–110, and 1–103 have been found in PD, DLB, and MSA patients' brains. In Parkinsonism, excess misfolded α-synuclein overwhelms the proteasome degradation system, resulting in truncated protein production and accumulation in the mitochondria and endoplasmic reticulum.
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α-synuclein (αS) is an abundant, neuronal protein that assembles into fibrillar pathological inclusions in a spectrum of neurodegenerative diseases that include Lewy body diseases (LBD) and Multiple System Atrophy (MSA). The cellular and regional distributions of pathological inclusions vary widely between different synucleinopathies contributing to the spectrum of clinical presentations. Extensive cleavage within the carboxy (C)-terminal region of αS is associated with inclusion formation, although the events leading to these modifications and the implications for pathobiology are of ongoing study. αS preformed fibrils can induce prion-like spread of αS pathology in both in vitro and animal models of disease. Using C truncation-specific antibodies, we demonstrated here that prion-like cellular uptake and processing of αS preformed fibrils resulted in two major cleavages at residues 103 and 114. A third cleavage product (122 αS) accumulated upon application of lysosomal protease inhibitors. In vitro, both 1-103 and 1-114 αS polymerized rapidly and extensively in isolation and in the presence of full-length αS. 1-103 αS also demonstrated more extensive aggregation when expressed in cultured cells. Furthermore, we used novel antibodies to αS cleaved at residue Glu114, to assess x-114 αS pathology in postmortem brain tissue from patients with LBD and MSA, as well as three different transgenic αS mouse models of prion-like induction. The distribution of x-114 αS pathology was distinct from that of overall αS pathology. These studies reveal the cellular formation and behavior of αS C-truncated at residues 114 and 103 as well as the disease dependent distribution of x-114 αS pathology.
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¹: Present address: Neurodegenerative Diseases Lab., Vall d'Hebron Research Institute, Mediterranean Building, First Floor, Lab. 102, Pg. Vall d'Hebron 119-129, 08035 Barcelona, Spain.

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Neurodegeneration, Neuroprotection, and Disease-Oriented NeuroscienceResearch Paperα-synuclein phosphorylation and truncation are normal events in the adult human brain

Abstract

Highlights

Section snippets

Human brain samples

α-synuclein levels in human samples

Discussion

Acknowledgments

J Biol Chem

Lancet

J Biol Chem

Neuron

Neurosci Lett

Exp Neurol

J Biol Chem

Exp Neurol

J Biol Chem

Brain Res

J Biol Chem

Anal Biochem

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

Brain Res Mol Brain Res

Neurobiol Aging

J Biol Chem

Neurosci Lett

Brain Res Mol Brain Res

Expression of alpha-synuclein in different brain parts of adult and aged rats

J Physiol Pharmacol

Staging/typing of Lewy body related alpha-synuclein pathology: a study of the BrainNet Europe Consortium

Acta Neuropathol

Assessment of alpha-synuclein pathology: a study of the BrainNet Europe Consortium

J Neuropathol Exp Neurol

Identification and characterization of a new alpha-synuclein isoform and its role in Lewy body diseases

Neurogenetics

Low alpha-synuclein 126 mRNA levels in dementia with Lewy bodies and Alzheimer disease

Neuroreport

Differential expression of alpha-synuclein isoforms in dementia with Lewy bodies

Neuropathol Appl Neurobiol

The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease

J Neurochem

Neurodegeneration, Neuroprotection, and Disease-Oriented Neuroscience
Research Paper
α-synuclein phosphorylation and truncation are normal events in the adult human brain