A pilot study on the immune cell proteome of long COVID patients shows changes to physiological pathways similar to those in myalgic encephalomyelitis/chronic fatigue syndrome.

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.

Towards a Better Understanding of the Complexities of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Long COVID.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex condition arising in susceptible people, predominantly following viral infection, but also other stressful events. The susceptibility factors discussed here are both genetic and environmental although not well understood. While the dysfunctional physiology in ME/CFS is becoming clearer, understanding has been hampered by different combinations of symptoms in each affected person. A common core set of mainly neurological symptoms forms the modern clinical case definition, in the absence of an accessible molecular diagnostic test. This landscape has prompted interest in whether ME/CFS patients can be classified into a particular phenotype/subtype that might assist better management of their illness and suggest preferred therapeutic options. Currently, the same promising drugs, nutraceuticals, or behavioral therapies available can be beneficial, have no effect, or be detrimental to each individual patient. We have shown that individuals with the same disease profile exhibit unique molecular changes and physiological responses to stress, exercise and even vaccination. Key features of ME/CFS discussed here are the possible mechanisms determining the shift of an immune/inflammatory response from transient to chronic in ME/CFS, and how the brain and CNS manifests the neurological symptoms, likely with activation of its specific immune system and resulting neuroinflammation. The many cases of the post viral ME/CFS-like condition, Long COVID, following SARS-CoV-2 infection, and the intense research interest and investment in understanding this condition, provide exciting opportunities for the development of new therapeutics that will benefit ME/CFS patients.

Dynamic Epigenetic Changes during a Relapse and Recovery Cycle in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex disease with variable severity. Patients experience frequent relapses where symptoms increase in severity, leaving them with a marked reduction in quality of life. Previous work has investigated molecular differences between ME/CFS patients and healthy controls, but not the dynamic changes specific to each individual patient. We applied precision medicine here to map genomic changes in two selected ME/CFS patients through a period that contained a relapse recovery cycle. DNA was isolated from two patients and a healthy age/gender matched control at regular intervals and captured the patient relapse in each case. Reduced representation DNA methylation sequencing profiles were obtained spanning the relapse recovery cycle. Both patients showed a significantly larger methylome variability (10-20-fold) through the period of sampling compared with the control. During the relapse, changes in the methylome profiles of the two patients were detected in regulatory-active regions of the genome that were associated, respectively, with 157 and 127 downstream genes, indicating disturbed metabolic, immune and inflammatory functions. Severe health relapses in the ME/CFS patients resulted in functionally important changes in their DNA methylomes that, while differing between the two patients, led to very similar compromised physiology. DNA methylation as a signature of disease variability in ongoing ME/CFS may have practical applications for strategies to decrease relapse frequency.

Effect of soluble amyloid precursor protein-alpha on adult hippocampal neurogenesis in a mouse model of Alzheimer's disease.

Soluble amyloid precursor protein-alpha (sAPPα) is a regulator of neuronal and memory mechanisms, while also having neurogenic and neuroprotective effects in the brain. As adult hippocampal neurogenesis is impaired in Alzheimer's disease, we tested the hypothesis that sAPPα delivery would rescue adult hippocampal neurogenesis in an APP/PS1 mouse model of Alzheimer's disease. An adeno-associated virus-9 (AAV9) encoding murine sAPPα was injected into the hippocampus of 8-month-old wild-type and APP/PS1 mice, and later two different thymidine analogues (XdU) were systemically injected to label adult-born cells at different time points after viral transduction. The proliferation of adult-born cells, cell survival after eight weeks, and cell differentiation into either neurons or astrocytes was studied. Proliferation was impaired in APP/PS1 mice but was restored to wild-type levels by viral expression of sAPPα. In contrast, sAPPα overexpression failed to rescue the survival of XdU+-labelled cells that was impaired in APP/PS1 mice, although it did cause a significant increase in the area density of astrocytes in the granule cell layer across both genotypes. Finally, viral expression of sAPPα reduced amyloid-beta plaque load in APP/PS1 mice in the dentate gyrus and somatosensory cortex. These data add further evidence that increased levels of sAPPα could be therapeutic for the cognitive decline in AD, in part through restoration of the proliferation of neural progenitor cells in adults.

Lentivirus-Mediated Expression of Human Secreted Amyloid Precursor Protein-Alpha Promotes Long-Term Induction of Neuroprotective Genes and Pathways in a Mouse Model of Alzheimer's Disease.

BACKGROUND: Secreted amyloid precursor protein-alpha (sAPPα) can enhance memory and is neurotrophic and neuroprotective across a range of disease-associated insults, including amyloid-ß toxicity. In a significant step toward validating sAPPα as a therapeutic for Alzheimer's disease (AD), we demonstrated that long-term overexpression of human sAPPα (for 8 months) in a mouse model of amyloidosis (APP/PS1) could prevent the behavioral and electrophysiological deficits that develop in these mice. OBJECTIVE: To explore the underlying molecular mechanisms responsible for the significant physiological and behavioral improvements observed in sAPPα-treated APP/PS1 mice. METHODS: We assessed the long-term effects on the hippocampal transcriptome following continuous lentiviral delivery of sAPPα or empty-vector to male APP/PS1 mice and wild-type controls using Affymetrix Mouse Transcriptome Assays. Data analysis was carried out within the Affymetrix Transcriptome Analysis Console and an integrated analysis of the resulting transcriptomic data was performed with Ingenuity Pathway analysis (IPA). RESULTS: Mouse transcriptome assays revealed expected AD-associated gene expression changes in empty-vector APP/PS1 mice, providing validation of the assays used for the analysis. By contrast, there were specific sAPPα-associated gene expression profiles which included increases in key neuroprotective genes such as Decorin, betaine-GABA transporter and protocadherin beta-5, subsequently validated by qRT-PCR. An integrated biological pathways analysis highlighted regulation of GABA receptor signaling, cell survival and inflammatory responses. Furthermore, upstream gene regulatory analysis implicated sAPPα activation of Interleukin-4, which can counteract inflammatory changes in AD. CONCLUSION: This study identified key molecular processes that likely underpin the long-term neuroprotective and therapeutic effects of increasing sAPPα levels in vivo.

Treatment of beta amyloid 1-42 (Aß(1-42))-induced basal forebrain cholinergic damage by a non-classical estrogen signaling activator in vivo.

In Alzheimer's disease (AD), there is a loss in cholinergic innervation targets of basal forebrain which has been implicated in substantial cognitive decline. Amyloid beta peptide (Aß(1-42)) accumulates in AD that is highly toxic for basal forebrain cholinergic (BFC) neurons. Although the gonadal steroid estradiol is neuroprotective, the administration is associated with risk of off-target effects. Previous findings suggested that non-classical estradiol action on intracellular signaling pathways has ameliorative potential without estrogenic side effects. After Aß(1-42) injection into mouse basal forebrain, a single dose of 4-estren-3α, 17ß-diol (estren), the non-classical estradiol pathway activator, restored loss of cholinergic cortical projections and also attenuated the Aß(1-42)-induced learning deficits. Estren rapidly and directly phosphorylates c-AMP-response-element-binding-protein and extracellular-signal-regulated-kinase-1/2 in BFC neurons and restores the cholinergic fibers via estrogen receptor-α. These findings indicated that selective activation of non-classical intracellular estrogen signaling has a potential to treat the damage of cholinergic neurons in AD.

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome.

The decoding release factor (RF) triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre (PTC) of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure: the anticodon loop and the loop at the tip of helix alpha5. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.

Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents.

The secreted fragment of the amyloid precursor protein (sAPPalpha) generated following cleavage by alpha-secretase is an important mediator of cell function and is both neurotrophic and neuroprotective. HEK 293T cells have been stably integrated with a fragment of the APP gene to produce and secrete either sAPPalpha, or the alternative cleavage product sAPPbeta. Heparin binding domains on the proteins have been utilised to develop a one-step fast-performance-liquid-chromatography (FPLC) purification of sAPPs from the conditioned media. Immunoblotting analyses with a sAPP specific antibody coupled with highly sensitive silver staining techniques have validated the expression and purification strategy. Functional activity of the purified fragments was demonstrated by their ability to protect COS-7 and SH-SY5Y (neuroblastoma) cells against the adverse effects of glucose deprivation in a cell viability assay. The purified sAPPs also activated the NFkappaB transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPPalpha the more potent activator as expected. The simple protocol to produce these mammalian expressed proteins will facilitate their use as potential neuropharmacological reagents in the elucidation of biochemical pathways modulated by sAPPs, and in the study of Alzheimer's disease mechanisms in general.

Dopamine D1/D5 receptor activation fails to initiate an activity-independent late-phase LTP in rat hippocampus.

The role of dopamine in the hippocampus remains poorly defined. Numerous studies have suggested that it acts as a neuromodulator of late-phase long-term potentiation (L-LTP) in CA1, while other reports controversially indicate that D1/D5 receptor (D1/D5R) activation may directly initiate activity-independent LTP. We have further investigated this putative role of dopamine in area CA1 in rat hippocampal slices using field potential recording techniques. Application of the dopamine D1/D5 receptor agonists SKF 38393 and 6-bromo-APB at 100 microM for 20 min did not induce an activity-independent L-LTP. Varying the incubation conditions still did not permit either SKF 38393 or an alternative D1/D5R agonist, 6-chloro-PB, to induce L-LTP. To further determine if intracellular mechanisms, which may act to limit the expression of LTP, were preventing D1/D5R-induced L-LTP expression, we inhibited protein phosphatase 1 activity by reducing cyclin-dependent kinase 5 (cdk5) inhibition of inhibitor 1. Inhibition of cdk5 by roscovitine (10 microM, 40 min) did not facilitate the ability of SKF 38393 to induce L-LTP in CA1. Biochemical experiments confirmed that the concentration of agonist used significantly elevated intracellular cAMP levels, suggesting that effective D1/D5R activation was achieved. Furthermore, coactivation with NMDA receptors (NMDAR) resulted in a synergistic increase in cAMP. These findings demonstrate that D1/D5R activation in CA1 initiates intracellular second messenger accumulation, but that this is insufficient to induce an activity-independent L-LTP.

Molecular mimicry in the decoding of translational stop signals.

Molecular mimicry was a concept that was revived as we understood more about the ligands that bound to the active center of the ribosome, and the characteristics of the active center itself. It has been particularly useful for the termination phase of protein synthesis, because for many years this major process seemed not only to be out of step) with the initiation and elongation phases but also there were no common features of the process between eubacteria and eukaryotes. As the facts that supported molecular mimicry emerged, it was seen that the protein factors that facilitated polypeptide chain release when the decoding of an mRNA was complete had common features with the ligands involved in the other phases. Moreover, now common features and mechanisms began to emerge between the eubacterial and eukaryotic RFs and suddenly there seemed to be remarkable synergy between the external ligands and commonality in at least some features of the mechanistic prnciples. Almost 10 years after molecular mimicry took hold as a framework concept, we can now see that this idea is probably too simple. For example, structural mimicry can be apparent if there are extensive conformational changes either in the ribosome active center or in the ligand itself or, most likely, both. Early indications are that the bacterial RF may indeed undergo extensive conformational changes from its solution structure to achieve this accommodation. Thus, as important if not more important than structural and functional mimicry among the ligands, might be their accomodation of a common single active center made up of at least three parts to carry out a complex series of reactions. One part of the ribosomal active center is committed to decoding, a second is committed to the chemistry of putting the protein together and releasing it, and a third part, perhaps residing in the subdomains, is committed to binding ligands so that they can perform their respective single or multiple functions. It might be more accurate to regard the decoding RF as the cuckoo taking over the nest that was crafted and honed through evolution by another, the tRNA. A somewhat ungainly RF, perhaps bigger in dimensions than the tRNA, is able, nevertheless, like the cuckoo, to maneuvre into the nest. Perhaps it pushes the nest a little out of shape, but is still able to use the site for its own functions of stop signal decoding and for facilitating the release of the polypeptide. The term molecular mimicry has been dominant in the literature for a period of important advances in the understanding of protein synthesis. When the first structures of the ribosome appeared, the concept survived and was seen to be valid still. Now, we are at the stage of understanding the more detailed molecular interactions between ligands and the rRNA in particular, and how subtle changes in localized spatial orientations of atoms occur within these interactions. The simplicity of the original concept of mimicry will inevitably be blurred by this more detailed analysis. Nevertheless, it has provided a significant set of principles that allowed development of experimental programs to enhance our understanding of the dynamic events at this remarkable active site at the interface between the two subunits of this fascinating cell organelle, the ribosome.

Mapping functionally important motifs SPF and GGQ of the decoding release factor RF2 to the Escherichia coli ribosome by hydroxyl radical footprinting. Implications for macromolecular mimicry and structural changes in RF2.

The function of the decoding release factor (RF) in translation termination is to couple cognate recognition of the stop codon in the mRNA with hydrolysis of the completed polypeptide from its covalently linked tRNA. For this to occur, the RF must interact with specific A-site components of the active centers within both the small and large ribosomal subunits. In this work, we have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor RF2, during translation termination. In the presence of the cognate UGA stop codon, residues flanking the universally conserved (250)GGQ(252) motif of RF2 were each shown to footprint to the large ribosomal subunit, specifically to conserved elements of the peptidyltransferase and GTPase-associated centers. In contrast, residues that flank the putative "peptide anticodon" of RF2, (205)SPF(207), were shown to make a footprint in the small ribosomal subunit at positions within well characterized 16 S rRNA motifs in the vicinity of the decoding center. Within the recently solved crystal structure of E. coli RF2, the GGQ and SPF motifs are separated by 23 A only, a distance that is incompatible with the observed cleavage sites that are up to 100 A apart. Our data suggest that RF2 may undergo gross conformational changes upon ribosome binding, the implications of which are discussed in terms of the mechanism of RF-mediated termination.