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Of those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ~ 10% develop the chronic post-viral debilitating condition, long COVID (LC). Although LC is a heterogeneous condition, about half of cases have typical post-viral fatigue with onset and symptoms that are very similar to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). A key question is whether these conditions are closely related. ME/CFS is a post-stressor fatigue condition that arises from multiple triggers. To investigate the pathophysiology of LC, a pilot study of patients (n = 6) and healthy controls (n = 5) has used quantitative proteomics to discover changes in peripheral blood mononuclear cell (PBMC) proteins. A principal component analysis separated all long COVID patients from healthy controls. Analysis of 3131 proteins identified 162 proteins differentially regulated, of which 37 were related to immune functions, and 21 to mitochondrial functions. Markov cluster analysis identified clusters involved in immune system processes, and two aspects of gene expression-spliceosome and transcription. These results were compared with an earlier dataset of 346 differentially regulated proteins in PBMC’s from ME/CFS patients (n = 9) analysed by the same methodology. There were overlapping protein clusters and enriched molecular pathways particularly in immune functions, suggesting the two conditions have similar immune pathophysiology as a prominent feature, and mitochondrial functions involved in energy production were affected in both conditions.

Post-viral conditions, Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Long COVID (LC), share > 95% of their symptoms, but the connection between disturbances in their underlying molecular biology is unclear. This study investigates DNA methylation patterns in peripheral blood mononuclear cells (PBMC) from patients with ME/CFS, LC, and healthy controls (HC). Reduced Representation Bisulphite Sequencing (RRBS) was applied to the DNA of age- and sex-matched cohorts: ME/CFS (n = 5), LC (n = 5), and HC (n = 5). The global DNA methylomes of the three cohorts were similar and spread equally across all chromosomes, except the sex chromosomes, but there were distinct minor changes in the exons of the disease cohorts towards more hypermethylation. A principal component analysis (PCA) analysing significant methylation changes (p < 0.05) separated the ME/CFS, LC, and HC cohorts into three distinct clusters. Analysis with a limit of >10% methylation difference and at p < 0.05 identified 214 Differentially Methylated Fragments (DMF) in ME/CFS, and 429 in LC compared to HC. Of these, 118 DMFs were common to both cohorts. Those in promoters and exons were mainly hypermethylated, with a minority hypomethylated. There were rarer examples with either no change in methylation in ME/CFS but a change in LC, or a methylation change in ME/CFS but in the opposite direction in LC. The differential methylation in a number of fragments was significantly greater in the LC cohort than in the ME/CFS cohort. Our data reveal a generally shared epigenetic makeup between ME/CFS and LC but with specific, distinct changes. Differences between the two cohorts likely reflect the stage of the disease from onset (LC 1 year vs. ME/CFS 12 years), but specific changes imposed by the SARS-CoV-2 virus in the case of the LC patients cannot be discounted. These findings provide a foundation for further studies with larger cohorts at the same disease stage and for functional analyses to establish clinical relevance.

Post-exertional malaise (PEM) is a defining symptom of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), yet its molecular underpinnings remain elusive. This study investigated the temporal–longitudinal DNA methylation changes associated with PEM using a structured two-day maximum repeated effort cardiopulmonary exercise testing (CPET) protocol involving pre- and two post-exercise blood samplings from five ME/CFS patients. Cardiopulmonary measurements revealed complex heterogeneous profiles among the patients compared to typical healthy controls, and VO2 peak indicated all patients had poor normative fitness. The switch to anaerobic metabolism occurred at a lower workload in some patients on Day Two of the test. Reduced Representation Bisulphite Sequencing followed by analysis with Differential Methylation Analysis Package-version 2 (DMAP2) identified differentially methylated fragments (DMFs) present in the DNA genomes of all five ME/CFS patients through the exercise test compared with ‘before exercise’. With further filtering for >10% methylation differences, there were early DMFs (0–24 h after first exercise test) and late DMFs between (24–48 h after the second exercise test), as well as DMFs that changed gradually (between 0 and 48 h). Of these, 98% were ME/CFS-specific, compared with the two healthy controls accompanying the longitudinal study. Principal component analysis illustrated the three distinct clusters at the 0 h, 24 h, and 48 h timepoints, but with heterogeneity among the patients within the clusters, highlighting dynamic methylation responses to exertion in individual patients. There were 24 ME/CFS-specific DMFs at gene promoter fragments that revealed distinct patterns of temporal methylation across the timepoints. Functional enrichment of ME-specific DMFs revealed pathways involved in endothelial function, morphogenesis, inflammation, and immune regulation. These findings uncovered temporally dynamic epigenetic changes in stress/immune functions in ME/CFS during PEM and suggest molecular signatures with potential for diagnosis and of mechanistic significance.

Secreted amyloid precursor protein alpha (sAPP) processed from a parent human brain protein, APP, can modulate learning and memory. It has potential for development of a therapy preventing, delaying, or even reversing Alzheimer’s disease. In this study a comprehensive analysis to understand how it affects the transcriptome and proteome of the human neuron was undertaken. Human iPSC-derived glutamatergic neurons in culture were exposed to 1 nM sAPP over a time course and changes in the transcriptome and proteome were identified with RNA sequencing and SWATH-MS respectively. A large subset (~30%) of differentially expressed transcripts and proteins were functionally involved with the molecular biology of learning and memory, consistent with reported links of sAPP to memory enhancement, as well as neurogenic, neurotrophic and neuroprotective phenotypes in previous studies. Differentially expressed proteins included those encoded in previously identified Alzheimer’s risk genes, APP processing related proteins, proteins involved in synaptogenesis, neurotransmitters, receptors, synaptic vesicle proteins, cytoskeletal proteins, proteins involved in protein and organelle trafficking, and proteins important for cell signalling, transcriptional splicing, and functions of the proteosome and lysosome. We have identified a complex set of genes affected by sAPP, which may aid further investigation into the mechanism of how this neuroprotective protein affects memory formation and how it might be used as an Alzheimer’s disease therapy.

Alzheimer's disease (AD), the most common chronic neurodegenerative disorder, has complex neuropathology. The principal neuropathological hallmarks of the disease are the deposition of extracellular β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) comprised of hyperphosphorylated tau (p-tau) protein. These changes occur with neuroinflammation, a compromised blood-brain barrier (BBB) integrity, and neuronal synaptic dysfunction, all of which ultimately lead to neuronal cell loss and cognitive deficits in AD. Aβ was stereotaxically administered bilaterally into the CA1 region of the hippocampi of 18-month-old male C57BL/6 mice. This study aimed to characterize, utilizing immunohistochemistry and behavioral testing, the spatial and temporal effects of Aβ on a broad set of parameters characteristic of AD: p-tau, neuroinflammation, vascular pathology, pyramidal cell survival, and behavior. Three days after Aβ injection and before significant neuronal cell loss was detected, acute neuroinflammatory and vascular responses were observed. These responses included the up-regulation of glial fibrillary acidic protein (GFAP), cell adhesion molecule-1 (PECAM-1, also known as CD31), fibrinogen labeling, and an increased number of activated astrocytes and microglia in the CA1 region of the hippocampus. From day 7, there was significant pyramidal cell loss in the CA1 region of the hippocampus, and by 30 days, significant localized up-regulation of p-tau, GFAP, Iba-1, CD31, and alpha-smooth muscle actin (α-SMA) in the Aβ -injected mice compared with controls. These molecular changes in Aβ -injected mice were accompanied by cognitive deterioration, as demonstrated by long-term spatial memory impairment. This study is reporting a comprehensive examination of a complex set of parameters associated with intrahippocampal administration of Aβ in mice, their spatiotemporal interactions and combined contribution to the disease progression. We show that a single Aβ injection can reproduce aspects of the inflammatory, vascular, and p-tau induced pathology occurring in the AD human brain that lead to cognitive deficits.

Alzheimer’s disease (AD) is a neurodegenerative disorder with an increasing need for developing disease-modifying treatments as current therapies only provide marginal symptomatic relief. Recent evidence suggests the γ-aminobutyric acid (GABA) neurotransmitter system undergoes remodeling in AD, disrupting the excitatory/inhibitory (E/I) balance in the brain. Altered expression levels of K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), which are cation–chloride cotransporters (CCCs), have been implicated in disrupting GABAergic activity by regulating GABAA receptor signaling polarity in several neurological disorders, but these have not yet been explored in AD. NKCC1 and KCC2 regulate intracellular chloride [Cl−]i by accumulating and extruding Cl−, respectively. Increased NKCC1 expression in mature neurons has been reported in these disease conditions, and bumetanide, an NKCC1 inhibitor, is suggested to show potential therapeutic benefits. This study used primary mouse hippocampal neurons to explore if KCC2 and NKCC1 expression levels are altered following beta-amyloid (Aβ1-42) treatment and the potential neuroprotective effects of bumetanide. KCC2 and NKCC1 expression levels were also examined in 18-months-old male C57BL/6 mice following bilateral hippocampal Aβ1-42 stereotaxic injection. No change in KCC2 and NKCC1 expression levels were observed in mouse hippocampal neurons treated with 1 nM Aβ1-42, but NKCC1 expression increased 30-days post-Aβ1-42-injection in the CA1 region of the mouse hippocampus. Primary mouse hippocampal cultures were treated with 1 nM Aβ1-42 alone or with various concentrations of bumetanide (1 µM, 10 µM, 100 µM, 1 mM) to investigate the effect of the drug on cell viability. Aβ1-42 produced 53.1 ± 1.4% cell death after 5 days, and the addition of bumetanide did not reduce this. However, the drug at all concentrations significantly reduced cell viability, suggesting bumetanide is highly neurotoxic. In summary, these results suggest that chronic exposure to Aβ1-42 alters the balance of KCC2 and NKCC1 expression in a region-and layer-specific manner in mouse hippocampal tissue; therefore, this process most likely contributes to altered hippocampal E/I balance in this model. Furthermore, bumetanide induces hippocampal neurotoxicity, thus questioning its suitability for AD therapy. Further investigations are required to examine the effects of Aβ1-42 on KCC2 and NKCC1 expression and whether targeting CCCs might offer a therapeutic approach for AD.

Alzheimer’s disease (AD) is a complex and chronic neurodegenerative disorder that involves a progressive and severe decline in cognition and memory. During the last few decades a considerable amount of research has been done in order to better understand tau-pathology, inflammatory activity and neuronal synapse loss in AD, all of them contributing to cognitive decline. Early hippocampal network dysfunction is one of the main factors associated with cognitive decline in AD. Much has been published about amyloid-beta1-42 (Aβ1-42)-mediated excitotoxicity in AD. However, increasing evidence demonstrates that the remodeling of the inhibitory gamma-aminobutyric acid (GABAergic) system contributes to the excitatory/inhibitory (E/I) disruption in the AD hippocampus, but the underlying mechanisms are not well understood. In the present study, we show that hippocampal injection of Aβ1-42 is sufficient to induce cognitive deficits 7 days post-injection. We demonstrate using in vitro whole-cell patch-clamping an increased inhibitory GABAergic tonic conductance mediated by extrasynaptic type A GABA receptors (GABAARs), recorded in the CA1 region of the mouse hippocampus following Aβ1-42 micro injection. Such alterations in GABA neurotransmission and/or inhibitory GABAARs could have a significant impact on both hippocampal structure and function, causing E/I balance disruption and potentially contributing to cognitive deficits in AD.

Alzheimer’s Disease (AD) is the leading type of dementia worldwide. Despite an increasing burden of disease due to a rapidly ageing population, there is still a lack of complete understanding of the precise pathological mechanisms which drive its progression. Glutamate is the main excitatory neurotransmitter in the brain and plays an essential role in the normal function and excitability of neuronal networks. While previous studies have shown alterations in the function of the glutamatergic system in AD, the underlying etiology of beta amyloid (Aβ1-42) induced changes has not been explored. Here we have investigated the acute effects of stereotaxic hippocampal Aβ1-42 injection on specific glutamatergic receptors and transporters in the mouse hippocampus, using immunohistochemistry and confocal microscopy 3 days after Aβ1-42 injection in aged male C57BL/6 mice before the onset of neuronal cell death. We show that acute injection of Aβ1-42 is sufficient to induce cognitive deficits 3 days post-injection. We also report no significant changes in glutamate receptor subunits GluA1, GluA2, VGluT1, and VGluT2 in response to acute injection of Aβ1-42 when compared with the ACSF-vehicle injected mice. However, we observed increased expression in the DG hilus and ventral stratum (str.) granulosum, CA3 str. radiatum and str. oriens, and CA1 str. radiatum of the GluN1 subunit, and increased expression within the CA3 str. radiatum and decreased expression within the DG str. granulosum of the GluN2A subunit in Aβ1-42 injected mice compared to NC, and a similar trend observed when compared to ACSF injected mice. We also observed alterations in expression patterns of glutamatergic receptor subunits and transporters within specific layers of hippocampal subregions in response to a microinjection stimulus. These findings indicate that the pathological alterations in the glutamatergic system observed in AD are likely to be partially a result of both acute and chronic exposure to Aβ1-42 and implies a much more complex circuit mechanism associated with glutamatergic dysfunction than simply glutamate-mediated excitotoxic neuronal death.

Alzheimer’s disease (AD) is a neurodegenerative disease driven in large part by accumulated deposits in the brain of the amyloid precursor protein (APP) cleavage product amyloid-β peptide (Aβ). However, AD is also characterised by reductions in secreted amyloid precursor protein-alpha (sAPPα), an alternative cleavage product of APP. In contrast to the neurotoxicity of accumulated Αβ, sAPPα has many neuroprotective and neurotrophic properties. Increasing sAPPα levels has the potential to serve as a therapeutic treatment that mitigates the effects of Aβ and rescue cognitive function. Here we tested the hypothesis that lentivirus-mediated expression of a human sAPPα construct in a mouse model of AD (APPswe/PS1dE9), begun before the onset of plaque pathology, could prevent later behavioural and electrophysiological deficits. Male mice were given bilateral intra-hippocampal injections at 4 months of age and tested 8–10 months later. Transgenic mice expressing sAPPα performed significantly better than untreated littermates in all aspects of the spatial water maze task. Expression of sAPPα also resulted in partial rescue of long-term potentiation (LTP), tested in vitro. These improvements occurred in the absence of changes in amyloid pathology. Supporting these findings on LTP, lentiviral-mediated expression of sAPPα for 3 months from 10 months of age, or acute sAPPα treatment in hippocampal slices from 18 to 20 months old transgenic mice, completely reversed the deficits in LTP. Together these findings suggest that sAPPα has wide potential to act as either a preventative or restorative therapeutic treatment in AD by mitigating the effects of Aβ toxicity and enhancing cognitive reserve.

Secreted amyloid precursor protein-α (sAPPα) is a neuroprotective and memory-enhancing molecule, however, the mechanisms through which sAPPα promotes these effects are not well understood. Recently, we have shown that sAPPα enhances cell-surface expression of glutamate receptors. Activity-related cytoskeletal-associated protein Arc (Arg3.1) is an immediate early gene capable of modulating long-term potentiation, long-term depression and homeostatic plasticity through regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor localization. Accordingly, we hypothesized that sAPPα may enhance synaptic plasticity, in part, by the synthesis of Arc. Using primary cortical and hippocampal neuronal cultures we found that sAPPα (1 nM, 2 h) enhances levels of mRNA and protein. Arc protein levels were increased in both the neuronal somata and dendrites in a Ca /calmodulin-dependent protein kinase II-dependent manner. Additionally, dendritic Arc expression was dependent upon activation of mitogen-activated protein kinase and protein kinase G. The enhancement of dendritic Arc protein was significantly reduced by antagonism of -methyl-D-aspartate (NMDA) and nicotinic acetylcholine (α7nACh) receptors, and fully eliminated by dual application of these antagonists. This effect was further corroborated in area CA1 of acute hippocampal slices. These data suggest sAPPα-regulated plasticity within hippocampal neurons is mediated by cooperation of NMDA and α7nACh receptors to engage a cascade of signal transduction molecules to enhance the transcription and translation of Arc.