Biology subjects

McHugh, T.

Publications and source records attributed to McHugh, T..

17 recordsLinked to original sources

bioRxiv2026

Cerebellar induced VTA plasticity underlies chronic stress-induced depression-like behaviors

Synaptic regulation is a key mechanism underlying neural circuit homeostasis and plasticity. We previously showed that projections from the deep cerebellar nuclei (DCN) to the ventral tegmental area (VTAp-DCN neurons) contribute to depression-like behaviors following chronic restraint stress (RS), but the mechanisms by which these outputs induce downstream long-term functional changes remain unknown. Here, we show that chronic RS induces an activity-dependent reduction in vesicular glutamate transporter 2 (VGLUT2) expression in VTAp-DCN axons, resulting in decreased miniature excitatory postsynaptic current frequency in dorsolateral VTA dopaminergic neurons and reduced phasic dopamine release in the nucleus accumbens. Notably, VGLUT2 reduction temporally coincides with the emergence of depression-like behaviors. Consistent with this, targeted VGLUT2 overexpression in VTAp-DCN neurons rescues synaptic transmission and dopamine signaling and prevents depression-like behaviors following chronic RS. These findings reveal a stress-induced plasticity mechanism in the cerebellar-VTA circuit linking chronic stress to depression-like behaviors.

neuroscience↗

bioRxiv2026

RNA degradation by DIS3 is a necessary step in the resolution of backtracked transcription complexes

The RNA exosome is known to participate in transcription, but the contribution of its ribonuclease activities to this process remains unclear. Here we investigated the role of DIS3, one of the exosome ribonucleases, in transcription by RNA polymerase II (RNAPII). Rapid depletion of DIS3 reduced RNA synthesis and induced RNAPII elongation defects that were exacerbated by UV irradiation, a treatment that generates transcription-blocking DNA lesions and promotes RNAPII backtracking. Notably, DIS3 itself was redistributed following UV irradiation in a manner that closely paralleled RNAPII dynamics, which suggested that DIS3 acts in concert with the transcription machinery. We also investigated whether RNA degradation by DIS3 was required for transcription elongation and found that the 3-5 exoribonucleolytic activity of DIS3, but not its endonucleolytic activity, is essential for efficient transcription elongation. More specifically, DIS3 degrades the 3 ends of backtracked RNA, as shown by sequencing of RNA fragments released by TFIIS-induced transcript cleavage in vitro. This DIS3-dependent degradation of backtracked RNA is critical for resolving stalled RNAPII complexes and enabling productive transcription elongation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/703186v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@c080dorg.highwire.dtl.DTLVardef@1e4b375org.highwire.dtl.DTLVardef@1c1f575org.highwire.dtl.DTLVardef@d9d640_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDIS3 depletion leads to severely reduced rates of RNA synthesis in human cells. C_LIO_LITranscription-blocking lesions reveal a critical role for DIS3 in RNA polymerase II elongation. C_LIO_LIThe 3 to 5 exoribonuclease activity of DIS3 is necessary for transcription elongation. C_LIO_LIDIS3-mediated degradation of backtracked RNA is required for the resolution of stalled transcription complexes. C_LI

molecular biology↗

bioRxiv2025

Investigation of Neurons and Microglia from APOE3 Induced Pluripotent Stem Cells using Data-Independent Acquisitions on a ZenoTOF 7600

Induced pluripotent stem cell (iPSC)-derived neurons and microglia are valuable human models for studying neurodegenerative diseases. Specifically, the apolipoprotein E4 (APOE4) gene is a major genetic risk factor for late-onset Alzheimers disease. Apolipoprotein E (APOE) alleles E2, E3 and E4 can be beneficial, neutral, or increase the risk of Alzheimers disease (AD). Here, we developed a proteomic workflow using data-independent acquisitions to provide a quantitative mass spectrometric proteome analysis, and proteomic screening assays for brain-specific cell types derived from iPSC. Protein groups were quantified in APOE3 neurons and microglia, respectively, with [~]80% overlap. Cell type-specific markers and enriched pathways reflected the specialized functions of each cell type, such as synaptic signaling in neurons and immune and inflammatory responses in microglia. The neuron-specific markers included proteins APP, CALB1, CALB2, DLGs, GAP43, NEFL, MAPs; while microglial markers included proteins AIF1, CDs, MMP9, and ITGAM. Ultimately, the combination of robust iPSC differentiation and sensitive proteomic screening assays described here provides a valuable platform for probing the cellular mechanisms underlying neurological disorders. SignificanceThe quantification of dysregulated proteins and pathways in patient-derived neurons and microglia can provide insights into disease etiology and progression. More broadly, this DIA approach enables deep proteome profiling of unique iPSC-derived cell models, increasing their utility for investigating disease biology and therapeutic development. We focused on iPSC models from two important cell types of the brain, excitatory neurons and microglia. We integrated the proteomes of these two cell types. These tools provide robust biological and mass spectrometric screening tools for future therapeutic interventions using disease-relevant human brain cell types or brain organoid models. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/686128v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@d7dorg.highwire.dtl.DTLVardef@17594d3org.highwire.dtl.DTLVardef@7dd894org.highwire.dtl.DTLVardef@1606db_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPresentation of a proteomic workflow using data-independent acquisitions to monitor and screen proteomes of iPSC-derived brain cell types. C_LIO_LIQuick MS Assays to determine protein profiles of iPSC-derived neurons and microglia. C_LIO_LICharacterization of different cell type proteomes from APOE3 iPSCs. C_LIO_LIRevealing of neuron-specific markers and microglia-specific markers by mass spectrometry. C_LIO_LIStep-by-step instructions for the set-up of the DIA-MS assays C_LI

neuroscience↗

bioRxiv2025

Tetramerisation governs SALL transcription factor function in development and disease

Spalt-like (SALL) proteins are C2H2 zinc-finger transcription factors important for embryogenesis, with mutations in SALL1 and SALL4 causing rare congenital disorders Townes-Brocks and Okihiro syndromes, respectively. While SALL proteins are known to associate with one another, the biological significance of the resulting complexes is unknown. Here we define a conserved glutamine-rich region that mediates SALL1/4 homo- and heterotetramerisation and find that complex formation is indispensable for DNA binding. Modelling a patient mutation that abolishes SALL4 multimerisation led to gene misregulation and, in mice, embryonic lethality, therefore phenocopying a complete Sall4 knockout. Furthermore, a common disease-causing SALL1 truncation, which retains multimerisation but lacks DNA-binding domains, sequesters SALL4 into heterotetramers that are defective in DNA binding, thereby providing a mechanistic explanation for the dominant-negative effects of many Townes-Brocks mutations. Together, our findings establish tetramerisation as a prerequisite for SALL function, linking complex formation to developmental gene regulation and human disease.

molecular biology↗

bioRxiv2024

PLK1-Mediated Phosphorylation Cascade Activates the Mis18 Complex to Ensure Centromere Inheritance

Accurate chromosome segregation requires the attachment of spindle microtubules to centromeres, which are epigenetically defined by the enrichment of CENP-A nucleosomes. During DNA replication, existing CENP-A nucleosomes undergo dilution as they get redistributed among the two DNA strands. To preserve centromere identity, CENP-A levels must be restored in a cell-cycle controlled manner orchestrated by the Mis18 complex. Here we provide a comprehensive mechanistic basis for PLK1-mediated licensing of CENP-A loading. We demonstrate that PLK1 interacts with Mis18 and Mis18BP1 subunits of the Mis18 complex by recognising self-primed phosphorylations of Mis18 (S54) and Mis18BP1 (T78 and S93) through its Polo-box binding domain. Disrupting these PLK1 phosphorylations perturbed the centromere recruitment of HJURP and new CENP-A loading. Biochemical and functional analyses show that phosphorylation of Mis18 and subsequent PLK1 binding is required to activate the Mis18/{beta} complex for robust Mis18/{beta}-HJURP interaction. Thus, our study reveals key molecular events underpinning the licensing role of PLK1 in ensuring accurate centromere inheritance. One-Sentence SummaryPLK1 phosphorylation cascade licenses CENP-A loading by facilitating HJURP centromere recruitment via Mis18/{beta} activation.

cell biology↗

bioRxiv2023

Gamma sensory stimulation and effects on the brain

Findings by the Tsai lab and others 1-8 demonstrate that 40 Hz frequency sensory stimulation induces electrophysiological responses and attenuates pathology in mouse models of Alzheimers disease (AD). A recent study in Nature Neurosciene 9 concluded that the stimulation does not affect endogenous gamma oscillations or amyloid burden. We welcome research investigating 40 Hz sensory stimulation, and the article by Soula et al enhances our understanding of the brains electrophysiological response to 40Hz. However, we respectfully suggest that the data in Soula et al are consistent with a neuronal response to 40 Hz, which we further support with new data in humans. Moreover we contend the non-significant effects on amyloid are due to technical limitations of the study.

neuroscience↗

bioRxiv2023

Hawkes process modeling quantifies complicated firing behaviors of cortical neurons during sleep and wakefulness

Despite the importance of sleep to the cerebral cortex, how much sleep changes cortical neuronal firing remains unclear due to complicated firing behaviors. Here we quantified firing of cortical neurons using Hawkes process modeling that can model sequential random events exhibiting temporal clusters. "Intensity" is a parameter of Hawkes process that defines the probability of an event occurring. We defined the appearance of repetitive firing as the firing intensity corresponding to "intensity" in Hawkes process. Firing patterns were quantified by the magnitude of firing intensity, the time constant of firing intensity, and the background firing intensity. The higher the magnitude of firing intensity, the higher the likelihood that the spike will continue. The larger the time constant of firing intensity, the longer the repetitive firing lasts. The higher the background firing intensity, the more likely neurons fire randomly. The magnitude of firing intensity was inversely proportional to the time constant of firing intensity, and non-REM sleep increased the magnitude of firing intensity and decreased the time constant of firing intensity. The background firing intensity was not affected by the sleep/wake state. Our findings suggest that the cortex is organized such that neurons with a higher probability of repetitive firing have shorter repetitive firing periods. In addition, our results suggest that repetitive firing is ordered to become high frequency and short term during non-REM sleep, while unregulated components of firing are independent of the sleep/wake state in the cortex. Hawkes process modeling of firing will reveal novel properties of the brain.

neuroscience↗

bioRxiv2023

MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation

Correlative evidence has suggested that DNA methylation promotes the formation of transcriptionally silent heterochromatin. Accordingly, the methyl-CpG binding domain protein MeCP2 is often portrayed as a constituent of heterochromatin. This interpretation has been reinforced by the use of mouse cells as an experimental system for studying the mammalian epigenome, as heterochromatin, DNA methylation and MeCP2 colocalise in prominent foci. The findings presented here revise this view. We show that focal localisation of MeCP2 in mice is independent of heterochromatin, as DNA methylation-dependent MeCP2 foci persist even when the signature heterochromatin histone mark H3K9me3 is absent and heterochromatin protein HP1 is diffuse. Contrary to the proposal that MeCP2 forms condensates at mouse heterochromatic foci via liquid-liquid phase transition, the short methyl-CpG binding domain, which lacks the disordered domains thought to be required for condensation, is sufficient to target foci in mouse cells. Importantly, we find that the formation of MeCP2 foci in mice is highly atypical, as they are indetectable in 14 out of 16 other mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which can interbreed with Mus musculus but lacks its highly methylated pericentric satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form nuclear condensates and its localisation is independent of heterochromatin formation. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns and is typically euchromatic.

cell biology↗

bioRxiv2023

Phosphorylation controls spatial and temporal activities of motor-PRC1 complexes to complete mitosis

During mitosis, spindle architecture alters as chromosomes segregate to daughter cells. The microtubule crosslinker Protein Required for Cytokinesis 1 (PRC1) is essential for spindle stability, chromosome segregation and completion of cytokinesis, but how it recruits motors to the central spindle to coordinate the segregation of chromosomes is unknown. Here, we combine structural and cell biology approaches to show that the human CENP-E motor, which is essential for chromosome capture and alignment by microtubules, binds to PRC1 through a conserved hydrophobic motif. This binding mechanism is also used by Kinesin-4 Kif4A:PRC1. Using in vitro reconstitution, we demonstrate that CENP-E slides antiparallel PRC1-crosslinked microtubules. We find that the regulation of CENP-E -PRC1 interaction is spatially and temporally coupled with relocalization to overlapping microtubules in anaphase. Finally, we demonstrate that the PRC1:microtubule motor interaction is essential in anaphase to control chromosome partitioning, retain central spindle integrity and ensure cytokinesis. Taken together our findings reveal the molecular basis for the cell cycle regulation of motor-PRC1 complexes to couple chromosome segregation and cytokinesis.

biochemistry↗

bioRxiv2023

Dentate neurogenesis mediates cognitive flexibility via modulation of the hippocampal longitudinal-axis network

Adult neurogenesis endows the hippocampus with unparalleled neural plasticity, essential for intricate cognitive functions. However, the manner in which sparse newborn neurons (NBNs) modulate neural activities and subsequently shape behavior remains enigmatic. Utilizing a newly engineered NBN-Tetanus Toxin mouse model, we non-invasively silenced NBNs and confirmed their crucial role in cognitive flexibility, as demonstrated through reversal learning in the Morris water maze and the Go/Nogo task in operant learning. Pairing task-based functional MRI (tb-fMRI) with operant learning revealed a dorsal hippocampal hyperactivation during Nogo task, implying that hippocampal hyperexcitability might underlie the observed behavioral deficits. Notably, resting-state fMRI (rs-fMRI) revealed enhanced functional connectivity between the dorsal and ventral dentate gyrus following NBN silencing. Further exploration of PV+ interneurons and mossy cells activities highlighted NBNs integral part in preserving the excitation/inhibition balance within the hippocampus. Our findings emphasize how the neural plasticity driven by NBNs extensively modulates the hippocampus, sculpting cognitive flexibility.

neuroscience↗

bioRxiv2021

Centromere identity is dependent on nuclear spatial organization

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-ACnp1 incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-ACnp1 overexpression bypasses heterochromatin dependency, suggesting heterochromatin ensures exposure to conditions or locations permissive for CENP-ACnp1 assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-ACnp1 incorporation. We demonstrate that heterochromatin-independent de novo CENP-ACnp1 chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-ACnp1 assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation because target sequences are exposed to high levels of CENP-ACnp1 and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.

cell biology↗

bioRxiv2021

Molecular Basis for CPC-Sgo1 Interaction: Implications for Centromere Localisation and Function of the CPC

The Chromosomal Passenger Complex (CPC; consisting of Borealin, Survivin, INCENP and Aurora B kinase) and Shugoshin 1 (Sgo1) are key regulators of chromosome bi-orientation, a process essential for error-free chromosome segregation. Their functions rely on their ability to associate with centromeres. Two histone phosphorylations, histone H3 Thr3 (H3T3ph; directly recognised by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognised via Sgo1), together with CPCs intrinsic ability to bind nucleosome, facilitate CPC centromere recruitment. The molecular basis for CPC-Sgo1 binding and how their direct interaction influences CPC centromere localisation and function are lacking. Here, using an integrative structure-function approach, we show that the histone H3-like Sgo1 N-terminal tail interacts with Survivin acting as a hot-spot for CPC-Sgo1 assembly, while downstream Sgo1 residues, mainly with Borealin contributes for high affinity interaction. Disruption of the Sgo1 N-terminal tail-Survivin interaction abolished CPC-Sgo1 assembly in vitro and perturbed centromere localisation and function of CPC. Our findings provide evidence that CPC binding to Sgo1 and histone H3 N-terminal tail are mutually exclusive, suggesting that these interactions will likely take place in a spatially/temporally restricted manner and provide a rationale for the Sgo1-mediated kinetochore proximal centromere pool of CPC.

cell biology↗

bioRxiv2020

K+ efflux through postsynaptic NMDA receptors suppresses local astrocytic glutamate uptake

Glutamatergic transmission prompts K+ efflux through postsynaptic NMDA receptors. K+ depolarizes local presynaptic terminals, boosting glutamate release, but whether it also affecting glutamate uptake remains unknown. Here, we find that the pharmacological blockade, or conditional knockout, of NMDA receptors suppresses the progressive use- dependent increase in the amplitude and decay time of the astrocytic glutamate transporter current (IGluT), whereas blocking the astrocytic inward-rectifying K+ channels prevents the decay time increase only. Glutamate spot-uncaging reveals that local astrocyte depolarization, rather than extracellular K+ rises on their own, reduces the amplitude and prolong the decay of IGluT. Biophysical simulations confirm that local transient elevations of extracellular K+ can inhibit local glutamate uptake in fine astrocytic processes. Optical glutamate sensor imaging and a two-pathway test relate postsynaptic K+ efflux to enhanced extrasynaptic glutamate signaling. Thus, postsynaptic K+ efflux facilitates presynaptic release while reducing local glutamate uptake.

neuroscience↗

bioRxiv2020

Emergent properties of a mitotic Kif18b-MCAK-EB network

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. Prior work has identified key molecular players in this process, including the kinesin-18 Kif18b and the kinesin-13 Kif2C/MCAK, which both promote microtubule depolymerization. MCAK acts as a potent microtubule depolymerase diffusing short distances on microtubules, while Kif18b is a mitotic processive motor with weak depolymerase activity. However the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 act in an integrated manner to potently promote microtubule depolymerization. We find Kif18b acts as a microtubule plus end delivery factor for its cargo MCAK, and that Kif18b also promotes EB accumulation to plus ends independently of lattice nucleotide state. Together, our work defines the mechanistic basis for a cooperative Kif18b-EB-MCAK network with emergent properties, that acts to efficiently shorten microtubules in mitosis.

biochemistry↗

bioRxiv2018

Microtubule end tethering of a processive Kinesin-8 motor Kif18b is required for spindle positioning

Mitotic spindle positioning specifies the plane of cell division during anaphase. Spindle orientation and positioning is therefore critical to ensure symmetric division in mitosis and asymmetric division during development. The control of astral microtubule length plays an essential role in positioning the spindle. Here we show using gene knockout that the Kinesin-8 Kif18b controls microtubule length to center the mitotic spindle at metaphase. Using an integrated approach, we reveal that Kif18b is a highly processive plus end-directed motor that uses a C-terminal non-motor microtubule-binding region to accumulate at growing microtubule plus ends. This region is regulated by phosphorylation to spatially control Kif18b accumulation at plus ends and is essential for Kif18b-dependent spindle positioning and regulation of microtubule length. Finally, we demonstrate that Kif18b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work reveals that Kif18b utilizes its motile properties to reach microtubule ends where it regulates astral microtubule length to ensure spindle centering.

cell biology↗

bioRxiv2017

Single molecule mechanics reveal Kif15 as an active molecular ratchet with acute strain sensitivity

Human Kif15 is a tetrameric kinesin-12 that contributes critically to bipolar spindle assembly in eukaryotes. Here we examine its single molecule mechanics. Under hindering loads, Kif15 steps predominantly towards microtubule plus ends, with its forestep:backstep ratio decreasing exponentially with load and stall occurring at ~6pN. Between steps, Kif15 binds stably, usually via a single head domain. By complete contrast, under assisting loads, Kif15 detaches rapidly, even in AMPPNP. Furthermore, Kif15 can autoinhibit, via an interaction requiring its C-terminus. Autoinhibited Kif15 binds microtubules nucleotide-independently, resists both hindering and assisting loads, and is further stabilized by Tpx2, which interacts with the Kif15 C-terminus. Our data reveal the mechanics of Kif15 to be extraordinarily sensitive to loading direction. When unloaded, it walks rapidly; when pulled forwards it slips and when pulled backwards it grips. We discuss the implications of this unique mechanical behaviour for the roles of Kif15 in spindle function.

cell biology↗

bioRxiv2017

Polymersomes Targeting Mononuclear Phagocytes

Mononuclear phagocytes such as monocytes, tissue-specific macrophages and dendritic cells are primary actors in both innate and adaptive immunity, as well as tissue homoeostasis. They have key roles in a range of physiological and pathological processes, so any strategy targeting these cells will have wide-ranging impact. These phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favouring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis, which is the most pandemic and one of the deadliest disease with one third of the worlds population infected, and 1.8 million deaths worldwide in 2015. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to both circulating monocytes and resident macrophages, using pH sensitive nanoscopic polymersomes made of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-co-poly(2-(di-isopropylamino)ethyl methacrylate) (PMPC-PDPA). Polymersome selectivity to mononuclear phagocytes is demonstrated and ascribed to the polymerised phosphorylcholine motifs affinity toward scavenger receptors. Finally, we demonstrate the successful exploitation of this targeting for the effective eradication of intracellular bacteria that cause tuberculosis Mycobacterium tuberculosis as well as other intracellular parasites including the Mycobacterium bovis, Mycobacterium marinum and the most common bacteria associated with antibiotic resistance, the Staphylococcus aureus.

immunology↗