Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases.

The abnormal aggregation and accumulation of alpha-synuclein (aSyn) in the brain is a defining hallmark of synucleinopathies. Various aSyn conformations and post-translationally modified forms accumulate in pathological inclusions and vary in abundance among these disorders. Relying on antibodies that have not been assessed for their ability to detect the diverse forms of aSyn may lead to inaccurate estimations of aSyn pathology in human brains or disease models. To address this challenge, we developed and characterized an expanded antibody panel that targets different sequences and post-translational modifications along the length of aSyn, and that recognizes all monomeric, oligomeric, and fibrillar aSyn conformations. Next, we profiled aSyn pathology across sporadic and familial Lewy body diseases (LBDs) and reveal heterogeneous forms of aSyn pathology, rich in Serine 129 phosphorylation, Tyrosine 39 nitration and N- and C-terminal tyrosine phosphorylations, scattered both to neurons and glia. In addition, we show that aSyn can become hyperphosphorylated during processes of aggregation and inclusion maturation in neuronal and animal models of aSyn seeding and spreading. The validation pipeline we describe for these antibodies paves the way for systematic investigations into aSyn pathological diversity in the human brain, peripheral tissues, as well as in cellular and animal models of synucleinopathies.

Prominent astrocytic alpha-synuclein pathology with unique post-translational modification signatures unveiled across Lewy body disorders.

Alpha-synuclein (aSyn) is a pre-synaptic monomeric protein that can form aggregates in neurons in Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB), and in oligodendrocytes in multiple system atrophy (MSA). Although aSyn in astrocytes has previously been described in PD, PDD and DLB, the biochemical properties and topographical distribution of astrocytic aSyn have not been studied in detail. Here, we present a systematic investigation of aSyn astrocytic pathology using an expanded antibody toolset covering the entire sequence and key post-translational modifications (PTMs) of aSyn in Lewy body disorders (LBDs) and in MSA. Astrocytic aSyn was detected in the limbic cortical regions of LBDs but were absent in main pathological regions of MSA. The astrocytic aSyn was revealed only with antibodies against the mid N-terminal and non-amyloid component (NAC) regions covering aSyn residues 34-99. The astroglial accumulations were negative to canonical aSyn aggregation markers, including p62, ubiquitin and aSyn pS129, but positive for phosphorylated and nitrated forms of aSyn at Tyrosine 39 (Y39), and not resistant to proteinase K. Our findings suggest that astrocytic aSyn accumulations represent a major part of aSyn pathology in LBDs and possess a distinct sequence and PTM signature that is characterized by both N- and C-terminal truncations and modifications at Y39. This is the first description that aSyn accumulations are made solely from N- and C-terminally cleaved aSyn species and the first report demonstrating that astrocytic aSyn is a mixture of Y39 phosphorylated and nitrated species. These observations underscore the importance of systematic characterization of aSyn accumulations in different cell types to capture the aSyn pathological diversity in the brain. Our findings combined with further studies on the role of astrocytic pathology in the progression of LBDs can pave the way towards identifying novel disease mechanisms and therapeutic targets.

Pathological Relevance of Post-Translationally Modified Alpha-Synuclein (pSer87, pSer129, nTyr39) in Idiopathic Parkinson's Disease and Multiple System Atrophy.

Aggregated alpha-synuclein (α-synuclein) is the main component of Lewy bodies (LBs), Lewy neurites (LNs), and glial cytoplasmic inclusions (GCIs), which are pathological hallmarks of idiopathic Parkinson's disease (IPD) and multiple system atrophy (MSA). Initiating factors that culminate in forming LBs/LNs/GCIs remain elusive. Several species of α-synuclein exist, including phosphorylated and nitrated forms. It is unclear which α-synuclein post-translational modifications (PTMs) appear within aggregates throughout disease pathology. Herein we aimed to establish the predominant α-synuclein PTMs in postmortem IPD and MSA pathology using immunohistochemistry. We examined the patterns of three α-synuclein PTMs (pS87, pS129, nY39) simultaneously in pathology-affected regions of 15 IPD cases, 5 MSA cases, and 6 neurologically normal controls. All antibodies recognized LBs, LNs, and GCIs, albeit to a variable extent. pS129 α-synuclein antibody was particularly immunopositive for LNs and synaptic dot-like structures, followed by nY39 α-synuclein antibody. GCIs, neuronal inclusions, and small threads were positive for nY39 α-synuclein in MSA. Quantification of the LB scores revealed that pS129 α-synuclein was the dominant and earliest α-synuclein PTM, followed by nY39 α-synuclein, while lower amounts of pSer87 α-synuclein appeared later in disease progression in PD. These results may have implications for novel biomarker and therapeutic developments.

Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity.

Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson's disease and other neurodegenerative diseases. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (PTMs) (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighboring PTMs. Although most pS129 antibodies showed good performance in detecting aSyn aggregates in cells, neurons and mouse brain tissue containing abundant aSyn pathology, they also showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or high molecular weight aSyn species. Our observations suggest that not all pS129 antibodies capture the biochemical and morphological diversity of aSyn pathology, and all should be used with the appropriate protein standards and controls when investigating aSyn under physiological conditions. Finally, our work underscores the need for more pS129 antibodies that are not sensitive to neighboring PTMs and more thorough characterization and validation of existing and new antibodies.