Biology subjects

Curtis, M. A.

Publications and source records attributed to Curtis, M. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Systematic screening identifies elevated neurovascular BCL-XL in Parkinson's disease.

BackgroundVascular cells are emerging as active players in Parkinsons disease (PD), yet their molecular contribution to -Synuclein (-Syn) pathology remains undefined. Here, we show that human brain pericytes respond in a distinct manner to unique -Syn strains, with systematic validation identifying BCL-XL as a potential regulator of the neurovascular unit in PD. MethodsPrimary human brain-derived pericytes were exposed to five recombinant -Syn strains (Fibrils, Fibrils-65, Fibrils-91, Fibrils-110, and Ribbons). Transcriptomic profiling identified differentially expressed genes (DEGs), which were validated at the protein level using multiplex immunocytochemistry and in situ labelling of post-mortem middle temporal gyrus (MTG) tissue microarrays from PD (n = 24) and neurologically normal (n = 24) cases. Results-Syn strain exposure produced 300 DEGs with limited overlap between strains. BCL-XL and CSNK1D were upregulated in -Syn-treated pericytes. In post-mortem PD tissue BCL-XL showed marked pericyte-specific elevation in the MTG and increased pericytic and microglial expression in the substantia nigra. ConclusionBCL-XL emerges as a potential regulator of pericyte and microglial resilience in PD, linking acute -Syn strain-specific responses in pericytes to broader neurovascular alterations. Its upregulation likely represents a generalised compensatory response to chronic -Syn-associated stress beyond individual strain effects, identifying BCL-XL as a possible therapeutic target within the neurovascular unit.

neuroscience↗

bioRxiv2025

Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington's disease human cerebral cortex

Huntingtons disease (HD) is a hereditary neurodegenerative condition caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein. The accumulation of Htt inclusion bodies is a pathological hallmark of HD and a common target for therapeutic strategies. However, the limited efficacy of treatments targeting the Htt protein highlights the need for a better understanding of the role of Htt inclusion bodies in HD pathogenesis. This study examined the heterogeneity of Htt inclusion body composition by co-labelling with three Htt epitope-specific antibodies to characterize Htt inclusion body immunophenotype. We then characterized the size and sub-cellular location of Htt inclusions with distinct immunophenotypes. Using multiplex immunohistochemistry, we also examined the ubiquitination profile of each immunophenotype. Our findings demonstrate that Htt inclusions have a range of immunophenotypes, with some labelled by only one of the three antibodies and others exhibiting co-labelling by several antibodies, thus demonstrating the heterogeneity in inclusion composition and structure. We outline evidence that inclusion bodies exclusively labelled with the EM48 antibody are small, non-nuclear, and more abundant in HD cases with increased CAG repeat length, higher Vonsattel grade, and earlier age of onset. We also find that Htt inclusion bodies labelled by multiple antibodies are more likely to be ubiquitinated, predominantly by K63-rather than K48-linked ubiquitin, suggesting preferential degradation by autophagy. Lastly, we show that ubiquitinated Htt inclusion bodies are more highly immunoreactive for ubiquilin 2 than p62. Our findings highlight the need for multiple antibodies to capture the full spectrum of Htt pathology in HD and imply that future studies should consider the diversity of inclusion body composition and structure when correlating pathology formation to neurodegeneration, clinical symptoms, or disease severity.

neuroscience↗

bioRxiv2024

The Porphyromonas gingivalis lipid A 1-phosphatase LpxE has unique features and requires a functional type IX secretion system for its activity

Porphyromonas gingivalis is a Gram-negative bacterium that plays a central role in the development of periodontal disease. It uses a type IX secretion system (T9SS) to export a range of virulence factors to the bacterial surface where they are attached to A-LPS, one of the two forms of lipopolysaccharide (LPS) produced in P. gingivalis, and then packaged into outer membrane vesicles (OMVs). We previously showed that 1-P dephosphorylation of the lipid A component of LPS is regulated by the T9SS outer membrane protein (OMP) PorV, and this is linked to membrane destabilisation and OMV blebbing/formation. In this study we aimed to extend this and investigate the role of other T9SS OMPs in OMV biogenesis. We examined gingipain activity, gingipain secretion, A-LPS production, OMV morphology, and lipid A structure in P. gingivalis W50 and T9SS OMP mutant strains, and our results support an essential role for these proteins in type IX secretion. In addition, we produced a lipid A 1-phosphatase ({Delta}lpxE) mutant and show that all T9SS OMPs are required for LpxE activity and correct vesicle formation. LpxE has a unique C-terminal extension, and we propose that a cargo protein exported by the T9SS can directly/indirectly interact with this and regulate LpxE activity. This study provides insight into a new mechanism that links type IX cargo sorting with OMV blebbing, which may also be present in other Bacteroidota that colonise the gut and oral cavity.

microbiology↗

bioRxiv2024

Neuronal TDP-43 aggregation drives changes in microglial morphology prior to immunophenotype in amyotrophic lateral sclerosis

Microglia are the innate immune cells of the brain with the capacity to react to damage or disease. Microglial reactions can be characterised in post-mortem tissues by assessing their pattern of protein expression, or immunophenotypes, and cell morphologies. We recently demonstrated that microglia have a phagocytic immunophenotype in early-stage ALS but transition to a dysfunctional immunophenotype by end stage, and that these states are driven by TAR DNA-binding protein 43 (TDP-43) aggregation in the human brain. However, it remains unclear how microglial morphologies are changed in ALS. Here we examine the relationship between microglial immunophenotypes and morphologies, and TDP-43 pathology in motor cortex tissue from people with ALS and from a TDP-43-driven ALS mouse model. Post-mortem human brain tissue from 10 control and 10 ALS cases was analysed alongside brain tissue from the bigenic NEFH-tTA/tetO-hTDP-43{Delta}NLS (rNLS) mouse model of ALS at distinct disease stages. Sections were immunohistochemically labelled for microglial markers (HLA-DR, CD68, and Iba1) and phosphorylated TDP-43 (pTDP-43). Single-cell microglial HLA-DR, CD68, and Iba1 average intensities, and morphological features (cell body area, process number, total outgrowth, and branch number) were measured using custom image analysis pipelines. In human ALS motor cortex, we identified a significant change in microglial morphologies from ramified to hypertrophic, which was associated with increased Iba1 and CD68 levels. In the rNLS mouse motor cortex, the microglial morphological changed from ramified to hypertrophic and increased Iba1 levels occurred in parallel with pTDP-43 aggregation, prior to increases in CD68 levels. Overall, the evidence presented in this study demonstrates that microglia change their morphologies prior to immunophenotype changes. These morphological changes may prime microglia near neurons with pTDP-43 aggregation for phagocytosis, in turn triggering immunophenotype changes; first, to a phagocytic state then to a dysfunctional one.

neuroscience↗

bioRxiv2024

Huntingtin is an RNA-binding protein and participates in NEAT1-mediated paraspeckles

AbstractHuntingtin protein, mutated in Huntington disease, is implicated in nucleic acid- mediated processes, yet evidence for direct huntingtin-nucleic acid interaction is limited. Here we show wildtype and mutant huntingtin co-purify with nucleic acids, primarily RNA, and interact directly with G-rich RNAs in in vitro assays. Huntingtin RNA immunoprecipitation sequencing from patient-derived fibroblasts and neuronal progenitor cells expressing wildtype and mutant huntingtin revealed NEAT1 as a significantly enriched transcript. Altered NEAT1 levels were evident in Huntingtons disease cells and postmortem brain tissues, and huntingtin knockdown decreased NEAT1 levels. Huntingtin co-localized with NEAT1 in paraspeckles, and we identified a high-affinity RNA motif preferred by huntingtin. This study highlights NEAT1 as a novel huntingtin interactor, demonstrating huntingtins involvement in RNA-mediated functions and paraspeckle regulation. One-Sentence SummaryHTT is an RNA-binding protein that interacts with G-rich sequences, including those in the paraspeckle lncRNA NEAT1.

cell biology↗

bioRxiv2023

Developmental pyrethroid exposure disrupts folate metabolism in mouse brain

Environmental and genetic risk factors, and their interactions, contribute significantly to the etiology of neurodevelopmental disorders (NDDs). Recent epidemiology studies have implicated pyrethroid pesticides as an environmental risk factor for autism and developmental delay. Our previous research showed that low-dose developmental exposure to the pyrethroid pesticide deltamethrin in mice caused male-biased changes in the brain and in NDD-relevant behaviors in adulthood. Here, we used a metabolomics approach to determine the broadest possible set of metabolic changes in the adult male mouse brain caused by low-dose pyrethroid exposure during development. Using a litter-based design, we exposed mouse dams during pregnancy and lactation to deltamethrin (3 mg/kg or vehicle every 3 days) at a concentration well below the EPA-determined benchmark dose used for regulatory guidance. We raised male offspring to adulthood and collected whole brain samples for untargeted high-resolution metabolomics analysis. Developmentally exposed mice had disruptions in 116 metabolites which clustered into pathways for folate biosynthesis, retinol metabolism, and tryptophan metabolism. As a cross-validation, we integrated metabolomics and transcriptomics data from the same samples, which confirmed previous findings of altered dopamine signaling. These results suggest that pyrethroid exposure during development leads to disruptions in metabolism in the adult brain, which may inform both prevention and therapeutic strategies. HighlightsO_LIDevelopmental pyrethroid exposure disrupts brain metabolism in adulthood C_LIO_LIExposure disrupts metabolic pathways for folate, retinol, and tryptophan C_LIO_LIExposure disrupts genetic and metabolic pathways for dopamine signaling C_LI

neuroscience↗

bioRxiv2023

Developmental pyrethroid exposure disrupts molecular pathways for circadian rhythms and synaptic plasticity in mouse brain.

Neurodevelopmental disorders (NDDs) are a category of pervasive disorders of the developing nervous system with few or no recognized biomarkers. A significant portion of the risk for NDDs, including attention deficit hyperactivity disorder (ADHD), is contributed by the environment, and exposure to pyrethroid pesticides during pregnancy has been identified as a potential risk factor for NDD in the unborn child. We recently showed that low-dose developmental exposure to the pyrethroid pesticide deltamethrin in mice causes male-biased changes to ADHD- and NDD-relevant behaviors as well as the striatal dopamine system. Here, we used an integrated multiomics approach to determine the broadest possible set of biological changes in the mouse brain caused by developmental pyrethroid exposure (DPE). Using a litter-based, split-sample design, we exposed mouse dams during pregnancy and lactation to deltamethrin (3 mg/kg or vehicle every 3 days) at a concentration well below the EPA-determined benchmark dose used for regulatory guidance. We raised male offspring to adulthood, euthanized them, and pulverized and divided whole brain samples for split-sample transcriptomics, kinomics and multiomics integration. Transcriptome analysis revealed alterations to multiple canonical clock genes, and kinome analysis revealed changes in the activity of multiple kinases involved in synaptic plasticity, including the mitogen-activated protein (MAP) kinase ERK. Multiomics integration revealed a dysregulated protein-protein interaction network containing primary clusters for MAP kinase cascades, regulation of apoptosis, and synaptic function. These results demonstrate that DPE causes a multi-modal biophenotype in the brain relevant to ADHD and identifies new potential mechanisms of action. NEW & NOTEWORTHYHere, we provide the first evidence that low-dose developmental exposure to the pyrethroid pesticide, deltamethrin, results in molecular disruptions in the adult mouse brain in pathways regulating circadian rhythms and neuronal growth (MAP kinase). This same exposure causes a neurodevelopmental disorder (NDD) relevant behavioral changes in adult mice, making these findings relevant to the prevention of NDDs.

neuroscience↗

bioRxiv2023

Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

Microglia, the innate immune cells of the brain, are activated by damage or disease. In mouse models of amyotrophic lateral sclerosis (ALS), microglia shift from neurotrophic to neurotoxic states with disease progression. It remains unclear how human microglia change relative to the TAR DNA-binding protein 43 (TDP-43) aggregation that occurs in 97% of ALS cases. Here we examine spatial relationships between microglial activation and TDP-43 pathology in brain tissue from people with ALS and from a TDP-43-driven ALS mouse model. Post-mortem human brain tissue from the Neurological Foundation Human Brain Bank was obtained from 10 control and 10 ALS cases in parallel with brain tissue from a bigenic NFFH-tTA/tetO-hTDP-43{Delta}NLS (rNLS) mouse model of ALS at disease onset, early disease, and late disease stages. The spatiotemporal relationship between microglial activation and ALS pathology was determined by investigating microglial functional marker expression in brain regions with low and high TDP-43 burden at end-stage human disease: hippocampus and motor cortex, respectively. Sections were immunohistochemically labelled with a two-round multiplexed antibody panel against; microglial functional markers (L-ferritin, HLA-DR, CD74, CD68, and Iba1), a neuronal marker (NeuN), an astrocyte marker (GFAP), and pathological phosphorylated TDP-43 (pTDP-43). Single-cell levels of microglial functional markers were quantified using custom analysis pipelines and mapped to anatomical regions and ALS pathology. We identified a significant increase in microglial Iba1 and CD68 expression in the human ALS motor cortex, with microglial CD68 being significantly correlated with pTDP-43 pathology load. We also identified two subpopulations of microglia enriched in the ALS motor cortex that were defined by high L-ferritin expression. A similar pattern of microglial changes was observed in the rNLS mouse, with an increase first in CD68 and then in L-ferritin expression, with both occurring only after pTDP-43 inclusions were detectable. Our data strongly suggest that microglia are phagocytic at early-stage ALS but transition to a dysfunctional state at end-stage disease, and that these functional states are driven by pTDP-43 aggregation. Overall, these findings enhance our understanding of microglial phenotypes and function in ALS.

neuroscience↗

bioRxiv2023

Distribution of ubiquilin 2 and TDP-43 aggregates throughout the CNS in UBQLN2 p.T487I-linked amyotrophic lateral sclerosis and frontotemporal dementia

Mutations in the UBQLN2 gene cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The neuropathology of such UBQLN2-linked cases of ALS/FTD is characterised by aggregates of the ubiquilin 2 protein in addition to aggregates of the transactive response DNA-binding protein of 43 kDa (TDP-43). ALS and FTD without UBQLN2 mutations are also characterised by TDP-43 aggregates, that may or may not colocalise with wildtype ubiquilin 2. Despite this, the relative contributions of TDP-43 and ubiquilin 2 to disease pathogenesis remain largely under-characterised, as does their relative deposition as aggregates across the central nervous system (CNS). Here we conducted multiplex immunohistochemistry of three UBQLN2 p.T487I-linked ALS/FTD cases, three non-UBQLN2-linked (sporadic) ALS cases, and eight non-neurodegenerative disease controls, covering 40 CNS regions. We then quantified ubiquilin 2 aggregates, TDP-43 aggregates, and aggregates containing both proteins in regions of interest to determine how UBQLN2-linked and non-UBQLN2-linked proteinopathy differ. We find that ubiquilin 2 aggregates that are negative for TDP-43 are predominantly small and punctate, and are abundant in the hippocampal formation, spinal cord, all tested regions of neocortex, medulla, and substantia nigra in UBQLN2-linked ALS/FTD but not sporadic ALS. Curiously, the striatum harboured small punctate ubiquilin 2 aggregates in all cases examined, while large diffuse striatal ubiquilin 2 aggregates were specific to UBQLN2-linked ALS/FTD. Overall, ubiquilin 2 is mainly deposited in clinically unaffected regions throughout the CNS such that symptomology in UBQLN2-linked cases maps best to the aggregation of TDP-43.

neuroscience↗

bioRxiv2023

Differential exon usage and cryptic exon profiles of TDP-43 loss of function in amyotrophic lateral sclerosis brain tissue

TDP-43 dysfunction is a molecular hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major hypothesis of TDP-43 dysfunction in disease is the loss of normal nuclear function, resulting in impaired RNA regulation and the emergence of cryptic exons. Cryptic exons and exon changes are emerging as promising markers of lost TDP-43 function in addition to revealing biological pathways involved in neurodegeneration in ALS/FTD. In this brief report, we identified markers of TDP-43 loss of function by depleting TARDBP from post-mortem human brain pericytes, a manipulable in vitro primary human brain cell model, and identifying differential exon usage events with bulk RNA-sequencing analysis. We present these data in an interactive database (https://www.scotterlab.auckland.ac.nz/research-themes/tdp43-lof-db-v2/) together with seven other TDP-43-depletion datasets we meta-analysed previously, for user analysis of differential expression and splicing signatures. Differential exon usage events that were validated by qPCR were then compiled into a differential exon usage panel with other well-established TDP-43 loss-of-function exon markers. This differential exon usage panel was investigated in ALS and control motor cortex tissue to verify whether, and to what extent, TDP-43 loss of function occurs in ALS. We find that profiles of TDP-43-regulated cryptic exons and changed exon usage discriminate ALS brain tissue from controls, verifying TDP-43 loss of function as a pathomechanism in ALS. We propose that TDP-43-regulated splicing markers with most predictive value for therapeutic intervention will be those based on splicing events that occur both in tissues/biofluids amenable to sampling, and in brain tissue susceptible to disease.

neuroscience↗

bioRxiv2022

Antagonistic roles of canonical and alternative RPA in tandem CAG repeat diseases

Tandem CAG repeat expansion mutations cause >15 neurodegenerative diseases, where ongoing expansions in patients brains are thought to drive disease onset and progression. Repeat length mutations will involve single-stranded DNAs prone to form mutagenic DNA structures. However, the involvement of single-stranded DNA binding proteins (SSBs) in the prevention or formation of repeat instability is poorly understood. Here, we assessed the role of two SSBs, canonical RPA (RPA1-RPA2-RPA3) and the related Alternative-RPA (Alt-RPA, RPA1-RPA4-RPA3), where the primate-specific RPA4 replaces RPA2. RPA is essential for all forms of DNA metabolism, while Alt-RPA has undefined functions. RPA and Alt-RPA are upregulated 2- and 10-fold, respectively, in brains of Huntington disease (HD) and spinocerebellar ataxia type 1 (SCA1) patients. Correct repair of slipped-CAG DNA structures, intermediates of expansion mutations, is enhanced by RPA, but blocked by Alt-RPA. Slipped-DNAs are bound and melted more efficiently by RPA than by Alt-RPA. Removal of excess slipped-DNAs by FAN1 nuclease is enhanced by RPA, but blocked by Alt-RPA. Protein-protein interactomes (BioID) reveal unique and shared partners of RPA and Alt-RPA, including proteins involved in CAG instability and known modifiers of HD and SCA1 disease. RPA overexpression inhibits rampant CAG expansions in SCA1 mouse brains, coinciding with improved neuron morphology and rescued motor phenotypes. Thus, SSBs are involved in repeat length mutations, where Alt-RPA antagonistically blocks RPA from suppressing CAG expansions and hence pathogenesis. The processing of repeat length mutations is one example by which an Alt-RPA{leftrightarrow}RPA antagonistic interaction can affect outcomes, illuminating questions as to which of the many processes mediated by canonical RPA may also be modulated by Alt-RPA.

genetics↗

bioRxiv2022

Human pericytes degrade alpha-synuclein aggregates in a strain-dependent manner

Parkinsons disease (PD) is a progressive, neurodegenerative disorder characterised by the abnormal accumulation of -synuclein (-syn) aggregates. Central to disease progression is the gradual spread of pathological -syn. -syn aggregation is closely linked to progressive neuron loss. As such, clearance of -syn aggregates may slow the progression of PD and lead to less severe symptoms. Evidence that non-neuronal cells play a role in PD and other synucleinopathies such as Lewy body dementia and multiple system atrophy are increasing. Our previous work has shown that pericytes -- vascular mural cells that regulate the blood-brain barrier -- contain -syn aggregates in human PD brains. Here, we demonstrate that pericytes efficiently internalise fibrillar -syn irrespective of being in a monoculture or mixed neuronal cell culture. Pericytes efficiently break down -syn aggregates in vitro, with clear differences in the number of -syn aggregates/cell and average aggregate size when comparing five pure -syn strains (Fibrils, Ribbons, fibrils65, fibrils91 and fibrils110). Furthermore, pericytes derived from PD brains have a less uniform response than those derived from control brains. Our results highlight the vital role brain vasculature may play in reducing -syn burden in PD.

neuroscience↗

bioRxiv2022

N-terminal mutant Huntingtin deposition correlates with CAG repeat length and disease onset, but not neuronal loss in Huntington's disease

Huntingtons disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake a high-throughput screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs). Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age-matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabelled human brain tissue. The Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with high-throughput analysis. Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. Significant associations were found between the number of Htt aggregates and both age of disease onset, and CAG repeat length in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and Htt cortical aggregate deposition is associated with the onset of clinical symptoms. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD are not specific for Htt in human brain tissue, thereby highlighting the need for validated means of Htt detection to support drug development for HD.

neuroscience↗

bioRxiv2022

Hemin availability induces coordinated DNA methylation and gene expression changes in Porphyromonas gingivalis

Periodontal disease is a common chronic inflammatory disease. Porphyromonas gingivalis is an important bacterium in the development of the disease and expresses a variety of virulence determinants. Hemin (iron [III] protopotphyrin IX), an essential nutrient of this organism, whose concentration increases with increasing inflammation, is a global regulator of virulence in P. gingivalis: high hemin levels increase expression of several virulence determinants. However, the mechanism through which hemin influences bacterial gene expression is poorly understood. Bacterial DNA methylation has the potential to fulfil this mechanistic role. Here, we characterised the methylome of P. gingivalis, and compared its variation to transcriptomic changes in response to changes in hemin concentration. Gene expression and DNA methylation profiling of P. gingivalis W50 was performed, following continuous culture in chemostats with excess or limited hemin, using Illumina RNA-Seq and Nanopore DNA sequencing. DNA methylation quantification was carried out for Dam/Dcm motifs and all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC) base pair modifications. Differential expression and methylation in response to excess hemin availability are presented after multiple testing correction (FDR 5%). In excess hemin there were 161 over- and 268 under-expressed genes compared to limited hemin. Genes under-expressed in excess hemin were involved in iron recruitment (the hemophore HmuY) and transport (TonB-dependent receptors), and those over-expressed were involved in iron-sulphur cluster binding. Hemin-dependent differentially methylation was observed for the Dam GATC motif and all-context 6mA and 5mC, with 36, 49 and 47 signals, respectively. Coordinated genome-wide differential expression and methylation effects were observed in 6 genes encoding a Ppx/GppA family phosphatase, a lactate utilization protein, a 4-alpha-glucanotransferase, two ABC transporter proteins, and a hypothetical protein HMPREF1322_RS00730. The findings indicate that altered genome methylation occurs in response to the availability of hemin and give insights into the molecular mechanisms of regulation of virulence in this bacterium. Author SummaryDNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis, an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the gene regulatory processes underlying these effects remain unknown. To this end, we profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin conditions that reflect conditions associated with health and disease, respectively. Notably, we also detected differential DNA methylation signatures for the Dam GATC motif and both all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC) in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease.

genomics↗

bioRxiv2022

Hippocampal protein aggregation signatures fully distinguish pathogenic and wildtype UBQLN2 in amyotrophic lateral sclerosis

Mutations in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD) characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial ALS or FTD cases not caused by UBQLN2 mutations, particularly C9orf72-linked cases. This makes the mechanistic role of ubiquilin 2 mutations and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 41 genotypically diverse ALS cases with or without FTD, including five cases with UBQLN2 variants (resulting in p.S222G, p.P497H, p.P506S, and two cases with p.T487I). Using multiplexed (5-label) fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43 (pTDP-43), dipeptide repeat aggregates, and p62, in the hippocampus of controls (n=5), or ALS with or without FTD in sporadic (n=20), unknown familial (n=3), SOD1-linked (n=1), FUS-linked (n=1), C9orf72-linked (n=5), and UBQLN2-linked (n=5) cases. We differentiate between i) ubiquilin 2 aggregation together with pTDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation promoted by UBQLN2 gene mutations that cause ALS/FTD. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in ALS with or without FTD, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.

neuroscience↗

bioRxiv2021

Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation

Escherichia coli is a Gram-negative bacterium that colonizes the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and some commensal E. coli strains. It can degrade mucins in the intestine, promotes biofilm maturation and it is a major determinant of infection in virulent strains, although how it carries out these functions is not well understood. Here we examine SslE from the E. coli Waksman and H10407 strains and reveal that it has a novel and dynamic structure. In response to acidification within mature biofilms we show how SslE forms a unique functional aggregate that interacts with cellulose and regulates the distribution of exopolysaccharides in macrocolony biofilms. Our data indicates that the spatial organization of SslE polymers and local pH are critical for biofilm maturation and SslE is a key factor that drives persistence of SslE-secreting bacteria during acidic stress.

microbiology↗

bioRxiv2020

The essential Porphyromonas gingivalis type IX secretion system component PorZ delivers anionic-lipopolysaccharide to the PorU sortase for transpeptidase processing of cargos

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from phylum Bacteroidetes invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of selected cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ, and found that it consists of two {beta}-propeller moieties followed by a CTD. In this study, we performed structure-based modelling suggesting that PorZ is a carbohydrate-binding protein. We found that recombinant PorZ specifically binds A-LPS. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of the A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the attachment complex composed of PorU, PorZ, and the integral OM {beta}-barrel proteins PorV and PorQ, which is involved in post-translational modification and retention of T9SS cargos on the bacterial surface. Author summaryBacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis. Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS- bound PorZ interacts with sortase with significantly greater affinity, facilitating modification of cargo proteins by the cell-surface attachment complex of the T9SS.

microbiology↗

bioRxiv2019

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

Phosphorylation of the N-terminal domain of the Huntingtin (HTT) protein (at T3, S13, and S16) has emerged as a key regulator of HTT stability, clearance, localization, aggregation and toxicity. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that specifically and efficiently phosphorylates both wild-type and mutant full-length or N-terminal fragments of HTT in vitro (S13/S16) and in cell/ neuronal cultures (S13). We show that overexpression of TBK1 in mammalian cells, primary neurons and a Caenorhabditis elegans model of Huntingtons Disease (HD) increases mutant HTTex1 phosphorylation, lowers its levels, increases its nuclear localization and significantly reduces its aggregation and cytotoxicity. Our mechanistic studies demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTTex1 aggregation and an increase in autophagic flux. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/869586v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1368abeorg.highwire.dtl.DTLVardef@1cecff4org.highwire.dtl.DTLVardef@b6b7c5org.highwire.dtl.DTLVardef@1daf53d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

neuroscience↗