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Novel conformation-selective alpha-synuclein antibodies raised against different in vitro fibril forms show distinct patterns of Lewy pathology in Parkinson's disease.

ABSTRACT: AIMS:The aim of this study was to test the hypothesis that different conformations of misfolded ?-synuclein (?-syn) are present in Parkinson's disease (PD) brain. METHODS:Using two previously characterized conformations of ?-syn fibrils, we generated new conformation-selective ?-syn monoclonal antibodies (mAbs). We then interrogated multiple brain regions in a well-characterized autopsy cohort of PD patients (n = 49) with these mAbs, Syn7015 and Syn9029. RESULTS:Syn7015 detects Lewy bodies (LBs) and Lewy neurites (LNs) formed by pathological ?-syn in all brain regions tested, and is particularly sensitive to LNs and small Lewy dots, inclusions believed to form early in the disease. Further, we observed colocalization between Syn7015 and an early marker of ?-syn pathology formation, phospho-Ser129-?-syn, and a lack of extensive colocalization with markers of more mature pathology. In comparison, Syn9029 detects Lewy pathology in all regions examined, but indicates significantly fewer LNs than Syn7015. In addition, colocalization of Syn9029 with later markers of ?-syn pathology maturation (ubiquitin and P62) suggests that the pathology detected by Syn9029 is older. Semiquantitative scoring of both LN and LB pathology in nine brain regions further established this trend, with Syn7015 LN scores consistently higher than Syn9029 LN scores. CONCLUSIONS:Our data indicate that different conformations of ?-syn pathology are present in PD brain and correspond to different stages of maturity for Lewy pathology. Regional analysis of Syn7015 and Syn9029 immunostaining also provides support for the Braak hypothesis that ?-syn pathology advances through the brain.

SUBMITTER: Covell DJ

PROVIDER: S-EPMC5632188 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Novel conformation-selective alpha-synuclein antibodies raised against different in vitro fibril forms show distinct patterns of Lewy pathology in Parkinson's disease.

Covell D J DJ Robinson J L JL Akhtar R S RS Grossman M M Weintraub D D Bucklin H M HM Pitkin R M RM Riddle D D Yousef A A Trojanowski J Q JQ Lee V M-Y VM

Neuropathology and applied neurobiology 20170515 7

<h4>Aims</h4>The aim of this study was to test the hypothesis that different conformations of misfolded α-synuclein (α-syn) are present in Parkinson's disease (PD) brain.<h4>Methods</h4>Using two previously characterized conformations of α-syn fibrils, we generated new conformation-selective α-syn monoclonal antibodies (mAbs). We then interrogated multiple brain regions in a well-characterized autopsy cohort of PD patients (n = 49) with these mAbs, Syn7015 and Syn9029.<h4>Results</h4>Syn7015 det ...[more]

PMID: 28386933

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Project description:Lewy body disorders are characterized by the emergence of α-synucleinopathy in many parts of the central and peripheral nervous systems, including in the telencephalon. Dense α-synuclein+ pathology appears in regio inferior of the hippocampus in both Parkinson's disease and dementia with Lewy bodies and may disturb cognitive function. The preformed α-synuclein fibril model of Parkinson's disease is growing in use, given its potential for seeding the self-propagating spread of α-synucleinopathy throughout the mammalian brain. Although it is often assumed that the spread occurs through neuroanatomical connections, this is generally not examined vis-à-vis the uptake and transport of tract-tracers infused at precisely the same stereotaxic coordinates. As the neuronal connections of the hippocampus are historically well defined, we examined the first-order spread of α-synucleinopathy three months following fibril infusions centered in the mouse regio inferior (CA2+CA3), and contrasted this to retrograde and anterograde transport of the established tract-tracers FluoroGold and biotinylated dextran amines (BDA). Massive hippocampal α-synucleinopathy was insufficient to elicit memory deficits or loss of cells and synaptic markers in this model of early disease processes. However, dense α-synuclein+ inclusions in the fascia dentata were negatively correlated with memory capacity. A modest compensatory increase in synaptophysin was evident in the stratum radiatum of cornu Ammonis in fibril-infused animals, and synaptophysin expression correlated inversely with memory function in fibril but not PBS-infused mice. No changes in synapsin I/II expression were observed. The spread of α-synucleinopathy was somewhat, but not entirely consistent with FluoroGold and BDA axonal transport, suggesting that variables other than innervation density also contribute to the materialization of α-synucleinopathy. For example, layer II entorhinal neurons of the perforant pathway exhibited somal α-synuclein+ inclusions as well as retrogradely labeled FluoroGold+ somata. However, some afferent brain regions displayed dense retrograde FluoroGold label and no α-synuclein+ inclusions (e.g. medial septum/diagonal band), supporting the selective vulnerability hypothesis. The pattern of inclusions on the contralateral side was consistent with specific spread through commissural connections (e.g. stratum pyramidale of CA3), but again, not all commissural projections exhibited α-synucleinopathy (e.g. hilar mossy cells). The topographical extent of inclusions is displayed here in high-resolution images that afford viewers a rich opportunity to dissect the potential spread of pathology through neural circuitry. Finally, the results of this expository study were leveraged to highlight the challenges and limitations of working with preformed α-synuclein fibrils.

Project description:Differentiating clinically between Parkinson's disease (PD) and the atypical parkinsonian syndromes of Progressive supranuclear palsy (PSP), corticobasal syndrome (CBS) and multiple system atrophy (MSA) is challenging but crucial for patient management and recruitment into clinical trials. Because PD (and the related disorder Dementia with Lewy bodies (DLB)) and MSA are characterised by the deposition of aggregated forms of ?-synuclein protein (?-syn) in the brain, whereas CBS and PSP are tauopathies, we have developed immunoassays to detect levels of total and oligomeric forms of ?-syn, and phosphorylated and phosphorylated oligomeric forms of ?-syn, within body fluids, in an attempt to find a biomarker that will differentiate between these disorders. Levels of these 4 different forms of ?-syn were measured in post mortem samples of ventricular cerebrospinal fluid (CSF) obtained from 76 patients with PD, DLB, PSP or MSA, and in 20 healthy controls. Mean CSF levels of total and oligomeric ?-syn, and phosphorylated ?-syn, did not vary significantly between the diagnostic groups, whereas mean CSF levels of phosphorylated oligomeric ?-syn did differ significantly (p<0.001) amongst the different diagnostic groups. Although all 4 measures of ?-syn were higher in patients with MSA compared to all other diagnostic groups, these were only significantly raised (p<0.001) in MSA compared to all other diagnostic groups, for phosphorylated oligomeric forms of ?-syn. This suggests that this particular assay may have utility in differentiating MSA from control subject and patients with other ?-synucleinopathies. However, it does not appear to be of help in distinguishing patients with PD and DLB from those with PSP or from control subjects. Western blots show that the principal form of ?-syn within CSF is phosphorylated, and the finding that the phosphorylated oligomeric ?-syn immunoassay appears to be the most informative of the 4 assays would be consistent with this observation.

Dataset Information

Novel conformation-selective alpha-synuclein antibodies raised against different in vitro fibril forms show distinct patterns of Lewy pathology in Parkinson's disease.

Publications

Novel conformation-selective alpha-synuclein antibodies raised against different in vitro fibril forms show distinct patterns of Lewy pathology in Parkinson's disease.

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