Biology subjects

Newton, J.

Publications and source records attributed to Newton, J..

12 recordsLinked to original sources

bioRxiv2026

Dual MMP-9/12 Inhibition with AZD1236 Confers Neurovascular Protection and Reduces Post-Stroke Pain in Experimental Stroke Models.

Background: Stroke remains a leading cause of death and disability worldwide. Matrix metalloproteinases (MMPs), particularly MMP-9 and MMP-12, contribute to early blood-brain barrier (BBB) disruption, neuroinflammation, haemorrhagic transformation, and intracerebral haemorrhage (ICH). Intravenous thrombolysis is the only widely used pharmacological therapy for acute ischaemic stroke, but its utility is limited by narrow eligibility criteria and haemorrhagic risk. Inhibition of MMPs in the acute phase may offer a complementary neurovascular protective strategy. Methods: AZD1236, a selective dual MMP-9/-12 inhibitor, was evaluated in transient and permanent middle cerebral artery occlusion models and in a collagenase-induced ICH model in young, aged, obese, and female mice. Drug or vehicle was administered 2-6 hours after stroke onset. Outcomes included infarct or haematoma volume, BBB integrity, neurological function, and pain-related behaviours. Results: AZD1236 given within 2-4 hours after ischaemic or haemorrhagic insult significantly reduced infarct and haematoma volumes, improved short- and long-term neurological scores, and preserved BBB integrity, whereas treatment at 6 hours was largely ineffective. AZD1236 also attenuated the development of post-stroke mechanical allodynia and thermal hyperalgesia. Mechanistically, treatment reduced MMP-9 and MMP-12 activity, increased tight junction protein expression, and dampened inflammatory responses. Conclusions: Dual inhibition of MMP-9/-12 with AZD1236 confers robust neurovascular protection and mitigates post-stroke pain across clinically relevant models of ischaemic and haemorrhagic stroke. These findings provide a strong preclinical rationale for clinical evaluation of dual MMP-9/12 inhibition as an adjunctive neuroprotective strategy for acute stroke.

neuroscience↗

bioRxiv2026

NPC1 deficiency engages a lysosome - genome - immune program linked to neurodegeneration and cellular aging signatures

Lysosomal dysfunction is a prominent feature of neurodegeneration and aging, yet how primary defects in lysosomal trafficking are converted into progressive cellular decline remains poorly understood. Niemann Pick disease type C (NPC), caused by impaired NPC1 dependent cholesterol export, provides a genetically defined model to address this question. Here, we show that NPC1 deficiency activates a lysosome, genome, immune axis linking cholesterol trafficking failure to neurodegeneration and hallmarks of cellular aging. In Npc1 mutant mice, NPC1 loss triggered DNA damage, neuroinflammation, microglial and astrocytic activation, Purkinje neuron degeneration, and motor dysfunction. Consistently, NPC patient-derived fibroblasts exhibited mitochondrial abnormalities and widespread DNA double-strand breaks. Genome-wide DNA break mapping and transcriptomic analyses revealed extensive genomic instability at regulatory regions, including enrichment of DNA breaks at transcription start sites and G quadruplex associated loci, accompanied by widespread transcriptional reprogramming, activation of innate immune pathways, disruption of fibroblast identity, and induction of cellular aging signatures. We further identify Fingolimod, an FDA approved sphingosine - 1 phosphate receptor modulator, as a potent modifier of this disease network. Fingolimod improved lysosomal cholesterol trafficking, increased LAMP1 abundance, attenuated STING associated inflammatory signaling, normalized mitochondrial function, reduced neuroinflammatory and neurodegenerative phenotypes in Npc1 mutant mice, and broadly shifted disease-associated transcriptional programs toward a healthier state. Extending these findings beyond NPC, Fingolimod improved age-associated phenotypes in C. elegans and prolonged lifespan in aged male mice. Together, these findings identify genome instability and chronic innate immune activation as major downstream consequences of lysosomal cholesterol trafficking failure and establish Fingolimod as a clinically actionable modulator of lysosomal dysfunction, neurodegeneration, and aging-related decline.

molecular biology↗

bioRxiv2025

eDNA on the go: a direct comparison of fixed and vehicle mounted airborne eDNA sampling methods for terrestrial vertebrate species detection at large spatial scales

Air is receiving increasing recognition as a biologically rich source of taxonomically diverse environmental DNA (eDNA). Multiple proof-of-concept studies have explored air as a medium for the detection of single terrestrial species, and even entire terrestrial communities, including mammals, non-anemophilous plants and insects. Airborne eDNA has been sampled using various stationary devices but if we can access it using mobile collection methods, we can rapidly assess biodiversity at large spatial scales. We therefore compared passive eDNA filters deployed for short (30 mins) and long periods (24 hrs) with filters attached to cars that sampled transects through natural and human-modified environments. Our metabarcoding procedure detected 49 vertebrate taxa (33 birds, 15 mammals and 1 amphibian) from all three eDNA air sampling approaches combined, with 29 of these taxa detected in samples collected using the mobile method. The total number of taxa, or proportions of unique taxa detected, did not differ by sampling method, land use or day. Community composition was not significantly impacted by sampling method or sampling day but differed significantly between land uses. We propose that our car-mounted air eDNA sampler could be a game changer for large scale, rapid biomonitoring efforts.

ecology↗

bioRxiv2025

Remodelling of supernumerary leaflet primordia leads to bicuspid aortic valve (BAV) caused by loss of primary cilia

AimsBicuspid aortic valve (BAV), where two valve leaflets are found instead of the usual three, affects 1-2% of the general population and is associated with significant morbidity and mortality. Despite its frequency, the majority of cases remain unexplained. This is, at least in part, because there are two types of valve leaflet primordia: endocardial cushions and intercalated valve swellings (ICVS). Moreover, multiple progenitors make distinct contribution to the formation of these primordia. Genomic studies in mouse and human have suggested a correlation between BAV and malfunctional primary cilia. However, the precise requirement for cilia during early embryonic valvulogenesis remains unknown. Methods and resultsHere, we disrupted primary cilia by deleting the ciliary gene Ift88 in the main progenitor cells forming the aortic valve using specific Cre drivers: Wnt1-Cre for neural crest cells, Isl1-Cre for second heart field cells (SHF); Tie2-Cre for endocardial-derived cells and Tnnt2-Cre for direct-differentiating SHF in the ICVS. Loss of Ift88, and thus primary cilia, from neural crest cells and endocardium did not impact aortic valve formation. However, primary cilia are essential in SHF cells for aortic valve leaflet formation, with over half of Ift88f/f;Isl1-Cre mutants presenting with BAV. As the valve leaflets are forming, 50% of the Ift88f/f;Isl1-Cre mutants have two small leaflets in the position of the usual posterior leaflet, meaning that at this stage the aortic valve is quadricuspid, which then remodels to BAV by E15.5. Mechanistic studies demonstrate premature differentiation of SHF cells as the ICVS form, leading to the formation of a broadened ICVS that forms two posterior leaflet precursors. This abnormality in the formation of the ICVS is associated with disruption of Notch-Jag1 signalling pathway, with Jag1f/f;Isl1-Cre mutants presenting with a similar phenotype. ConclusionsThese data show that primary cilia, via the Notch-Jag1 signalling pathway, regulate differentiation of SHF cells in the aortic valve primordia. Additionally, we identify a mechanistic link between the developmental basis of quadricuspid and bicuspid arterial valve leaflets. Translational PerspectiveSeveral genomic studies in human and mouse have suggested that disruption of cilia-related genes may be a significant cause of CHD. Although there is limited data from animal models to suggest a link between cilia and bicuspid aortic valve (BAV), the mechanisms underpinning BAV formation during early valvulogenesis have not been described. Here, we established a potential mechanism underpinning BAV formation, highlighting a role for primary cilia in a subset of valve interstitial cells (VIC) derived from second heart field progenitors. Loss of cilia altered VIC differentiation and valvulogenesis. This study confirms that disruption of cilial formation and/or function can lead to arterial valve defects and could pave the way to finding therapies for patient benefit.

developmental biology↗

bioRxiv2024

Identification of an Outbreak of Bivalve Transmissible Neoplasia in Soft-Shell Clams (Mya arenaria) in the Puget Sound Using Hemolymph and eDNA Surveys

Bivalve transmissible neoplasia (BTN) is one of three known types of naturally transmissible cancer-- cancers in which the whole cancer cells move from individual to individual, spreading through natural populations. BTN is a lethal leukemia-like cancer that has been observed throughout soft-shell clam (Mya arenaria) populations on the east coast of North America, with two distinct sublineages circulating at low enzootic levels in New England, USA, and Prince Edward Island, Canada. Major cancer outbreaks likely due to Mya arenaria BTN (MarBTN) were reported in 1980s and the 2000s and the disease has been observed since the 1970s, but it has not been observed in populations of this clam species on the US west coast. In 2022, we collected soft-shell clams from several sites in Puget Sound, Washington, USA, and unexpectedly found high prevalence of BTN in two sites (Triangle Cove on Camano Island and near Stanwood in South Skagit Bay). Prevalence of BTN increased in subsequent years, reaching >75% in both sites in 2024, while it was not observed in other sites, suggesting the early stages of a severe disease outbreak following recent introduction. We observed that these cancer cells contain several somatic transposing insertion sites found only in the USA-sublineage of MarBTN, showing that it likely was recently transplanted from New England to this location. We then developed a sensitive environmental DNA (eDNA) assay, using qPCR to target somatic mutations in the MarBTN mitogenome, and showed that MarBTN can be detected in seawater at Triangle Cove, as well as several kilometers outside of the cove. We then used this assay to survey 50 sites throughout Puget Sound, confirming that the disease can be detected at high levels at Triangle Cove and South Skagit Bay, and showing that it extends beyond these known sites. However, while normal soft-shell clam mtDNA was widely detected, MarBTN was undetectable throughout most of Puget Sound and currently remains limited to the South Skagit Bay area and north Port Susan. These results identify a previously unknown severe outbreak of a transmissible cancer due to long-distance transplantation of disease from another ocean, and they demonstrate the utility of eDNA methods to track the spread of BTN through the environment.

ecology↗

bioRxiv2024

Spinal cord perfusion impairments in the M83 mouse model of Parkinson's disease

Metabolism and bioenergetics in the central nervous system play important roles in the pathophysiology of Parkinsons disease (PD). Here, we employed a multimodal imaging approach to assess oxygenation changes in the spinal cord of a transgenic M83 murine model of PD in comparison to non-transgenic littermates at 9-12 months-of-age. A lower oxygen saturation (SO2)SVOT was detected in vivo with spiral volumetric optoacoustic tomography (SVOT) in the spinal cord of M83 mice compared to non-transgenic littermate mice. Ex-vivo high-field T1-weighted magnetic resonance imaging (MRI) and immunostaining for alpha-synuclein (phospho-S129) and vascular organisation (CD31 and GLUT1) were used to investigate the nature of the abnormalities detected via in vivo imaging. Ex-vivo analysis showed that the vascular network in the spinal cord was not impaired in the spinal cord of M83 mice. Ex-vivo MRI assisted with deep learning-based automatic segmentation showed no volumetric atrophy in the spinal cord of M83 mice compared to non-transgenic littermates, whereas nuclear alpha-synuclein phosphorylated at Ser129 site could be linked to early pathology and metabolic dysfunction. The proposed and validated non-invasive high-resolution imaging tool to study oxygen saturation in the spinal cord of PD mice holds promise for assessing early changes preceding motor deficits in PD mice.

neuroscience↗

bioRxiv2024

A disease similarity approach identifies short-lived Niemann-Pick type C disease mice with accelerated brain aging as a novel mouse model for Alzheimer's disease and aging research

Since its first description in 1906 by Dr. Alois Alzheimer, Alzheimers disease (AD) has been the most common type of dementia. Initially thought to be caused by age-associated accumulation of plaques, in recent years, research has increasingly associated AD with lysosomal storage and metabolic disorders, and the explanation of its pathogenesis has shifted from amyloid and tau accumulation to oxidative stress and impaired lipid and glucose metabolism aggravated by hypoxic conditions. However, the underlying mechanisms linking those cellular processes and conditions to disease progression have yet to be defined. Here, we applied a disease similarity approach to identify unknown molecular targets of AD by using transcriptomic data from congenital diseases known to increase AD risk, namely Down Syndrome, Niemann Pick Disease Type C (NPC), and Mucopolysaccharidoses I. We uncovered common pathways, hub genes, and miRNAs across in vitro and in vivo models of these diseases as potential molecular targets for neuroprotection and amelioration of AD pathology, many of which have never been associated with AD. We then investigated common molecular alterations in brain samples from an NPC disease mouse model by juxtaposing them with brain samples of both human and mouse models of AD. Detailed phenotypic and molecular analyses revealed that the NPCmut mouse model can serve as a potential short-lived in vivo model for AD research and for understanding molecular factors affecting brain aging. This research represents the first comprehensive approach to congenital disease association with neurodegeneration and a new perspective on AD research while highlighting shortcomings and lack of correlation in diverse in vitro models. Considering the lack of an AD mouse model that recapitulates the physiological hallmarks of brain aging, the characterization of a short-lived NPC mouse model will further accelerate the research in these fields and offer a unique model for understanding the molecular mechanisms of AD from a perspective of accelerated brain aging.

genetics↗

bioRxiv2022

Cell-autonomous immune dysfunction driven by disrupted autophagy in C9orf72-ALS iPSC-derived microglia contributes to neurodegeneration

The most common genetic mutation found in familial and sporadic amyotrophic lateral sclerosis (ALS), as well as fronto-temporal dementia (FTD), is a repeat expansion in the C9orf72 gene. C9orf72 is highly expressed in human myeloid cells, and although neuroinflammation and microglial pathology are widely found in ALS/FTD, the underlying mechanisms are poorly understood. Here, using human induced pluripotent stem cell-derived microglia-like cells (hiPSC-MG) harbouring C9orf72 mutation (mC9-MG) together with gene-corrected isogenic controls (isoC9-MG) and C9ORF72 knock-out hiPSC-MG (C9KO-MG), we show that reduced C9ORF72 protein is associated with impaired phagocytosis and an exaggerated inflammatory response upon stimulation with lipopolysaccharide, driven by sustained activation of NLRP3 inflammasome and NF-{kappa}B signalling. Analysis of the hiPSC-MG C9ORF72 interactome revealed an association of C9ORF72 with key regulators of autophagy, a process involved in the homeostatic regulation of the innate immune response. We found impaired initiation of autophagy in C9KO-MG and mC9-MG. Furthermore, through motor neuron-microglial (MN-MG) co-culture studies, we identified that autophagy deficit in mC9-MG led to increased vulnerability of C9 MNs to excitotoxic stimulus. Pharmacological activation of autophagy ameliorated the sustained activation of NLRP3 inflammasome and NF-{kappa}B signalling, reversed the phagocytic deficit found in mC9-MG and also reduced MN death in MN-MG co-cultures. We validated these findings in blood-derived macrophages from people with C9orf72 mutation. Our results reveal an important role for C9ORF72 in regulating microglial immune homeostasis and identify dysregulation in human myeloid cells as a contributor to neurodegeneration in ALS/FTD. TeaserDisrupted autophagy led immune activation in microglia results in enhanced motor neuronal death in C9orf72-ALS.

neuroscience↗

bioRxiv2020

Visualising Cholesterol in Brain by On-Tissue Derivatisation and Quantitative Mass Spectrometry Imaging

Despite being a critical molecule for neurobiology and brain health, mass spectrometry imaging (MSI) of cholesterol has been under reported compared to other lipids, due to the difficulty in ionising the sterol molecule. In the present work we have employed an on-tissue enzyme-assisted derivatisation strategy to improve detection of cholesterol in brain tissue sections. We report distribution and levels of cholesterol across specific brain structures of the mouse brain, in a model of Niemann-Pick type C1 (NPC1) disease, and during brain development. MSI revealed how cholesterol changes during development and that in the adult is highest in pons and medulla of the brain stem. Cholesterol was significantly reduced in the corpus callosum and other brain regions in the Npc1 null mouse, confirming hypomyelination at the molecular level. Our study demonstrates the potential of MSI to the study of sterols in neuroscience.

biochemistry↗

bioRxiv2020

Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain

Long Interspersed Nuclear Element-1 (LINE-1 or L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as mRNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, disordered N-terminal region of ORF1. Using microscopy and NMR spectroscopy, we demonstrate that ORF1 forms liquid droplets in vitro in a salt-dependent manner and that interactions between its N-terminal region and coiled-coil domain are necessary for phase separation. Mutations disrupting blocks of charged residues within the N-terminus impair phase separation while some mutations within the coiled-coil domain enhance phase separation. Demixing of the L1 particle from the cytosol may provide a mechanism to protect the L1 transcript from degradation. Statement of significanceOver half of the human genome is comprised of repetitive sequences. The Long Interspersed Nuclear Element-1 (L1) is an autonomous mobile DNA element that can alter its genomic location, resulting in genomic instability and DNA damage. L1 encodes two proteins that are required for this function: the ORF1 RNA chaperone and the enzymatic ORF2. Here, we demonstrate that ORF1 forms liquid-liquid phase separated states in vitro, which is mediated by electrostatic interactions between the conserved, disordered N-terminus and coiled-coil domain. This work provides a framework to explore how L1 phase separation may enhance the ability of the retrotransposable element to colonize the genome by preventing degradation of the L1 transcript and evasion of host immune responses.

biophysics↗

bioRxiv2020

Loss of Caspase-8 function in combination with SMAC mimetic treatment sensitizes Head and Neck Squamous Carcinoma to radiation through induction of necroptosis

Caspase-8 (CASP8) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCC), and mutations of CASP8 are associated with poor overall survival. The distribution of these mutations in HNSCC suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a unique cell death mechanism. Here, we evaluated how CASP8 regulates necroptosis in HSNCC using cell line models and syngeneic mouse xenografts. In vitro, knockdown of CASP8 rendered HNSCCs susceptible to necroptosis induced by a second mitochondria-derived activator of caspase (SMAC) mimetic, Birinapant, when combined with pan-caspase inhibitors Z-VAD-FMK or Emricasan. Strikingly, inhibition of CASP8 function via knockdown or Emricasan treatment was associated with enhanced radiation killing by Birinapant through induction of necroptosis. In a syngeneic mouse model of oral cancer, Birinapant, particularly when combined with radiation delayed tumor growth and enhanced survival under CASP8 loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine-protein kinase-3 (RIP3), a key enzyme in the necroptosis pathway was crucial in determining susceptibility to this mode of death. Although an in vitro screen revealed that many HNSCC cell lines were resistant to necroptosis due to low levels of RIP3, patient tumors maintain RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be a relevant therapeutic approach in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained. SignificanceCASP8 status regulates necroptotic death in HNSCC and this pathway can be exploited therapeutically.

cancer biology↗

bioRxiv2018

Imaging Oxysterols in Mouse Brain by On-Tissue Derivatisation-Robotic Liquid Micro-Extraction Surface Analysis-Liquid Chromatography Mass Spectrometry

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatisation in combination with micro-liquid-extraction for surface analysis and liquid chromatography - mass spectrometry to image sterols in tissue slices (10 {micro}m) of mouse brain. The method provides sterolomic analysis at 400 {micro}m spot diameter with a limit of quantification of 0.01 ng/mm2. It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low abundance and difficult to ionise sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild type and cholesterol 24S-hydroxylase knock-out mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues.\n\nSignificanceThe brain is a remarkably complex organ and cholesterol homeostasis underpins brain function. It is known that cholesterol is not evenly distributed across different brain regions, however, the precise map of cholesterol metabolism in the brain remains unclear. If cholesterol metabolism is to be correlated with brain function it is essential to generate such a map. Here we describe an advanced mass spectrometry imaging platform to reveal spatial cholesterol metabolism in situ at 400 {micro}m resolution on 10 {micro}m tissue slices from mouse brain. We mapped, not only cholesterol, but also other biologically active sterols arising from cholesterol turnover in both wild type and mice lacking cholesterol 24-hydroxylase (Cyp46a1), the major cholesterol metabolising enzyme.

neuroscience↗