Biology subjects

Lashuel, H. A.

Publications and source records attributed to Lashuel, H. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Autophagy activator AA-20 engages CDK5 to promote lipophagy and improve neurodegeneration-associated phenotypes

Lipophagy, the selective degradation of lipid droplets (LDs) by autophagy, has the potential to ameliorate neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD), wherein LD accumulation has been linked to the exacerbation of the proteinopathy component of neurodegeneration. Pharmacological lipophagy inducers remain scarce. We previously identified AA-20 as a novel autophagy activator that acts independently of mTORC1 inhibition, thereby providing a means of stimulating autophagy while avoiding the immunosuppressive side effects associated with direct mTORC1 inhibition. Prior work also showed that AA-20 treatment reduces PolyQ aggregate levels in several C. elegans tissues. Here, we demonstrate that AA-20 enhances LD recruitment to autophagosomes and promotes LD degradation via lysosomal acid lipase activation in murine hippocampal neuronal cells, consistent with lipophagy activation. In human Swedish Amyloid Precursor Protein mutated cerebral organoids, two weeks of AA-20 treatment normalized aberrant lipidomic profiles by selectively reducing various disease-associated lipids, while also correcting the AD-associated electrical hyperexcitability phenotype. Moreover, we identified cyclin-dependent kinase 5 (CDK5), a neuronal kinase linked to AD hyperexcitability and PD pathology, as a target of AA-20. AA-20-induced autophagy and lipid clearance in HT22 cells require CDK5. In a neuronal seeding model of -synuclein pathology, AA-20 reduced pS129--synuclein levels. Collectively, these findings indicate AA-20 can promote lipophagic clearance of LDs and mitigate multiple cellular phenotypes associated with lipidopathy and proteinopathy in neurodegenerative disease models.

molecular biology↗

bioRxiv2026

De novo Design of Polymorph-Specific Binders Targeting α-Synuclein Fibrils

Alpha-synuclein (aSyn) aggregation into amyloid fibrils is a hallmark of synucleinopathies, a group of neurodegenerative disorders that includes Parkinsons disease. Beyond the central role of fibrils in disease pathology and propagation, distinct aSyn fibril polymorphs have been associated with different synucleinopathies, presenting opportunities for differential diagnosis and structure-based disease-specific phenotyping. Here, we present AmyBind, a computational pipeline for the de novo design of mini-protein binders that target polymorph-specific structural features of amyloid fibrils. Using this approach, we developed two aSyn fibril-specific binders, one of which exhibited polymorph-specificity. Neither binder interfered with fibril elongation in vitro, consistent with their designed binding mode on lateral fibril surfaces. In cell-based models, both binders showed polymorph-specific colocalization but did not affect fibril uptake or seeding, indicating their utility as fibril-selective labeling agents in cellular contexts. These findings demonstrate the feasibility of structure-guided, polymorph-specific amyloid targeting and provide a foundation for developing early-stage diagnostics and therapeutics for synucleinopathies and other amyloid-related diseases.

bioengineering↗

bioRxiv2026

α-Synuclein Facilitates Spontaneous Dopamine Release in a Calcium- and Phosphorylation-Dependent Manner

-Synuclein (aSyn) is central to Parkinsons disease pathogenesis, yet its native physiological role at the presynapse remain poorly defined. Here, super-resolution imaging in dopaminergic neurons reveals that endogenous aSyn localises within nanometres of L-type voltage-gated calcium channels (LTCC), with closer proximity under both spontaneous neuronal activity and stimulated conditions compared to when extracellular calcium is chelated. Blocking Ca2+/calmodulin-dependent kinase II (CaMKII) reduces aSyn clustering at LTCC under spontaneous activity, suggesting that calcium entry and downstream calcium-dependent kinase activity contribute to aSyn localisation. Moreover, quantitative single-molecule analyses indicate that calcium increases the abundance of both total and serine129 phosphorylated (pS129) aSyn in synaptosomes under spontaneous conditions, and NMR analysis reveals that both calcium and S129 phosphorylation increase the binding affinity of aSyn to synaptic vesicles. Functional assays further demonstrate that LTCC blockade elevates intracellular DA levels exclusively in the presence of aSyn under spontaneous but not stimulated conditions. Finally, biochemical fractionation and multi-colour single-molecule imaging reveal that aSyn preferentially associates with small vesicles that are not obligately coupled to full-fusion associated recycling pools. These results suggest that aSyn acts as a calcium- and phosphorylation-regulated modulator of spontaneous DA release through pathways that are largely independent of full-fusion recycling mechanisms, and that pS129 aSyn is not solely a pathological marker but may also reflects physiological regulation. Together, these insights provide a framework for understanding how therapeutic strategies targeting aSyn may impact its normal synaptic functions.

neuroscience↗

bioRxiv2026

Ultrasensitive Detection of Alpha-Synuclein Oligomers in Human Plasma Using Optimized Nano-QuIC

Early diagnosis of Parkinsons disease (PD) is critical, as clinical symptoms typically emerge only after substantial neuronal loss. While -synuclein (-Syn) oligomers in blood are promising biomarkers for early detection, their clinical utility is limited by their low abundance and the presence of inhibitory components in the plasma matrix. To address these limitations, we tailored the Nanoparticle-enhanced Quaking-Induced Conversion (Nano-QuIC) platform specifically for the ultrasensitive detection of -Syn oligomers in human plasma. We identified critical reaction determinants by investigating buffer pH, ionic strength, detergent types, and shaking conditions. Furthermore, the integration of silica nanoparticles (siNPs) proved essential in mitigating plasma matrix interference, ensuring robust and reproducible protein aggregation. Under these optimized conditions, the assay achieved a detection limit of 100 pg/mL for -Syn oligomers spiked into human plasma. These results demonstrate that our adapted Nano-QuIC platform provides a highly sensitive and minimally invasive method for detecting pathological -Syn species, offering a significant advancement toward the development of early-stage PD diagnostics.

bioengineering↗

bioRxiv2025

Recapitulating Parkinson's pathology in human iPSC dopaminergic neurons reveals new mechanistic insights into Lewy body formation and heterogeneity.

The accumulation of alpha-synuclein (aSyn) into intraneuronal inclusions of heterogeneous morphology, known as Lewy bodies (LB), is one of the defining diagnostic features of Parkinsons disease (PD); yet, our understanding of the mechanisms underpinning their formation and heterogeneity remains incomplete. Here, we present a human isogenic iPSC-derived dopaminergic neuron (iDA) model that faithfully recapitulates the diverse biochemical, morphological, and ultrastructural features of LB neuropathology in PD. Unlike other iDA seeding models, our model does not rely on aSyn overexpression, mutations, or genetic engineering, making it a more physiologically relevant system for studying PD. We demonstrate that the iDA model accurately reproduces the temporal relationships between neuritic and cell-body aSyn pathology, recapitulating the full biochemical spectrum, post-translational modifications (PTM), and morphological diversity of aSyn aggregates found in human PD tissue. Moreover, our work provides critical insight into how different pathways to aSyn fibrillization and the complex interaction between aSyn fibrils and membranous organelles influence the morphological diversity of LB-like inclusions. This model represents a versatile platform for investigating the mechanisms of pathology formation, maturation, and neuronal dysfunction, as well as supporting the development of diagnostics that capture the diversity of aSyn pathology in PD and related synucleinopathies.

neuroscience↗

bioRxiv2025

Defined human tri-lineage brain microtissues

Microglia are the immune cells of the central nervous system and are thought to be key players in both physiological and disease conditions. Several microglial features are poorly conserved between mice and human, such as the function of the neurodegeneration-associated immune receptor Trem2. Induced pluripotent stem cell (iPSC)-derived microglia offer a powerful opportunity to generate and study human microglia. However, human iPSC-derived microglia often exhibit activated phenotypes in vitro, and assessing their impact on other brain cell types remains challenging due to limitations in current co-culture systems. Here, we developed fully defined brain microtissues, composed of human iPSC-derived neurons, astrocytes, and microglia, co-cultured in 2D or 3D formats. Our microtissues are stable and self-sufficient over time, requiring no exogenous cytokines or growth factors. All three cell types exhibit morphologies characteristic of their in vivo environment and show functional properties. Co-cultured microglia develop more homeostatic phenotypes compared to microglia exposed to exogenous cytokines. Hence, these tri-cultures provide a unique approach to investigate cell-cell interactions between brain cell types. We found that astrocytes and not neurons are sufficient for microglial survival and maturation, and that astrocyte-derived M-CSF is essential for microglial survival. Single-cell and single-nucleus RNA sequencing analyses nominated a network of reciprocal communication between cell types. Brain microtissues faithfully recapitulated pathogenic -synuclein seeding and aggregation, suggesting their usefulness as human cell models to study not only normal but also pathological cell biological processes.

neuroscience↗

bioRxiv2025

A Microfluidic Platform for Whole-Membrane Integrity Profiling in Live Neuronal Cells

Structural and functional compromise of the cellular membrane are central mechanisms in the pathogenesis of numerous diseases, including neurodegenerative disorders such as Alzheimers and Parkinsons disease. However, existing techniques for assessing membrane integrity often lack the ability to provide dynamic, whole-cell measurements and are limited to localized damage detection or population-level analysis. There is a growing need for methods capable of monitoring membrane integrity over time at the single-cell level and across the entire membrane surface. In this study, we present a microfluidic platform for real-time, label-free assessment of membrane integrity by analyzing dielectric properties. We apply this system to investigate how different aggregated forms of -Synuclein (aSyn), a protein that plays a central role in the pathogenesis of Parkinsons disease and disrupts neuronal membranes. Our platform integrates electrokinetic microdevices with 3D microelectrodes and imaging, enabling continuous analysis of up to 30 live neuronal cells per hour under flow. By measuring electrorotation responses, we quantify changes in plasma membrane capacitance in response to monomeric, oligomeric, and fibrillar aSyn. This approach allows direct, time-resolved comparison of membrane-disruptive effects across different aSyn conformations with single-cell resolution and whole-membrane sensitivity.

bioengineering↗

bioRxiv2025

Mapping the Structural Landscape of Amyloid Fibrils to Guide Polymorph-Specific Therapeutics

Amyloid fibrils are pathological hallmarks of neurodegenerative diseases and central contributors to their progression, representing promising targets for disease-modifying interventions. However, limited access to patient-derived fibrils and the inability to reproduce pathological folds in vitro hinder the development of fibril-specific ligands. Here, we present FibrilSite, a computational pipeline that identifies geometric and physicochemical similarities across fibril surfaces and demonstrate its utility to identify structural features that distinguish different fibril polymorphs. Our analysis uncovered conserved and polymorph-specific features within alpha-synuclein fibrils. Notably, one site was conserved between ex vivo multiple system atrophy and in vitro H50Q mutant fibrils, suggesting the latters potential utility in drug discovery. Druggability predictions further prioritized ligandable sites across fibrils. Together, our findings establish a structure-based framework for identifying disease-relevant features and mapping them onto suitable in vitro models, guiding the rational development of polymorph-specific diagnostics and therapeutics for amyloid-related disorders with improved translational potential.

bioinformatics↗

bioRxiv2024

Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

Alpha-synuclein (aSyn) post-translational modifications (PTMs), particularly phosphorylation at serine 129 and C-terminal truncations, are highly enriched in Lewy bodies (LBs), Lewy neurites, and other types of aSyn pathological aggregates in the brain of patients with Parkinsons disease (PD) and other synucleinopathies. However, our knowledge about the precise role of PTMs in regulating the different stages of pathology formation, neurodegeneration, and aSyn pathology spreading remains incomplete. In this work, we applied a systematic approach to address this knowledge gap with an emphasis on mapping and elucidating the role of post-fibrillization C-terminal aSyn truncations in regulating the uptake, processing, seeding activity, and formation of LB-like inclusions and maturations in a well-established neuronal seeding model that recapitulates all the stages leading to LB formation and neurodegeneration. Our work shows that C-terminal cleavage of aSyn fibrils at multiple sites is a conserved process that occurs rapidly after and during the formation of intracellular LB-like aSyn inclusions in all neuronal seeding models. Interestingly, blocking the cleavage of internalized fibrils does not influence their seeding activity, whereas inhibiting the enzymes that regulate the cleavage of newly formed fibrils (e.g., calpains 1 and 2) significantly alters the formation of LB-like inclusions. We also show that C-terminal truncations, in combination with other PTMs, play a crucial role in regulating the interactome and remodeling of newly formed aSyn fibrils, including their shortening, lateral association, and packing during LB formation and maturation. Altogether, our results demonstrate that post-fibrillization C-terminal truncations have a differential role at different stages of aSyn aggregation and pathology formation. These insights, combined with the abundance of truncated aSyn species in LBs, have significant implications in understanding aSyn pathological diversity and developing therapeutic strategies targeting the C-terminus of aSyn or its proteolytic processing.

neuroscience↗

bioRxiv2023

Post-fibrillization nitration of alpha-synuclein abolishes its seeding activity and pathology formation in primary neurons and in vivo

Increasing evidence points to post-translational modifications (PTMs) as key regulators of alpha-synuclein (-Syn) function in health and disease. However, whether these PTMs occur before or after -Syn pathology formation and their role in regulating -Syn toxicity remain unclear. In this study, we demonstrate that post-fibrillization nitration of -Syn fibrils induced their fragmentation, modified their surface and dynamic properties but not their structure, and nearly abolished their seeding activity in primary neurons and in vivo. Furthermore, we show that the dynamic and surface properties of the fibrils, rather than simply their length, are important determinants of -Syn fibril seeding activity. Altogether, our work demonstrates that post-aggregation modifications of -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 -Syn aggregates in the brain are pathogenic.

neuroscience↗

bioRxiv2022

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 Parkinsons disease (PD), Parkinsons disease with dementia (PDD) and dementia with Lewy bodies (DLB), and in oligodendrocytes in multiple system atrophy (MSA). Although the accumulation of aSyn in astrocytes has previously been described in PD, PDD and DLB, the biochemical properties of aSyn in this type of pathology and its topographical distribution have not been studied in detail. Here, we present a systematic investigation of aSyn astrocytic pathology, using an expanded toolset of antibodies covering the entire sequence and known post-translational modifications (PTMs) of aSyn in Lewy body (LB) disorders, including sporadic PD, PDD, DLB, familial PD with SNCA G51D mutation and SNCA duplication, and in MSA. Astrocytic aSyn was mainly detected in the limbic cortical regions of LB disorders, but were absent in key pathological regions of MSA. These astrocytic aSyn accumulations were detected only with aSyn 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 mostly not resistant to proteinase K. Our findings suggest that astrocytic aSyn accumulations are a major part of aSyn pathology in LB disorders, and possess a distinct sequence and PTM signature that is characterized by both N- and C-terminal truncations and modifications at Y39. To the best of our knowledge, this is the first description of aSyn accumulation made solely from N- and C-terminally cleaved aSyn species and the first report demonstrating that astrocytic aSyn exists as a mixture of Y39 phosphorylated and nitrated species. These observations underscore the critical importance of systematic characterization of aSyn accumulation in different cell types as a necessary step to capturing the diversity of aSyn species and pathology in the brain. Our findings combined with further studies on the role of astrocytic pathology in the progression of LB disorders can pave the way towards identifying novel disease mechanisms and therapeutic targets.

neuroscience↗

bioRxiv2022

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. Different aSyn conformations and post-translationally modified forms accumulate in pathological inclusions and vary in abundance across synucleinopathies. Relying on antibodies that have not been assessed for their ability to capture the diversity of aSyn species may not provide an accurate estimation of aSyn pathology in human brains or disease models. To address this challenge, we developed and characterised an expanded antibody panel that targets different sequences and post-translational modifications along the length of aSyn and recognises all three aSyn conformations (monomeric, oligomeric, fibrillar). Next, we profiled aSyn pathology across sporadic and familial Lewy body diseases (LBDs) and reveal heterogeneously modified aSyn pathologies rich in Serine 129 phosphorylation but also in Tyrosine 39 nitration and N- and C-terminal tyrosine phosphorylations, scattered to neurons and glia. We also show that aSyn may become hyperphosphorylated during the aggregation and inclusion maturation processes in neuronal and animal models of aSyn aggregation and spreading. The antibody validation pipeline we describe here paves the way for more systematic investigations of aSyn pathological diversity in the human brain and peripheral tissues, and in cellular and animal models of synucleinopathies.

neuroscience↗

bioRxiv2022

N-terminal mutant Huntingtin deposition correlates with CAG repeat length and disease onset, but not neuronal loss in Huntington's disease

Huntingtons disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake a high-throughput screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs). Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age-matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabelled human brain tissue. The Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with high-throughput analysis. Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. Significant associations were found between the number of Htt aggregates and both age of disease onset, and CAG repeat length in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and Htt cortical aggregate deposition is associated with the onset of clinical symptoms. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD are not specific for Htt in human brain tissue, thereby highlighting the need for validated means of Htt detection to support drug development for HD.

neuroscience↗

bioRxiv2022

Neighbouring modifications interfere with the detection of phosphorylated alpha-synuclein at Serine 129: Revisiting the specificity of pS129 antibodies

Alpha-synuclein (aSyn) within Lewy bodies, Lewy neurites, and other pathological hallmarks of Parkinsons 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.

neuroscience↗

bioRxiv2022

Pathological relevance of post-translationally modified alpha-synuclein (pSer87, pSer129, nTyr39) in idiopathic Parkinson's disease and Multiple System Atrophy.

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

neuroscience↗

bioRxiv2021

A novel mutation (E83Q) unlocks the pathogenicity of human alpha-synuclein fibrils and recapitulates its pathological diversity

A novel mutation (E83Q), the first in the NAC domain of alpha-synuclein (aSyn), was recently identified in a patient with dementia with Lewy bodies. We investigated the effects of this mutation on the aggregation of aSyn monomers and the structure, morphology, dynamic, and seeding activity of the aSyn fibrils in neurons. We found that it dramatically accelerates aSyn fibrillization and results in the formation of fibrils with distinct structural and dynamic properties. In cells, this mutation is associated with higher levels of aSyn, accumulation of pS129, and increased toxicity. In a neuronal seeding model of Lewy bodies (LB) formation, the E83Q mutation significantly enhances the internalization of fibrils into neurons, induce higher seeding activity and results in the formation of diverse aSyn pathologies, including the formation of LB-like inclusions that recapitulate the immunohistochemical and morphological features of brainstem LBs observed in PD patient brains. TeaserA novel mutation (E83Q) exacerbates alpha-synuclein aggregation and toxicity and reproduces PD pathological diversity.

neuroscience↗

bioRxiv2021

Correlative light and electron microscopy reveals that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington's disease

Huntingtons disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (i) multivesicular bodies (MVBs), or amphisomes and (ii) autolysosomes or residual bodies. The organelles were often found in close proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.

neuroscience↗

bioRxiv2021

Investigating crosstalk among PTMs provides novel insight into the structural basis underlying the differential effects of Nt17 PTMs on mutant Httex1 aggregation

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 Huntingtons disease. Our groups 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/432155v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@15dbf84org.highwire.dtl.DTLVardef@1c8a50corg.highwire.dtl.DTLVardef@1cbe0c6org.highwire.dtl.DTLVardef@120e0a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗