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Increasing evidence points to post-translational modifications (PTMs) as key regulators of alpha-synuclein (α-Syn) function in health and disease. However, whether these PTMs occur before or after α-Syn pathology formation and their role in regulating α-Syn toxicity remain unclear. In this study, we demonstrate that post-fibrillization nitration of α-Syn fibrils induced their fragmentation, modified their surface and dynamic properties but not their structure, and nearly abolished their seeding activity in primary neurons andin vivo. Furthermore, we show that the dynamic and surface properties of the fibrils, rather than simply their length, are important determinants of α-Syn fibril seeding activity. Altogether, our work demonstrates that post-aggregation modifications of α-Syn may provide novel approaches to target a central process that contributes to pathology formation and disease progression. Finally, our results suggest that the pattern of PTMs on pathological aggregates, rather than simply their presence, could be a key determinant of their toxicity and neurodegeneration. This calls for reconsidering current approaches relying solely on quantifying and correlating the level of pathology to assess the efficacy of novel therapies, as not all α-Syn aggregates in the brain are pathogenic.

Huntington’s disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer’s disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer’s disease patients.

The lack of detailed insight into the structure of aggregates formed by the huntingtin protein (HTT) has hampered the efforts to develop therapeutics and diagnostics targeting pathology formation in the brain of patients with Huntington's disease. To address this knowledge gap, we investigated the structural properties of in vitro-generated fibrils from exon1 of the huntingtin protein by cryogenic electron microscopy and single-particle analyses. We show that wildtype and mutant exon1 of the huntingtin protein form nonhelical fibrils with a polyglutamine amyloid core composed of beta hairpins with unique characteristics that have not been previously observed with other amyloid filaments. The stacks of beta-hairpins form long planar beta-sheets (protofilaments) which combine inter- and intramolecular interactions, with variable stacking angles and occasional out-of-register states of individual beta-hairpins. These features and the propensity of protofilaments to undergo lateral association result in a high degree of fibril polymorphisms, including fibrils composed of varying numbers of protofilaments. Our results allow us to speculate on how the fianking domains are organized around the polyglutamine core of the fibril and provide insight into how they might affect the huntingtin fibril structure and polymorphism. The removal of the first 17 amino acids at the N-terminus resulted in surprising intra-fibril structural heterogeneity and reduced fibril's propensity to lateral associations. Overall, this work provides valuable insights that could help guide future mechanistic studies to elucidate the sequence and structural determinants of huntingtin aggregation, as well as for cryo-EM and structural studies of fibrils derived from huntingtin protein and other disease-associated polyglutamine-containing proteins.

Post‐translational modifications (PTMs) within the first 17 amino acids (Nt17) of exon 1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntington's disease (HD). In this paper, we describe a new and simple methodology for producing milligram quantities of highly pure wild‐type or mutant Httex1 proteins that are site‐specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK), and 2) the development of an efficient methodology for producing recombinant native Httex1 proteins by using a SUMO‐fusion expression and purification strategy.[26] As a proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site‐specifically phosphorylated and fluorescently or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 or HTT structure, aggregation, interactome, and function(s) in health and disease., The tools presented in this paper offer new opportunities to enlarge the range of potential methods and experimental approaches that can be used to study the mechanisms by which Nt17 phosphorylation influences the properties and functions of Httex1 in health and disease. (NMR depiction is adapted from J. Am. Chem. Soc. 2017, 139, 1168).

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer’s disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau normal functions and its pathogenic properties in Tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. In this work, we aimed to address this knowledge gap and determine the relative contribution of phosphorylation of one or several of the five tyrosine residues in Tau (Y18, Y29, Y197, Y310 and Y394) to the regulation of its biophysical, aggregation and functional properties. Towards this goal, we used a combination of site-specific mutagenesis andin vitrophosphorylation by c-Abl kinase to generate Tau species phosphorylated at all tyrosine residues, all tyrosine residues except Y310 or Y394 (pTau-Y310F, pTau-Y394F) and Tau phosphorylated only at Y310 or Y394 (4F\pY310 or 4F\pY394). Our results show that phosphorylation at all five tyrosine residues, multiple N-terminal tyrosine residues (Y18, Y29 and Y197) or site-specific phosphorylation at residue Y310, itself located in the microtubule-binding and aggregation-prone domain of Tau, was sufficient to abolish Tau aggregation and inhibit its microtubule- and lipid-binding properties. NMR studies demonstrated that these effects were mediated by a local decrease in β−sheet propensity of the PHF6 domain. Our findings underscore the unique role of Y310 phosphorylation in the regulation of Tau aggregation, microtubule and lipid interactions and highlight the importance of conducting further studies to elucidate its role in the regulation of Tau normal functions and its pathogenic properties.

Alpha-synuclein (aSyn) within Lewy bodies, Lewy neurites, and other pathological hallmarks of Parkinson’s disease and synucleinopathies have consistently been shown to accumulate in aggregated and phosphorylated forms of the protein, predominantly at Serine 129 (S129). Antibodies against phosphorylated S129 (pS129) have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues. However, most of the antibodies and immunoassays aimed at detecting pS129-aSyn were developed based on the assumption that neighbouring post-translational modifications (PTMs) either do not co-occur with pS129 or do not influence its detection. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal 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 neighbouring PTMs. However, consistent with previous reports, most pS129 antibodies showed cross-reactivity towards other proteins and often detected low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or High Molecular Weight aggregates of aSyn. Our observations suggest that the pS129 antibodies do not capture the biochemical and morphological diversity of aSyn pathology. They also underscore the need for more specific pS129 antibodies, more thorough characterization and validation of existing antibodies, and the use of the appropriate protein standards and controls in future studies.

The lack of detailed insight into the structure of aggregates formed by the huntingtin protein has hampered efforts to develop therapeutics and diagnostics targeting pathology formation in the brain of patients with Huntington’s disease. To address this knowledge gap, we investigated the structural properties of in vitro generated fibrils from exon1 of the huntingtin protein by electron cryo-microscopy and single-particle analysis. We show that wildtype and mutant exon1 of the huntingtin protein form non-helical fibrils with a polygultamine amyloid core composed of β-hairpins with unique characteristics that have not been previously observed with other amyloid filaments. The stacks of β-hairpins form long planar β- sheets (protofilaments) with variable stacking angle and occasional out-of-register state of individual β-hairpins. These features and the propensity of protofilament to undergo lateral association results in a high degree of fibril polymorphism, including fibrils composed of varying numbers of protofilaments. Our results also represent the first direct observation of how the flanking domains are organized around the polyglutamine core of the fibril and provide insight into how they might affect huntingtin fibril structure, polymorphism, and stacking of β-hairpins within its core structure. Removal of the first 17 amino acids at the N-terminus resulted in surprising intra-fibril structural heterogeneity and reduced fibril’s propensity to lateral associations. Overall, this work provides valuable insights that could guide future mechanistic studies to elucidate the sequence and structural determinants of huntingtin aggregation, as well as cryo-EM and structural studies of fibrils derived from huntingtin proteins and other disease-associated polyglutamine-containing proteins.

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington’s disease. Our group’s previous studies suggested that the Nt17 PTM code is a combinatorial code that involves a complex interplay between different PTMs. Here, we expand on these studies by investigating the effect of methionine 8 oxidation (oxM8) and crosstalk between this PTM and either lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1. We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mutant Httex1 aggregates. This delay in aggregation kinetics could be attributed to the transient accumulation of oligomeric aggregates, which disappear upon the formation of Httex1 oxM8 fibrils. Interestingly, the presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization, whereas the presence of oxM8 did not influence the aggregation inhibitory effect of pT3. To gain insight into the structural basis underlying these proteins’ aggregation properties, we investigated the impact of each PTM and the combination of these PTMs on the conformational properties of the Nt17 peptide by circular dichroism spectroscopy and molecular dynamics simulation. These studies show that M8 oxidation decreases the helicity of the Nt17 in the presence or absence of PTMs and provides novel insight into the structural basis underlying the effects of different PTMs on mutant Httex1 aggregation. PTMs that lower the mutant Httex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and phosphorylation at Serine 13) result in stabilization and increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation of mutant Httex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mutant Httex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mutant Httex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mutant Httex1 aggregation.TOC Figure

Phosphorylation of the N‐terminal domain of the huntingtin (HTT) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK‐binding kinase 1 (TBK1), that efficiently phosphorylates full‐length and N‐terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo. TBK1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK1‐mediated neuroprotective effects are due to phosphorylation‐dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation., Phosphorylation of the N‐terminal domain of Huntingtin (HTT) by TANK‐binding kinase 1 (TBK1) promotes its autophagic clearance and reduce its aggregation and cytotoxicity, suggesting new strategies for neuroprotective therapies.

Objectives: To examine whether preadmission use of nonaspirin nonsteroidal anti-inflammatory drugs (NSAIDs) influenced 30-day stroke mortality. Methods: We conducted a nationwide population-based cohort study. Using medical databases, we identified all first-time stroke hospitalizations in Denmark between 2004 and 2012 (n = 100,043) and subsequent mortality. We categorized NSAID use as current (prescription redemption within 60 days before hospital admission), former, and nonuse. Current use was further classified as new or long-term use. Cox regression was used to compute hazard ratios (HRs) of death within 30 days, controlling for potential confounding through multivariable adjustment and propensity score matching. Results: The adjusted HR of death for ischemic stroke was 1.19 (95% confidence interval [CI]: 1.02–1.38) for current users of selective cyclooxygenase (COX)-2 inhibitors compared with nonusers, driven by the effect among new users (1.42, 95% CI: 1.14–1.77). Comparing the different COX-2 inhibitors, the HR was driven by new use of older traditional COX-2 inhibitors (1.42, 95% CI: 1.14–1.78) among which it was 1.53 (95% CI: 1.02–2.28) for etodolac and 1.28 (95% CI: 0.98–1.68) for diclofenac. The propensity score–matched analysis supported the association between older COX-2 inhibitors and ischemic stroke mortality. There was no association for former users. Mortality from intracerebral hemorrhage was not associated with use of nonselective NSAIDs or COX-2 inhibitors. Conclusions: Preadmission use of COX-2 inhibitors was associated with increased 30-day mortality after ischemic stroke, but not hemorrhagic stroke. Use of nonselective NSAIDs at time of admission was not associated with mortality from ischemic stroke or intracerebral hemorrhage.

Huntington's disease is a fatal neurodegenerative disorder resulting from a CAG repeat expansion in the first exon of the gene encoding the Huntingtin protein (Htt). Phosphorylation of this protein region (Httex1) has been shown to play important roles in regulating the structure, toxicity, and cellular properties of N-terminal fragments and full-length Htt. However, increasing evidence suggests that phosphomimetic substitutions in Htt result in inconsistent findings and do not reproduce all aspects of true phosphorylation. Here, we investigated the effects of bona fide phosphorylation at Ser-13 or Ser-16 on the structure, aggregation, membrane binding, and subcellular properties of the Httex1-Q18A variant and compared these effects with those of phosphomimetic substitutions. We show that phosphorylation at either Ser-13 and/or Ser-16 or phosphomimetic substitutions at both these residues inhibit the aggregation of mutant Httex1, but that only phosphorylation strongly disrupts the amphipathic α-helix of the N terminus and prompts the internalization and nuclear targeting of preformed Httex1 aggregates. In synthetic peptides, phosphorylation at Ser-13, Ser-16, or both residues strongly disrupted the amphipathic α-helix of the N-terminal 17 residues (Nt17) of Httex1 and Nt17 membrane binding. Experiments with peptides bearing different combinations of phosphorylation sites within Nt17 revealed a phosphorylation-dependent switch that regulates the Httex1 structure, involving cross-talk between phosphorylation at Thr-3 and Ser-13 or Ser-16. Our results provide crucial insights into the role of phosphorylation in regulating Httex1 structure and function, and underscore the critical importance of identifying the enzymes responsible for regulating Htt phosphorylation, and their potential as therapeutic targets for managing Huntington's disease.

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.

Phosphorylation of the N-terminal domain of the Huntingtin (HTT) protein (at T3, S13, and S16) has emerged as a key regulator of HTT stability, clearance, localization, aggregation and toxicity. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that specifically and efficiently phosphorylates both wild-type and mutant full-length or N-terminal fragments of HTT in vitro (S13/S16) and in cell/ neuronal cultures (S13). We show that overexpression of TBK1 in mammalian cells, primary neurons and a Caenorhabditis elegans model of Huntington’s Disease (HD) increases mutant HTTex1 phosphorylation, lowers its levels, increases its nuclear localization and significantly reduces its aggregation and cytotoxicity. Our mechanistic studies demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTTex1 aggregation and an increase in autophagic flux. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD.Graphical abstract

Increasing evidence suggests that the process of alpha‐synuclein (α‐syn) aggregation from monomers into amyloid fibrils and Lewy bodies, via oligomeric intermediates plays an essential role in the pathogenesis of different synucleinopathies, including Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies (DLB). However, the nature of the toxic species and the mechanisms by which they contribute to neurotoxicity and disease progression remain elusive. Over the past two decades, significant efforts and resources have been invested in studies aimed at identifying and targeting toxic species along the pathway of α‐syn fibrillization. Although this approach has helped to advance the field and provide insights into the biological properties and toxicity of different α‐syn species, many of the fundamental questions regarding the role of α‐syn aggregation in PD remain unanswered, and no therapeutic compounds targeting α‐syn aggregates have passed clinical trials. Several factors have contributed to this slow progress, including the complexity of the aggregation pathways and the heterogeneity and dynamic nature of α‐syn aggregates. In the majority of experiment, the α‐syn samples used contain mixtures of α‐syn species that exist in equilibrium and their ratio changes upon modifying experimental conditions. The failure to quantitatively account for the distribution of different α‐syn species in different studies has contributed not only to experimental irreproducibility but also to misinterpretation of results and misdirection of valuable resources. Towards addressing these challenges and improving experimental reproducibility in Parkinson's research, we describe here a simple centrifugation‐based filtration protocol for the isolation, quantification and assessment of the distribution of α‐syn monomers, oligomers and fibrils, in heterogeneous α‐syn samples of increasing complexity. The protocol is simple, does not require any special instrumentation and can be performed rapidly on multiple samples using small volumes. Here, we present and discuss several examples that illustrate the applications of this protocol and how it could contribute to improving the reproducibility of experiments aimed at elucidating the structural basis of α‐syn aggregation, seeding activity, toxicity and pathology spreading. This protocol is applicable, with slight modifications, to other amyloid‐forming proteins., We describe here a simple protocol for the isolation, quantification and assessment of the distribution of different types of α‐syn species, such as monomers, oligomers and fibrils, in α‐syn samples. The protocol is simple, does not require any special instrumentation and can be performed rapidly on multiple samples using small volumes. We believe that the application of this protocol by the α‐syn research community could contribute significantly to improving the reproducibility of experiments aimed at elucidating the structural basis of α‐syn aggregation, seeding activity, toxicity and pathology spreading, and facilitate the development of α‐synuclein‐based biomarkers and therapeutics. Read the Editorial Highlight for this article on page https://doi.org/10.1111/jnc.14973.

Significance The ability of proteins to self-assemble into different types of fibrils with distinct morphologies has been linked to the pathological and clinical heterogeneity of amyloid diseases such as Alzheimer’s disease and Parkinson’s disease. Here, we describe nanoparticles (NPs) that efficiently label amyloid fibrils produced in vitro or isolated from postmortem tissues, under hydrating conditions and in such a way as to unmask their polymorphism and morphological features. Using these NPs, we show that pathological aggregates exhibit exceptional morphological homogeneity compared with amyloid fibrils produced in vitro, consistent with the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. These advances pave the way for elucidating the structural basis of amyloid strains and toxicity., Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer’s disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

As an intrinsically disordered protein, monomeric alpha synuclein (aSyn) constantly reconfigures and probes the conformational space. Long-range interactions across the protein maintain its solubility and mediate this dynamic flexibility, but also provide residual structure. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. By using calcium to perturb the conformational ensemble, we observe differences in structure and intra-molecular dynamics between two aSyn C-terminal variants, D121A and pS129, and the aSyn familial disease mutants, A30P, E46K, H50Q, G51D, A53T and A53E, compared to wild-type (WT) aSyn. We observe that the more exposed the N-terminus and the beginning of the NAC region are, the more aggregation prone monomeric aSyn conformations become. N-terminus exposure occurs upon release of C-terminus interactions when calcium binds, but the level of exposure is specific to the aSyn mutation present. There was no correlation between single charge alterations, calcium affinity, or the number of ions bound on aSyn’s aggregation propensity, indicating that sequence or post-translation modification (PTM)-specific conformational differences between the N- and C-termini and the specific local environment mediate aggregation propensity instead. Understanding aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein, to stabilise aSyn in non-aggregation prone conformations, by either preserving long-range interactions between the N- and C-termini or by protecting the N-terminus from exposure.

Although converging evidence point to alpha-synuclein (a-syn) aggregation and Lewy body (LB) formation as central events in Parkinson's disease (PD), the molecular mechanisms that regulate these processes and their role in disease pathogenesis remain poorly understood. Herein, we applied an integrative biochemical, structural and imaging approach to elucidate the sequence, molecular and cellular mechanisms that regulate LB formation in primary neurons. Our results establish that post-fibrillization C-terminal truncation mediated by calpains 1 and 2 and potentially other enzymes, plays critical roles in regulating a-syn seeding, fibrillization and orchestrates many of the events associated with LB formation and maturation. These findings combined with the abundance of a-syn truncated species in LBs and pathological a-syn aggregates have significant implications for ongoing efforts to develop therapeutic strategies based on targeting the C-terminus of a-syn or proteolytic processing of this region.

Chronic stress and associated heightened glucocorticoid levels are risk factors for depression, a common non-motor symptom in Parkinson’s disease (PD). However, how heightened glucocorticoids influence PD neuropathology [alpha-synuclein (α-Syn) containing Lewy pathology and neurodegeneration] and disease progression is unclear. To address this knowledge gap, we investigated the impact of chronic corticosterone administration on α-Syn pathology, neurodegeneration, behavior and mitochondrial function in a mouse model of α-Syn pathology spreading after intracerebral injection of α-Syn preformed fibrils (PFFs). Our results demonstrate that heightened corticosterone aggravates neurodegeneration and α-Syn pathology spreading, intriguingly to specific brain regions, such as the entorhinal cortex. Corticosterone-treatment abolished distinct physiological adaptations after PFF-injection and induced differential physiological and behavioral consequences. Taken together, our work points to elevated glucocorticoids as a risk factor for the development of the neuropathological hallmarks of PD. Strategies aimed at reducing glucocorticoid levels might slow down pathology spreading and disease progression in synucleinopathy.

Phosphorylation of exon1 of the Huntingtin protein (Httex1) has been shown to play important roles in regulating the structure, toxicity and cellular properties of N-terminal fragments and the full-length Huntingtin protein. Here, we investigated and compared the effect of bona fide phosphorylation at S13 and/or S16 on the structure, aggregation, membrane binding, and subcellular properties of mutant Httex1-Q18A. We show that serine phosphorylation at either S13 or S16 strongly disrupts the amphipathic α-helix of the N-terminus, inhibits the aggregation of mutant Httex1 and prompts the internalization and nuclear targeting of Httex1 preformed aggregates. In synthetic peptides phosphorylation at S13 and/or S16 strongly disrupted the amphipathic α-helix of the N-terminal 17 residues (Nt17) of Httex1 and Nt17 membrane binding. Our studies on peptides bearing a different combination of phosphorylation sites within Nt17 revealed a novel phosphorylation-dependent switch for regulating the structure of Httex1 involving crosstalk between phosphorylation at T3 and S13 or S16. Together, our results provide novel insights into the role of phosphorylation in regulating Httex1 structure and function in health and disease and underscore the critical importance of identifying enzymes responsible for regulating Htt phosphorylation and their potential as therapeutic targets for the treatment of Huntington’s disease.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of intraneuronal inclusions consisting of aggregated and post-translationally modified α-synuclein (α-syn). Despite advances in the chemical synthesis of α-syn and other proteins, the generation of site-specifically nitrated synthetic proteins has not been reported. Consequently, it has not been possible to determine the roles of nitration at specific residues in regulating the physiological and pathogenic properties of α-syn. Here we report, for the first time, the site-specific incorporation of 3-nitrotyrosine at different regions of α-syn using native chemical ligation combined with a novel desulfurization strategy. This strategy enabled us to investigate the role of nitration at single or multiple tyrosine residues in regulating α-syn structure, membrane binding, oligomerization, and fibrils formation. We demonstrate that different site-specifically nitrated α-syn species exhibit distinct structural and aggregation properties and exhibit reduced affinity to negatively charged vesicle membranes. We provide evidence that intermolecular interactions between the N- and C-terminal regions of α-syn play critical roles in mediating nitration-induced α-syn oligomerization. For example, when Y39 is not available for nitration (Y39F and Y39/125F), the extent of cross-linking is limited mostly to dimer formation, whereas mutants in which Y39 along with one or multiple C-terminal tyrosines (Y125F, Y133F, Y136F and Y133/136F) can still undergo nitration readily to form higher-order oligomers. Our semisynthetic strategy for generating site-specifically nitrated proteins opens up new possibilities for investigating the role of nitration in regulating protein structure and function in health and disease.

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.

Significance The findings in this manuscript report on the identification of a posttranslational modification in the huntingtin protein (phosphorylation on residue T3 in the N17 region of the protein), which can revert the conformational effects of the Huntington’s disease (HD) mutation itself on the huntingtin protein and inhibit its aggregation properties in vitro. Using the first ultrasensitive immunoassay for a posttranslational modification of huntingtin protein, we demonstrate that pT3 levels are decreased in mutant huntingtin in preclinical models as well as in clinically relevant samples from HD patients. These findings are of high significance to Huntington’s disease biology, provide insights into mechanisms of Huntington’s disease pathogenesis, and open new opportunities for the development of therapeutics and diagnostics for Huntington’s disease., Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantitative assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein associated with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclinical and clinical samples. We find that T3 phosphorylation is greatly reduced in samples from Huntington’s disease models and in Huntington’s disease patients, and we provide evidence that bona-fide T3 phosphorylation alters Huntingtin exon 1 protein conformation and aggregation properties. These findings have significant implications for both mechanisms of disease pathogenesis and the development of therapeutics and diagnostics for Huntington’s disease.

The presence of expanded poly-glutamine (polyQ) repeats in proteins is directly linked to the pathogenesis of several neurodegenerative diseases, including Huntington’s disease. However, the molecular and structural basis underlying the increased toxicity of aggregates formed by proteins containing expanded polyQ repeats remain poorly understood, in part due to the size and morphological heterogeneity of the aggregates they form in vitro. To address this knowledge gap and technical limitations, we investigated the structural, mechanical and morphological properties of fibrillar aggregates at the single molecule and nanometer scale using the first exon of the Huntingtin protein as a model system (Exon1). Our findings demonstrate a direct correlation of the morphological and mechanical properties of Exon1 aggregates with their structural organization at the single aggregate and nanometric scale and provide novel insights into the molecular and structural basis of Huntingtin Exon1 aggregation and toxicity.

Huntington's disease (HD) is a fatal genetic neurodegenerative disorder caused by a CAG expansion gene which is translated into a polyglutamine stretch within the first exon of the Huntingtin protein (Htt). HD patients suffer from motor impairments, cognitive decline and depression. A hallmark of HD pathogenesis is the loss of neurons in the striatum and cortex which is closely linked to the formation of large cytoplasmic and especially nuclear aggregates composed of various N-terminal fragments of the mutant Huntingtin protein. In HD patient brains, Htt abnormally aggregates due to the expansion of its polyQ tract (more than 37 glutamines). Moreover, the severity of the disease increases with the number of glutamines, and the age of onset lowers as the polyQ tract expands. Several experimental observations suggest that overexpression of exon 1 of the mutant Huntingtin protein (Httex1) alone in transgenic mice is sufficient to reproduce HD pathology. Httex1, among other N-terminal Htt fragments, has also been consistently found in post-mortem HD brains. However, the molecular determinants of Httex1 aggregation and toxicity in HD remain unknown. Httex1 undergoes a wide range of post-translational modifications (PTMs) (such as phosphorylation, acetylation, ubiquitination and sumoylation) which were shown to modulate the toxicity and aggregation properties of the protein. Nevertheless, a comprehensive understanding of the effects of these PTMs on the biophysical and biochemical properties of Httex1 remains challenging since: 1) production and purification of recombinant mutant Httex1 protein is complicated by its high aggregation propensity; 2) most of the enzymes involved in regulating Httex1 PTMs remain unknown. In this study, we developed a novel semisynthetic strategy for the production of the highly aggregation-prone mutant Htt. Using specific chemical ligation and desulfurization conditions; we were able to produce for the first time, tag-free and pure mutant Httex1 in mg quantities. This advance will enable for the first time investigation of mutant Httex1 in the absence of large protein fusions, thus allowing for accurate determination of the role of the polyQ repeat length and post-translational modifications in modulating the aggregation kinetics and toxicity. Furthermore, the availability of highly pure WT and post-translationally modified mutant Httex1 proteins should facilitate structural studies aimed at determining the structural basis of Htt misfolding, aggregation and the identification and validation of novel HD biomarkers.

Huntington’s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch. The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content ( 42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of our strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility 45 of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation. Interestingly, acetylation at K6, but not at K9 or K15, reversed the inhibitory effect of T3 phosphorylation. Together, our results provide novel insight into the role of Nt17 post-translational modifications in regulating the structure and aggregation of Httex1 and suggest that its aggregation and possibly its function(s) are controlled by regulatory mechanisms involving crosstalk between different PTMs.

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays an important role in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity.HighlightsThe Nt17 and polyQ domains synergistically promote Httex1 aggregation.The Nt17 domain is a key determinant of the lateral association and morphology of fibrils.The Nt17 domain and conformation regulate the nuclear/cytoplasmic distribution and toxicity of Httex1.Nt17 conformation is a key determinant of Httex1 fibril membrane interaction and cellular uptake.Nt17 serves as one of the master regulators of Httex1 aggregation, cellular uptake and toxicity.Graphical abstractThe Nt17 domain: A master switch of Httex1 aggregation, uptake, subcellular localization and neurotoxicity.In this paper, we showed that 1) the Nt17 and polyQ domains synergistically promote Httex1 aggregation; 2) the Nt17 domain is a key determinant of the lateral association and morphology of fibrils in vitro, 3) Nt17 conformation is a key determinant of Httex1 fibril membrane interaction and cellular uptake in primary neurons; 4) the Nt17 domain and conformation regulate the nuclear/cytoplasmic distribution and toxicity of Httex1 in primary neurons.The figure was created with Biorender and https://www.vectorstock.com/royalty-free-vector/icon-on-and-off-toggle-switch-button-white-design-vector-30148026