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Welcome to OXION, Universities of Oxford, Cambridge, London and MRC Harwell
Functional genomic and proteomic analysis reveals disruption of myelin-related genes and translation in a mouse model of early life neglect.
Early life neglect is an important public health problem which can lead to lasting psychological dysfunction. Good animal models are necessary to understand the mechanisms responsible for the behavioral and anatomical pathology that results. We recently described a novel model of early life neglect, maternal separation with early weaning (MSEW), that produces behavioral changes in the mouse that persist into adulthood. To begin to understand the mechanism by which MSEW leads to these changes we applied cDNA microarray, next-generation RNA-sequencing (RNA-seq), label-free proteomics, multiple reaction monitoring (MRM) proteomics, and methylation analysis to tissue samples obtained from medial prefrontal cortex to determine the molecular changes induced by MSEW that persist into adulthood. The results show that MSEW leads to dysregulation of markers of mature oligodendrocytes and genes involved in protein translation and other categories, an apparent downward biasing of translation, and methylation changes in the promoter regions of selected dysregulated genes. These findings are likely to prove useful in understanding the mechanism by which early life neglect affects brain structure, cognition, and behavior.
Parallel declines in cognition, motivation, and locomotion in aging mice: association with immune gene upregulation in the medial prefrontal cortex.
Aging in humans is associated with parallel changes in cognition, motivation, and motoric performance. Based on the human aging literature, we hypothesized that this constellation of age-related changes is mediated by the medial prefrontal cortex and that it would be observed in aging mice. Toward this end, we performed detailed assessments of cognition, motivation, and motoric behavior in aging mice. We assessed behavioral and cognitive performance in C57Bl/6 mice aged 6, 18, and 24 months, and followed this with microarray analysis of tissue from the medial prefrontal cortex and analysis of serum cytokine levels. Multivariate modeling of these data suggested that the age-related changes in cognition, motivation, motor performance, and prefrontal immune gene expression were highly correlated. Peripheral cytokine levels were also correlated with these variables, but less strongly than measures of prefrontal immune gene upregulation. To determine whether the observed immune gene expression changes were due to prefrontal microglial cells, we isolated CD11b-positive cells from the prefrontal cortex and subject them to next-generation RNA sequencing. Many of the immune changes present in whole medial prefrontal cortex were enriched in this cell population. These data suggest that, as in humans, cognition, motivation, and motoric performance in the mouse change together with age and are strongly associated with CNS immune gene upregulation.
PKA phosphorylation of NDE1 is DISC1/PDE4 dependent and modulates its interaction with LIS1 and NDEL1.
Nuclear distribution factor E-homolog 1 (NDE1), Lissencephaly 1 (LIS1), and NDE-like 1 (NDEL1) together participate in essential neurodevelopmental processes, including neuronal precursor proliferation and differentiation, neuronal migration, and neurite outgrowth. NDE1/LIS1/NDEL1 interacts with Disrupted in Schizophrenia 1 (DISC1) and the cAMP-hydrolyzing enzyme phosphodiesterase 4 (PDE4). DISC1, PDE4, NDE1, and NDEL1 have each been implicated as genetic risk factors for major mental illness. Here, we demonstrate that DISC1 and PDE4 modulate NDE1 phosphorylation by cAMP-dependent protein kinase A (PKA) and identify a novel PKA substrate site on NDE1 at threonine-131 (T131). Homology modeling predicts that phosphorylation at T131 modulates NDE1-LIS1 and NDE1-NDEL1 interactions, which we confirm experimentally. DISC1-PDE4 interaction thus modulates organization of the NDE1/NDEL1/LIS1 complex. T131-phosphorylated NDE1 is present at the postsynaptic density, in proximal axons, within the nucleus, and at the centrosome where it becomes substantially enriched during mitosis. Mutation of the NDE1 T131 site to mimic PKA phosphorylation inhibits neurite outgrowth. Thus PKA-dependent phosphorylation of the NDE1/LIS1/NDEL1 complex is DISC1-PDE4 modulated and likely to regulate its neural functions.
NDE1 and NDEL1: multimerisation, alternate splicing and DISC1 interaction.
Nuclear Distribution Factor E Homolog 1 (NDE1) and NDE-Like 1 (NDEL1) are highly homologous mammalian proteins. However, whereas NDEL1 is well studied, there is remarkably little known about NDE1. We demonstrate the presence of multiple isoforms of both NDE1 and NDEL1 in the brain, showing that NDE1 binds directly to multiple isoforms of Disrupted in Schizophrenia 1 (DISC1), and to itself. We also show that NDE1 can complex with NDEL1. Together these results predict a high degree of complexity of DISC1-mediated regulation of neuronal activity.
DISC1, PDE4B, and NDE1 at the centrosome and synapse.
Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at synapses in cultured neurons. NDE1 is phosphorylated by cAMP-dependant Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.
A multiregional proteomic survey of the postnatal human brain.
Detailed observations of transcriptional, translational and post-translational events in the human brain are essential to improving our understanding of its development, function and vulnerability to disease. Here, we exploited label-free quantitative tandem mass-spectrometry to create an in-depth proteomic survey of regions of the postnatal human brain, ranging in age from early infancy to adulthood. Integration of protein data with existing matched whole-transcriptome sequencing (RNA-seq) from the BrainSpan project revealed varied patterns of protein-RNA relationships, with generally increased magnitudes of protein abundance differences between brain regions compared to RNA. Many of the differences amplified in protein data were reflective of cytoarchitectural and functional variation between brain regions. Comparing structurally similar cortical regions revealed significant differences in the abundances of receptor-associated and resident plasma membrane proteins that were not readily observed in the RNA expression data.
Maternal separation with early weaning: a rodent model providing novel insights into neglect associated developmental deficits.
Child neglect is the most prevalent form of child maltreatment in the United States, and poses a serious public health concern. Children who survive such episodes go on to experience long-lasting psychological and behavioral problems, including higher rates of post-traumatic stress disorder symptoms, depression, alcohol and drug abuse, attention-deficit/hyperactivity disorder, and cognitive deficits. To date, most research into the causes of these life-long problems has focused on well-established targets such as stress responsive systems, including the hypothalamus-pituitary-adrenal axis. Using the maternal separation and early weaning model, we have attempted to provide comprehensive molecular profiling of a model of early-life neglect in an organism amenable to genomic manipulation: the mouse. In this article, we report new findings generated with this model using chromatin immunoprecipitation sequencing, diffuse tensor magnetic resonance imaging, and behavioral analyses. We also review the validity of the maternal separation and early weaning model, which reflects behavioral deficits observed in neglected humans including hyperactivity, anxiety, and attentional deficits. Finally, we summarize the molecular characterization of these animals, including RNA profiling and label-free proteomics, which highlight protein translation and myelination as novel pathways of interest.
VGF as a biomarker and therapeutic target in neurodegenerative and psychiatric diseases.
Neurosecretory protein VGF (non-acronymic) belongs to the granin family of neuropeptides. VGF and VGF-derived peptides have been repeatedly identified in well-powered and well-designed multi-omic studies as dysregulated in neurodegenerative and psychiatric diseases. New therapeutics is urgently needed for these devastating and costly diseases, as are new biomarkers to improve disease diagnosis and mechanistic understanding. From a list of 537 genes involved in Alzheimer's disease pathogenesis, VGF was highlighted by the Accelerating Medicines Partnership in Alzheimer's disease as the potential therapeutic target of greatest interest. VGF levels are consistently decreased in brain tissue and CSF samples from patients with Alzheimer's disease compared to controls, and its levels correlate with disease severity and Alzheimer's disease pathology. In the brain, VGF exists as multiple functional VGF-derived peptides. Full-length human VGF1-615 undergoes proteolytic processing by prohormone convertases and other proteases in the regulated secretory pathway to produce at least 12 active VGF-derived peptides. In cell and animal models, these VGF-derived peptides have been linked to energy balance regulation, neurogenesis, synaptogenesis, learning and memory, and depression-related behaviours throughout development and adulthood. The C-terminal VGF-derived peptides, TLQP-62 (VGF554-615) and TLQP-21 (VGF554-574) have differential effects on Alzheimer's disease pathogenesis, neuronal and microglial activity, and learning and memory. TLQP-62 activates neuronal cell-surface receptors and regulates long-term hippocampal memory formation. TLQP-62 also prevents immune-mediated memory impairment, depression-like and anxiety-like behaviours in mice. TLQP-21 binds to microglial cell-surface receptors, triggering microglial chemotaxis and phagocytosis. These actions were reported to reduce amyloid-β plaques and decrease neuritic dystrophy in a transgenic mouse model of familial Alzheimer's disease. Expression differences of VGF-derived peptides have also been associated with frontotemporal lobar dementias, amyotrophic lateral sclerosis, Lewy body diseases, Huntington's disease, pain, schizophrenia, bipolar disorder, depression and antidepressant response. This review summarizes current knowledge and highlights questions for future investigation regarding the roles of VGF and its dysregulation in neurodegenerative and psychiatric disease. Finally, the potential of VGF and VGF-derived peptides as biomarkers and novel therapeutic targets for neurodegenerative and psychiatric diseases is highlighted.
Synaptic proteins associated with cognitive performance and neuropathology in older humans revealed by multiplexed fractionated proteomics.
Alzheimer's disease (AD) is defined by the presence of abundant amyloid-β (Aβ) and tau neuropathology. While this neuropathology is necessary for AD diagnosis, it is not sufficient for causing cognitive impairment. Up to one third of community dwelling older adults harbor intermediate to high levels of AD neuropathology at death yet demonstrate no significant cognitive impairment. Conversely, there are individuals who exhibit dementia with no gross explanatory neuropathology. In prior studies, synapse loss correlated with cognitive impairment. To understand how synaptic composition changes in relation to neuropathology and cognition, multiplexed liquid chromatography mass-spectrometry was used to quantify enriched synaptic proteins from the parietal association cortex of 100 subjects with contrasting levels of AD pathology and cognitive performance. 123 unique proteins were significantly associated with diagnostic category. Functional analysis showed enrichment of serotonin release and oxidative phosphorylation categories in normal (cognitively unimpaired, low neuropathology) and "resilient" (unimpaired despite AD pathology) individuals. In contrast, frail individuals, (low pathology, impaired cognition) showed a metabolic shift towards glycolysis and increased presence of proteasome subunits.
Isoform-Level Interpretation of High-Throughput Proteomics Data Enabled by Deep Integration with RNA-seq.
Cellular control of gene expression is a complex process that is subject to multiple levels of regulation, but ultimately it is the protein produced that determines the biosynthetic state of the cell. One way that a cell can regulate the protein output from each gene is by expressing alternate isoforms with distinct amino acid sequences. These isoforms may exhibit differences in localization and binding interactions that can have profound functional implications. High-throughput liquid chromatography tandem mass spectrometry proteomics (LC-MS/MS) relies on enzymatic digestion and has lower coverage and sensitivity than transcriptomic profiling methods such as RNA-seq. Digestion results in predictable fragmentation of a protein, which can limit the generation of peptides capable of distinguishing between isoforms. Here we exploit transcript-level expression from RNA-seq to set prior likelihoods and enable protein isoform abundances to be directly estimated from LC-MS/MS, an approach derived from the principle that most genes appear to be expressed as a single dominant isoform in a given cell type or tissue. Through this deep integration of RNA-seq and LC-MS/MS data from the same sample, we show that a principal isoform can be identified in >80% of gene products in homogeneous HEK293 cell culture and >70% of proteins detected in complex human brain tissue. We demonstrate that the incorporation of translatome data from ribosome profiling further refines this process. Defining isoforms in experiments with matched RNA-seq/translatome and proteomic data increases the functional relevance of such data sets and will further broaden our understanding of multilevel control of gene expression.
Proteomic Approaches for the Discovery of Biofluid Biomarkers of Neurodegenerative Dementias.
Neurodegenerative dementias are highly complex disorders driven by vicious cycles of intersecting pathophysiologies. While most can be definitively diagnosed by the presence of disease-specific pathology in the brain at postmortem examination, clinical disease presentations often involve substantially overlapping cognitive, behavioral, and functional impairment profiles that hamper accurate diagnosis of the specific disease. As global demographics shift towards an aging population in developed countries, clinicians need more sensitive and specific diagnostic tools to appropriately diagnose, monitor, and treat neurodegenerative conditions. This review is intended as an overview of how modern proteomic techniques (liquid chromatography mass spectrometry (LC-MS/MS) and advanced capture-based technologies) may contribute to the discovery and establishment of better biofluid biomarkers for neurodegenerative disease, and the limitations of these techniques. The review highlights some of the more interesting technical innovations and common themes in the field but is not intended to be an exhaustive systematic review of studies to date. Finally, we discuss clear reporting principles that should be integrated into all studies going forward to ensure data is presented in sufficient detail to allow meaningful comparisons across studies.
The technical reliability and biotemporal stability of cerebrospinal fluid biomarkers for profiling multiple pathophysiologies in Alzheimer's disease.
OBJECTIVE: Alzheimer's disease (AD) is a complex neurodegenerative disease driven by multiple interacting pathophysiological processes that ultimately results in synaptic loss, neuronal death, and dementia. We implemented a fit-for-purpose modeled approach to qualify a broad selection of commercially available immunoassays and evaluate the biotemporal stability of analytes across five pathophysiological domains of interest in AD, including core amyloid-β (Aβ) and tau AD biomarkers, neurodegeneration, inflammation/immune modulation, neurovascular injury, and metabolism/oxidative stress. METHODS: Paired baseline and eight-week CSFs from twenty participants in a clinical drug trial for mild cognitive impairment (MCI) or mild dementia due to AD were used to evaluate sensitivity, intra-assay precision, inter-assay replicability, and eight-week biotemporal stability for sixty unique analytes measured with commercially available single- and multi-plex ELISA assays. Coefficients of variation (CV) were calculated, and intraclass correlation and Wilcoxon signed rank tests were applied. RESULTS: We identified 32 biomarker candidates with good to excellent performance characteristics according to assay technical performance and CSF analyte biotemporal stability cut-off criteria. These included: 1) the core AD biomarkers Aβ1-42, Aβ1-40, Aβ1-38, and total tau; 2) non-Aβ, non-tau neurodegeneration markers NfL and FABP3; 3) inflammation/immune modulation markers IL-6, IL-7, IL-8, IL-12/23p40, IL-15, IL-16, MCP-1, MDC, MIP-1β, and YKL-40; 4) neurovascular markers Flt-1, ICAM-1, MMP-1, MMP-2, MMP-3, MMP-10, PlGF, VCAM-1, VEGF, VEGF-C, and VEGF-D; and 5) metabolism/oxidative stress markers 24-OHC, adiponectin, leptin, soluble insulin receptor, and 8-OHdG. CONCLUSIONS: Assays for these CSF analytes demonstrate consistent sensitivity, reliability, and biotemporally stability for use in a multiple pathophysiological CSF biomarker panel to profile AD. Their qualification enables further investigation for use in AD diagnosis, staging and progression, disease mechanism profiling, and clinical trials.
Mass spectrometry in cerebrospinal fluid uncovers association of glycolysis biomarkers with Alzheimer's disease in a large clinical sample.
Alzheimer's disease (AD) is a complex and heterogeneous neurodegenerative disorder with contributions from multiple pathophysiological pathways. One of the long-recognized and important features of AD is disrupted cerebral glucose metabolism, but the underlying molecular basis remains unclear. In this study, unbiased mass spectrometry was used to survey CSF from a large clinical cohort, comparing patients who are either cognitively unimpaired (CU; n = 68), suffering from mild-cognitive impairment or dementia from AD (MCI-AD, n = 95; DEM-AD, n = 72), or other causes (MCI-other, n = 77; DEM-other, n = 23), or Normal Pressure Hydrocephalus (NPH, n = 57). The results revealed changes related to altered glucose metabolism. In particular, two glycolytic enzymes, pyruvate kinase (PKM) and aldolase A (ALDOA), were found to be upregulated in CSF from patients with AD compared to those with other neurological conditions. Increases in full-length PKM and ALDOA levels in CSF were confirmed with immunoblotting. Levels of these enzymes furthermore correlated negatively with CSF glucose in matching CSF samples. PKM levels were also found to be increased in AD in publicly available brain-tissue data. These results indicate that ALDOA and PKM may act as technically-robust potential biomarkers of glucose metabolism dysregulation in AD.
BERNN: Enhancing classification of Liquid Chromatography Mass Spectrometry data with batch effect removal neural networks.
Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful method for profiling complex biological samples. However, batch effects typically arise from differences in sample processing protocols, experimental conditions, and data acquisition techniques, significantly impacting the interpretability of results. Correcting batch effects is crucial for the reproducibility of omics research, but current methods are not optimal for the removal of batch effects without compressing the genuine biological variation under study. We propose a suite of Batch Effect Removal Neural Networks (BERNN) to remove batch effects in large LC-MS experiments, with the goal of maximizing sample classification performance between conditions. More importantly, these models must efficiently generalize in batches not seen during training. A comparison of batch effect correction methods across five diverse datasets demonstrated that BERNN models consistently showed the strongest sample classification performance. However, the model producing the greatest classification improvements did not always perform best in terms of batch effect removal. Finally, we show that the overcorrection of batch effects resulted in the loss of some essential biological variability. These findings highlight the importance of balancing batch effect removal while preserving valuable biological diversity in large-scale LC-MS experiments.
Interrogation of the human cortical peptidome uncovers cell-type specific signatures of cognitive resilience against Alzheimer's disease.
Alzheimer's disease (AD) is characterised by age-related cognitive decline. Brain accumulation of amyloid-β plaques and tau tangles is required for a neuropathological AD diagnosis, yet up to one-third of AD-pathology positive community-dwelling elderly adults experience no symptoms of cognitive decline during life. Conversely, some exhibit chronic cognitive impairment in absence of measurable neuropathology, prompting interest into cognitive resilience-retained cognition despite significant neuropathology-and cognitive frailty-impaired cognition despite low neuropathology. Synapse loss is widespread within the AD-dementia, but not AD-resilient, brain. Recent evidence points towards critical roles for synaptic proteins, such as neurosecretory VGF, in cognitive resilience. However, VGF and related proteins often signal as peptide derivatives. Here, nontryptic peptidomic mass spectrometry was performed on 102 post-mortem cortical samples from individuals across cognitive and neuropathological spectra. Neuropeptide signalling proteoforms derived from VGF, somatostatin (SST) and protachykinin-1 (TAC1) showed higher abundance in AD-resilient than AD-dementia brain, whereas signalling proteoforms of cholecystokinin (CCK) and chromogranin (CHG) A/B and multiple cytoskeletal molecules were enriched in frail vs control brain. Integrating our data with publicly available single nuclear RNA sequencing (snRNA-seq) showed enrichment of cognition-related genes in defined cell-types with established links to cognitive resilience, including SST interneurons and excitatory intratelencephalic cells.
Plasma VEGFA and PGF impact longitudinal tau and cognition in preclinical Alzheimer's disease.
Vascular dysfunction is increasingly recognized as an important contributor to the pathogenesis of Alzheimer's disease. Alterations in vascular endothelial growth factor (VEGF) pathways have been implicated as potential mechanisms. However, the specific impact of VEGF proteins in preclinical Alzheimer's disease and their relationships with other Alzheimer's disease and vascular pathologies during this critical early period remain to be elucidated. We included 317 older adults from the Harvard Aging Brain Study, a cohort of individuals who were cognitively unimpaired at baseline and followed longitudinally for up to 12 years. Baseline VEGF family protein levels (VEGFA, VEGFC, VEGFD, PGF and FLT1) were measured in fasting plasma using high-sensitivity immunoassays. Using linear mixed effects models, we examined the interactive effects of baseline plasma VEGF proteins and amyloid PET burden (Pittsburgh Compound-B) on longitudinal cognition (Preclinical Alzheimer Cognitive Composite-5). We further investigated if effects on cognition were mediated by early neocortical tau accumulation (flortaucipir PET burden in the inferior temporal cortex) or hippocampal atrophy. Lastly, we examined the impact of adjusting for baseline cardiovascular risk score or white matter hyperintensity volume. Baseline plasma VEGFA and PGF each showed a significant interaction with amyloid burden on prospective cognitive decline. Specifically, low VEGFA and high PGF were associated with greater cognitive decline in individuals with elevated amyloid, i.e. those on the Alzheimer's disease continuum. Concordantly, low VEGFA and high PGF were associated with accelerated longitudinal tau accumulation in those with elevated amyloid. Moderated mediation analyses confirmed that accelerated tau accumulation fully mediated the effects of low VEGFA and partially mediated (31%) the effects of high PGF on faster amyloid-related cognitive decline. The effects of VEGFA and PGF on tau and cognition remained significant after adjusting for cardiovascular risk score or white matter hyperintensity volume. There were concordant but non-significant associations with longitudinal hippocampal atrophy. Together, our findings implicate low VEGFA and high PGF in accelerating early neocortical tau pathology and cognitive decline in preclinical Alzheimer's disease. Additionally, our results underscore the potential of these minimally-invasive plasma biomarkers to inform the risk of Alzheimer's disease progression in the preclinical population. Importantly, VEGFA and PGF appear to capture distinct effects from vascular risks and cerebrovascular injury. This highlights their potential as new therapeutic targets, in combination with anti-amyloid and traditional vascular risk reduction therapies, to slow the trajectory of preclinical Alzheimer's disease and delay or prevent the onset of cognitive decline.
Evaluation of serological lateral flow assays for severe acute respiratory syndrome coronavirus-2.
BACKGROUND: COVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed. METHODS: We evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays' performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence. RESULTS: Combined IgG + IgM sensitivities ranged from 33.9 to 94.6%, while combined specificities ranged from 92.6 to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG + IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG + IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 μg/mL), followed by a similar LOD of 1.5 μg/mL for CareHealth, Cellex, KHB, and Vivachek. CONCLUSION: We provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.
