A New Chemoenzymatic Semisynthetic Approach Provides Insight into the Role of Phosphorylation beyond Exon1 of Huntingtin and Reveals N-Terminal Fragment Length-Dependent Distinct Mechanisms of Aggregation

Huntington’s disease is a neurodegenerative dis- order caused by the expansion of a polyglutamine repeat (>36Q) in the N-terminal domain of the huntingtin protein (Htt), which renders the protein or fragments thereof more prone to aggregate and form inclusions. Although several Htt N-terminal fragments of different lengths have been identified within Htt inclusions, most studies on the mechanisms, sequence, and structural determinants of Htt aggregation have focused on the Httexon1 (Httex1). Herein, we investigated the aggregation properties of mutant N- terminal Htt fragments of various lengths (Htt171, Htt140, and Htt104) in comparison to mutant Httex1 (mHttex1). We also present a new chemoenzymatic semisynthetic strategy that enables site-specific phosphorylation of Htt beyond Httex1. These advances yielded insights into how post-translational modifications (PTMs) and structured domains beyond Httex1 influence aggregation mechanisms, kinetics, and fibril morphology of longer N-terminal Htt fragments. We demonstrate that phosphorylation at T107 significantly slows the aggregation of mHtt171, whereas phosphorylation at T107 and S116 accelerates the aggregation, underscoring the importance of crosstalk between different PTMs. The mHtt171 proteins aggregate via a different mechanism and form oligomers and fibrillar aggregates with morphological properties that are distinct from that of mHttex1. These observations suggest that different N-terminal fragments could have distinct aggregation mechanisms and that a single polyQ-targeting antiaggregation strategy may not effectively inhibit the aggregation of all N-terminal Htt fragments. Finally, our results underscore the need for further studies to investigate the aggregation mechanisms of Htt fragments and how the various fragments interact with each other and influence Htt toxicity and disease progression.

Exploring the role of post-translational modifications in regulating α-synuclein interactions by studying the effects of phosphorylation on nanobody binding

Anass Chiki, Farah El Turk, Bruno Claude Daniel Fauvet, Juan Kiwi, Hilal Lashuel

Intracellular deposits of α-synuclein in the form of Lewy bodies are major hallmarks of Parkinson’s disease (PD) and a range of related neurodegenerative disorders. Post-translational modifications (PTMs) of α-synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C-terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post-mortem PD patients, and its level appears to correlate with disease progression. Phosphorylation at the neighboring tyrosine residue Y125 has also been shown to protect against α-synuclein toxicity in a Drosophila model of PD. In the present study we address the potential roles of C-terminal phosphorylation in modulating the interaction of α-synuclein with other protein partners, using a single domain antibody fragment (NbSyn87) that binds to the C-terminal region of α-synuclein with nanomolar affinity. The results reveal that phosphorylation at S129 has negligible effect on the binding affinity of NbSyn87 to α-synuclein while phosphorylation at Y125, only four residues away, decreases the binding affinity by a factor of 400. These findings show that, despite the fact that α-synuclein is intrinsically disordered in solution, selective phosphorylation can modulate significantly its interactions with other molecules, and suggest how this particular form of modification could play a key role in regulating the normal and aberrant function of α-synuclein.

2018

Capturing the heterogeneity of alpha-synuclein pathology in synucleinopathies

Melek Firat Altay

Synucleinopathies such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy (MSA) are detrimental neurodegenerative diseases. Alpha-synuclein (aSyn), the main aggregating protein found in the pathological inclusions of these ailments, exists in numerous modified forms and conformations, leading to neuropathological heterogeneity in synucleinopathies. The functional, pathological and clinical implications of this heterogeneity remain unclear. Responding this knowledge gap requires the development of novel toolsets capable of capturing the biochemical and structural diversity of aSyn pathology in the brain and peripheral tissues.To address this challenge, we describe in Chapter 2 the development and characterisation of antibodies that cover all three regions and post-translational modifications (PTMs) of aSyn. We assess these antibodies through a comprehensive validation pipeline, and select the best performing antibody subset to capture the neuropathological diversity of aSyn across Lewy body diseases (LBDs). We demonstrate that these antibodies could be used to achieve an in-depth characterization of the biochemical and morphological diversity of aSyn aggregates in the neuronal and in vivo models of aSyn seeding. Our results suggest that aSyn pathology is rich in Serine 129 phosphorylation (pS129) and in previously under-described PTMs, including nitration and phosphorylation at Tyrosine 39 (nY39, pY39) and C-terminal tyrosine phosphorylations (pY133, pY136) across neuronal and glial populations in LBDs. We also report that aSyn is hyperphosphorylated in the cellular and in vivo seeding models. In Chapter 3, we demonstrate the power of our antibodies to achieve an unprecedented analysis of astrocytic aSyn pathology in LBDs. We characterise the biochemical properties, post-translational modifications and the aggregation state of astrocytic aSyn accumulations in the limbic regions across sporadic and familial LBDs. The astrocytic aSyn is revealed distinctively by antibodies against the late amino-terminus (N-terminus) and non-amyloid-beta component (NAC) region of aSyn, but not by the extreme N-terminal or carboxy-terminal (C-terminal) antibodies, suggesting that these aSyn species are truncated at both ends. The astrocytic aSyn accumulations are negative for the key markers of Lewy bodies (aSyn pS129, Amytracker, p62- and ubiquitin-positivity and proteinase K resistance), and positive for aSyn nY39 and pY39. These findings suggest that the astrocytic accumulations may be composed of non-fibrillar, possibly oligomeric conformers of aSyn. Chapter 4 focuses on deciphering the biochemical and morphological signatures of aSyn pathology in MSA. The antibodies described in Chapter 2 are applied to high and low pathology regions of MSA brains by immunohistochemistry (IHC). We also biochemically profile the aSyn species, and report an abundance of aSyn pS129-positive glial and neuronal inclusions that correspond to insoluble high molecular weight species by Western blot. We detect an enrichment of insoluble and truncated aSyn in the MSA cerebellum. By IHC, there is an enhancement of aSyn nY39, pY136 and of other aSyn PTMs.Collectively, the new tools and findings described here may pave the way for future studies elucidating the role of these neuronal and glial aSyn proteoforms in disease pathogenesis and propagation in synucleinopathies.

EPFL2022

Capturing the heterogeneity of alpha-synuclein pathology in synucleinopathies

Melek Firat Altay

EPFL2022