Biology subjects

Henley, J.

Publications and source records attributed to Henley, J..

14 recordsLinked to original sources

bioRxiv2026

MFF SUMOylation regulates neuronal mitochondrial morphology and facilitates AMPK-induced mitochondrial fragmentation

Mitochondrial dysfunction and fragmentation in neurons are characteristics of many neurodegenerative conditions. AMP-activated protein kinase (AMPK) is a cellular energy sensor which is hyperactivated in Alzheimers disease. Under stress conditions, AMPK-mediates phosphorylation of the outer mitochondrial membrane protein mitochondrial fission factor (MFF), leading to SUMOylation at Lys151. MFF SUMOylation promotes fission and subsequent mitochondrial fragmentation. Here, using primary neuronal culture, we investigate the role of MFF SUMOylation in mitochondrial morphology and function. We show that MFF SUMOylation is required to maintain neuronal mitochondrial length under basal conditions in axons and dendrites. Using the AMPK activator (AICAR) to induce mitochondrial fission, show that preventing MFF SUMOylation reduces AICAR-induced fragmentation of dendritic mitochondria. These findings reconcile observations of AMPK hyperactivation and mitochondrial fragmentation by revealing the AMPK-MFF-SUMO pathway to be crucial in neuronal mitochondrial fission.

neuroscience↗

bioRxiv2026

Disease-associated mutations in GluK2 disrupt compartment-specific regulatory mechanisms controlling kainate receptor membrane trafficking.

Missense mutations in the Grik2 gene that encodes the kainate receptor subunit GluK2 are associated with neurodevelopmental disorders, but how individual mutations perturb receptor regulation remains unclear. To investigate how disease-associated GluK2 mutations affect receptor trafficking and signalling we used lentiviral approaches to knockdown and replace endogenous GluK2 with tagged wild-type or mutant GluK2 constructs in primary rat neuronal cultures. We show that two disease-associated GluK2 mutations disrupt receptor trafficking through distinct molecular mechanisms. The neurodevelopmental disorder-associated A657T mutation impairs receptor folding and assembly, resulting in endoplasmic reticulum retention, proteasome-dependent degradation, and severely reduced surface expression. In contrast, the C-terminal mutation M867I, identified in patients with autism, markedly reduces PKC-dependent phosphorylation at S868. This does not impede maturation but uncouples GluK2 surface redistribution from activity-dependent signalling. Thus, pathogenic GluK2 variants selectively disrupt either receptor biogenesis or post-translational regulatory integration. Functionally, these mutations differentially alter downstream Ca{superscript 2} signalling responses, linking molecular defects to altered receptor output. These findings establish the GluK2 C-terminus as a critical control node for synaptic receptor homeostasis and illustrate how discrete mutations can uncouple distinct layers of membrane protein regulation.

neuroscience↗

bioRxiv2025

A cross-environment comparison of nontuberculous mycobacterial diversity

Nontuberculous mycobacteria (NTM) are a group of environmental bacteria that encompass nearly 200 described species, some of which can cause chronic pulmonary infection in humans. What makes these infections unique is that they are environmentally acquired, yet there remains a limited understanding of how different environments contribute to potential pathogen exposure. Here, we use new and existing marker gene datasets to compare the amounts and types of NTM across three environments known to harbor mycobacteria, surface waters, soil, and household plumbing biofilms, to better understand potential pathogen occurrence in each environment. We used 16S rRNA gene sequencing, in tandem with mycobacterial-specific marker gene sequencing to characterize variation in the relative abundances of the genus Mycobacterium and specific mycobacterial taxa across the three environments, with a focus on a clinically significant NTM. We found that household plumbing biofilms contained both the highest relative abundance of the genus Mycobacterium (on average, 13.7% of bacteria were members of the genus), as well as the highest occurrence of clinically relevant species detected (Mycobacterium avium, Mycobacterium abscessus), compared to surface waters and soil. Although mycobacteria are ubiquitous across many different environments, mycobacterial diversity is highly variable between environments with clinically relevant species largely restricted to household plumbing biofilms, information that is critical for understanding the ecology and epidemiology of NTM disease. ImportanceNontuberculous mycobacteria, or NTM, are a diverse group of bacteria within the genus Mycobacterium that are common in many environments. While most members of the genus pose little threat to human health, a handful of species, namely the Mycobacterium avium complex, M. abscessus, and M. mucogenicum, can cause severe and prolonged lung infections. These environmentally acquired infections are on the rise in the United States and around the world, yet we still dont have a good understanding of which environment types pose the greatest risk of infection to susceptible populations. Our study used cultivation independent approaches to identify the specific NTM taxa found in over 1,000 samples from three potentially important environmental reservoirs - surface waters, soils, and household plumbing systems, to determine which of these environments are most likely to harbor NTM of clinical significance. Our results highlight the high degree of variability in the types of NTM taxa detected in different environments (including extensive novel diversity within the genus) and show that household plumbing biofilms are likely the most important reservoir and subsequent route of transmission for clinically significant NTM.

microbiology↗

bioRxiv2023

SUMOylation of MFF is required for stress-induced mitochondrial fission

Mitochondrial fission regulates mitochondrial morphology, function, mitophagy and apoptosis. Fission is mediated by the GTPase dynamin related protein-1 (DRP1) and its recruitment to the outer mitochondrial membrane by DRP1 receptors. Mitochondrial fission factor (MFF) is considered the major pro-fission receptor, whereas the mitochondrial dynamics proteins (MiD49/51) sequester inactive DRP1 and facilitate the MFF-DRP1 interaction by forming a trimeric DRP1-MiD-MFF complex. Here, we identify MFF as a target of poly-SUMOylation at a single residue (Lys151). Following bioenergetic stress, AMPK phosphorylates MFF to promote its SUMOylation, a critical step in stress-induced fragmentation. MFF SUMOylation is not required for DRP1 recruitment from the cytosol but causes a rearrangement of the trimeric fission complex to displace MiD proteins. This alleviates MiD inhibition of DRP1 to facilitate formation of a fission-competent complex. Thus, our data demonstrate that MFF SUMOylation fine-tunes the ratio of MiD to DRP1 for the dynamic control of stress-induced mitochondrial fragmentation.

cell biology↗

bioRxiv2022

Intercellular Mitochondrial Transfer as a Rescue Mechanism in Response to Protein Import Failure

Mitochondria are the powerhouses of eukaryotic cells, composed mostly of nuclear-encoded proteins imported from the cytosol. Thus, problems with the import machinery will disrupt their regenerative capacity and the cells energy (ATP) supplies-particularly troublesome for energy demanding cells like neurons and myocytes. Unsurprisingly then, dysfunctional import is implicated in disease. This study explores the consequences of import failure in mammalian cells; wherein, blocking the import machinery has profound effects on mitochondrial ultra-structure and dynamics, but, surprisingly, does not impact import. The explanation is an astonishing response involving intercellular mitochondrial transfer via tunnelling nanotubes: for the import of healthy mitochondria and jettisoning of those with jammed import sites. These observations support the existence of a widespread mechanism for the rescue of mitochondrial protein import failure. One-Sentence SummaryA mitochondrial import rescue mechanism involving intercellular mitochondrial transport through tunneling nanotubes (TNTs).

cell biology↗

bioRxiv2022

Perturbation of the mitochondrial import machinery by disease prone Tau affects organelle morphology and reduces neuronal complexity

Protein import into mitochondria is an intricate and highly conserved process essential for organellar biogenesis, and maintenance of its structure and function. Defects in the import apparatus impact the assembly of the respiratory chain and ATP synthase complexes required for oxidative phosphorylation, compromising the supply of ATP to the cytosol. The consequences of reduced bioenergetic function are particularly severe for cells with high energetic demands, such as neurons. However, relatively little is known about how defective import contributes to neurodegeneration, or how aggregation prone toxic proteins, characteristic of neurodegenerative disease, impact mitochondrial import efficiency. Here, we used HeLa cells to investigate how expressing Tau, or a disease-causing variant, affects mitochondrial import activity, morphology, and function. We found that a variant associated with frontotemporal dementia (TauP301L), but not the native version, colocalises with mitochondria, associating with TOM40-the protein-channel component of the outer membrane import complex. Interestingly, TauP301L production had no discernible effect on overall mitochondrial import function, despite associating with TOM40 and altering mitochondrial morphology. This raised suspicions of a rescue mechanism manifested by the appearance of microtubule and actin containing tunnelling nanotubes (TNTs), used to recruit healthy mitochondria from neighbouring cells and/ or dispose of mitochondria containing aggregated Tau. Furthermore, in primary neuronal cultures TauP301L induces morphological changes that resemble a neurodegeneration-like phenotype-also mirrored in cells where the import sites are blocked artificially. These results reveal an intriguing link between the production of aggregation prone protein variants, such as TauP301L and others, with the mitochondrial protein import machinery relevant to neurodegenerative disease.

cell biology↗

bioRxiv2022

GluK2 Q/R editing regulates kainate receptor signalling and synaptic AMPA receptor expression and function.

Q/R editing of the kainate receptor (KAR) subunit GluK2 radically alters properties of recombinant KARs, but the effects in vivo remain largely unexplored. We compared GluK2 editing-deficient mice that express [~]95% unedited GluK2(Q) to wild-type counterparts that express [~]85% edited GluK2(R). At mossy fibre-CA3 (MF-CA3) synapses GluK2(Q) mice displayed enhanced postsynaptic KAR function and increased KAR-mediated presynaptic facilitation, demonstrating heightened ionotropic function. Conversely, there was reduced metabotropic KAR function, assessed by KAR-mediated afterhyperpolarization currents, in GluK2(Q) mice. GluK2(Q) mice had fewer GluA1- and GluA3-containing AMPA receptors (AMPARs) and reduced postsynaptic AMPAR currents at both MF-CA3 and CA1-Schaffer collateral synapses. Moreover, long-term potentiation of AMPAR-mediated transmission at CA1-Schaffer collateral synapses was reduced in GluK2(Q) mice. These findings suggest that GluK2 Q/R editing influences ionotropic/metabotropic balance of KAR signalling to regulate synaptic expression of AMPARs and plasticity.

neuroscience↗

bioRxiv2022

The NanoLuc Assay System for Accurate Real-Time Monitoring of Mitochondrial Protein Import within Intact Mammalian Cells

Only a few proteins (13 in humans) are encoded by the mammalian mitochondrial genome. Therefore, the other mitochondrial resident proteins (>1000) must be recruited via specialised import pathways. Protein import is critical for mitochondrial biogenesis and bioenergetic function and health; loss of function has been implicated with a wide range of pathologies. Despite this, our understanding of the kinetic and dynamics of import is somewhat limited, particularly within mammalian cells. Here, we report an adaptation of an assay system, established previously to monitor mitochondrial import into isolated yeast mitochondria, to quantitatively monitor mitochondrial import inside mammalian cells. The reporting is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a recombinant precursor protein destined for import. Following import successively through the TOM complex of the outer membrane and the TIM23 complex of the inner membrane, the complementary fragments combine to form an active luciferase. The resultant luminescent signal provides a sensitive, accurate, free of noise and continuous measure of protein import, enabling mathematical model fitting to identify and understand the steps that make up import. This advance allows detailed mechanistic examination of the transport process in live cells. In addition, the assay will enable characterisation of the protein import when the machinery is challenged; for example, in situations associated with disease. Moreover, the assay is compatible with high throughput for large data set collection and kinetic modelling, as well as for drug screening and characterisation. Our set-up also has the potential to be adapted for the analysis of alternative transport systems and different cell types, and even for multicellular model organisms.

biochemistry↗

bioRxiv2022

GABA-receptors are a new druggable target for limiting disease severity, lung viral load, and death in SARS-CoV-2 infected mice

GABA-receptors (GABA-Rs) are well-known neurotransmitter receptors in the central nervous system. GABA-Rs are also expressed by immune cells and lung epithelial cells and GABA-R agonists/potentiators reduce inflammatory immune cell activities and limit acute lung injuries. Notably, plasma GABA levels are reduced in hospitalized COVID-19 patients. Hence, GABA-R agonists may have therapeutic potential for treating COVID-19. Here, we show that oral GABA treatment initiated just after SARS-CoV-2 infection, or 2 days later near the peak of lung viral load, reduced disease severity, lung coefficient index, and death rates in K18-hACE2 mice. GABA-treated mice had a reduced viral load in their lungs and displayed shifts in their serum cytokine and chemokine levels that are associated with better outcomes in COVID-19 patients. Thus, GABA-R activation had multiple beneficial effects in this mouse model which are also desirable for the treatment of COVID-19. A number of GABA-R agonists are safe for human use and can be readily tested in clinical trials with COVID-19 patients. We also discuss their potential for limiting COVID-19-associated neuroinflammation.

microbiology↗

bioRxiv2021

Neutrophil-epithelial interactions augment infectivity and pro-inflammatory responses to SARS-CoV-2 infection

In response to viral infection, neutrophils release inflammatory mediators as part of the innate immune response, contributing to pathogen clearance through virus internalization and killing. Pre-existing co- morbidities correlating to incidence of severe COVID-19 are associated with chronic airway neutrophilia. Furthermore, examination of COVID-19 explanted lung tissue revealed a series of epithelial pathologies associated with the infiltration and activation of neutrophils, indicating neutrophil activity in response to SARS- CoV-2 infection. To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory responses to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. SARS-CoV-2 infection of the airway epithelium alone does not result in a notable pro-inflammatory response from the epithelium. The addition of neutrophils induces the release of proinflammatory cytokines and stimulates a significantly augmented pro-inflammatory response subsequent SARS-CoV-2 infection. The resulting inflammatory response is polarized with differential release from the apical and basolateral side of the epithelium. Additionally, the integrity of the epithelial barrier is impaired with notable epithelial damage and infection of basal stem cells. This study reveals a key role for neutrophil-epithelial interactions in determining inflammation and infectivity in response to SARS-CoV-2 infection.

cell biology↗

bioRxiv2019

Protective role of the deSUMOylating enzyme SENP3 in myocardial ischaemia-reperfusion injury

Interruption of blood supply to the heart is a leading cause of death and disability. However, the molecular events that occur during heart ischaemia, and how these changes prime consequent cell death upon reperfusion, are poorly understood. Protein SUMOylation is a post-translational modification that has been strongly implicated in the protection of cells against a variety of stressors, including ischaemia-reperfusion. In particular, the SUMO2/3-specific protease SENP3 has emerged as an important determinant of cell survival after ischaemic infarct. Here, we used the Langendorff perfusion model to examine changes in the levels and localisation of SUMOylated target proteins and SENP3 in whole heart. We observed a 50% loss of SENP3 from the cytosolic fraction of hearts after preconditioning, a 90% loss after ischaemia and an 80% loss after ischaemia-reperfusion. To examine these effects further, we performed ischaemia and ischaemia-reperfusion experiments in the cardiomyocyte H9C2 cell line. Similar to whole hearts, ischaemia induced a decrease in cytosolic SENP3. Furthermore, shRNA-mediated knockdown of SENP3 led to an increase in the rate of cell death upon reperfusion. Together, our results indicate that cardiac ischaemia dramatically alter levels of SENP3 and suggest that this may a mechanism to promote cell survival after ischaemia-reperfusion in heart.

cell biology↗

bioRxiv2019

Parkin-mediated ubiquitination contributes to the constitutive turnover of mitochondrial fission factor (Mff)

The mitochondrial outer membrane protein Mitochondrial Fission Factor (Mff) plays a key role in both physiological and pathological fission. It is well established that in stressed or functionally impaired mitochondria the PINK1 recruits the ubiquitin ligase Parkin which ubiquitinates Mff to facilitate the removal of defective mitochondria and maintain the integrity mitochondrial network. Here we show that, in addition to this clearance pathway, Parkin also ubiquitinates Mff in a PINK1-dependent manner under basal, non-stressed conditions to regulate constitutive Mff turnover. We further show that removing Parkin with shRNA knockdown does not completely prevent Mff ubiquitination under these conditions indicating that at least one other ubiquitin ligase contributes to Mff proteostasis. These data demonstrate that Parkin plays a role in physiological maintenance of mitochondrial membrane protein composition in healthy mitochondria through constitutive low-level activation.

cell biology↗

bioRxiv2018

ADAR2-mediated Q/R editing of GluK2 regulates kainate receptor upscaling in response to suppression of synaptic activity

Kainate receptors (KARs) regulate neuronal excitability and network function. Most KARs contain the subunit GluK2 and the properties of these receptors are determined in part by ADAR2-mediated mRNA editing of GluK2 that changes a genomically encoded glutamine (Q) to arginine (R). Suppression of synaptic activity reduces ADAR2-dependent Q/R editing of GluK2 with a consequential increase in GluK2-containing KAR surface expression. However, the mechanism underlying this reduction in GluK2 editing has not been addressed. Here we show that induction of KAR upscaling results in proteasomal degradation of ADAR2, which reduces GluK2 Q/R editing. Because KARs incorporating unedited GluK2(Q) assemble and exit the ER more efficiently this leads to an upscaling of KAR surface expression. Consistent with this, we demonstrate that partial ADAR2 knockdown phenocopies and occludes KAR upscaling. Moreover, we show that although the AMPAR subunit GluA2 also undergoes ADAR2-dependent Q/R editing, this process does not mediate AMPAR upscaling. These data demonstrate that activity-dependent regulation of ADAR2 proteostasis and GluK2 Q/R editing are key determinants of KAR, but not AMPAR, trafficking and upscaling.\n\nSummary statementSynaptic suppression promotes proteasomal degradation of the mRNA-editing enzyme ADAR2. Decreased ADAR2 levels reduce Q/R editing of the kainate receptor subunit GluK2 leading to enhanced surface expression and homeostatic upscaling.

neuroscience↗

bioRxiv2018

ADAR2 mediated Q/R editing of GluK2 regulates homeostatic plasticity of kainate receptors

Kainate receptors (KARs) are heteromeric glutamate-gated ion channels that regulate neuronal excitability and network function in the brain. Most KARs contain the subunit GluK2 and the precise properties of these GluK2-containing KARs are determined by additional factors including ADAR2-mediated mRNA editing of a single codon that changes a genomically encoded glutamine (Q) to arginine (R) in the pore-lining region of GluK2. ADAR2-dependent Q/R editing of GluK2 is dynamically regulated during homeostatic plasticity (scaling) elicited by suppression of synaptic activity with TTX. Here we show that TTX decreases levels of ADAR2 by enhancing its proteasomal degradation. This selectively reduces the numbers of GluK2 subunits that are edited and increases the surface expression of GluK2-containing KARs. Furthermore, we show that partial ADAR2 knockdown phenocopies and occludes TTX-induced scaling of KARs. These data indicate that activity-dependent regulation of ADAR2 proteostasis and GluK2 Q/R editing provides a mechanism for KAR homeostatic plasticity.

neuroscience↗