Aβ Treatment Research Articles

NEK7 induces lactylation in Alzheimer's disease to promote pyroptosis in BV-2 cells.

Alzheimer's disease (AD), an age-related neurodegenerative disorder, is characterized by irreversible brain tissue degeneration. The amyloid-β (Aβ) cascade hypothesis stands as the predominant paradigm explaining AD pathogenesis. This study aimed to elucidate the mechanisms underlying Aβ-induced pyroptosis in AD. AD models were established using amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice and Aβ-treated BV-2 cells (5 µM, 24h). NEK7 expression was evaluated in vitro and in vivo. Cell pyroptosis was assessed before and after NEK7 expression was inhibited in BV-2 cells. Adeno-associated virus (AAV) vectors carrying short hairpin RNA (shRNA) against NEK7 (AAV-sh-NEK7) were administered to mice to knockdown NEK7 in vivo. Spatial learning and memory abilities were evaluated using the Morris water maze test. The interaction between NEK7 and histone H4 lysine 12 lactylation (H4K12la) were then investigated. The results suggested that NEK7 expression was markedly elevated in both in vitro and in vivo AD models. Treatment with Aβ significantly reduced cell viability and enhanced pyroptosis in BV-2 cells; these effects were reversed by inhibiting NEK7. Furthermore, AD mice with NEK7 knockdown exhibited shorter escape latencies and increased time spent in the target quadrant, suggesting that NEK7 inhibition improved cognitive function and memory retention. Mechanistically, Aβ treatment induced histone lactylation in BV-2 cells, and suppression of lactylation attenuated NEK7 transcriptional activity and mRNA levels. In summary, elevated NEK7 expression promoted histone lactylation in BV-2 cells, thereby facilitating pyroptosis. Inhibition of NEK7 conferred protection against Aβ-induced cellular damage and enhanced cognitive performance and memory retention in AD model mice. Collectively, targeting NEK7 represents a potential therapeutic strategy for alleviating AD symptoms.

Primary cortical cell tri-culture to study effects of amyloid-β on microglia function and neuroinflammatory response.

Microglia play a critical role in neurodegenerative disorders, such as Alzheimer's disease, where alterations in microglial function may result in pathogenic amyloid-β (Aβ) accumulation, chronic neuroinflammation, and deleterious effects on neuronal function. However, studying these complex factors in vivo, where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of critical cell types in the same culture. We employed a rat primary tri-culture (neurons, astrocytes, and microglia) model and compared it to co-culture (neurons and astrocytes) and mono-culture (microglia) to study microglial function (i.e., motility and Aβ clearance) and proteomic response to exogenous Aβ. The cultures were exposed to fluorescently-labeled Aβ (FITC-Aβ) particles for varying durations. Epifluorescence microscopy images were analyzed to quantify the number of FITC-Aβ particles and assess cytomorphological features. Cytokine profiles from conditioned media were obtained. Live-cell imaging was employed to extract microglia motility parameters. FITC-Aβ particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-Aβ particles, as confirmed via epifluorescence and confocal microscopy. FITC-Aβ treatment significantly increased microglia size, but had no significant effect on neuronal surface coverage or astrocyte size. Upon FITC-Aβ treatment, there was a significant increase in proinflammatory cytokines in tri-culture, but not in co-culture. Aβ treatment altered microglia motility evident as a swarming-like motion. The results suggest that neuron-astrocyte-microglia interactions influence microglia function and highlight the utility of the tri-culture model for studies of neuroinflammation, neurodegeneration, and cell-cell communication.

Keep neuroinflammation in mind when addressing Alzheimer's disease: A microglia perspective.

This commentary offers a detailed examination of a newly published paper on the effects of small molecule decoys of amyloid-β (Aβ) aggregation on microglial activation. It was discovered that the NSC16224 decoy peptide inhibited proinflammatory cytokines TNFα and IL6 release from microglia in response to Aβ40 and Aβ42 treatment. The research addresses the potential of blocking a sequence of events that lead to the progression of Alzheimer's disease (AD). Here, we discuss the significance of these results in neuroinflammation, highlighting the greater implications for how decoy peptides would be interesting for the research and development of new drugs for AD therapy.

Amyloid PET detects the deposition of brain Aβ earlier than CSF fluid biomarkers.

Understanding the relationship between amyloid beta (Aβ) positron emission tomography (PET) and Aβ cerebrospinal fluid (CSF) biomarkers will define their potential utility in Aβ treatment. Few population-based or neuropathologic comparisons have been reported. Participants 50+ years with Aβ PET and Aβ CSF biomarkers (phosphorylated tau [p-tau]181/Aβ42, n=505, and Aβ42/40, n=54) were included from the Mayo Clinic Study on Aging. From these participants, an autopsy subgroup was identified (n=47). The relationships of Aβ PET and Aβ CSF biomarkers were assessed cross-sectionally in all participants and longitudinally in autopsy data. Cross-sectionally, more participants were Aβ PET+ versus Aβ CSF- than Aβ PET- versus Aβ CSF+ with an incremental effect when using Aβ PET regions selected for early Aβ deposition. The sensitivity for the first detection of Thal phase≥1 in longitudinal data was higher for Aβ PET (89%) than p-tau181/Aβ42 (64%). Aβ PET can detect earlier cortical Aβ deposition than Aβ CSF biomarkers. Aβ PET+ versus Aβ CSF- findings are several-fold greater using regional Aβ PET analyses and in peri-threshold-standardized uptake value ratio participants. Amyloid beta (Aβ) positron emission tomography (PET) has greater sensitivity for Aβ deposition than Aβ cerebrospinal fluid (CSF) in early Aβ development. A population-based sample of participants (n=505) with PET and CSF tests was used. Cortical regions showing early Aβ on Aβ PET were also used in these analyses. Neuropathology was used to validate detection of Aβ by Aβ PET and Aβ CSF biomarkers.

Uncovering Plaque-Glia Niches in Human Alzheimer's Disease Brains Using Spatial Transcriptomics.

Amyloid-beta (Aβ) plaques and surrounding glial activation are prominent histopathological hallmarks of Alzheimer's Disease (AD). However, it is unclear how Aβ plaques interact with surrounding glial cells in the human brain. Here, we applied spatial transcriptomics (ST) and immunohistochemistry (IHC) for Aβ, GFAP, and IBA1 to acquire data from 258,987 ST spots within 78 postmortem brain sections of 21 individuals. By coupling ST and adjacent-section IHC, we showed that low Aβ spots exhibit transcriptomic profiles indicative of greater neuronal loss than high Aβ spots, and high-glia spots present transcriptomic changes indicative of more significant inflammation and neurodegeneration. Furthermore, we observed that this ST glial response bears signatures of reported mouse gene modules of plaque-induced genes (PIG), oligodendrocyte (OLIG) response, disease-associated microglia (DAM), and disease-associated astrocytes (DAA), as well as different microglia (MG) states identified in human AD brains, indicating that multiple glial cell states arise around plaques and contribute to local immune response. We then validated the observed effects of Aβ on cell apoptosis and plaque-surrounding glia on inflammation and synaptic loss using IHC. In addition, transcriptomic changes of iPSC-derived microglia-like cells upon short-interval Aβ treatment mimic the ST glial response and mirror the reported activated MG states. Our results demonstrate an exacerbation of synaptic and neuronal loss in low-Aβ or high-glia areas, indicating that microglia response to Aβ-oligomers likely initiates glial activation in plaque-glia niches. Our study lays the groundwork for future pathology genomics studies, opening the door for investigating pathological heterogeneity and causal effects in neurodegenerative diseases.

Alzheimer's disease linked Aβ42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling.

Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimer's disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling.

Comprehensive characterization of the neurogenic and neuroprotective action of a novel TrkB agonist using mouse and human stem cell models of Alzheimer’s disease

BackgroundNeural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimer’s disease (AD) pathology. Addressing these impairments by using neurotrophic factors is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations.MethodsHere, we use a combination of mouse and human stem cell models for comprehensive in-vitro characterization of a novel neurogenic compound, focusing on the brain-derived neurotrophic factor (BDNF) pathway. The ability of ENT-A011, a steroidal dehydroepiandrosterone derivative, to activate the tyrosine receptor kinase B (TrkB) receptor was tested through western blotting in NIH-3T3 cells and its neurogenic and neuroprotective action were assessed through proliferation, cell death and Amyloid-β (Aβ) toxicity assays in mouse primary adult hippocampal NSCs, mouse embryonic cortical NSCs and neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. RNA-seq profiling was used to assess if the compound acts through the same gene network as BDNF in human NPCs.ResultsENT-A011 was able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Aβ-induced cell death, acting selectively through TrkB activation. The compound was able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from Αβ toxicity and prevent synapse loss after Aβ treatment. ENT-A011 successfully induces proliferation and prevents cell death after Aβ toxicity in human NPCs, acting through a core gene network shared with BDNF as shown through RNA-seq.ConclusionsOur work characterizes a novel BDNF mimetic with preferable pharmacological properties and neurogenic and neuroprotective actions in Alzheimer’s disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing.

Increased expression of SLC25A18 is associated with Alzheimer’s disease and is involved in Aβ42-induced mitochondrial dysfunction and apoptosis in neuronal cells

Alzheimer’s disease (AD) is currently one of the most serious public health concerns in the world. However, the best approach to treat AD has yet to be discovered, implying that we must continue to work hard to find new AD target genes. In this study, we further analysed Gene Expression Omnibus (GEO) data and discovered that the expression of the Mitochondria glutamate carrier SLC25A18 is associated with AD by screening the differentially expressed genes in different regions of the brains of Alzheimer’s disease patients. To verify the expression of SLC25A18 during Alzheimer’s disease development, we analysed animal models (5×FAD transgenic AD animal model, chemically induced AD animal model, natural ageing animal model), and the results showed that the expression of SLC25A18 was increased in animal models of AD. Further investigation of the different regions found that SLC25A18 expression was elevated in the EC, TeA, and CA3, and expressed in neurons. Next, We found that Aβ42 treatment elevated SLC25A18 expression in Neuro 2A cells. Reducing SLC25A18 expression attenuated mitochondrial dysfunction and neuronal apoptosis caused by Aβ42. Overexpression of SLC25A18 increased ATP and intracellular superoxide anions but decreased mitochondrial membrane potential. The results indicate that SLC25A18 affects mitochondrial function and neuronal apoptosis, and is related to AD, which makes it a potential target for treating brain dysfunction.

Hsa_circ_0049472 contributed to amyloid-beta peptide-induced neurotoxicity, apoptosis and inflammation via regulating PI3K-AKT signaling pathway by interacting with miR-22–3p/ZNF217 axis

BackgroundCircular RNAs (circRNAs) exhibited important roles in Alzheimer’s disease (AD). Here, we focused on the dysregulation of hsa_circ_0049472 (circ_0049472) and potential functions in SK-N-SH cells with amyloid-beta peptide (Aβ) treatment in AD. MethodsRNA expression was detected by real-time quantitative PCR. Cell viability and proliferation were measured by MTS and Edu assays. Flow cytometry was used for apoptosis detection, and cell inflammation was assessed using enzyme-linked immunosorbent assay. Target interaction was validated by dual-luciferase reporter assay and RNA immunoprecipitation assay. Protein expression and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) pathway were examined by Immunoblotting. ResultsAβ treatment inhibited cell viability and proliferation of SK-N-SH cells, but enhanced apoptosis rate, apoptosis protein levels (Bcl2-associated X protein and cleaved-caspase-3) and inflammatory cytokines (interleukin −6, IL-1β, tumor necrosis factor-α). Then, circ_0049472 expression was shown to be upregulated in response to Aβ stimulation and knockdown of circ_0049472 has ameliorated Aβ-induced cell injury. Circ_0049472 was identified as a sponge for miR-22–3p, and miR-22–3p inhibition reversed the regulation of circ_0049472 knockdown in Aβ-treated cells. Furthermore, ZNF217 acted as a target of miR-22–3p and circ_0049472 could regulate ZNF217 expression via binding to miR-22–3p. Overexpression of miR-22–3p abated Aβ-induced apoptosis and inflammation via downregulating ZNF217. Furthermore, Aβ reduced proteins levels of p-PI3K and p-AKT, and this inhibition of PI3K-AKT pathway was restored by the regulation of circ_0049472/miR-22–3p/ZNF217 axis. ConclusionCirc_0049472 was involved in Aβ-induced neural injury by regulating miR-22–3p/ZNF217 axis to affect PI3K-AKT pathway. This study has discovered an innovative mechanism for AD.

TLR4/Rac1/NLRP3 Pathway Mediates Amyloid-β-Induced Neuroinflammation in Alzheimer's Disease.

Neuroinflammation plays a crucial part in the initial onset and progression of Alzheimer's disease (AD). NLRP3 inflammasome was demonstrated to get involved in amyloid-β (Aβ)-induced neuroinflammation. However, the mechanism of Aβ-triggered activation of NLRP3 inflammasome remains poorly understood. Based on our previous data, the study aimed to identify the downstream signals that bridge the activation of TLR4 and NLRP3 inflammasome associated with Aβ. BV-2 cells were transfected with TLR4siRNA or pretreated with a CLI-095 or NSC23766, followed by Aβ1-42 treatment. APP/PS1 mice were injected intraperitoneally with CLI-095 or NSC23766. NLRP3 inflammasome and microglia activation was detected with immunostaining and western blot. G-LISA and Rac1 pull-down activation test were performed to investigate the activation of Rac1. Real-time PCR and ELISA were used to detect the inflammatory cytokines. Aβ plaques were assessed by western blotting and immunofluorescence staining. Morris water maze test was conducted to determine the spatial memory in mice. Rac1 and NLRP3 inflammasome were activated by Aβ in both in vitro and in vivo experiments. Inhibition of TLR4 reduced the activity of Rac1 and NLRP3 inflammasome induced by Aβ1-42. Furthermore, inhibition of Rac1 blocked NLRP3 inflammasome activation mediated by TLR4. Blocking the pathway by CLI095 or NSC23766 suppressed Aβ1-42-triggered activation of microglia, reduced the expression of pro-inflammatory mediators and ameliorated the cognition deficits in APP/PS1 mice. Our study demonstrated that TLR4/Rac1/NLRP3 pathway mediated Aβ-induced neuroinflammation, which unveiled a novel pathway and key contributors underlying the pathogenic mechanism of Aβ.

Neuroprotective effect of emodin on Aβ25-35-induced cytotoxicity in PC12 cells involves Nrf2/GPX4 and TLR4/p-NF-κB/NLRP3 pathways

The present study aims to investigate the neuroprotective effects of emodin in Alzheimer’s disease (AD). PC12 cells were used to explore the underlying mechanism and were incubated with Aβ25-35 for 24 h as the model group, incubated with emodin at different concentrations (2.5, 5, 10 μM) as the drug administration groups. The content of MDA and the enzymic activities of CAT, GSH-Px were detected by the corresponding commercial kits. The ROS level in Aβ25-35 induced cells was decreased by emodin dose-dependently, but the MMP in these cells were elevated. The expressions of AChE, TLR4, p-NF-κB, NLRP3, IL-1β, and TNF-α in PC12 cells were increased by Aβ25-35 treatment, the expressions of Nrf2, HO-1, GPX4, xCT were decreased, all the levels of expressions were reversed by emodin. Besides, ultraviolet spectrophotometry and infrared spectrophotometry were ultilized to ascertain the production of emodin-Fe (Ⅱ) complex. The FerroOrange results showed that emodin reduced free Fe2+ in cells. The immunofluorescent intensities of Nrf2, GPX4, and p-NF-κB offered direct visible evidence for emodin’s multi-targets in AD treatment. Collectively, emodin could inhibit the activity of AChE and exert neuroprotective effects against AD through antioxidant, anti-ferroptotic, anti-inflammatory properties via Nrf2/GPX4 and TLR4/p-NF-κB/NLRP3 pathways.

Potential neuroprotective effects of 2-hydroxypropyl-β cyclodextrin against amyloid β (1-42)-induced neurotoxicity on the rat hippocampus

The neurodegenerative mechanisms of Alzheimer’s disease (AD) are not fully understood, but it is believed that amyloid beta (Aβ) peptide causes oxidative stress, neuroinflammation, and disrupts metabotropic glutamate receptor 5 (mGluR5) signaling by interacting with cholesterol and caveolin-1 (Cav-1) in pathogenic lipid rafts. This study examined the effect of 2-hydroxypropyl-β-cyclodextrin (HP-CD) on cholesterol, oxidative stress (total oxidant status), neuroinflammation (TNF-α), and mGluR5 signaling molecules such as PKCβ1, PKCβ2, ERK1/2, CREB, BDNF, and NGF in Aβ (1-42)-induced neurotoxicity. The Sprague-Dawley rats were divided into four groups: control (saline), Aβ (1-42), HP-CD (100 mg/kg), and Aβ (1-42) + HP-CD (100 mg/kg). All groups received bilateral stereotaxic injections of Aβ (1–42) or saline into the hippocampus. After surgery, HP-CD was administered intraperitoneally (ip) for 7 days. Cholesterol, TNF-α, and TOS levels were measured in synaptosomes isolated from hippocampus tissue using spectrophotometry, fluorometry, and enzyme immunoassay, respectively. The gene expressions of Cav-1, mGluR5, PKCβ1, PKCβ2, ERK1/2, CREB, BDNF, and NGF in hippocampus tissue were evaluated using reverse transcription PCR after real-time PCR analysis. Treatment with Aβ (1-42) significantly elevated cholesterol, TOS, TNF-α, Cav-1, PKCβ2, and ERK1/2 levels. Additionally, mGluR5, CREB, and BDNF levels were shown to be lowered. HP-CD reduced cholesterol, TOS, and TNF-α levels while increasing mGluR5, CREB, and BDNF in response to Aβ (1–42) treatment. These findings indicate that HP-CD may have neuroprotective activity due to the decreased levels of cholesterol, oxidative stress, and neuroinflammation, as well as upregulated levels of mGluR5, CREB, and BDNF.

Restoring capillary-to-arteriole signaling in Alzheimer's Disease

There is growing evidence for vascular contributions to cognitive impairments in Alzheimer’s Disease (AD). Functional hyperemia, which refers to local increases in blood flow in response to local increases in brain activity, is impaired early in patients and animal models of AD. The mechanisms underlying functional hyperemia are referred to as neurovascular coupling (NVC). A key player in NVC is the inward rectifying K+ channel (Kir2.1) in capillary endothelial cells (cECs). Activation of Kir2.1 channels initiates a retrograde hyperpolarizing signal which causes upstream arterioles to dilate and increase local blood flow. Studies have shown that Kir2.1 channel function is impaired in the 5xFAD mouse model for AD, and administration of Kir2.1 cofactor, phosphatidylinositol 4,5-bisphosphate (PIP2), was suffcient to restore channel function and functional hyperemia deficits. The objective of this study is to characterize the effect of AD on NVC. Since AD is characterized by the deposition of amyloid beta (Aβ), we hypothesize that Aβ is impairing Kir2.1 function in cECs at an early stage of the disease. Using 6-month-old female and male 5xFAD mice, we performed our ex vivo capillary-parenchymal arteriole (CaPA) technique to investigate NVC responses by directly stimulating capillaries and recording upstream arteriolar dilation in real time. We further tested the direct effect of Aβ oligomers (200 nM) and reactive oxygen species (ROS) scavengers (superoxide dismutase 120 U/mL + catalase 1000 U/mL), to probe their effect on the NVC response. Finally, to test the ability of PIP2 to rescue the NVC response, systemic administration via intraperitoneal injection was performed one hour before CaPA experiment. Consistent with a NVC impairment in AD, capillary stimulation with 10 mM K+ failed to evoke upstream arteriolar dilation in 5xFAD mice at an early stage, and this impairment was recapitulated in wild type mice after treatment with Aβ. Importantly, Aβ only caused a modest arteriole constriction (20.11% ± 5.24 %) but did not impair its ability to dilate in response to direct stimulation with 10 mM K+, suggesting an effect on cECs. ROS scavenging during Aβ treatment prevented NVC impairment as 10 mM K+ capillary stimulation induced an upstream arteriolar dilation of 59% of max dilation. Finally, systemic administration of PIP2 also rescued the NVC response to the levels observed in control conditions. Taken together, our results suggest that Aβ is causing impairment in NVC via increasing ROS production, specifically at the capillary endothelial level. This increase in ROS is likely leading to PIP2 degradation and causing Kir2.1 channel dysfunction. Administration of PIP2 was suffcient to rescue the NVC response, suggesting a possible strategy for rescuing functional hyperemia in AD. This study was supported by a research grant from the Center for Women’s Health Research located at the University of Colorado Anschutz Medical Campus; 2 research grants from the University of Pennsylvania Orphan Disease Center in partnership with the cureCADASIL (2019 and 2022), the National Institute of Neurological Disorders and Stroke RF1/R01NS129022, and the National Heart, Lung, and Blood Institute R01HL136636 to FD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anti-inflammatory and Neuroprotective α-Pyrones from a Marine-Derived Strain of the Fungus Arthrinium arundinis and Their Heterologous Expression.

Fungal linear polyketides, such as α-pyrones with a 6-alkenyl chain, have been a rich source of biologically active compounds. Two new (1 and 2) and four known (3-6) 6-alkenylpyrone polyketides were isolated from a marine-derived strain of the fungus Arthrinium arundinis. Their structures were determined based on extensive spectroscopic analysis. The biosynthetic gene cluster (alt) for alternapyrones was identified from A. arundinis ZSDS-F3 and validated by heterologous expression in Aspergillus nidulans A1145 ΔSTΔEM, which revealed that the cytochrome P450 monooxygenase Alt2' could convert the methyl group 26-CH3 to a carboxyl group to produce 4 from 3. Another cytochrome P450 monooxygenase, Alt3', catalyzed successive hydroxylation, epoxidation, and oxidation steps to produce 1, 2, 5, and 6 from 4. Alternapyrone G (1) not only suppressed M1 polarization in lipopolysaccharide (LPS)-stimulated BV2 microglia but also stimulated dendrite regeneration and neuronal survival after Aβ treatment, suggesting alternapyrone G may be utilized as a privileged scaffold for Alzheimer's disease drug discovery.

Dual Role of NMDAR Containing NR2A and NR2B Subunits in Alzheimer's Disease.

Alzheimer's disease (AD) is the main cause of dementia worldwide. Given that learning and memory are impaired in this pathology, NMDA receptors (NMDARs) appear as key players in the onset and progression of the disease. NMDARs are glutamate receptors, mainly located at the post-synapse, which regulate voltage-dependent influx of calcium into the neurons. They are heterotetramers, and there are different subunits that can be part of the receptors, which are usually composed of two obligatory GluN1 subunits plus either two NR2A or two NR2B subunits. NR2A are mostly located at the synapse, and their activation is involved in the expression of pro-survival genes. Conversely, NR2B are mainly extrasynaptic, and their activation has been related to cell death and neurodegeneration. Thus, activation of NR2A and/or inactivation of NR2B-containing NMDARS has been proposed as a therapeutic strategy to treat AD. Here, we wanted to investigate the main differences between both subunits signalling in neuronal primary cultures of the cortex and hippocampus. It has been observed that Aβ induces a significant increase in calcium release and also in MAPK phosphorylation signalling in NR2B-containing NMDAR in cortical and hippocampal neurons. However, while NR2A-containing NMDAR decreases neuronal death and favours cell viability after Aβ treatment, NR2B-containing NMDAR shows higher levels of cytotoxicity and low levels of neuronal survival. Finally, it has been detected that NMDAR has no effect on pTau axonal transport. The present results demonstrate a different role between GluNA and GluNB subunits in neurodegenerative diseases such as Alzheimer's.

Simple ammonium salt and sigma-1 receptor ligand dipentylammonium provides neuroprotective effects in cell culture and Caenorhabditis elegans models of Alzheimer's disease

The sigma-1 receptor (σ-1R), a chaperone protein located at the mitochondria-associated membrane (MAM) of the endoplasmic reticulum, can interact with and modify the signaling pathways of various proteins, thereby modulating many disease pathologies, including Alzheimer's disease (AD). The σ-1R ligand dipentylammonium (DPA) was analyzed for its anti-AD properties using PC12 cells (in vitro) and Caenorhabditis elegans (in vivo) models along with molecular docking (in silico) analysis. DPA at 1 and 10 µM concentrations was able to significantly potentiate NGF-induced neurite growth length by 137.7 ± 12.0 and 187.8 ± 16.4, respectively, when compared to the control 76.9 ± 7.4. DPA also regulated neurite damage caused by Aβ(25−35) treatment in differentiated PC12 cells by improving cell viability and neurite length. In C. elegans, DPA could significantly extend the median and maximum lifespan of Aβ transgenic strain CL2006 without impacting wild-type nematodes. Additionally, it could significantly reduce the paralysis phenotype of another Aβ transgenic strain, CL4176, thereby improving the overall health in AD pathogenesis. This effect depended on σ-1R, as DPA could not modulate the lifespan of σ-1R mutant TM3443. This was further confirmed using agonist PRE084 and antagonist BD1047, wherein the agonist alone could extend the lifespan of CL2006, while the antagonist suppressed the effect of DPA in CL2006. Interestingly, neither had an TM3443. Further, molecular docking analysis showed that DPA had a similar binding affinity as that of PRE084, BD1047 and pentazocine against the σ-1R receptor in humans and C. elegans, which collectively suggests the anti-AD properties of DPA.

Ceftriaxone Modulates Ubiquitination of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid Receptors to Improve Long-Term Potentiation Impairment Induced by Exogenous β-Amyloid in a Glutamate Transporter-1 Dependent Manner.

Α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are crucial for properties of synaptic plasticity, such as long-term potentiation (LTP). LTP impairment can occur early in the onset of Alzheimer's disease (AD). The downregulation or decreased abundance of AMPAR expression in the postsynaptic membrane is closely associated with LTP impairment. Ceftriaxone (Cef) can improve LTP impairment in the early stages of AD in a mouse model. The purpose of this study was to explore the mechanism underlying this process from the aspects of AMPAR expression and ubiquitination degree. In this study, we found that β-amyloid (Aβ) treatment induced hippocampal LTP impairment and AMPAR downregulation and ubiquitination. Cef pretreatment ameliorated Aβ-induced hippocampal LTP impairment, reduced AMPAR ubiquitination, and increased AMPAR expression, especially in the plasma membrane, in Aβ-treated mice. Administration of USP46 siRNA and DHK (a specific blocker of glutamate transporter-1) significantly inhibited the above effects of Cef, suggesting a role for anti-AMPAR ubiquitination and upregulation of glutamate transporter-1 (GLT-1) in the Cef-induced improvements mentioned above. The above findings demonstrate that pretreatment with Cef effectively mitigated Aβ-induced impairment of hippocampal LTP by suppressing the ubiquitination process of AMPARs in a GLT-1-dependent manner. These results provide novel insights into the underlying mechanisms elucidating the anti-AD by Cef.

Alzheimer's Amyloid-β Accelerates Cell Senescence and Suppresses SIRT1 in Human Neural Stem Cells.

As a lifelong source of neurons, neural stem cells (NSCs) serve multiple crucial functions in the brain. The senescence of NSCs may be associated with the onset and progression of Alzheimer's disease (AD). Our study reveals a noteworthy finding, indicating that the AD-associated pathogenic protein amyloid-β (Aβ) substantially enhances senescence-related characteristics of human NSCs. These characteristics encompass the enhanced expression of p16 and p21, the upregulation of genes associated with the senescence-associated secretory phenotype (SASP), increased SA-β-gal activity, and the activation of the DNA damage response. Further studies revealed that Aβ treatment significantly downregulates the SIRT1 protein which plays a crucial role in regulating the aging process and decreases downstream PGC-1α and FOXO3. Subsequently, we found that SIRT1 overexpression significantly alleviates a range of Aβ-induced senescent markers in human NSCs. Taken together, our results uncover that Aβ accelerates cellular senescence in human NSCs, making SIRT1 a highly promising therapeutic target for senescent NSCs which may contribute to age-related neurodegenerative diseases, including AD.

Interferon Regulatory Factor 5 Regulates the Phagocytosis of Microglia and Alleviate Alzheimer's Pathology.

Microglia play a critical role in the pathophysiology of Alzheimer's disease. They are involved in Aβ-induced neuroinflammatory responses, regulating the production of inflammatory mediators. Interferon regulatory factor 5 (IRF5) plays a central role in inflammatory diseases in the periphery, the role of which in central nervous system remains elusive. This study aimed to investigate the role of IRF5 in Aβ-induced neuroinflammation and the progression of Aβ pathology. We found that Aβ1-42 oligomers significantly increased the level of IRF5 in BV2 microglia. The levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly upregulated with Aβ treatment. IRF5 knockdown with siRNA in microglia significantly reduced the expression of these proinflammatory factors induced by Aβ and promoted Aβ phagocytosis. Besides, LC3 upregulation and p62 downregulation were observed in IRF5 knockdown microglia. This was also validated in APP/PS1 mice with IRF5 knockdown, leading to reduced Aβ levels in the brain. We conclude that IRF5 mediates Aβ-induced microglial inflammatory responses. IRF5 knockdown attenuated Aβ-induced inflammatory responses and promoted the phagocytosis and autophagy of Aβ by microglia.

Age-Dependent and Aβ-Induced Dynamic Changes in the Subcellular Localization of HMGB1 in Neurons and Microglia in the Brains of an Animal Model of Alzheimer's Disease.

HMGB1 is a prototypical danger-associated molecular pattern (DAMP) molecule that co-localizes with amyloid beta (Aβ) in the brains of patients with Alzheimer's disease. HMGB1 levels are significantly higher in the cerebrospinal fluid of patients. However, the cellular and subcellular distribution of HMGB1 in relation to the pathology of Alzheimer's disease has not yet been studied in detail. Here, we investigated whether HMGB1 protein levels in brain tissue homogenates (frontal cortex and striatum) and sera from Tg-APP/PS1 mice, along with its cellular and subcellular localization in those regions, differed. Total HMGB1 levels were increased in the frontal cortices of aged wildtype (7.5 M) mice compared to young (3.5 M) mice, whereas total HMGB1 levels in the frontal cortices of Tg-APP/PS1 mice (7.5 M) were significantly lower than those in age-matched wildtype mice. In contrast, total serum HMGB1 levels were enhanced in aged wildtype (7.5 M) mice and Tg-APP/PS1 mice (7.5 M). Further analysis indicated that nuclear HMGB1 levels in the frontal cortices of Tg-APP/PS1 mice were significantly reduced compared to those in age-matched wildtype controls, and cytosolic HMGB1 levels were also significantly decreased. Triple-fluorescence immunohistochemical analysis indicated that HMGB1 appeared as a ring shape in the cytoplasm of most neurons and microglia in the frontal cortices of 9.5 M Tg-APP/PS1 mice, indicating that nuclear HMGB1 is reduced by aging and in Tg-APP/PS1 mice. Consistent with these observations, Aβ treatment of both primary cortical neuron and primary microglial cultures increased HMGB1 secretion in the media, in an Aβ-dose-dependent manner. Our results indicate that nuclear HMGB1 might be translocated from the nucleus to the cytoplasm in both neurons and microglia in the brains of Tg-APP/PS1 mice, and that it may subsequently be secreted extracellularly.