RESUMO
Secreted amyloid precursor protein alpha (sAPPα), processed from a parent mammalian brain protein, amyloid precursor protein, can modulate learning and memory. Recently it has been shown to modulate the transcriptome and proteome of human neurons, including proteins with neurological functions. Here, we analysed whether the acute administration of sAPPα facilitated changes in the proteome and secretome of mouse primary astrocytes in culture. Astrocytes contribute to the neuronal processes of neurogenesis, synaptogenesis and synaptic plasticity. Cortical mouse astrocytes in culture were exposed to 1 nM sAPPα, and changes in both the whole-cell proteome (2 h) and the secretome (6 h) were identified with Sequential Window Acquisition of All Theoretical Fragment Ion Spectra-Mass Spectrometry (SWATH-MS). Differentially regulated proteins were identified in both the cellular proteome and secretome that are involved with neurologically related functions of the normal physiology of the brain and central nervous system. Groups of proteins have a relationship to APP and have roles in the modulation of cell morphology, vesicle dynamics and the myelin sheath. Some are related to pathways containing proteins whose genes have been previously implicated in Alzheimer's disease (AD). The secretome is also enriched in proteins related to Insulin Growth Factor 2 (IGF2) signaling and the extracellular matrix (ECM). There is the promise that a more specific investigation of these proteins will help to understand the mechanisms of how sAPPα signaling affects memory formation.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Camundongos , Animais , Humanos , Precursor de Proteína beta-Amiloide/metabolismo , Proteoma/metabolismo , Astrócitos/metabolismo , Secretoma , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Mamíferos/metabolismoRESUMO
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.
Assuntos
COVID-19 , Síndrome de Fadiga Crônica , Humanos , Síndrome de Fadiga Crônica/etiologia , Síndrome de Fadiga Crônica/terapia , Síndrome de Fadiga Crônica/diagnóstico , Síndrome de COVID-19 Pós-Aguda , SARS-CoV-2 , CausalidadeRESUMO
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.
Assuntos
Síndrome de Fadiga Crônica , Epigênese Genética , Epigenômica , Síndrome de Fadiga Crônica/genética , Síndrome de Fadiga Crônica/metabolismo , Humanos , Qualidade de Vida , RecidivaRESUMO
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 (Aß1-42) 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 Aß1-42 stereotaxic injection. No change in KCC2 and NKCC1 expression levels were observed in mouse hippocampal neurons treated with 1 nM Aß1-42, but NKCC1 expression increased 30-days post-Aß1-42-injection in the CA1 region of the mouse hippocampus. Primary mouse hippocampal cultures were treated with 1 nM Aß1-42 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. Aß1-42 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 Aß1-42 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 Aß1-42 on KCC2 and NKCC1 expression and whether targeting CCCs might offer a therapeutic approach for AD.
Assuntos
Bumetanida , Hipocampo , Membro 2 da Família 12 de Carreador de Soluto , Simportadores , Peptídeos beta-Amiloides , Animais , Bumetanida/metabolismo , Bumetanida/farmacologia , Cloretos/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismoRESUMO
The Maillard reaction, a spontaneous 'one pot' reaction between amino acids and reducing sugars that occurs at low reactant concentrations and low temperatures, is a good candidate for having played a role in the origin of life on the Earth. In view of the probability that RNA and DNA were preceded by an evolutionary forerunner with a more straightforward prebiotic synthesis, it is a testament to the prescience of Oró and colleagues that, in 1975, they drew attention to the Maillard reaction, in particular evidence that melanoidin polymers (the end-product of the reaction) contain ' heterocyclic nitrogen compounds similar to the nitrogenous bases' (Nissenbaum in J Mol Evol 6:253-270, 1975). Indeed, reports of the Maillard reaction product, 2-Acetyl-6-(Hydroxymethyl)-5,6-Dihydro-4H-Pyridinone (AHDP), with a structure reminiscent of the pyrimidine nucleobase uracil, suggest the Maillard reaction might have played a key role in the synthesis of components of a proto-RNA polymer, with AHDP and two structurally related products predicted to be similar to uracil in the latter's ability to form non-standard base pair interactions. It is possible that the primary function of these interactions was to allow molecules such as AHDP to separate out of the prebiotic chemical clutter. If this were the case, catalysis, and coding-made possible by the polymerization of proto-nucleoside monomers into linear sequence strings-would have been evolving properties.
Assuntos
Compostos Heterocíclicos , Reação de Maillard , Aminoácidos , Catálise , Origem da Vida , PolimerizaçãoRESUMO
Secreted amyloid precursor protein-alpha (sAPPα) has growth factor-like properties and can modulate long-term potentiation (LTP) and memory. Here, we demonstrate that exposure to sAPPα converts short-lasting LTP into protein-synthesis-dependent late LTP in hippocampal slices from male rats. sAPPß had no discernable effect. We hypothesized that sAPPα facilitated LTP via regulated glutamate receptor trafficking and de novo protein synthesis. We found using a linear mixed model that sAPPα stimulated trafficking of GluA2-lacking AMPARs, as well as NMDARs to the extrasynaptic cell surface, in a calcium/calmodulin-dependent kinase II and protein kinase G-dependent manner. Both cell surface receptor accumulation and LTP facilitation were present even after sAPPα washout and inhibition of receptor trafficking or protein synthesis prevented all these effects. Direct visualization of newly synthesized proteins (FUNCAT-PLA) confirmed the ability of sAPPα to stimulate de novo protein synthesis and revealed GluA1 as one of the upregulated proteins. Therefore, sAPPα generates a coordinated synthesis and trafficking of glutamate receptors to the cell surface that facilitate LTP.SIGNIFICANCE STATEMENT Secreted amyloid precursor protein-alpha (sAPPα) is a neurotrophic and neuroprotective protein that can promote synaptic plasticity and memory, yet the molecular mechanisms underlying these effects are still not well understood. Here, we show that sAPPα facilitates long-term potentiation (LTP) in a concentration-dependent fashion through cellular processes involving de novo protein synthesis and trafficking of both GluA2-lacking AMPARs and NMDARs to the extrasynaptic cell surface. sAPPα also enhances GluA1, but not GluA2, synthesis. The trafficking effects, along with the LTP facilitation, persist after sAPPα washout, revealing a metaplastic capability of exogenous sAPPα administration. sAPPα thus facilitates LTP through coordinated activation of protein synthesis and trafficking of glutamate receptors to the cell surface, where they are positioned for priming LTP.
Assuntos
Precursor de Proteína beta-Amiloide/farmacologia , Hipocampo/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Biossíntese de Proteínas/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Alzheimer's disease (AD) is the leading type of dementia worldwide. With an increasing burden of an aging population coupled with the lack of any foreseeable cure, AD warrants the current intense research effort on the toxic effects of an increased concentration of beta-amyloid (Aß) in the brain. Glutamate is the main excitatory brain neurotransmitter and it plays an essential role in the function and health of neurons and neuronal excitability. While previous studies have shown alterations in expression of glutamatergic signaling components in AD, the underlying mechanisms of these changes are not well understood. This is the first comprehensive anatomical study to characterize the subregion- and cell layer-specific long-term effect of Aß1-42 on the expression of specific glutamate receptors and transporters in the mouse hippocampus, using immunohistochemistry with confocal microscopy. Outcomes are examined 30 days after Aß1-42 stereotactic injection in aged male C57BL/6 mice. We report significant decreases in density of the glutamate receptor subunit GluA1 and the vesicular glutamate transporter (VGluT) 1 in the conus ammonis 1 region of the hippocampus in the Aß1-42 injected mice compared with artificial cerebrospinal fluid injected and naïve controls, notably in the stratum oriens and stratum radiatum. GluA1 subunit density also decreased within the dentate gyrus dorsal stratum moleculare in Aß1-42 injected mice compared with artificial cerebrospinal fluid injected controls. These changes are consistent with findings previously reported in the human AD hippocampus. By contrast, glutamate receptor subunits GluA2, GluN1, GluN2A, and VGluT2 showed no changes in expression. These findings indicate that Aß1-42 induces brain region and layer specific expression changes of the glutamatergic receptors and transporters, suggesting complex and spatial vulnerability of this pathway during development of AD neuropathology. Read the Editorial Highlight for this article on page 7. Cover Image for this issue: https://doi.org/10.1111/jnc.14763.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Hipocampo/metabolismo , Fragmentos de Peptídeos/toxicidade , Receptores de AMPA/biossíntese , Proteína Vesicular 1 de Transporte de Glutamato/biossíntese , Peptídeos beta-Amiloides/farmacologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/metabolismo , Região CA3 Hipocampal/efeitos dos fármacos , Região CA3 Hipocampal/metabolismo , Giro Denteado/efeitos dos fármacos , Giro Denteado/metabolismo , Hipocampo/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/farmacologia , Receptores de AMPA/genética , Proteína Vesicular 1 de Transporte de Glutamato/genéticaRESUMO
When a stop codon is at the 80S ribosomal A site, there are six nucleotides (+4 to +9) downstream that are inferred to be occupying the mRNA channel. We examined the influence of these downstream nucleotides on translation termination success or failure in mammalian cells at the three stop codons. The expected hierarchy in the intrinsic fidelity of the stop codons (UAA>UAG>>UGA) was observed, with highly influential effects on termination readthrough mediated by nucleotides at position +4 and position +8. A more complex influence was observed from the nucleotides at positions +5 and +6. The weakest termination contexts were most affected by increases or decreases in the concentration of the decoding release factor (eRF1), indicating that eRF1 binding to these signals was rate-limiting. When termination efficiency was significantly reduced by cognate suppressor tRNAs, the observed influence of downstream nucleotides was maintained. There was a positive correlation between experimentally measured signal strength and frequency of the signal in eukaryotic genomes, particularly in Saccharomyces cerevisiae and Drosophila melanogaster. We propose that termination efficiency is not only influenced by interrogation of the stop signal directly by the release factor, but also by downstream ribosomal interactions with the mRNA nucleotides in the entry channel.
Assuntos
Códon de Terminação , Terminação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Animais , Células COS , Chlorocebus aethiops , Drosophila melanogaster/genética , Células HEK293 , Humanos , Nucleotídeos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
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 (Aß1-42)-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 Aß1-42 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 Aß1-42 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.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Região CA1 Hipocampal/metabolismo , Hipocampo/metabolismo , Células Piramidais/metabolismo , Ácido gama-Aminobutírico/metabolismo , Doença de Alzheimer/metabolismo , Animais , Masculino , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Translational stop codons, UAA, UAG, and UGA, form an integral part of the universal genetic code. They are of significant interest today for their underlying fundamental role in terminating protein synthesis, but also for their potential utilisation for programmed alternative translation events. In diverse organisms, UAA has wide usage, but it is puzzling that the high fidelity UAG is selected against and yet UGA, vulnerable to suppression, is widely used, particularly in those archaeal and bacterial genomes with a high GC content. In canonical protein synthesis, stop codons are interpreted by protein release factors that structurally and functionally mimic decoding tRNAs and occupy the decoding site on the ribosome. The release factors make close contact with the decoding complex through multiple interactions. Correct interactions cause conformational changes resulting in new and enhanced contacts with the ribosome, particularly between specific bases in the mRNA and rRNA. The base following the stop codon (fourth or +4 base) may strongly influence decoding efficiency, facilitating alternative non-canonical events like frameshifting or selenocysteine incorporation. The fourth base is drawn into the decoding site with a compacted stop codon in the eukaryotic termination complex. Surprisingly, mRNA sequences upstream and downstream of this core tetranucleotide signal have a significant influence on the strength of the signal. Since nine bases downstream of the stop codon are within the mRNA channel, their interactions with rRNA, and r-proteins may affect efficiency. With this understanding, it is now possible to design stop signals of desired strength for specific applied purposes.
Assuntos
Códon de Terminação/genética , Ribossomos/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSym(R7A) is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNA(phe) from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSym(R7A), suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSym(R7A)-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSym(R7A) excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSym(R7A) transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSym(R7A) transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Sequências Repetitivas Dispersas , Percepção de Quorum , Ribossomos/ultraestrutura , Sequência de Bases , Sítios de Ligação , Técnicas de Transferência de Genes , Ilhas Genômicas , Espectrometria de Massas , Mesorhizobium/metabolismo , Plantas/microbiologia , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Rhizobium/metabolismo , Ribossomos/química , Simbiose , Fatores de Transcrição , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/metabolismoRESUMO
Secreted amyloid precursor protein-α (sAPPα) is a neurotrophic and neuroprotective molecule which can enhance learning and synaptic plasticity. Aging is associated with memory decline and impaired long-term potentiation (LTP). SAPPα therefore has potential as a nootropic agent which could be used to offset age-related cognitive decline. In this study we investigated the effects of sAPPα on spatial memory tasks and LTP in aged and young Long-Evans rats. Two hippocampus-dependent tasks were employed to measure spatial memory that is susceptible to impairments during aging. Aged rats showed a mild deficit in the novel object location task, but memory was significantly enhanced by bilateral intrahippocampal injections of sAPPα. There was no effect on the performance of young animals. In the watermaze task, however, sAPPα did not alleviate age-related decline in spatial memory. In subsequent electrophysiological experiments, LTP was impaired in slices from aged animals, but plasticity was rescued in a concentration-dependent manner by exogenous sAPPα administration. In contrast, LTP was impaired in young animals by sAPPα. Overall, these data support the hypothesis that sAPPα has therapeutic potential as a treatment for age-related cognitive decline.
Assuntos
Envelhecimento/fisiologia , Precursor de Proteína beta-Amiloide/farmacologia , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Memória Espacial/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Ratos , Ratos Long-Evans , Memória Espacial/fisiologiaRESUMO
BACKGROUND: Differential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of Alzheimer's disease, or secreted amyloid precursor protein-alpha (sAPPα), which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity. The underlying molecular mechanisms recruited by sAPPα that underpin these considerable cellular effects are not well elucidated. As these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sAPPα in rat hippocampal organotypic slice cultures. Slices were exposed to 1 nM sAPPα or phosphate buffered saline for 15 min, 2 h or 24 h and sAPPα-associated gene expression profiles were produced for each time-point using Affymetrix Rat Gene 1.0 ST arrays (moderated t-test using Limma: p < 0.05, and fold change ± 1.15). RESULTS: Treatment of organotypic hippocampal slice cultures with 1 nM sAPPα induced temporally distinct gene expression profiles, including mRNA and microRNA associated with Alzheimer's disease. Having demonstrated that treatment with human recombinant sAPPα was protective against N-methyl d-aspartate-induced toxicity, we next explored the sAPPα-induced gene expression profiles. Ingenuity Pathway Analysis predicted that short-term exposure to sAPPα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors (AP-1, Egr1), modulation of the chromatin environment, and apparent activation of the constitutive transcription factors CREB and NF-κB. Importantly, dynamic regulation of NF-κB appears to be integral to the transcriptional response across all time-points. In contrast, medium and long exposure to sAPPα resulted in an overall downregulation of gene expression. While these results suggest commonality between sAPPα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. The gene networks formed following medium and long exposure to sAPPα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sAPPα. CONCLUSIONS: Our results demonstrate that sAPPα rapidly and persistently regulates gene expression in rat hippocampus. This regulation is multi-level, temporally specific and is likely to underpin the neuroprotective effects of sAPPα.
Assuntos
Precursor de Proteína beta-Amiloide/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HEK293 , Hipocampo/citologia , Hipocampo/patologia , Humanos , Técnicas In Vitro , Inflamação/genética , Inflamação/patologia , Masculino , N-Metilaspartato/toxicidade , Neurogênese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
COVID-19 , Síndrome de Fadiga Crônica , Viroses , Humanos , Leucócitos Mononucleares/metabolismo , Proteoma/metabolismo , Síndrome de COVID-19 Pós-Aguda , Projetos Piloto , SARS-CoV-2 , COVID-19/metabolismo , Viroses/metabolismoRESUMO
Amyloid precursor protein (APP) is an integral membrane glycoprotein present at high levels in nerve cells. Two soluble secreted forms, sAPPα and sAPPß, are processed from APP by two mutually exclusive proteolytic pathways. sAPPα shows a range of neuroprotective and growth factor properties, including reduction of neuronal injury and improvement in memory performance, in contrast to the generally less potent sAPPß. In addition, sAPPα has been shown to increase the proliferation of both embryonic neural stem cells and neural progenitor cells (NPCs) derived from the subventricular zone (SVZ) of the adult brain. However, an effect of sAPPα (or sAPPß) on adult hippocampal progenitor cell proliferation and differentiation has not previously been observed. In this study, we examined the effect of both the α- and ß-cleaved ectodomains of sAPP on adult NPCs isolated from the subgranular zone (SGZ) of the rat hippocampus in the presence or absence of depolarizing conditions. Assays were performed to examine the effect of sAPPα and sAPPß on SGZ-derived adult NPC proliferation in parallel with SVZ-derived cells and on differentiation with SGZ-derived cells. We observed both sAPPα and sAPPß increased the proliferation of SGZ-derived NPCs in vitro. Further, treatment of SGZ-derived NPCs with either sAPPα or sAPPß increased the number of cells expressing the astrocytic marker GFAP and promoted cell survival. The effect on differential fate was observed in both the presence and absence of depolarizing conditions. Thus, both sAPPα and sAPPß exert a complex range of effects on SGZ-derived adult NPCs, including increasing NPC proliferation, maintaining cell viability, yet promoting glial over neuronal differentiation. These findings provide the first direct support for the secreted forms of APP regulating SGZ-derived NPCs, and raise the possibility some or all of the effects may have therapeutic benefit in models of neurological disease.
Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Hipocampo/citologia , Neurônios/fisiologia , Células-Tronco Adultas/fisiologia , Análise de Variância , Animais , Bromodesoxiuridina/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Masculino , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The two codon-specific eubacterial release factors (RF1: UAA/UAG and RF2: UAA/UGA) have specific tripeptide motifs (PXT/SPF) within an exposed recognition loop shown in recent structures to interact with stop codons during protein synthesis termination. The motifs have been inferred to be critical for codon specificity, but this study shows that they are insufficient to determine specificity alone. Swapping the motifs or the entire loop between factors resulted in a loss of codon recognition rather than a switch of codon specificity. From a study of chimeric eubacterial RF1/RF2 recognition loops and an atypical shorter variant in Caenorhabditis elegans mitochondrial RF1 that lacks the classical tripeptide motif PXT, key determinants throughout the whole loop have been defined. It reveals that more than one configuration of the recognition loop based on specific sequence and size can achieve the same desired codon specificity. This study has provided unexpected insight into why a combination of the two factors is necessary in eubacteria to exclude recognition of UGG as stop.
Assuntos
Bactérias/metabolismo , Bactérias/genética , Sequência de Bases , Códon/metabolismo , Códon de Terminação/metabolismo , Eubacterium/genética , Eubacterium/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Biossíntese de Proteínas , Estrutura Terciária de Proteína/genética , Sensibilidade e EspecificidadeRESUMO
Vertebrate mitochondria use stop codons UAA and UAG decoded by the release factor (RF) MTRF1L and two reassigned arginine codons, AGA and AGG. A second highly conserved RF-like factor, MTRF1, which evolved from a gene duplication of an ancestral mitochondrial RF1 and not a RF2, is a good candidate for recognizing the nonstandard codons. MTRF1 differs from other RFs by having insertions in the two external loops important for stop codon recognition (tip of helix alpha5 and recognition loop) and by having key substitutions that are involved in stop codon interactions in eubacterial RF/ribosome structures. These changes may allow recognition of the larger purine base in the first position of AGA/G and, uniquely for RFs, only of G at position 2. In contrast, residues that support A and G recognition in the third position in RF1 are conserved as would be required for recognition of AGA and AGG. Since an assay with vertebrate mitochondrial ribosomes has not been established, we modified Escherichia coli RF1 at the helix alpha5 and recognition loop regions to mimic MTRF1. There was loss of peptidyl-tRNA hydrolysis activity with standard stop codons beginning with U (e.g., UAG), but a gain of activity with codons beginning with A (AAG in particular). A lower level of activity with AGA could be enhanced by solvent modification. These observations imply that MTRF1 has the characteristics to recognize A as the first base of a stop codon as would be required to decode the nonstandard codons AGA and AGG.
Assuntos
Códon de Terminação , Biologia Computacional , Mitocôndrias/genética , Fatores de Terminação de Peptídeos/genética , Vertebrados/genética , Animais , Arginina/genética , Códon/genética , Códon de Terminação/genética , Sequência Conservada , Duplicação Gênica , Humanos , Proteínas Mitocondriais/genética , Terminação Traducional da Cadeia Peptídica/genética , Ornitorrinco/genética , Biossíntese de ProteínasRESUMO
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.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , NeurogêneseRESUMO
INTRODUCTION: Early intervention in Alzheimer's disease (AD) requires the development of an easily administered test that is able to identify those at risk. Focusing on microRNA robustly detected in plasma and standardizing the analysis strategy, we sought to identify disease-stage specific biomarkers. METHODS: Using TaqMan microfluidics arrays and a statistical consensus approach, we assessed plasma levels of 185 neurodegeneration-related microRNA, in cohorts of cognitively normal amyloid ß-positive (CN-Aß+), mild cognitive impairment (MCI), and Alzheimer's disease (AD) participants, relative to their respective controls. RESULTS: Distinct disease stage microRNA biomarkers were identified, shown to predict membership of the groups (area under the curve [AUC] >0.8) and were altered dynamically with AD progression in a longitudinal study. Bioinformatics demonstrated that these microRNA target known AD-related pathways, such as the Phosphoinositide 3-kinase (PI3K-Akt) signalling pathway. Furthermore, a significant correlation was found between miR-27a-3p, miR-27b-3p, and miR-324-5p and amyloid beta load. DISCUSSION: Our results show that microRNA signatures alter throughout the progression of AD, reflect the underlying disease pathology, and may prove to be useful diagnostic markers.
RESUMO
The canonical view of the maintenance of long-term potentiation (LTP), a widely accepted experimental model for memory processes, is that new gene transcription contributes to its consolidation; however, the gene networks involved are unknown. To address this issue, we have used high-density Rat 230.2 Affymetrix arrays to establish a set of genes induced 20-min post-LTP, and using Ingenuity Pathway network analysis tools we have investigated how these early responding genes are interrelated. This analysis identified LTP-induced regulatory networks in which the transcription factors (TFs) nuclear factor-KB and serum response factor, which, to date, have not been widely recognized as coordinating the early gene response, play a key role alongside the more well-known TFs cyclic AMP response element-binding protein, and early growth response 1. Analysis of gene-regulatory promoter sites and chromosomal locations of the genes within the dataset reinforced the importance of these molecules in the early gene response and predicted that the coordinated action might arise from gene clustering on particular chromosomes. We have also identified a transcription-based response that affects mitogen-activated protein kinase signaling pathways and protein synthesis during the stabilization of the LTP response. Furthermore, evidence from biological function, networks, and regulatory analyses showed convergence on genes related to development, proliferation, and neurogenesis, suggesting that these functions are regulated early following LTP induction. This raises the interesting possibility that LTP-related gene expression plays a role in both synaptic reorganization and neurogenesis.