Accumulation of amyloid-like Aβ1–42 in AEL (autophagy–endosomal–lysosomal) vesicles: potential implications for plaque biogenesis

Abnormal accumulation of Aβ (amyloid β) within AEL (autophagy–endosomal–lysosomal) vesicles is a prominent neuropathological feature of AD (Alzheimer''s disease), but the mechanism of accumulation within vesicles is not clear. We express secretory forms of human Aβ_1–40 or Aβ_1–42 in Drosophila neurons and observe preferential localization of Aβ_1–42 within AEL vesicles. In young animals, Aβ_1–42 appears to associate with plasma membrane, whereas Aβ_1–40 does not, suggesting that recycling endocytosis may underlie its routing to AEL vesicles. Aβ_1–40, in contrast, appears to partially localize in extracellular spaces in whole brain and is preferentially secreted by cultured neurons. As animals become older, AEL vesicles become dysfunctional, enlarge and their turnover appears delayed. Genetic inhibition of AEL function results in decreased Aβ_1–42 accumulation. In samples from older animals, Aβ_1–42 is broadly distributed within neurons, but only the Aβ_1–42 within dysfunctional AEL vesicles appears to be in an amyloid-like state. Moreover, the Aβ_1–42-containing AEL vesicles share properties with AD-like extracellular plaques. They appear to be able to relocate to extracellular spaces either as a consequence of age-dependent neurodegeneration or a non-neurodegenerative separation from host neurons by plasma membrane infolding. We propose that dysfunctional AEL vesicles may thus be the source of amyloid-like plaque accumulation in Aβ_1–42-expressing Drosophila with potential relevance for AD.     

Oridonin Attenuates Aβ1–42-Induced Neuroinflammation and Inhibits NF-κB Pathway

Neuroinflammation induced by beta-amyloid (Aβ) plays a critical role in the pathogenesis of Alzheimer’s disease (AD), and inhibiting Aβ-induced neuroinflammation serves as a potential strategy for the treatment of AD. Oridonin (Ori), a compound of Rabdosia rubescens, has been shown to exert anti-inflammatory effects. In this study, we demonstrated that Ori inhibited glial activation and decreased the release of inflammatory cytokines in the hippocampus of Aβ_1–42-induced AD mice. In addition, Ori inhibited the NF-κB pathway and Aβ_1–42-induced apoptosis. Furthermore, Ori could attenuate memory deficits in Aβ_1–42-induced AD mice. In conclusion, our study demonstrated that Ori inhibited the neuroinflammation and attenuated memory deficits induced by Aβ_1–42, suggesting that Ori might be a promising candidate for AD treatment.     

Protection of TGF-β1 against Neuroinflammation and Neurodegeneration in Aβ1–42-Induced Alzheimer’s Disease Model Rats

Neuroinflammation has been reported to be associated with Alzheimer’s disease (AD) pathogenesis. Neuroinflammation is generally considered as an outcome of glial activation; however, we recently demonstrated that T helper (Th)17 cells, a subpopulation of proinflammatory CD4+ T cells, are also involved in AD pathogenesis. Transforming growth factor (TGF)-β1, a cytokine that can be expressed in the brain, can be immunosuppressive, but its effects on lymphocyte-mediated neuroinflammation in AD pathogenesis have not been well addressed. In the current study we administered TGF-β1 via intracerebroventricle (ICV) and intranasal (IN) routes in AD model rats to investigate its antiinflammatory and neuroprotective effects. The AD rat model was prepared by bilateral hippocampal injection of amyloid-β (Aβ)_1–42. TGF-β1 was administered via ICV one hour prior to Aβ_1–42 injection or via both nares seven days after Aβ_1–42 injection. ICV administration of TGF-β1 before Aβ_1–42 injection remarkably ameliorated Aβ_1–42-induced neurodegeneration and prevented Aβ_1–42-induced increases in glia-derived proinflammatory mediators (TNF-α, IL-1β and iNOS), as well as T cell-derived proinflammatory cytokines (IFN-γ, IL-2, IL-17 and IL-22), in the hypothalamus, serum or cerebrospinal fluid (CSF) in a concentration-dependent manner. TGF-β1 pretreatment also prevented Aβ_1–42-induced decreases in the neurotrophic factors, IGF-1, GDNF and BDNF, and in the antiinflammatory cytokine, IL-10. Similarly, IN administration of TGF-β1 after Aβ_1–42 injection reduced neurodegeneration, elevation of proinflammatory mediators and cytokines, and reduction of neurotrophic and antiinflammatory factors, in the hypothalamus, serum or CSF. These findings suggest that TGF-β1 suppresses glial and T cell-mediated neuroinflammation and thereby alleviates AD-related neurodegeneration. The effectiveness of IN administered TGF-β1 in reducing Aβ_1–42 neurotoxicity suggests a possible therapeutic approach in patients with AD.     

Triptolide Inhibited Cytotoxicity of Differentiated PC12 Cells Induced by Amyloid-Beta25–35 via the Autophagy Pathway

Evidence shows that an abnormal deposition of amyloid beta-peptide_25–35 (Aβ_25–35) was the primary cause of the pathogenesis of Alzheimer’s disease (AD). And the elimination of Aβ_25–35 is considered an important target for the treatment of AD. Triptolide (TP), isolated from Tripterygium wilfordii Hook.f. (TWHF), has been shown to possess a broad spectrum of biological profiles, including neurotrophic and neuroprotective effects. In our study investigating the effect and potential mechanism of triptolide on cytotoxicity of differentiated rat pheochromocytoma cell line (the PC12 cell line is often used as a neuronal developmental model) induced by Amyloid-Beta_25–35 (Aβ_25–35), we used 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay, flow cytometry, Western blot, and acridine orange staining to detect whether triptolide could inhibit Aβ_25–35–induced cell apoptosis. We focused on the potential role of the autophagy pathway in Aβ_25–35-treated differentiated PC12 cells. Our experiments show that cell viability is significantly decreased, and the apoptosis increased in Aβ_25–35-treated differentiated PC12 cells. Meanwhile, Aβ_25–35 treatment increased the expression of microtubule-associated protein light chain 3 II (LC3 II), which indicates an activation of autophagy. However, triptolide could protect differentiated PC12 cells against Aβ_25–35-induced cytotoxicity and attenuate Aβ_25–35-induced differentiated PC12 cell apoptosis. Triptolide could also suppress the level of autophagy. In order to assess the effect of autophagy on the protective effects of triptolide in differentiated PC12 cells treated with Aβ_25–35, we used 3-Methyladenine (3-MA, an autophagy inhibitor) and rapamycin (an autophagy activator). MTT assay showed that 3-MA elevated cell viability compared with the Aβ_25–35-treated group and rapamycin inhibits the protection of triptolide. These results suggest that triptolide will repair the neurological damage in AD caused by deposition of Aβ_25–35 via the autophagy pathway, all of which may provide an exciting view of the potential application of triptolide or TWHF as a future research for AD.     

Linking Aβ42-Induced Hyperexcitability to Neurodegeneration,Learning and Motor Deficits,and a Shorter Lifespan in an Alzheimer’s Model

Alzheimer’s disease (AD) is the most prevalent form of dementia in the elderly. β-amyloid (Aβ) accumulation in the brain is thought to be a primary event leading to eventual cognitive and motor dysfunction in AD. Aβ has been shown to promote neuronal hyperactivity, which is consistent with enhanced seizure activity in mouse models and AD patients. Little, however, is known about whether, and how, increased excitability contributes to downstream pathologies of AD. Here, we show that overexpression of human Aβ42 in a Drosophila model indeed induces increased neuronal activity. We found that the underlying mechanism involves the selective degradation of the A-type K+ channel, Kv4. An age-dependent loss of Kv4 leads to an increased probability of AP firing. Interestingly, we find that loss of Kv4 alone results in learning and locomotion defects, as well as a shortened lifespan. To test whether the Aβ42-induced increase in neuronal excitability contributes to, or exacerbates, downstream pathologies, we transgenically over-expressed Kv4 to near wild-type levels in Aβ42-expressing animals. We show that restoration of Kv4 attenuated age-dependent learning and locomotor deficits, slowed the onset of neurodegeneration, and partially rescued premature death seen in Aβ42-expressing animals. We conclude that Aβ42-induced hyperactivity plays a critical role in the age-dependent cognitive and motor decline of this Aβ42-Drosophila model, and possibly in AD.     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

Enhanced Aβ1–40 Production in Endothelial Cells Stimulated with Fibrillar Aβ1–42

Amyloid accumulation in the brain of Alzheimer’s patients results from altered processing of the 39- to 43-amino acid amyloid β protein (Aβ). The mechanisms for the elevated amyloid (Aβ_1–42) are considered to be over-expression of the amyloid precursor protein (APP), enhanced cleavage of APP to Aβ, and decreased clearance of Aβ from the central nervous system (CNS). We report herein studies of Aβ stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving Aβ_1–42 fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of Aβ_1–40 by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that Aβ_1–40 accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar Aβ_1–42 treatment involving eNOS upregulation and increased autophagy are also reported.     

The Molecular Mechanism of Amyloid β42 Peptide Toxicity: The Role of Sphingosine Kinase-1 and Mitochondrial Sirtuins

Our study focused on the relationship between amyloid β 1–42 (Aβ), sphingosine kinases (SphKs) and mitochondrial sirtuins in regulating cell fate. SphK1 is a key enzyme involved in maintaining sphingolipid rheostat in the brain. Deregulation of the sphingolipid metabolism may play a crucial role in the pathogenesis of Alzheimer’s disease (AD). Mitochondrial function and mitochondrial deacetylases, i.e. sirtuins (Sirt3,-4,-5), are also important for cell viability. In this study, we evaluated the interaction between Aβ_1–42, SphKs and Sirts in cell survival/death, and we examined several compounds to indicate possible target(s) for a strategy protecting against cytotoxicity of Aβ_1–42. PC12 cells were subjected to Aβ_1–42 oligomers and SphK inhibitor SKI II for 24–96 h. Our data indicated that Aβ_1–42 enhanced SphK1 expression and activity after 24 h, but down-regulated them after 96 h and had no effect on Sphk2. Aβ_1–42 and SKI II induced free radical formation, disturbed the balance between pro- and anti-apoptotic proteins and evoked cell death. Simultaneously, up-regulation of anti-oxidative enzymes catalase and superoxide dismutase 2 was observed. Moreover, the total protein level of glycogen synthase kinase-3β was decreased. Aβ_1–42 significantly increased the level of mitochondrial proteins: apoptosis-inducing factor AIF and Sirt3, -4, -5. By using several pharmacologically active compounds we showed that p53 protein plays a significant role at very early stages of Aβ_1–42 toxicity. However, during prolonged exposure to Aβ_1–42, the activation of caspases, MEK/ERK, and alterations in mitochondrial permeability transition pores were additional factors leading to cell death. Moreover, SphK product, sphingosine-1-phosphate (S1P), and Sirt activators and antioxidants, resveratrol and quercetin, significantly enhanced viability of cells subjected to Aβ_1–42. Our data indicated that p53 protein and inhibition of SphKs may be early key events responsible for cell death evoked by Aβ_1–42. We suggest that activation of S1P-dependent signalling and Sirts may offer a promising cytoprotective strategy.     

Molecular insights into the primary nucleation of polymorphic amyloid β dimers in DOPC lipid bilayer membrane

Alzheimer''s disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid β (Aβ) plaques in the brain that consists of Aβ oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aβ oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic “α‐helix/random coil” and “fibril‐like” Aβ dimers on 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aβ_1–42 “fibril‐like” dimers and impedes Aβ_1–42 “α‐helix/random coil” dimers. The N‐termini domains within Aβ_1–42 dimers play a role in Aβ aggregation in membrane milieus. In addition, the aromatic π–π interactions (involving residues F19 and F20 in Aβ_1–42) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aβ_1–42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aβ_1–42 dimer, due to relatively large contacts between Aβ_1–42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early‐stage Aβ_1–42 dimers have distinct impacts on DOPC membrane.     

Molecular Plasticity Regulates Oligomerization and Cytotoxicity of the Multipeptide-length Amyloid-β Peptide Pool

Current therapeutic approaches under development for Alzheimer disease, including γ-secretase modulating therapy, aim at increasing the production of Aβ_1–38 and Aβ_1–40 at the cost of longer Aβ peptides. Here, we consider the aggregation of Aβ_1–38 and Aβ_1–43 in addition to Aβ_1–40 and Aβ_1–42, in particular their behavior in mixtures representing the complex in vivo Aβ pool. We demonstrate that Aβ_1–38 and Aβ_1–43 aggregate similar to Aβ_1–40 and Aβ_1–42, respectively, but display a variation in the kinetics of assembly and toxicity due to differences in short timescale conformational plasticity. In biologically relevant mixtures of Aβ, Aβ_1–38 and Aβ_1–43 significantly affect the behaviors of Aβ_1–40 and Aβ_1–42. The short timescale conformational flexibility of Aβ_1–38 is suggested to be responsible for enhancing toxicity of Aβ_1–40 while exerting a cyto-protective effect on Aβ_1–42. Our results indicate that the complex in vivo Aβ peptide array and variations thereof is critical in Alzheimer disease, which can influence the selection of current and new therapeutic strategies.     

The Role of Heat Shock Protein 70 in the Protective Effect of YC-1 on β-Amyloid-Induced Toxicity in Differentiated PC12 Cells

Neurodegenerative brain disorders such as Alzheimer’s disease (AD) have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70) plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC), on Aβ_25–35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aβ_25–35 (10 µM) significantly increased p25 protein production in a pattern that was consistent with the increase in μ-calpain expression. Moreover, Aβ_25–35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5–10 µM) prevented the cell death induced by Aβ_25–35. In addition, YC-1 (1, 10 µM) significantly blocked Aβ_25–35-induced μ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5–20 µM) alone or combined with Aβ_25–35 (10 µM) significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM) or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM) did not affect the neuroprotective effect of YC-1 against Aβ_25–35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aβ_25–35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.     

Cytotoxic effects of GM1 ganglioside and amyloid β-peptide on mouse embryonic neural stem cells

AD (Alzheimer’s disease) is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Aβs (amyloid β-peptides) in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between G_M1 ganglioside and Aβs. Several reports have documented the bifunctional roles of Aβs in NSCs (neural stem cells), but the precise effects of G_M1 and Aβ on NSCs have not yet been clarified. We evaluated the effect of G_M1 and Aβ-(1–40) on mouse NECs (neuroepithelial cells), which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either G_M1 or Aβ-(1–40). On the contrary, a decreased number of NECs were cultured in the presence of a combination of G_M1 and Aβ-(1–40). The exogenously added G_M1 and Aβ-(1–40) were confirmed to incorporate into NECs. The Ras–MAPK (mitogen-activated protein kinase) pathway, important for cell proliferation, was intact in NECs simultaneously treated with G_M1 and Aβ-(1–40), but caspase 3 was activated. NECs treated with G_M1 and Aβ-(1–40) were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, an indicator of cell death. It was found that G_M1 and Aβ-(1–40) interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Aβ-(1–40) and G_M1 co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.     

Neurotransmitter receptor and time dependence of the synaptic plasticity disrupting actions of Alzheimer's disease Aβ in vivo

Many endogenous factors influence the time course and extent of the detrimental effects of amyloid β-protein (Aβ) on synaptic function. Here, we assessed the impact of varying endogenous glutamatergic and cholinergic transmission by pharmacological means on the disruption of plasticity at hippocampal CA3-to-CA1 synapses in the anaesthetized rat. NMDA receptors (NMDARs) are considered critical in mediating Aβ-induced inhibition of long-term potentiation (LTP). However, intracerebroventricular injection of Aβ_1–42 inhibited not only NMDAR-dependent LTP but also voltage-activated Ca²⁺-dependent LTP induced by strong conditioning stimulation during NMDAR blockade. On the other hand, another form of NMDAR-independent synaptic plasticity, endogenous acetylcholine-induced muscarinic receptor-dependent long-term enhancement, was not hindered by Aβ_1–42. Interestingly, augmenting endogenous acetylcholine activation of nicotinic receptors prior to the injection of Aβ_1–42 prevented the inhibition of NMDAR-dependent LTP, whereas the same intervention when introduced after the infusion of Aβ was ineffective. We also examined the duration of action of Aβ, including water soluble Aβ from Alzheimer''s disease (AD) brain. Remarkably, the inhibition of LTP induction caused by a single injection of sodium dodecyl sulfate-stable Aβ dimer-containing AD brain extract persisted for at least a week. These findings highlight the need to increase our understanding of non-NMDAR mechanisms and of developing novel means of overcoming, rather than just preventing, the deleterious synaptic actions of Aβ.     

Roles of receptor‐interacting protein kinase 1 in SH‐SY5Y cells with beta amyloid‐induced neurotoxicity

Alzheimer''s disease (AD), the major cause of dementia, affects the elderly population worldwide. Previous studies have shown that depletion of receptor‐interacting protein kinase 1 (RIPK1) expression reverted the AD phenotype in murine AD models. Necroptosis, executed by mixed lineage kinase domain‐like (MLKL) protein and activated by RIPK1 and RIPK3, has been shown to be involved in AD. However, the role of RIPK1 in beta‐amyloid (Aβ)‐induced necroptosis is not yet fully understood. In this study, we explored the role of RIPK1 in the SH‐SY5Y human neuroblastoma cells treated with Aβ 1–40 or Aβ 1–42. We showed that Aβ‐induced neuronal cell death was independent of apoptosis and autophagy pathways. Further analyses depicted that activation of RIPK1/MLKL‐dependant necroptosis pathway was observed in vitro. We demonstrated that inhibition of RIPK1 expression rescued the cells from Aβ‐induced neuronal cell death and ectopic expression of RIPK1 was found to enhance the stability of the endogenous APP. In summary, our findings demonstrated that Aβ can potentially drive necroptosis in an RIPK1‐MLKL‐dependent manner, proposing that RIPK1 plays an important role in the pathogenesis of AD.     

Methyllycaconitine Alleviates Amyloid-β Peptides-Induced Cytotoxicity in SH-SY5Y Cells

Alzheimer''s disease (AD) is a chronic progressive neurodegenerative disorder. As the most common form of dementia, it affects more than 35 million people worldwide and is increasing. Excessive extracellular deposition of amyloid-β peptide (Aβ) is a pathologic feature of AD. Accumulating evidence indicates that macroautophagy is involved in the pathogenesis of AD, but its exact role is still unclear. Although major findings on the molecular mechanisms have been reported, there are still no effective treatments to prevent, halt, or reverse Alzheimer''s disease. In this study, we investigated whether Aβ_25–35 could trigger an autophagy process and inhibit the growth of SH-SY5Y cells. Furthermore, we examined the effect of methyllycaconitine (MLA) on the cytotoxity of Aβ_25–35. MLA had a protective effect against cytotoxity of Aβ, which may be related to its inhibition of Aβ-induced autophagy and the involvement of the mammalian target of rapamycin pathway. Moreover, MLA had a good safety profile. MLA treatment may be a promising therapeutic tool for AD.     

Ginsenoside Rg1 Decreases Aβ1–42 Level by Upregulating PPARγ and IDE Expression in the Hippocampus of a Rat Model of Alzheimer's Disease

Background and Purpose

The present study was designed to examine the effects of ginsenoside Rg1 on expression of peroxisome proliferator-activated receptor γ (PPARγ) and insulin-degrading enzyme (IDE) in the hippocampus of rat model of Alzheimer''s disease (AD) to determine how ginsenoside Rg1 (Rg1) decreases Aβ levels in AD.

Experimental Approach

Experimental AD was induced in rats by a bilateral injection of 10 µg soluble beta-amyloid peptide 1–42 (Aβ_1–42) into the CA1 region of the hippocampus, and the rats were treated with Rg1 (10 mg·kg⁻¹, intraperitoneally) for 28 days. The Morris water maze was used to test spatial learning and memory performance. Hematoxylin-eosin staining was performed to analyze the hippocampal histopathological damage. Immunohistochemistry, western blotting, and real-time PCR were used to detect Aβ_1–42, PPARγ, and insulin-degrading enzyme (IDE) expression in the hippocampus.

Key Results

Injection of soluble Aβ_1–42 into the hippocampus led to significant dysfunction of learning and memory, hippocampal histopathological abnormalities and increased Aβ_1–42 levels in the hippocampus. Rg1 treatment significantly improved learning and memory function, attenuated hippocampal histopathological abnormalities, reduced Aβ_1–42 levels and increased PPARγ and IDE expression in the hippocampus; these effects of Rg1 could be effectively inhibited by GW9662, a PPARγ antagonist.

Conclusions and Implications

Given that PPARγ can upregulate IDE expression and IDE can degrade Aβ_1–42, these results indicate that Rg1 can increase IDE expression in the hippocampus by upregulating PPARγ, leading to decreased Aβ levels, attenuated hippocampal histopathological abnormalities and improved learning and memory in a rat model of AD.     

Truncated Amyloid-β(11–40/42) from Alzheimer Disease Binds Cu2+ with a Femtomolar Affinity and Influences Fiber Assembly

Alzheimer disease coincides with the formation of extracellular amyloid plaques composed of the amyloid-β (Aβ) peptide. Aβ is typically 40 residues long (Aβ_(1–40)) but can have variable C and N termini. Naturally occurring N-terminally truncated Aβ_(11–40/42) is found in the cerebrospinal fluid and has a similar abundance to Aβ_(1–42), constituting one-fifth of the plaque load. Based on its specific N-terminal sequence we hypothesized that truncated Aβ_(11–40/42) would have an elevated affinity for Cu²⁺. Various spectroscopic techniques, complemented with transmission electron microscopy, were used to determine the properties of the Cu²⁺-Aβ_(11–40/42) interaction and how Cu²⁺ influences amyloid fiber formation. We show that Cu²⁺-Aβ_(11–40) forms a tetragonal complex with a 34 ± 5 fm dissociation constant at pH 7.4. This affinity is 3 orders of magnitude tighter than Cu²⁺ binding to Aβ_(1–40/42) and more than an order of magnitude tighter than that of serum albumin, the extracellular Cu²⁺ transport protein. Furthermore, Aβ_(11–40/42) forms fibers twice as fast as Aβ_(1–40) with a very different morphology, forming bundles of very short amyloid rods. Substoichiometric Cu²⁺ drastically perturbs Aβ_(11–40/42) assembly, stabilizing much longer fibers. The very tight fm affinity of Cu²⁺ for Aβ_(11–40/42) explains the high levels of Cu²⁺ observed in Alzheimer disease plaques.     

Oridonin Attenuates Synaptic Loss and Cognitive Deficits in an Aβ1–42-Induced Mouse Model of Alzheimer’s Disease

Synaptic loss induced by beta-amyloid (Aβ) plays a critical role in the pathophysiology of Alzheimer’s disease (AD), but the mechanisms underlying this process remain unknown. In this study, we found that oridonin (Ori) rescued synaptic loss induced by Aβ_1–42 in vivo and in vitro and attenuated the alterations in dendritic structure and spine density observed in the hippocampus of AD mice. In addition, Ori increased the expression of PSD-95 and synaptophysin and promoted mitochondrial activity in the synaptosomes of AD mice. Ori also activated the BDNF/TrkB/CREB signaling pathway in the hippocampus of AD mice. Furthermore, in the Morris water maze test, Ori reduced latency and searching distance and increased the number of platform crosses in AD mice. These data suggest that Ori might prevent synaptic loss and improve behavioral symptoms in Aβ_1–42-induced AD mice.     

A Physically-Modified Saline Suppresses Neuronal Apoptosis,Attenuates Tau Phosphorylation and Protects Memory in an Animal Model of Alzheimer's Disease

Alzheimer''s disease (AD), the leading cause of dementia in the aging population, is characterized by the presence of neuritic plaques, neurofibrillary tangles and extensive neuronal apoptosis. Neuritic plaques are mainly composed of aggregates of amyloid-β (Aβ) protein while neurofibrillary tangles are composed of the hyperphosphorylated tau protein. Despite intense investigations, no effective therapy is currently available to halt the progression of this disease. Here, we have undertaken a novel approach to attenuate apoptosis and tau phosphorylation in cultured neuronal cells and in a transgenic animal model of AD. RNS60 is a 0.9% saline solution containing oxygenated nanobubbles that is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. In our experiments, fibrillar Aβ1-42, but not the reverse peptide Aβ42-1, induced apoptosis and cell death in human SHSY5Y neuronal cells. RNS60, but not NS (normal saline), RNS10.3 (TCP-modified saline without excess oxygen) or PNS60 (saline containing excess oxygen without TCP modification), attenuated Aβ(1–42)-induced cell death. RNS60 inhibited neuronal cell death via activation of the type 1A phosphatidylinositol-3 (PI-3) kinase – Akt – BAD pathway. Furthermore, RNS60 also decreased Aβ(1–42)-induced tau phosphorylation via (PI-3 kinase – Akt)-mediated inhibition of GSK-3β. Similarly, RNS60 treatment suppressed neuronal apoptosis, attenuated Tau phosphorylation, inhibited glial activation, and reduced the burden of Aβ in the hippocampus and protected memory and learning in 5XFAD transgenic mouse model of AD. Therefore, RNS60 may be a promising pharmaceutical candidate in halting or delaying the progression of AD.