The angiotensin-(1-7)/MasR axis improves pneumonia caused by Pseudomonas aeruginosa: Extending the therapeutic window for antibiotic therapy.

Pseudomonas aeruginosa is a frequent cause of antimicrobial-resistant hospital-acquired pneumonia, especially in critically ill patients. Inflammation triggered by P. aeruginosa infection is necessary for bacterial clearance but must be spatially and temporally regulated to prevent further tissue damage and bacterial dissemination. Emerging data have shed light on the pro-resolving actions of angiotensin-(1-7) [Ang-(1-7)] signaling through the G protein-coupled receptor Mas (MasR) during infections. Herein, we investigated the role of the Ang-(1-7)/Mas axis in pneumonia caused by P. aeruginosa by using genetic and pharmacological approach and found that Mas receptor-deficient animals developed a more severe form of pneumonia showing higher neutrophilic infiltration into the airways, bacterial load, cytokines, and chemokines production and more severe pulmonary damage. Conversely, treatment of pseudomonas-infected mice with Ang-(1-7) was able to decrease neutrophilic infiltration in airways and lungs, local and systemic levels of pro-inflammatory cytokines and chemokines, and increase the efferocytosis rates, mitigating lung damage/dysfunction caused by infection. Notably, the therapeutic association of Ang-(1-7) with antibiotics improved the survival rates of mice subjected to lethal inoculum of P. aeruginosa, extending the therapeutic window for imipenem. Mechanistically, Ang-(1-7) increased phagocytosis of bacteria by neutrophils and macrophages to accelerate pathogen clearance. Altogether, harnessing the Ang-(1-7) pathway during infection is a potential strategy for the development of host-directed therapies to promote mechanisms of resistance and resilience to pneumonia.

A naturally occurring 22-amino acid fragment of human hemoglobin A inhibits autophagy and HIV-1.

Autophagy is an evolutionarily ancient catabolic pathway and has recently emerged as an integral part of the innate immune system. While the core machinery of autophagy is well defined, the physiological regulation of autophagy is less understood. Here, we identify a C-terminal fragment of human hemoglobin A (HBA1, amino acids 111-132) in human bone marrow as a fast-acting non-inflammatory inhibitor of autophagy initiation. It is proteolytically released from full-length HBA1 by cathepsin E, trypsin or pepsin. Biochemical characterization revealed that HBA1(111-132) has an in vitro stability of 52 min in human plasma and adopts a flexible monomeric conformation in solution. Structure-activity relationship studies revealed that the C-terminal 13 amino acids of HBA1(120-132) are sufficient to inhibit autophagy, two charged amino acids (D127, K128) mediate solubility, and two serines (S125, S132) are required for function. Successful viruses like human immunodeficiency virus 1 (HIV-1) evolved strategies to subvert autophagy for virion production. Our results show that HBA1(120-132) reduced virus yields of lab-adapted and primary HIV-1. Summarizing, our data identifies naturally occurring HBA1(111-132) as a physiological, non-inflammatory antagonist of autophagy. Optimized derivatives of HBA1(111-132) may offer perspectives to restrict autophagy-dependent viruses.

Nanoscale Structural Characterization of Amyloid ß 1-42 Oligomers and Fibrils Grown in the Presence of Fatty Acids.

Mono- and polyunsaturated fatty acids (FAs) are broadly used as food supplements. However, their effect on the aggregation of amyloidogenic proteins remains unclear. In this study, we investigated the effect of a large number of mono- and polyunsaturated, as well as fully saturated FAs on the aggregation of amyloid ß1-42 (Aß1-42) peptide. A progressive aggregation of this peptide is the expected molecular cause of Alzheimer's disease (AD), one of the most common neurodegenerative pathologies in the world. We found that arachidonic and stearic acids delayed the aggregation of Aß1-42. Using Nano-Infrared spectroscopy, we found that FAs caused very little if any changes in the secondary structure of Aß1-42 oligomers and fibrils formed at different stages of protein aggregation. However, the analyzed mono- and polyunsaturated, as well as fully saturated FAs uniquely altered the toxicity of Aß1-42 fibrils. We found a direct relationship between the degree of FAs unsaturation and toxicity of Aß1-42 fibrils formed in their presence. Specifically, with an increase in the degree of unsaturation, the toxicity Aß1-42/FA fibrils increased. These results indicate that fully saturated or monounsaturated FAs could be used to decrease the toxicity of amyloid aggregates and, consequently, decelerate the development of AD.

Protein Kinase C-Delta Mediates Cell Cycle Reentry and Apoptosis Induced by Amyloid-Beta Peptide in Post-Mitotic Cortical Neurons.

Amyloid-beta peptide (Aß) is a neurotoxic constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. The detailed mechanisms by which protein kinase C-delta (PKCδ) contributes to Aß toxicity is not yet entirely understood. Using fully differentiated primary rat cortical neurons, we found that inhibition of Aß25-35-induced PKCδ increased cell viability with restoration of neuronal morphology. Using cyclin D1, proliferating cell nuclear antigen (PCNA), and histone H3 phosphorylated at Ser-10 (p-Histone H3) as the respective markers for the G1-, S-, and G2/M-phases, PKCδ inhibition mitigated cell cycle reentry (CCR) and subsequent caspase-3 cleavage induced by both Aß25-35 and Aß1-42 in the post-mitotic cortical neurons. Upstream of PKCδ, signal transducers and activators of transcription (STAT)-3 mediated PKCδ induction, CCR, and caspase-3 cleavage upon Aß exposure. Downstream of PKCδ, aberrant neuronal CCR was triggered by overactivating cyclin-dependent kinase-5 (CDK5) via calpain2-dependent p35 cleavage into p25. Finally, PKCδ and CDK5 also contributed to Aß25-35 induction of p53-upregulated modulator of apoptosis (PUMA) in cortical neurons. Together, we demonstrated that, in the post-mitotic neurons exposed to Aßs, STAT3-dependent PKCδ expression triggers calpain2-mediated p35 cleavage into p25 to overactivate CDK5, thus leading to aberrant CCR, PUMA induction, caspase-3 cleavage, and ultimately apoptosis.

Alzheimer's Disease as a Membrane Dysfunction Tauopathy? New Insights into the Amyloid Cascade Hypothesis.

The amyloid cascade hypothesis postulates that extracellular deposits of amyloid ß (Aß) are the primary and initial cause leading to the full development of Alzheimer's disease (AD) with intracellular neurofibrillary tangles; however, the details of this mechanism have not been fully described until now. Our preliminary data, coming from our day-to-day neuropathology practice, show that the primary location of the hyperphosphorylated tau protein is in the vicinity of the cell membrane of dystrophic neurites. This observation inspired us to formulate a hypothesis that presumes an interaction between low-density lipoprotein receptor-related protein 1 (LRP1) and fibrillar aggregates of, particularly, Aß42 anchored at the periphery of neuritic plaques, making internalization of the LRP1-Aß42 complex infeasible and, thus, causing membrane dysfunction, leading to the tauopathy characterized by intracellular accumulation and hyperphosphorylation of the tau protein. Understanding AD as a membrane dysfunction tauopathy may draw attention to new treatment approaches not only targeting Aß42 production but also, perhaps paradoxically, preventing the formation of LRP1-Aß42.

SIRT1 Regulates Mitochondrial Damage in N2a Cells Treated with the Prion Protein Fragment 106-126 via PGC-1α-TFAM-Mediated Mitochondrial Biogenesis.

Mitochondrial damage is an early and key marker of neuronal damage in prion diseases. As a process involved in mitochondrial quality control, mitochondrial biogenesis regulates mitochondrial homeostasis in neurons and promotes neuron health by increasing the number of effective mitochondria in the cytoplasm. Sirtuin 1 (SIRT1) is a NAD+-dependent deacetylase that regulates neuronal mitochondrial biogenesis and quality control in neurodegenerative diseases via deacetylation of a variety of substrates. In a cellular model of prion diseases, we found that both SIRT1 protein levels and deacetylase activity decreased, and SIRT1 overexpression and activation significantly ameliorated mitochondrial morphological damage and dysfunction caused by the neurotoxic peptide PrP106-126. Moreover, we found that mitochondrial biogenesis was impaired, and SIRT1 overexpression and activation alleviated PrP106-126-induced impairment of mitochondrial biogenesis in N2a cells. Further studies in PrP106-126-treated N2a cells revealed that SIRT1 regulates mitochondrial biogenesis through the PGC-1α-TFAM pathway. Finally, we showed that resveratrol resolved PrP106-126-induced mitochondrial dysfunction and cell apoptosis by promoting mitochondrial biogenesis through activation of the SIRT1-dependent PGC-1α/TFAM signaling pathway in N2a cells. Taken together, our findings further describe SIRT1 regulation of mitochondrial biogenesis and improve our understanding of mitochondria-related pathogenesis in prion diseases. Our findings support further investigation of SIRT1 as a potential target for therapeutic intervention of prion diseases.

Deciphering the Molecular Dance: Exploring the Dynamic Interplay Between Mouse Insulin B9-23 Peptides and their Variants.

Type 1 diabetes results from the autoimmune destruction of pancreatic insulin-producing ß-cells, primarily targeted by autoreactive T cells that recognize insulin B9-23 peptides as antigens. Using drift tube ion mobility spectrometry-mass spectrometry, transmission electron microscopy, and two-dimensional infrared spectroscopy, we characterized mouse insulin 1 B9-23 (Ins1 B9-23), insulin 2 B9-23 (Ins2 B9-23), along with two of their mutants, Ins2 B9-23 Y16A and Ins2 B9-23 C19S. Our findings indicate that Ins1 B9-23 and the Ins2 Y16A mutant exhibit rapid fibril formation, whereas Ins2 B9-23 and the Ins2 C19S mutant show slower fibrillization and a structural rearrangement from globular protofibrils to fibrillar aggregates. These differences in aggregation behaviors also manifest in interactions with (-)epigallocatechin gallate (EGCG), a canonical amyloid inhibitor. EGCG effectively disrupts the fibrils formed by Ins1 B9-23 and the Y16A mutant. However, it proves ineffective in preventing fibril formation of Ins2 B9-23 and the C19S mutant. These results establish a strong correlation between the aggregation behaviors of these peptides and their divergent effects on anti-islet autoimmunity.

The final walk with preptin.

Preptin, a 34-amino acid peptide derived from pro-IGF2, is believed to influence various physiological processes, including insulin secretion and the regulation of bone metabolism. Despite its recognized involvement, the precise physiological role of preptin remains enigmatic. To address this knowledge gap, we synthesized 16 analogs of preptin, spanning a spectrum from full-length forms to fragments, and conducted comprehensive comparative activity evaluations alongside native human, mouse and rat preptin. Our study aimed to elucidate the physiological role of preptin. Contrary to previous indications of broad biological activity, our thorough analyses across diverse cell types revealed no significant biological activity associated with preptin or its analogs. This suggests that the associations of preptin with various diseases or tissue-specific abundance fluctuations may be influenced by factors beyond preptin itself, such as higher levels of IGF2 or IGF2 proforms present in tissues. In conclusion, our findings challenge the conventional notion of preptin as an isolated biologically active molecule and underscore the complexity of its interactions within biological systems. Rather than acting independently, the observed effects of preptin may arise from experimental conditions, elevated preptin concentrations, or interactions with related molecules such as IGF2.

Microglia either promote or restrain TRAIL-mediated excitotoxicity caused by Aß1-42 oligomers.

BACKGROUND: Alzheimer's disease (AD) features progressive neurodegeneration and microglial activation that results in dementia and cognitive decline. The release of soluble amyloid (Aß) oligomers into the extracellular space is an early feature of AD pathology. This can promote excitotoxicity and microglial activation. Microglia can adopt several activation states with various functional outcomes. Protective microglial activation states have been identified in response to Aß plaque pathology in vivo. However, the role of microglia and immune mediators in neurotoxicity induced by soluble Aß oligomers is unclear. Further, there remains a need to identify druggable molecular targets that promote protective microglial states to slow or prevent the progression of AD. METHODS: Hippocampal entorhinal brain slice culture (HEBSC) was employed to study mechanisms of Aß1-42 oligomer-induced neurotoxicity as well as the role of microglia. The roles of glutamate hyperexcitation and immune signaling in Aß-induced neurotoxicity were assessed using MK801 and neutralizing antibodies to the TNF-related apoptosis-inducing ligand (TRAIL) respectively. Microglial activation state was manipulated using Gi-hM4di designer receptor exclusively activated by designer drugs (DREADDs), microglial depletion with the colony-stimulating factor 1 receptor (CSF1R) antagonist PLX3397, and microglial repopulation (PLX3397 withdrawal). Proteomic changes were assessed by LC-MS/MS in microglia isolated from control, repopulated, or Aß-treated HEBSCs. RESULTS: Neurotoxicity induced by soluble Aß1-42 oligomers involves glutamatergic hyperexcitation caused by the proinflammatory mediator and death receptor ligand TRAIL. Microglia were found to have the ability to both promote and restrain Aß-induced toxicity. Induction of microglial Gi-signaling with hM4di to prevent pro-inflammatory activation blunted Aß neurotoxicity, while microglial depletion with CSF1R antagonism worsened neurotoxicity caused by Aß as well as TRAIL. HEBSCs with repopulated microglia, however, showed a near complete resistance to Aß-induced neurotoxicity. Comparison of microglial proteomes revealed that repopulated microglia have a baseline anti-inflammatory and trophic phenotype with a predicted pathway activation that is nearly opposite that of Aß-exposed microglia. mTORC2 and IRF7 were identified as potential targets for intervention. CONCLUSION: Microglia are key mediators of both protection and neurodegeneration in response to Aß. Polarizing microglia toward a protective state could be used as a preventative strategy against Aß-induced neurotoxicity.

Near-infrared laser diode mitigates Aß1-42-induced neurodegeneration in cortical neurons.

Alzheimer's disease, a prevalent neurodegenerative condition primarily affecting older adults, remains incurable. Its principle pathological hallmark is the accelerated accumulation of amyloid ß (Aß) protein. This study investigates the potential of photobiomodulation using near infrared light to counteract Aß1-42-induced synaptic degeneration and neurotoxicity. We focused on the effect of 808 nm near-infrared laser diode (LD) on Aß1-42 cytotoxicity in primary cultured cortical neurons. We assessed cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, observing substantial benefits from LD irradiation with a power of 10 mW and a dose of 30 J. Cells exposed to Aß1-42 exhibited morphological changes indicative of synaptic damage and a significant decrease in the number of postsynaptic density protein-95 (PSD-95) contacts, which were significantly improved with near-infrared LD therapy. Furthermore, this therapy reduced Aß and phosphorylated tau (P-tau) protein accumulation. Additionally, near-infrared LD irradiation substantially lessened the Aß1-42-induced rise in glial fibrillary acid protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBA1) in astrocytes and microglia. Remarkably, near-infrared LD irradiation effectively inhibited phosphorylation of key proteins involved in Aß1-42-induced necroptosis, namely Receptor-interacting protein kinase-3 (RIP3) and Mixed Lineage Kinase domain-Like protein (MLKL). Our findings suggest that near-infrared LD treatment significantly reduces neurodegeneration by reducing glial overactivation and neuronal necroptosis triggered by Aß1-42. Thus, near-infrared LD treatment emerges as a promising approach for slowing or treating Alzheimer's disease, offering new avenues in its management.

Identification of Molecular Correlations of GSDMD with Pyroptosis inAlzheimer's Disease.

AIM: An analysis of bioinformatics and cell experiments was performed to verify the relationship between gasdermin D (GSDMD), an executive protein of pyroptosis, and Alzheimer's disease (AD). METHODS: The training set GSE33000 was utilized to identify differentially expressed genes (DEGs) in both the AD group and control group, as well as in the GSDMD protein high/low expression group. Subsequently, the weighted gene co-expression network analysis (WGCNA) and the least absolute shrinkage and selection operator (LASSO) regression analysis were conducted, followed by the selection of the key genes for the subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The association between GSDMD and AD was assessed and confirmed in the training set GSE33000, as well as in the validation sets GSE5281 and GSE48350. Immunofluorescence (IF) was employed to detect the myelin basic protein (MBP), a distinctive protein found in the rat oligodendrocytes (OLN-93 cells). A range of concentrations (1-15 µmol/L) of ß-amyloid 1-42 (Aß1-42) were exposed to the cells, and the subsequent observations were made regarding cell morphology. Additionally, the assessments were conducted to evaluate the cell viability, the lactate dehydrogenase (LDH) release, the cell membrane permeability, and the GSDMD protein expression. RESULTS: A total of 7,492 DEGs were screened using GSE33000. Subsequently, WGCNA analysis identified 19 genes that exhibited the strongest correlation with clinical traits in AD. Additionally, LASSO regression analysis identified 13 key genes, including GSDMD, AFF1, and ATOH8. Furthermore, the investigation revealed that the key genes were associated with cellular inflammation based on GO and KEGG analyses. Moreover, the area under the curve (AUC) values for the key genes in the training and validation sets were determined to be 0.95 and 0.70, respectively. Significantly, GSDMD demonstrated elevated levels of expression in AD across both datasets. The positivity of MBP expression in cells exceeded 95%. As the concentration of Aß1-42 action gradually escalated, the detrimental effects on cells progressively intensified, resulting in a gradual decline in cell survival rate, accompanied by an increase in lactate dehydrogenase release, cell membrane permeability, and GSDMD protein expression. CONCLUSION: The association between GSDMD and AD has been observed, and it has been found that Aß1-42 can induce a significant upregulation of GSDMD in OLN-93 cells. This suggests that Aß1-42 has the potential to induce cellular pyroptosis and can serve as a valuable cellular pyroptosis model for the study of AD.

Targeting the hydrophobic region of pyroglutamate-modified amyloid-ß by tyrocidine A prevents its nucleation-aggregation process and its "catalytic effect" on the Aßs aggregation.

Pyroglutamate (pE)-modified amyloid-ß (Aß) peptides play a crucial role in the development of Alzheimer's disease. pEAß3-42 can rapidly form oligomers that gradually elongate hydrophobic segments to form ß-sheet-rich amyloid intermediates, ultimately resulting in the formation of mature amyloid fibrils. pEAß3-42 can also catalyze the aggregation of Aß species and subsequently accelerate the formation of amyloid senile plaques. Considering the recent clinical success of the pEAß3-42-targeting antibody donanemab, molecules that strongly bind pEAß3-42 and prevent its aggregation and catalytic effect on Aßs may also provide potential therapeutic options for Alzheimer's disease. Here, we demonstrate that the natural antibiotic cyclopeptide tyrocidine A (TA) not only strongly inhibits the aggregation of Aß1-42 as previously reported, but also interacts with the hydrophobic C-terminus and middle domain of pEAß3-42 to maintain an unordered conformation, effectively impeding the formation of initial oligomers and subsequently halting the aggregation of pEAß3-42. Furthermore, TA can disrupt the "catalytic effect" of pEAß3-42 on amyloid aggregates, effectively suppressing Aß aggregation and ultimately preventing the pathological events induced by Aßs.

Repurposing Anidulafungin for Alzheimer's Disease via Fragment-Based Drug Discovery.

The misfolding and aggregation of beta-amyloid (Aß) peptides have been implicated as key pathogenic events in the early stages of Alzheimer's disease (AD). Inhibiting Aß aggregation represents a potential disease-modifying therapeutic approach to AD treatment. Previous studies have identified various molecules that inhibit Aß aggregation, some of which share common chemical substructures (fragments) that may be key to their inhibitory activity. Employing fragment-based drug discovery (FBDD) methods may facilitate the identification of these fragments, which can subsequently be used to screen new inhibitors and provide leads for further drug development. In this study, we used an in silico FBDD approach to identify 17 fragment clusters that are significantly enriched among Aß aggregation inhibitors. These fragments were then used to screen anti-infective agents, a promising drug class for repurposing against amyloid aggregation. This screening process identified 16 anti-infective drugs, 5 of which were chosen for further investigation. Among the 5 candidates, anidulafungin, an antifungal compound, showed high efficacy in inhibiting Aß aggregation in vitro. Kinetic analysis revealed that anidulafungin selectively blocks the primary nucleation step of Aß aggregation, substantially delaying Aß fibril formation. Cell viability assays demonstrated that anidulafungin can reduce the toxicity of oligomeric Aß on BV2 microglia cells. Molecular docking simulations predicted that anidulafungin interacted with various Aß species, including monomers, oligomers, and fibrils, potentially explaining its activity against Aß aggregation and toxicity. This study suggests that anidulafungin is a potential drug to be repurposed for AD, and FBDD is a promising approach for discovering drugs to combat Aß aggregation.

Unravelling heparin's enhancement of amyloid aggregation in a model peptide system.

A coarse-grained (CG) model for heparin, an anionic polysaccharide, was developed to investigate the mechanisms of heparin's enhancement of fibrillation in many amyloidogenic peptides. CG molecular dynamics simulations revealed that heparin, by forming contacts with the model amyloidogenic peptide, amyloid-ß's K16LVFFAE22 fragment (Aß16-22), promoted long-lived and highly beta-sheet-like domains in the peptide oligomers. Concomitantly, heparin-Aß16-22 contacts suppressed the entropy of mixing of the oligomers' beta-domains. Such oligomers could make better seeds for fibrillation, potentially contributing to heparin's fibril-enhancing behaviour. Additionally, reductions in heparin's flexibility led to delayed aggregation, and less ordered Aß16-22 oligomers, thus offering insights into the contrasting inhibition of fibrillation by the relatively rigid polysaccharide, chitosan.

Oat Beta-Glucan Dietary Intervention on Antioxidant Defense Parameters, Inflammatory Response and Angiotensin Signaling in the Testes of Rats with TNBS-Induced Colitis.

Male infertility represents a significant public health concern. There is a negative impact of inflammatory bowel diseases (IBDs) on the male reproductive system. The aim of this study was to investigate whether oat beta-glucan (OBG) with different molar mass can modulate parameters of antioxidant defense and inflammatory response in the testes of adult Sprague-Dawley rats with TNBS-induced colitis and whether the OBG intervention can modulate the inflammatory response in association with the RAS system. Results: higher testicular superoxide dismutase (SOD), glutathione reductase (GR) activities and glutathione (GSH) concentration, and lower testosterone (T) level and glutathione peroxidase (GPx) activity, were observed in rats with colitis than in healthy control ones. TNBS-induced colitis resulted in decreased the angiotensin 1-7 (ANG 1-7) level in the testes of rats fed with low-molar mass OBG compared to control animals. Conclusions: although colitis induced moderate pro-oxidant changes in the gonads, it seems plausible that dietary intervention with different fractions of oat beta-glucans mass may support the maintenance of reproductive homeostasis via the stimulation of the local antioxidant defense system.

Proteomic Analysis Reveals Physiological Activities of Aß Peptide for Alzheimer's Disease.

With the rapid progress in deciphering the pathogenesis of Alzheimer's disease (AD), it has been widely accepted that the accumulation of misfolded amyloid ß (Aß) in the brain could cause the neurodegeneration in AD. Although much evidence demonstrates the neurotoxicity of Aß, the role of Aß in the nervous system are complex. However, more comprehensive studies are needed to understand the physiological effect of Aß40 monomers in depth. To explore the physiological mechanism of Aß, we employed mass spectrometry to investigate the altered proteomic events induced by a lower submicromolar concentration of Aß. Human neuroblastoma SH-SY5Y cells were exposed to five different concentrations of Aß1-40 monomers and collected at four time points. The proteomic analysis revealed the time-course behavior of proteins involved in biological processes, such as RNA splicing, nuclear transport and protein localization. Further biological studies indicated that Aß40 monomers may activate PI3K/AKT signaling to regulate p-Tau, Ezrin and MAP2. These three proteins are associated with dendritic morphogenesis, neuronal polarity, synaptogenesis, axon establishment and axon elongation. Moreover, Aß40 monomers may regulate their physiological forms by inhibiting the expression of BACE1 and APP via activation of the ERK1/2 pathway. A comprehensive exploration of pathological and physiological mechanisms of Aß is beneficial for exploring novel treatment.

Hepatic Amyloid Beta-42-Metabolizing Proteins in Liver Steatosis and Metabolic Dysfunction-Associated Steatohepatitis.

Amyloid beta (Aß) plays a major role in the pathogenesis of Alzheimer's disease and, more recently, has been shown to protect against liver fibrosis. Therefore, we studied Aß-42 levels and the expression of genes involved in the generation, degradation, and transport of Aß proteins in liver samples from patients at different stages of metabolic dysfunction-associated liver disease (MASLD) and under steatotic conditions in vitro/in vivo. Amyloid precursor protein (APP), key Aß-metabolizing proteins, and Aß-42 were analyzed using RT-PCR, Western blotting, Luminex analysis in steatotic in vitro and fatty liver mouse models, and TaqMan qRT-PCR analysis in hepatic samples from patients with MASLD. Hepatocytes loaded with palmitic acid induced APP, presenilin, and neprilysin (NEP) expression, which was reversed by oleic acid. Increased APP and NEP, decreased BACE1, and unchanged Aß-42 protein levels were found in the steatotic mouse liver compared to the normal liver. Aß-42 concentrations were low in MASLD samples of patients with moderate to severe fibrosis compared to the livers of patients with mild or no MASLD. Consistent with the reduced Aß-42 levels, the mRNA expression of proteins involved in APP degradation (ADAM9/10/17, BACE2) and Aß-42 cleavage (MMP2/7/9, ACE) was increased. In the steatotic liver, the expression of APP- and Aß-metabolizing proteins is increased, most likely related to oxidative stress, but does not affect hepatic Aß-42 levels. Consistent with our previous findings, low Aß-42 levels in patients with liver fibrosis appear to be caused by the reduced production and enhanced non-amyloidogenic processing of APP.

Blood-brain barrier permeable carbon nano-assemblies for amyloid-ß clearance and neurotoxic attenuation.

Abnormal amyloid ß-protein (Aß42) fibrillation is a key event in Alzheimer's disease (AD), and photodynamic therapy (PDT) possesses great potential in modulating Aß42 self-assembly. However, the poor blood-brain barrier (BBB) penetration, low biocompatibility, and limited tissue penetration depth of existing photosensitizers limit the progress of photo-oxidation strategies. In this paper, novel indocyanine green-modified graphene quantum dot nano-assemblies (NBGQDs-ICGs) were synthesized based on a molecular assembly strategy of electrostatic interactions for PDT inhibition of Aß42 self-assembly process and decomposition of preformed fibrils under near-infrared light. Combining the small-size structure of graphene quantum dots and the near-infrared light-responsive properties of ICGs, the NBGQDs-ICGs could achieve BBB penetration under 808 nm irradiation. More importantly, the neuroprotective mechanism of NBGQDs-ICG was studied for the first time by AFM, which effectively weakened the adhesion of Aß42 aggregates to the cell surface by blocking the interaction between Aß42 and the cell membrane, and restored the mechanical stability and adhesion of the neuron membrane. Meanwhile, NBGQDs-ICG promoted phagocytosis of Aß42 by microglia. In addition, the good biocompatibility and stability ensured the biosafety of NBGQDs-ICG in future clinical applications. We anticipate that such multifunctional nanocomponents may provide promising avenues for the development of novel AD inhibitors.

Synthesis and mechanistic study of Aß42 C-terminus domain derived tetrapeptides that inhibit Alzheimer's Aß-aggregation-induced neurotoxicity.

Amyloid plaque formation in the brain is mainly responsible for the onset of Alzheimer's disease (AD). Structure-based peptides have gained importance in recent years, and rational design of the peptide sequences for the prevention of Aß-aggregation and related toxicity is imperative. In this study, we investigate the structural modification of tetrapeptides derived from the hydrophobic C-terminal region of Aß42 "VVIA-NH2" and its retro-sequence "AIVV-NH2." A preliminary screening of synthesized peptides through an MTT cell viability assay followed by a ThT fluorescence assay revealed a peptide 13 (Ala-Ile-Aib-Val-NH2) that showed protection against Aß-aggregation and associated neurotoxicity. The presence of the α-helix inducer "Aib" in peptide 13 manifested the conformational transition from cross-ß-sheets to α-helical content in Aß42. The absence of fibrils in electron microscopic analysis suggested the inhibitory potential of peptide 13. The HRMS, DLS, and ANS studies further confirmed the inhibitory activity of 13, and no cytotoxicity was observed. The structure-based peptide described herein is a promising amyloid-ß inhibitor and provides a new lead for the development of AD therapeutics.

Single-Molecule Characterization of Heterogeneous Oligomer Formation during Co-Aggregation of 40- and 42-Residue Amyloid-ß.

The two most abundant isoforms of amyloid-ß (Aß) are the 40- (Aß40) and 42-residue (Aß42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aß aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aß42 and Aß40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aß42 with a small amount of Aß40, while the second phase results mostly from aggregation of Aß40. We also found that the aggregation of Aß42 is slowed by Aß40 while the aggregation of Aß40 is accelerated by Aß42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aß42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aß40 in the co-oligomers in a 1:1 mixture of Aß42 and Aß40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aß42 and Aß40 (1:10), suggesting the critical role of Aß40 in oligomer formation and aggregation.