Biology subjects

Lashuel, H. A.

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

27 records · Page 2Linked to original sources

bioRxiv2021

Towards deciphering the Nt17 code: How the sequence and conformation of the first 17 amino acids in Huntingtin regulate the aggregation, cellular properties, and neurotoxicity of mutant Httex1

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. HighlightsO_LIThe Nt17 and polyQ domains synergistically promote Httex1 aggregation. C_LIO_LIThe Nt17 domain is a key determinant of the lateral association and morphology of fibrils. C_LIO_LIThe Nt17 domain and conformation regulate the nuclear/cytoplasmic distribution and toxicity of Httex1. C_LIO_LINt17 conformation is a key determinant of Httex1 fibril membrane interaction and cellular uptake. C_LIO_LINt17 serves as one of the master regulators of Httex1 aggregation, cellular uptake and toxicity. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/431207v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@7afd68org.highwire.dtl.DTLVardef@92c67aorg.highwire.dtl.DTLVardef@24823dorg.highwire.dtl.DTLVardef@e2a835_HPS_FORMAT_FIGEXP M_FIG The 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 C_FIG

neuroscience↗

bioRxiv2020

Site-specific phosphorylation of Huntingtin exon 1 recombinant proteins enabled by the discovery of novel kinases

Posttranslational modifications (PTMs) within the first 17 amino acids (Nt17) of exon1 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 Huntingtons disease (HD). In this manuscript, 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 using a SUMO-fusion expression and purification strategy. As proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site- specifically phosphorylated and fluorescently labeled 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 structure, aggregation, interactome and function(s) in health and disease.

biochemistry↗

bioRxiv2020

Characterization and validation of 15 α-synuclein conformation-specific antibodies using well-characterized preparations of α-synuclein monomers, fibrils and oligomers with distinct structures and morphology: How specific are the conformation-specific α-synuclein antibodies?

Increasing evidence suggests that alpha-synuclein (-syn) oligomers are obligate intermediates in the pathway involved in -syn fibrillization and Lewy body (LB) formation, and may also accumulate within LBs in Parkinsons disease (PD) and other synucleinopathies. Therefore, the development of tools and methods to detect and quantify -syn oligomers has become increasingly crucial for mechanistic studies to understand the role of these oligomers in PD, and to develop new diagnostic methods and therapies for PD and other synucleinopathies. The majority of these tools and methods rely primarily on the use of aggregation state-specific or conformation-specific antibodies. Given the impact of the data and knowledge generated using these antibodies on shaping the foundation and directions of -syn and PD research, it is crucial that these antibodies are thoroughly characterized, and their specificity or ability to capture diverse -syn species is tested and validated. Herein, we describe an antibody characterization and validation pipeline that allows a systematic investigation of the specificity of -syn antibodies using well-defined and well-characterized preparations of various -syn species, including monomers, fibrils, and different oligomer preparations that are characterized by distinct morphological, chemical and secondary structure properties. This pipeline was used to characterize 18 -syn antibodies, 16 of which have been reported as conformation- or oligomer-specific antibodies, using an array of techniques, including immunoblot analysis (slot blot and Western blot), a digital ELISA assay using single molecule array technology and surface plasmon resonance. Our results show that i) none of the antibodies tested are specific for one particular type of -syn species, including monomers, oligomers or fibrils; ii) all antibodies that were reported to be oligomer-specific also recognized fibrillar -syn; and iii) a few antibodies showed high specificity for oligomers and fibrils but did not bind to monomers. These findings suggest that the great majority of -syn aggregate-specific antibodies do not differentiate between oligomers and fibrils, thus highlighting the importance of exercising caution when interpreting results obtained using these antibodies. Our results also underscore the critical importance of the characterization and validation of antibodies before their use in mechanistic studies and as diagnostic and therapeutic agents. This will not only improve the quality and reproducibility of research and reduce costs but will also reduce the number of therapeutic antibody failures in the clinic.

neuroscience↗

bioRxiv2019

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington's disease

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 Huntingtons 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/869586v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1368abeorg.highwire.dtl.DTLVardef@1cecff4org.highwire.dtl.DTLVardef@b6b7c5org.highwire.dtl.DTLVardef@1daf53d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

neuroscience↗

bioRxiv2019

Site-specific hyperphosphorylation of tau inhibits its fibrillization in vitro, blocks its seeding capacity in cells, and disrupts its microtubule binding; Implications for the native state stabilization of tau

The consistent observation of aggregated phosopho-tau in the pathology of Alzheimers disease and other tauopathies has contributed to the emergence of a model where hyperphosphorylation of tau causes its disassociation from microtubules and subsequent pathological polymerization. However, the large number of possible phosphorylation sites in tau and lack of robust methods that enable the preparation of homogeneously phosphorylated tau species have made it difficult to validate this model. Herein, we applied a total chemical synthetic approach to site-specifically phosphorylate single (pS356) or multiple (pS356/pS262 and pS356/pS262/pS258) residues within the microtubule binding repeat domain (MTBD) of tau and show that hyperphosphorylation within the microtubule MTBD inhibits K18 tau 1) aggregation in vitro; 2) its seeding activity in cells, and 3) its ability to promote microtubule polymerization. The inhibition increased with the number of phosphorylated sites, with phosphorylation at S262 having the strongest effect. On the basis of these findings, we propose that targeting the kinases that regulate phosphorylation at these sites could provide a viable strategy to stabilize the native state of tau and inhibit its aggregation. Taken together, our results argue against the pathogenic hyperphosphorylation hypothesis and underscore the critical importance of revisiting the role of site-specific hyperphosphorylation of tau in regulating its function in health and disease.\n\nO_FIG O_LINKSMALLFIG WIDTH=159 HEIGHT=200 SRC=\"FIGDIR/small/772046v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (48K):\norg.highwire.dtl.DTLVardef@1328421org.highwire.dtl.DTLVardef@1809f73org.highwire.dtl.DTLVardef@16459a4org.highwire.dtl.DTLVardef@1393751_HPS_FORMAT_FIGEXP M_FIG Table of content C_FIG

neuroscience↗

bioRxiv2019

Extent of N-terminus exposure by altered long-range interactions of monomeric alpha-synuclein determines its aggregation propensity

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 aSyns 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.

biophysics↗

bioRxiv2018

The making of a Lewy body: the role of alpha-synuclein post-fibrillization modifications in regulating the formation and the maturation of pathological inclusions.

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.

neuroscience↗

bioRxiv2018

Chronic corticosterone enhancement aggravates alpha-synuclein brain spreading pathology and substantia nigra neurodegeneration in mice

Chronic stress and associated heightened glucocorticoid levels are risk factors for depression, a common non-motor symptom in Parkinsons disease (PD). However, how heightened glucocorticoids influence PD neuropathology [alpha-synuclein (-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 -Syn pathology, neurodegeneration, behavior and mitochondrial function in a mouse model of -Syn pathology spreading after intracerebral injection of -Syn preformed fibrils (PFFs). Our results demonstrate that heightened corticosterone aggravates neurodegeneration and -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.

neuroscience↗

bioRxiv2018

N-terminal phosphorylation of Huntingtin: A molecular switch for regulating Htt aggregation, helical conformation, internalization and nuclear targeting.

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 -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 -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 Huntingtons disease.

neuroscience↗