Biology subjects

Petry, S.

Publications and source records attributed to Petry, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Emergence of large-scale polar microtubule swarms for dense molecular transport

Microtubules (MTs) and their motor proteins collectively harness chemical energy to generate mechanical work, driving some of the most coordinated self-organized dynamics in living cells. The unique properties of these molecules also make them versatile building blocks of cytoskeletal active matter and biomimetic nanomachines that recapitulate cellular motility, emergent pattern formation, and motor-driven transport. However, these canonical systems use MTs of fixed length and do not incorporate the natural ability of MTs to grow and regenerate. Here, we go beyond these limits by using dynamic self-amplifying branched MT networks. Driven by kinesin-1 and cytoplasmic dynein activity, surface-gliding branched MT bundles undergo swarming that yields large-scale collective MT architectures with several sought-after features. They are polar and orientationally aligned, dense, span millimeter scales, and persist over hours. We then show that these features enable molecular transport along the swarm at unprecedented capacities, with up to six million motor complexes walking in parallel across millimeter-scale distances over hours. Our results introduce a new regime in cytoskeletal active matter in which the interplay between motor-driven activity and filament generation via branching leads to emergent polar order in proliferating swarms. Such emergent polarity makes these swarms suitable for engineering scalable transport nanotechnologies and programmable soft materials.

biophysics↗

bioRxiv2026

Capillary bundling of microtubules by condensates

The cytoskeleton organizes the cellular interior using cytoskeletal filaments that rely on bundling, usually executed by stable and ordered crosslinking proteins. Bundling often requires protein complexes with at least two defined microtubule binding regions, as present in many molecular motors. Here, we establish a mechanism of microtubule bundling based on capillary forces, analogous to how wet hair sticks together. We show using in vitro experiments and theory that condensates can bundle microtubules through capillary forces, wherein liquid-like capillary bridges form between microtubules and adhere them together through interfacial and wetting forces. We quantify the structure and dynamics of these capillary bundles using total internal reflection fluorescence microscopy, and directly measure the charge-dependent interfacial tensions of condensates on microtubules using atomic force microscopy. Lastly, we show that these capillary bridges provide viscous resistance to motor-driven microtubule sliding that is insensitive to the bulk protein concentration. Taken together, we provide a novel mechanism for how cytoskeletal filaments bundle: through condensate-mediated capillary forces.

biophysics↗

bioRxiv2025

Initial identification of genomic islands in Taylorella equigenitalis and Taylorella asinigenitalis and their distribution in isolates from around the world

Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), is endemic in several countries throughout the world and impacts the global movement of horses. It presents with symptoms ranging from non-clinical to endometritis, vaginitis, and acute infertility in females while males remain asymptomatic. Studies of the disease have focused primarily on understanding pathology, transmission dynamics, diagnostics, and phenotypic and genotypic characteristics. The goal of this study was to establish a more comprehensive global phylogenetic structure and generate reference-quality genomes for each major clade of the tree where such data were absent. Seven of these genomes along with 44 other publicly available genome assemblies, including Taylorella asinigenitalis, were then used to predict genomic islands. Exploration of the NCBI annotations in the genomic islands indicate links to virulence and ecological fitness. An additional 97 T. equigenitalis and 48 T. asinigenitalis short-read isolates from this study and 215 publicly available genomes were used to examine distribution of each genomic island across the phylogenetic tree for evidence of horizontal movement. The stability of these genomic islands in their natural environment was evaluated utilizing 141 isolates recovered from a recent CEM outbreak in the United States. The results obtained in this study demonstrate mobile genomic islands in T. equigenitalis genomes, sites of concern for their contribution to the potential virulence of the organism.

microbiology↗

bioRxiv2025

Sperm hyperactivation drives a circling-and-wandering migration strategy

During migration through the female reproductive tract, sperm undergo physiological changes known as capacitation, including a motility transition termed hyperactivation. Hyperactivation is essential for various aspects of fertilization, particularly effective migration within the tract. However, how hyperactivation facilitates this migration remains elusive. Here, we profiled bull sperm hyperactivation in Newtonian and complex fluids near microfluidic surfaces, mimicking generic swimming conditions in the tract. We identified three swim gaits: wandering (persistent random walks), circling, and an intriguing circling-and- wandering mode marked by stochastic transitions between the two. All gaits exhibit diffusive behavior over long time scales, with wandering showing a tenfold higher diffusivity than circling, and the diffusivity of circling-and-wandering falling in between. We found that while wandering sperm scatter from convex and concave surfaces, circling sperm become trapped around pillars, highlighting the distinctive nature of each phase. Additionally, stochastic simulations of swimming in porous media showed that as the geometrical complexity of the environment increases, circling-and-wandering outperforms either strategy alone in spreading through the media. Our findings suggest that while wandering promotes exploration and circling supports local exploitation, circling-and-wandering combines the strengths of both strategies by balancing exploration and exploitation to adapt motility, enhance migration, and potentially improve target search.

biophysics↗

bioRxiv2024

Rhodococcus parequi sp. nov., a new species isolated from equine farm soil closely related to the pathogen Rhodococcus equi.

We present the description of the new species, Rhodococcus (Prescottella) parequi, found during phylogenomic investigations of a global collection of strains identified as Rhodococcus (Prescottella) equi. Strain PAM 2766 was isolated from horse-breeding farm soil in Normandy, France, and was indistinguishable from R. equi based on the usual identification tests. Whole-genome phylogenetic analyses located PAM 2766 in the same Rhodococcus sublineage as R. equi, together with Rhodococcus agglutinans, Rhodococcus defluvii, Rhodococcus soli, Rhodococcus subtropicus, Rhodococcus spongiicola and Rhodococcus xishaensis. PAM 2766 is most closely related to, but sufficiently distinct from R. equi DSM 20307T to be considered as a separate species. Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) values are 88.60% and 92.35, respectively, well below the species cutoff. The PAM 2766 draft genome is [~]5.3 Mb in size with 68.98% G+C mol content. PAM 2766T is aerobic, non-motile, and produces smooth creamy to buff-coloured colonies very similar to those of R. equi. It phenotypically differs from the latter by the ability to grow at 5{degrees}C, a strongly positive urease test at 24 h, and specificities in the carbon and nitrogen source utilization profile as determined by phenotype microarray screens. Our data indicate that PAM 2766 belongs to a novel species, for which the name Rhodococcus parequi sp. nov. is proposed. R. parequi was avirulent in macrophage infection assays and is assumed to be non-pathogenic. The type strain is PAM 2766T (=CETC 30995T = NCTC 14987T).

microbiology↗

bioRxiv2024

In vitro biofilms of Clostridioides difficile undomesticated strains: Morphology and properties according to strain diversity

Clostridioides difficile is a Gram-positive, spore-forming, obligate anaerobe and an entero-pathogen representing a One Health problem. C. difficile infections are difficult to treat and recurrences frequent. C. difficile biofilms may play a role in persistence. We studied the biofilms formed in vitro by a collection of C. difficile undomesticated strains (n=27, 20 toxigenic and 7 non-toxigenic) of 11 PCR-ribotypes (WRT) and isolated from equids. After growth in rich BHIS medium supplemented with glucose for 48h, the biomass of biofilms stained by crystal violet, i. e. adhesive to the surface, was measured, while intact biofilms were observed in situ, by confocal laser scanning microscopy, to determine their total biovolume and 3D morphology. All biofilms were relatively similar, even though adhesive, stained biofilms of non-toxigenic strains of ribotype 009 significantly displayed highest biomass. These results suggested that strains of ribotype 009 were able to form a highly cohesive and polystyrene-adhesive biofilm than any other strains. 8 common, representative ribotypes in equids, including 6 clinically important ribotypes in humans, were then selected and one strain chosen for each of them. Their biofilms and planktonic cultures grown under the same conditions were compared for sporulation, toxin production and susceptibility to vancomycin at a highly inhibitory concentration (8 MIC). In both the biofilm and planktonic culture of all selected strains, sporulation was low and toxin production undetectable. The addition of vancomycin at T0=24h followed by a 24 hours exposure barely reduced the viability of neither planktonic cultures nor biofilms of any strains. On the contrary, for all selected strains, addition of vancomycin at T0=6h followed by a 24 hours exposure significantly and more efficiently reduced the viability of planktonic cultures than that of biofilms, which was not decreased at all in the case of strains of ribotype WRT 005, 009 and 035. The biofilms of these selected strains of eight ribotypes, compared to their planktonic cultures, therefore showed a higher tolerance to vancomycin, suggesting that they could play a role in persistence.

microbiology↗

bioRxiv2024

Clostridioides difficile in Equidae necropsied in Normandie, France from 2019 to 2021: common carriage of toxigenic strains and virulence of emerging ribotype 017

Clostridioides difficile is an anaerobic, spore-forming enteropathogen, which is much more extensively studied in humans than animals, despite growing evidence supporting its importance in One Health. We evaluated C. difficile occurrence, diversity, circulation and virulence in French Equidae (n=100) after their necropsy in northwestern France (Normandy), from 2019 to 2021. We systematically recovered all cecal contents and any watery (possibly diarrheal) intestinal contents. We isolated C. difficile strains, determined their toxin gene profile by PCR and established their PCR-ribotype according to the WEBRIBO database. We also performed free toxin detection. Twenty-seven Equidae were positive for C. difficile and twenty had a toxigenic strain, including one animal also colonized by a non-toxigenic strain. Toxigenic isolates belonged to eight common ribotypes: i) 078 and 126 (tcdA+ tcdB+ cdt+ according to multiplex PCR, i.e. able to produce toxin A, toxin B and binary toxin), ii) 005, 012, 020, AI-53 and FR227 (tcdA+ tcdB+), and iii) 017 (tcdB+). Non-toxigenic isolates were of ribotypes 009, 035 and 439. Ribotypes 017 and 009 were predominant (n=5). In two cases, Equidae of the same premises shared the same ribotype, either 020 or 009. Free toxins were detected in four animals: they displayed signs of diarrhea and a C. difficile of ribotype 126 (n=1) or 017 (n=3) as the only detected enteropathogen, suggesting a C. difficile infection (CDI). Three of them had received antibiotics. Two had died from an entero-toxic infection, for which C. difficile ribotype 017 was the only identified cause. French Equidae were found to display common pathogenic C. difficile, and ribotype 017 was highly virulent. These findings are of concern from a One Health perspective. ImportanceC. difficile, a major enteropathogen widely disseminated in the environment, is a One Health issue. Animals are raising concern as human contamination sources. Equidae are in close contact with humans and also develop post-antibiotic and healthcare-associated CDIs. The systematic survey of Equidae necropsied from 2019 to 2021 in the leading region for horse breeding in France, revealed that 20% harbored pathogenic strains. These belonged to clinically important ribotypes, raising the possibility of cross-species, possibly zoonotic transmission. Free toxins, which are rarely tested in animals, were detected in four animals with signs of diarrhea and a toxigenic C. difficile as the only identified enteropathogen, suggesting CDI. In two of them, C. difficile ribotype 017 was the only identified cause of entero-toxic disease and death. French Equidae could play a role in the dissemination of pathogenic C. difficile and notably ribotype 017. They should be surveilled carefully from a One Health perspective.

microbiology↗

bioRxiv2024

Boundary-Sensing Mechanism in Branched Microtubule Networks

The self-organization of cytoskeletal networks in confined geometries requires sensing and responding to mechanical cues at nanometer to micron scales that allow for dynamic adaptation. Here, we show that the branching of microtubules (MTs) via branching MT nucleation combined with dynamic instability constitutes a boundary-sensing mechanism within confined spaces. Using a nanotechnology platform, we observe the self-organization of a branched MT network in a channel featuring a narrow junction and a closed end. Our observations reveal that branching MT nucleation occurs in the post-narrowing region only if that region exceeds a certain length before it terminates at the channels closed end. The length-dependent occurrence of branching MT nucleation arises from the dynamic instability of existing MTs when they interact with the channels closed end, combined with the specific timescale required for new MTs to nucleate at a point distant from the closed end, creating a mechanical feedback. Increasing the concentration of the base branching factor TPX2 accelerates nucleation kinetics and thus tunes the minimum length scale needed for occurrence of branching MT nucleation. As such, this feedback not only allows for adaptation to the local geometry, but also allows for tunable formation of MT networks in narrow (micron and submicron scale) channels. However, while a high concentration of TPX2 increases the kinetic rate of branching MT nucleation, it also stabilizes MTs at the channels closed end leading to MT growth and nucleation in the reversed direction, and thus hinders boundary sensing. After experimental characterization of boundary-sensing feedback, we propose a minimal model and execute numerical simulations. We investigate how this feedback, wherein growing MTs dynamically sense their physical environment and provide nucleation sites for new MTs, sets a length/time scale that steers the architecture of MT networks in confined spaces. This "search- and-branch" mechanism has implications for the formation of MT networks during neuronal morphogenesis, including axonal growth and the formation of highly branched dendritic networks, as well as for plant development and MT-driven guidance in fungi, and engineering nanotechnologies.

bioengineering↗

bioRxiv2024

Mechanism of how the universal module XMAP215 γ-TuRC nucleates microtubules

It has become increasingly evident in recent years that nucleation of microtubules from a diverse set of MTOCs requires both the {gamma}-tubulin ring complex ({gamma}-TuRC) and the microtubule polymerase XMAP215. Despite their essentiality, little is known about how these nucleation factors interact and work together to generate microtubules. Using biochemical domain analysis of XMAP215 and structural approaches, we find that a sixth TOG domain in XMAP215 binds {gamma}-TuRC via {gamma}-tubulin as part of a broader interaction involving the C-terminal region. Moreover, TOG6 is required for XMAP215 to promote nucleation from {gamma}-TuRC to its full extent. Interestingly, we find that XMAP215 also depends strongly on TOG5 for microtubule lattice binding and nucleation. Accordingly, we report a cryo-EM structure of TOG5 bound to the microtubule lattice that reveals promotion of lateral interactions between tubulin dimers. Finally, we find that while XMAP215 constructs effects on nucleation are generally proportional to their effects on polymerization, formation of a direct complex with {gamma}-TuRC allows cooperative nucleation activity. Thus, we propose that XMAP215s C-terminal TOGs 5 and 6 play key roles in promoting nucleation by promoting formation of longitudinal and lateral bonds in {gamma}-TuRC templated nascent microtubules at cellular MTOCs.

biochemistry↗

bioRxiv2023

Conformational states of the microtubule nucleator, the γ-tubulin ring complex

Microtubules (MTs) perform essential functions in the cell, and it is critical that they are made at the correct cellular location and cell cycle stage. This nucleation process is catalyzed by the {gamma}-tubulin ring complex ({gamma}-TuRC), a cone-shaped protein complex composed of over 30 subunits. Despite recent insight into the structure of vertebrate {gamma}-TuRC, which shows that its diameter is wider than that of a MT, and that it exhibits little of the symmetry expected for an ideal MT template, the question of how {gamma}-TuRC achieves MT nucleation remains open. Here, we utilized single particle cryo-EM to identify two conformations of {gamma}-TuRC. The helix composed of 14 {gamma}-tubulins at the top of the {gamma}-TuRC cone undergoes substantial deformation, which is predominantly driven by bending of the hinge between the GRIP1 and GRIP2 domains of the {gamma}-tubulin complex proteins. However, surprisingly, this deformation does not remove the inherent asymmetry of {gamma}-TuRC. To further investigate the role of {gamma}-TuRC conformational change, we used cryo electron-tomography (cryo-ET) to obtain a 3D reconstruction of {gamma}-TuRC bound to a nucleated MT, providing insight into the post-nucleation state. Rigid-body fitting of our cryo-EM structures into this reconstruction suggests that the MT lattice is nucleated by spokes 2 through 14 of the {gamma}-tubulin helix, which entails spokes 13 and 14 becoming more structured than what is observed in apo {gamma}-TuRC. Together, our results allow us to propose a model for conformational changes in {gamma}-TuRC and how these may facilitate MT formation in a cell.

molecular biology↗

bioRxiv2023

HURP facilitates spindle assembly by stabilizing microtubules and working synergistically with TPX2

In large vertebrate spindles, the majority of microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein (HURP) is a microtubule-associated protein that has been implicated in spindle assembly, but its mode of action is yet to be defined. In this study, we show that HURP is necessary for RanGTP-induced branching microtubule nucleation in Xenopus egg extract. Specifically, HURP stabilizes the microtubule lattice to promote microtubule formation from {gamma}- TuRC. This function is shifted to promote branching microtubule nucleation in the presence of TPX2, another branching-promoting factor, as HURPs localization to microtubules is enhanced by TPX2 condensation. Lastly, we provide a structure of HURP on the microtubule lattice, revealing how HURP binding stabilizes the microtubule lattice. We propose a model in which HURP stabilizes microtubules during their formation, and TPX2 preferentially enriches HURP to microtubules to promote branching microtubule nucleation and thus spindle assembly.

molecular biology↗

bioRxiv2023

Building on-chip cytoskeletal circuits via branched microtubule networks

Controllable platforms to engineer robust cytoskeletal scaffolds have the potential to create novel on-chip nanotechnologies. Inspired by axons, we combined the branching microtubule (MT) nucleation pathway with microfabrication to develop "cytoskeletal circuits". This active matter platform allows control over the adaptive self-organization of uniformly polarized MT arrays via geometric features of microstructures designed within a microfluidic confinement. We build and characterize basic elements, including turns and divisions, as well as complex regulatory elements, such as biased division and MT diodes, to construct various MT architectures on a chip. Our platform could be used in diverse applications, ranging from efficient on-chip molecular transport to mechanical nano-actuators. Further, cytoskeletal circuits can serve as a tool to study how the physical environment contributes to MT architecture in living cells. SignificanceMicrotubules have essential functions within the cell, including providing a robust railroad for motor-driven cargo transport. The unique properties of microtubules have stimulated attempts to harness these characteristics for targeted delivery of molecular complexes, novel material design, and developing nanotechnologies with precision comparable to living organisms. However, Previous efforts mainly focused on microtubules with fixed length and layout and no controlled MT generation, setting a limit on designing MT architecture. In this study, we integrated nanofabrication with microtubule branching reactions borrowed directly from the cells toolkit to construct cytoskeletal circuits and generate microtubule architectures from scratch. That is, our system enables control over microtubule growth and autocatalytic nucleation on a microfluidic chip with micro/nanostructures patterned within.

bioengineering↗

bioRxiv2023

Impact of structural sampling, coupling scheme and state of interest on the energy transfer in CP29

The Qy and Bx excitation energy transfer (EET) in the minor light harvesting complex CP29 (LHCII B4.1) antenna complex of Pisum sativum was characterized using a computational approach. We applied Forster theory (FRET) and the transition density cube (TDC) method estimating the Coulombic coupling, based on a combination of classical molecular dynamics and QM/MM calculations. Employing TDC instead of FRET mostly affects the EET between chlorophylls (Chls) and carotenoids (Crts), as expected due to the Crts being spatially more challenging for FRET. Only between Chls, effects are found to be small (about only 0.1 EET efficiency change when introducing TDC instead of FRET). Effects of structural sampling were found to be small, illustrated by a small average standard deviation for the Qy state coupling elements (FRET/TDC: 0.97/0.94 cm-1). Due to the higher flexibility of the Bx state, the corresponding deviations are larger (FRET/TDC between Chl-Chl pairs: 17.58/22.67 cm-1, between Crt-Chl pairs: 62.58/31.63 cm-1). In summary, it was found for the Q band that the coupling between Chls varies only slightly depending on FRET or TDC, resulting in a minute effect on EET acceptor preference. In contrast, the coupling in the B band spectral region is found to be more affected. Here, the S2 (1Bu) states of the spatially challenging Crts may act as acceptors in addition to the Chl B states. Depending on FRET or TDC, several Chls show different Chl-to-Crt couplings. Interestingly, the EET between Chls or Crts in the B band is found to often outcompete the corresponding decay processes. The individual efficiencies for B band EET to Crts vary however strongly with the chosen coupling scheme (e.g., up to 0.29/0.99 FRET/TDC efficiency for the Chl a604/neoxanthin pair). Thus, the choice of coupling scheme must involve a consideration of the state of interest.

biophysics↗

bioRxiv2022

RanGTP regulates the augmin complex

Spindles are composed of microtubules that must nucleate at the right place and time during mitosis. Spindle microtubule nucleation is regulated by the GTPase Ran, which, through importin-{beta}, releases a gradient of spindle assembly factors (SAFs) centered at chromosomes. Branching MT nucleation generates most spindle MTs and requires the augmin complex. In Xenopus laevis, Ran can control branching through the SAF TPX2, TPX2 is non-essential in other organisms. Thus, how Ran regulates branching MT nucleation in the absence of TPX2 is unknown. Here, we use in vitro pulldowns and TIRF microscopy to show that augmin is itself a SAF. Augmin directly interacts with both importins through two nuclear localization sequences on the Haus8 subunit, which overlap the MT binding site. Moreover, Ran controls localization of augmin to MTs in both Xenopus egg extract and in vitro. By uncovering that RanGTP directly regulates augmin, we demonstrate how Ran controls branching MT nucleation and, thereby, spindle assembly and cell division.

biochemistry↗

bioRxiv2022

Integrated Model of the Vertebrate Augmin Complex

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which exponentially amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but understanding how augmin promotes branching has been hindered by a lack of structural information about the complex. Here, we report an integrated model of vertebrate augmin, combining cryo-electron microscopy, advanced protein structural prediction, and the visualization of fused bulky tags via negative stain electron microscopy. This strategy allowed us to identify the location and orientation of each subunit within the structure. Evolutionary analysis of augmins structure reveals that it is highly conserved across diverse eukaryotes, and that augmin contains a previously-unidentified microtubule binding site. Moreover, we identify homology with the kinetochore-localized NDC80 complex. This new model of the augmin complex provides insight towards the mechanism and evolution of branching microtubule nucleation.

molecular biology↗

bioRxiv2022

The conserved centrosomal motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC).

1.To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the {gamma}-tubulin ring complex ({gamma}TuRC). Although {gamma}TuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved {gamma}-tubulin nucleation activator ({gamma}TuNA) domain has been reported to increase the number of microtubules generated by {gamma}TuRCs. Here we utilize Xenopus egg extract and in vitro single molecule imaging assays to show that {gamma}TuNA activates microtubule nucleation in extract and directly activates {gamma}TuRC in vitro. Via mutation analysis, we find that {gamma}TuNA is an obligate dimer. Moreover, efficient dimerization as well as {gamma}TuNAs L70, F75, and L77 residues are required for binding to and activation of {gamma}TuRC. Finally, we find that {gamma}TuNAs activating effect opposes inhibitory regulation by stathmin. In sum, our study illuminates how {gamma}TuRC is controlled in space and time in order to build specific cytoskeletal structures.

biochemistry↗

bioRxiv2022

Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes

Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstituted microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and found that chromosomes alone can form spindles. We visualized microtubule nucleation at chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. The initial branches nucleate near and towards kinetochores, helping explain how kinetochores might be efficiently captured. By depleting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient around them that spatially biases branching nucleation. In the presence of motors, these branched networks are organized into multipolar spindles.

biophysics↗

bioRxiv2022

Widespread alterations in microRNA biogenesis in human Huntington's disease putamen

Altered microRNA (miRNA) expression is a common feature of Huntingtons disease (HD) and could participate in disease onset and progression. However, little is known about the underlying causes of miRNA disruption in HD. We and others have previously shown that mutant Huntingtin (mHTT) binds to Ago2, a central component of miRNA biogenesis, and disrupts mature miRNA levels. In this study, we sought to determine if miRNA maturation per se was compromised in HD. Towards this end, we characterized t major miRNA biogenesis pathway components and miRNA maturation products (pri-miRNA, pre-miRNA, and mature) in human HD (N=41, Vonsattel grades HD2-4) and healthy control (N=25) subjects. Notably, the striatum (putamen) and cortex (BA39) from the same individuals were analyzed in parallel. We show that Ago2, Drosha, and Dicer were strongly downregulated in human HD at early stages of disease. Using a panel of HD-related miRNAs (miR-10b, miR-196b, miR-132, miR-212, miR-127, miR-128), we uncovered various types of maturation defects in HD brain, the most prominent occurring at the pre-miRNA to mature miRNA maturation step. Consistent with earlier findings, we provide evidence that alterations in autophagy could participate in miRNA maturation defects. Notably, most changes occurred in the striatum, which is more prone to HTT aggregation and neurodegeneration. Likewise, we observed no significant alterations in miRNA biogenesis in human HD cortex and blood, strengthening tissue-specific effects. Overall, these data provide important clues into the underlying mechanisms behind miRNA alterations in HD-susceptible tissues. Further investigations are now required to understand the biological, diagnostic, and therapeutic implications of miRNA/RNAi biogenesis defects in HD and related neurodegenerative disorders.

neuroscience↗