Inhibition of metal-induced amyloid β-peptide aggregation by a blood–brain barrier permeable silica–cyclen nanochelator

Alzheimer''s disease (AD) is a neurodegenerative malady associated with amyloid β-peptide (Aβ) aggregation in the brain. Metal ions play important roles in Aβ aggregation and neurotoxicity. Metal chelators are potential therapeutic agents for AD because they could sequester metal ions from the Aβ aggregates and reverse the aggregation. The blood–brain barrier (BBB) is a major obstacle for drug delivery to AD patients. Herein, a nanoscale silica–cyclen composite combining cyclen as the metal chelator and silica nanoparticles as a carrier was reported. Silica–cyclen was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) and dynamic light scattering (DLS). The inhibitory effect of the silica–cyclen nanochelator on Zn²⁺- or Cu²⁺-induced Aβ aggregation was investigated by using a BCA protein assay and TEM. Similar to cyclen, silica–cyclen can effectively inhibit the Aβ aggregation and reduce the generation of reactive oxygen species induced by the Cu–Aβ₄₀ complex, thereby lessening the metal-induced Aβ toxicity against PC12 cells. In vivo studies indicate that the silica–cyclen nanochelator can cross the BBB, which may provide inspiration for the construction of novel Aβ inhibitors.

A BBB-passable nanoscale silica–cyclen chelator effectively reduces the metal-induced Aβ aggregates and related ROS, thereby decreasing the neurotoxicity of Aβ.     

Soluble TREM2 levels associate with conversion from mild cognitive impairment to Alzheimer’s disease

BACKGROUNDSoluble triggering receptor expressed on myeloid cells 2 (sTREM2) plays an important role in the clearance of pathological amyloid-β (Aβ) in Alzheimer’s disease (AD). This study aimed to explore sTREM2 as a central and peripheral predictor of the conversion from mild cognitive impairment (MCI) to AD.METHODSsTREM2 and Aβ_1–42 levels in cerebrospinal fluid (CSF) and florbetapir-PET (AV45) images were analyzed for healthy control (HCs), patients with MCI, and patients with AD from the ADNI database. Peripheral plasma sTREM2 and Aβ_1–42 levels were determined for our Neurology database of Ruijin Hospital for Alzheimer’s Disease (NRHAD) cohort, and patients with MCI were reevaluated at follow-up visits to assess for progression to AD. The association between CSF and plasma sTREM2 levels was analyzed in data from the Chinese Alzheimer’s Biomarker and Lifestyle (CABLE) database.RESULTSThe results showed that patients with MCI who had low levels of CSF sTREM2 and Aβ_1–42 were more likely to develop AD. Among participants with positive Aβ deposition, as assessed by AV45 imaging, elevated CSF sTREM2 levels were associated with a decreased risk of MCI-to-AD conversion. Meanwhile, in the NRHAD cohort, individuals in the MCI group with high sTREM2 levels in plasma were at a greater risk for AD, whereas low Aβ_1–42 with high sTREM2 levels in plasma were associated with a faster cognitive decline. In addition, CSF sTREM2 levels were highly correlated with plasma sTREM2 levels in the CABLE database.CONCLUSIONThese findings suggest that sTREM2 may be useful as a potential predictive biomarker of MCI-to-AD conversion.FUNDINGThis study was supported by grants from the National Natural Science Foundation of China (grant nos. 82001341, 82071415, 81873778, and 82201392); the Shanghai Sailing Program (grant no. 22YF1425100); and the China Postdoctoral Science Foundation funded project (grant no. 2021M702169).     

A Maitake (Grifola frondosa) polysaccharide ameliorates Alzheimer's disease-like pathology and cognitive impairments by enhancing microglial amyloid-β clearance

Alzheimer''s disease (AD) is characterized by the deposition of amyloid-β (Aβ) plaques, neuronal loss and neurofibrillary tangles. In addition, neuroinflammatory processes are thought to contribute to AD pathophysiology. Maitake (Grifola frondosa), an edible/medicinal mushroom, exhibits high nutritional value and contains a great amount of health-beneficial, bioactive compounds. It has been reported that proteo-β-glucan, a polysaccharide derived from Maitake (PGM), possesses strong immunomodulatory activities. However, whether PGM is responsible for the immunomodulatory and neuroprotection effects on APP_swe/PS1_ΔE9 (APP/PS1) transgenic mice, a widely used animal model of AD, remains unclear. In the present study, the results demonstrated that PGM could improve learning and memory impairment, attenuate neuron loss and histopathological abnormalities in APP/PS1 mice. In addition, PGM treatment could activate microglia and astrocytes and promote microglial recruitment to the Aβ plaques. Also, PGM could enhance Aβ phagocytosis, and thereby alleviate Aβ burden and the pathological changes in the cortex and hippocampus in APP/PS1 mice. Moreover, PGM showed no significant effect on mice body weight. In conclusion, these findings indicated that administration of PGM could improve memory impairment via immunomodulatory action, and dietary supplementation with PGM may provide potential benefits on brain aging related memory dysfunction.

PGM ameliorates AD-like pathology and cognitive impairments by enhancing microglial amyloid-β clearance.     

Monascus pigment rubropunctatin derivative FZU-H reduces Aβ(1-42)-induced neurotoxicity in Neuro-2A cells

Alzheimer''s disease (AD) is an extremely complex disease, characterized by several pathological features including oxidative stress and amyloid-β (Aβ) aggregation. Blockage of Aβ-induced injury has emerged as a potential therapeutic approach for AD. Our previous efforts resulted in the discovery of Monascus pigment rubropunctatin derivative FZU-H with potential neuroprotective effects. This novel lead compound significantly diminishes toxicity induced by Aβ(1-42) in Neuro-2A cells. Our further mechanism investigation revealed that FZU-H inhibited Aβ(1-42)-induced caspase-3 protein activation and the loss of mitochondrial membrane potential. In addition, treatment of FZU-H was proven to attenuate Aβ(1-42)-induced cell redox imbalance and Tau hyperphosphorylation which caused by okadaic acid in Neuro-2A cells. These results indicated that FZU-H shows promising neuroprotective effects for AD.

Monascus pigment rubropunctatin derivative FZU-H shows promising neuroprotective effects for AD.     

Endothelial LRP1 transports amyloid-β1–42 across the blood-brain barrier

According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor–related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-β (Aβ) brain accumulation and drives Alzheimer’s disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Aβ transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic Aβ clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slco1c1-CreER^T2 Lrp1^fl/fl mice) and used these mice to accurately evaluate LRP1-mediated Aβ BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [¹²⁵I] Aβ_1–42. Additionally, in the 5xFAD mouse model of AD, brain endothelial–specific Lrp1 deletion reduced plasma Aβ levels and elevated soluble brain Aβ, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Aβ elimination via the BBB. Together, our results suggest that receptor-mediated Aβ BBB clearance may be a potential target for treatment and prevention of Aβ brain accumulation in AD.     

The F19W mutation reduces the binding affinity of the transmembrane Aβ11–40 trimer to the membrane bilayer

Alzheimer''s disease is linked to the aggregation of the amyloid-β protein (Aβ) of 40 or 42 amino acids. Lipid membranes are known to modulate the rate and mechanisms of the Aβ aggregation. Point mutations in Aβ can alter these rates and mechanisms. In particular, experiments show that F19 mutations influence the aggregation rate, but maintain the fibril structures. Here, we used molecular dynamics simulations to examine the effect of the F19W mutation in the 3Aβ_11–40 trimer immersed in DPPC lipid bilayers submerged in aqueous solution. Substituting Phe by its closest (non-polar) aromatic amino acid Trp has a dramatic reduction in binding affinity to the phospholipid membrane (measured with respect to the solvated protein) compared to the wild type: the binding free energy of the protein–DPPC lipid bilayer increases by 40–50 kcal mol⁻¹ over the wild-type. This is accompanied by conformational changes and loss of salt bridges, as well as a more complex free energy surface, all indicative of a more flexible and less stable mutated trimer. These results suggest that the impact of mutations can be assessed, at least partially, by evaluating the interaction of the mutated peptides with the lipid membranes.

Dominant conformations of F19W 3Aβ_11–40 immersed in transmembrane DPPC lipid bilayer submerged in aqueous solution.     

Two statins and cromolyn as possible drugs against the cytotoxicity of Aβ(31–35) and Aβ(25–35) peptides: a comparative study by advanced computer simulation methods

In this work, possible effective mechanisms of cromolyn, atorvastatin and lovastatin on the cytotoxicity of Aβ(31–35) and Aβ(25–35) peptides were investigated by classical molecular dynamics and well-tempered metadynamics simulations. The results demonstrate that all the drugs affect the behavior of the peptides, such as their ability to aggregate, and alter their secondary structures and their affinity to a particular drug. Our findings from the computed properties suggest that the best drug candidate is lovastatin. This medicine inhibits peptide aggregation, adsorbs the peptides on the surface of the drug clusters, changes the secondary structure and binds to MET₃₅, which has been seen as the reason for the toxicity of the studied peptide sequences. Moreover, lovastatin is the drug which previously has demonstrated the strongest ability to penetrate the blood–brain barrier and makes lovastatin the most promising medicine among the three investigated drugs. Atorvastatin is also seen as a potential candidate if its penetration through the blood–brain barrier could be improved. Otherwise, its properties are even better than the ones demonstrated by lovastatin. Cromolyn appears to be less interesting as an anti-aggregant from the computational data, in comparison to the two statins.

In this work, possible effective mechanisms of cromolyn, atorvastatin and lovastatin on the cytotoxicity of Aβ(31–35) and Aβ(25–35) peptides were investigated by classical molecular dynamics and well-tempered metadynamics simulations.     

A promising new γ-secretase modulator for Alzheimer’s disease

Effective and safe treatments for Alzheimer’s disease (AD) have been an elusive target for scientists who have been working tirelessly to gain control over a disease that is affecting millions of people, with continually rising case numbers as the population ages. However, in this issue of JEM, Rynearson et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20202560) present a beacon of hope for this field with a preclinical evaluation of a potent and robust γ-secretase modulator (GSM).

One of the main neuropathological hallmarks of Alzheimer’s disease (AD) are amyloid-β (Aβ) plaques that accumulate in the brains of those afflicted with this disease. The amyloid hypothesis posits that increased production and/or decreased clearance of Aβ initiates a cascade of events that results in the accumulation of these plaques and eventually leads to neurofibrillary tangles, synaptic loss, and neuronal death manifesting as AD (Tanzi and Bertram, 2005). Aβ peptides of varying length are formed when the amyloid precursor protein (APP) is cleaved first by β-secretase (BACE1) and subsequently by γ-secretase. The length of Aβ formed is dependent on the position at which γ-secretase cuts the protein; the most predominant peptide formed is Aβ₄₀. The toxic variant that more readily aggregates into plaques is Aβ₄₂ (Tanzi and Bertram, 2005), and evidence from rodent and human studies shows that there is an increase in the ratio of Aβ₄₂/Aβ₄₀ in the disease setting (Jankowsky et al., 2004; Kwak et al., 2020). Shorter Aβ peptides, like Aβ₃₇ and Aβ₃₈, are nontoxic and are found to a lesser extent.Insights from Justyna A. Dobrowolska Zakaria and Robert J. Vassar.In this issue, Rynearson et al. (2021) present the results of a very thorough preclinical evaluation of several γ-secretase modulators (GSMs) from the pyridazine-derived class. Their goal was to find a GSM that could safely and effectively shift where the γ-secretase cleaved the C-terminal fragment of APP, so that less of the toxic Aβ₄₂ would be formed. One of the compounds presented, compound 2, showed promise for further clinical evaluation, but compound 3 may also be a viable option to pursue in the future. They tested these compounds in several animal models and with short (acute), medium (sub-chronic), and long-term (chronic) treatment. Initially, the researchers tested levels of plasma and brain Aβ₄₀ and Aβ₄₂ in mice that had received a 9-d oral treatment of compounds 2 and 3 at varying doses (10–50 mg/kg) and found that there was a dose-dependent decrease in Aβ₄₀ for both compounds. Brain Aβ₄₂ was below detection even at the lowest dose, and plasma Aβ₄₂ was significantly lowered starting from the lowest dose as well. Next, the researchers investigated the long-term treatment of compound 2 (the most promising of the compounds from this portfolio) in the presenilin APP (PSAPP) mouse model to determine its effects on Aβ deposition, how well this drug was tolerated, and whether it was safe in longer exposure settings. PSAPP mice have almost no Aβ deposition at 3 mo, but by 6 mo they have a significant number of plaques. Thus, by using this model, the researchers were able to test their compound in two scenarios: 1) would the drug prevent the formation of plaques (i.e., a prophylactic treatment) and 2) could the drug stop or reverse the accumulation of plaques after they had already started to form (disease-modifying treatment). Mice in the prophylactic group were 3 mo old, and those in the disease-modifying group were 6 mo old. All mice were treated for 3 mo. Analyses of the brains and plasma in the prophylactic group showed Aβ₄₀ and Aβ₄₂ were significantly decreased and Aβ₃₈ was increased. In the disease-modifying group, Aβ₄₂ was significantly decreased as well, and there were changes to Aβ₄₀ and Aβ₃₈ like in the prophylactic group, but these tests did not meet statistical significance. The authors performed necropsies of the mice that showed there was no obvious toxicity that had occurred as a result of the long-term treatment. They also tested the mutagenic potential of the drug in rats, and their tests confirmed it was not mutagenic. In both mice and rats treated with compound 2, Aβ₄₀ and Aβ₄₂ were positively correlated when measured in plasma, cerebrospinal fluid (CSF), or brain, indicating that, when determining effects of GSMs, Aβ peptides in biological fluids can be used as a surrogate biomarker of changes of these peptides in the brain.Following these studies, the researchers next assessed effects of compound 2 on Aβ₄₀ and Aβ₄₂ in a nonhuman primate (NHP) model of cynomolgus macaques over a range of 10–200 mg/kg of dosing, resulting in verified dose-dependent drug exposures. The maximum lowering (60–70%) of these two plasma Aβ peptides was achieved at the lowest dose of 10mg/kg, which was consistent with their rodent data. Unlike in rodents, there were no Aβ₃₈ changes in NHPs. Based on all the animal model data and human pharmacokinetic parameters, the researchers extrapolated that the 50% effective dose for humans would be 100 mg/d and this would have a 130-fold safety margin (Rynearson et al., 2021). Testing this compound on human participants is dependent on the results of the FDA’s review of the authors’ completed investigational new drug safety and toxicity studies.Over the past two decades, the vast majority of candidate therapeutics for AD have failed (Cummings et al., 2014). Some clinical trials have targeted BACE1 and γ-secretase using inhibitors of these proteases to prevent the formation of Aβ altogether. However, these two secretases both cleave other proteins, some with very important physiological functions, so having a unilateral decrease of their proteolytic activities has had unintended and unsafe effects, causing some of these clinical trials to be halted due to safety concerns (Coric et al., 2015; Egan et al., 2019; Henley et al., 2019). In this respect, GSMs, such as compound 2, hold an advantage, because unlike γ-secretase inhibitors, they still allow for γ-secretase to function as a protease of other proteins (Wagner et al., 2012) and do not present with any adverse effects on Notch signaling (Kounnas et al., 2010). Their mechanism of action is more targeted, with the goal being to alter the way APP is cut, thus shifting the cleavage to favor formation of the nontoxic Aβ₃₇ and Aβ₃₈ over the formation of Aβ₄₂ (see figure).(A) GSMs, such as compound 2, affect the presenilin-1 component of γ-secretase so that it cleaves APP preferentially to produce Aβ₃₇ and Aβ₃₈, thus decreasing production of the toxic Aβ₄₂. The goal of modulators is to not disturb γ-secretase proteolytic cleavage of Notch and over 100 other substrates of γ-secretase that have diverse and important physiological functions. (B) γ-secretase inhibitors (GSIs) are successful at decreasing all Aβ species, including the toxic form, by blocking γ-secretase cleavage of APP. However, this blanket inhibition has the undesired effect of blocking important and necessary physiological processing of Notch and many other substrates. Notch intracellular domain (NICD), for example, is critical in determinations of cell fate not only in embryonic development, but also in adult organisms, and perturbing Notch proteolysis has toxic effects with cancer implications. Figure created with BioRender.com.Additionally, in recent years it is becoming clearer that treatment of AD, particularly when focusing on decreasing production or increasing clearance of Aβ, may need to occur presymptomatically. In 2012, a pivotal study reported that humans with genetic mutations in APP or presenilin-1 (a component of γ-secretase) that lead to development of AD at an early age have amyloid deposits in their brains up to 15 yr before symptom onset (Bateman et al., 2012). Moreover, their CSF Aβ₄₂ concentrations started to decline even earlier than that: 25 yr before expected symptom onset! By the time a patient has noticeable symptoms, there is already a significant amyloid plaque burden throughout the brain that has caused a myriad of neurodegeneration. Most likely by this point, the trajectory of the disease is too far along, and continued neurodegeneration cannot be effectively halted by amyloid-targeted therapies, even if the therapies do decrease the patient’s current amyloid burden. Since Bateman’s report, particularly in the last several years, there has been a more marked shift in AD amyloid-based clinical trials away from patients with mild-to-moderate AD and toward targeting asymptomatic AD populations with no or low amyloid deposition (primary or secondary prevention, respectively) by identifying risk factors and biomarkers and enrolling those patients in studies (Crous-Bou et al., 2017). One large trial, the Dominantly Inherited Alzheimer Network Trials Unit, has enrolled presymptomatic mutation carriers that will eventually develop AD (Moulder et al., 2013). Additionally, secondary prevention trials, such as the Alzheimer’s Prevention Initiative, Anti-Amyloid Treatment in Asymptomatic Alzheimer’s Disease, and EARLY (A5) have emerged (Reiman et al., 2011; Sperling et al., 2014). These trials target older individuals that may be more predisposed to developing AD, for example, because they have one or two alleles of ApoE4 (which may enhance deposition or reduce clearance of Aβ). Participants of these trials undergo brain positron emission tomography imaging that indicates amyloid deposits are present even though they are still asymptomatic.The holy grail of stopping AD lies with “going earlier” into the prevention paradigm and testing potential therapeutics at a point in the disease where they stand a chance at being effective. Owing to this, compound 2 is an exciting potential new therapeutic in that preclinically it has shown that it can be used in rodents to prevent Aβ accumulation when administered before any initial Aβ is found in the brain. This is, therefore, a promising potential therapeutic to test in primary prevention trials. Further, compound 2 has also reduced Aβ₄₂ in rodents that already have plaque deposition, thus suggesting the possibility of it being tested in secondary prevention trials, as well.Compound 2 also may be a safer alternative to other therapeutics in its potential to avoid undesired effects on other γ-secretase substrates. Any AD therapeutic will require a high safety margin for chronic treatment, as it is becoming apparent that treatment to prevent AD would have to commence years, if not decades, before symptom onset, and most likely continue for the duration of the patient’s life. In this respect, compound 2 shows promise as well. Additionally, it is quite robust in reducing Aβ₄₂, as even in the lowest doses tested in NHPs ∼60% of the peptide was eliminated. From studies of an AD-protective APP mutation, A673T, we know that only a ∼30% reduction in amyloidogenic peptides is necessary to prevent the disease (Jonsson et al., 2012). Incorporating lower doses to achieve optimally safe Aβ₄₂ reduction could further eliminate any potential unexpected adverse effects.Rynearson et al. (2021) present a well-executed study that contributes significantly to the efforts of the AD field to discover successful disease-modifying AD therapies, of which none yet exist. We look forward to learning more about compound 2’s therapeutic potential in humans if it is approved by the FDA.     

Abeta42-Induced Neurodegeneration via an Age-Dependent Autophagic-Lysosomal Injury in Drosophila

The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD) remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Aβ_1–42) is believed to play a causative role in the development of AD. Here we expressed human Aβ_1–42 and amyloid beta 40 (Aβ_1–40) in Drosophila neurons. Aβ_1–42 but not Aβ_1–40 causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Aβ_1–42-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Aβ_1–42 expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Aβ_1–42 and Aβ_1–40, and reveal an Aβ_1–42-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.     

apoE isoform–specific disruption of amyloid β peptide clearance from mouse brain

Neurotoxic amyloid β peptide (Aβ) accumulates in the brains of individuals with Alzheimer disease (AD). The APOE4 allele is a major risk factor for sporadic AD and has been associated with increased brain parenchymal and vascular amyloid burden. How apoE isoforms influence Aβ accumulation in the brain has, however, remained unclear. Here, we have shown that apoE disrupts Aβ clearance across the mouse blood-brain barrier (BBB) in an isoform-specific manner (specifically, apoE4 had a greater disruptive effect than either apoE3 or apoE2). Aβ binding to apoE4 redirected the rapid clearance of free Aβ40/42 from the LDL receptor–related protein 1 (LRP1) to the VLDL receptor (VLDLR), which internalized apoE4 and Aβ-apoE4 complexes at the BBB more slowly than LRP1. In contrast, apoE2 and apoE3 as well as Aβ-apoE2 and Aβ-apoE3 complexes were cleared at the BBB via both VLDLR and LRP1 at a substantially faster rate than Aβ-apoE4 complexes. Astrocyte-secreted lipo-apoE2, lipo-apoE3, and lipo-apoE4 as well as their complexes with Aβ were cleared at the BBB by mechanisms similar to those of their respective lipid-poor isoforms but at 2- to 3-fold slower rates. Thus, apoE isoforms differentially regulate Aβ clearance from the brain, and this might contribute to the effects of APOE genotype on the disease process in both individuals with AD and animal models of AD.     

Atomistic investigation of an Iowa Amyloid-β trimer in aqueous solution

The self-assembly of Amyloid beta (Aβ) peptides are widely accepted to associate with Alzheimer''s disease (AD) via several proposed mechanisms. Because Aβ oligomers exist in a complicated environment consisting of various forms of Aβ, including oligomers, protofibrils, and fibrils, their structure has not been well understood. The negatively charged residue D23 is one of the critical residues of the Aβ peptide as it is located in the central hydrophobic domain of the Aβ N-terminal and forms a salt-bridge D23-K28, which helps stabilize the loop domain. In the familial Iowa (D23N) mutant, the total net charge of Aβ oligomers decreases, resulting in the decrease of electrostatic repulsion between D23N Aβ monomers and thus the increase in their self-aggregation rate. In this work, the impact of the D23N mutation on 3Aβ_11–40 trimer was characterized utilizing temperature replica exchange molecular dynamics (REMD) simulations. Our simulation reveals that D23N mutation significantly enhances the affinity between the constituting chains in the trimer, increases the β-content (especially in the sequence 21–23), and shifts the β-strand hydrophobic core from crossing arrangement to parallel arrangement, which is consistent with the increase in self-aggregation rate. Molecular docking indicates that the Aβ fibril-binding ligands bind to the D23N and WT forms at different poses. These compounds prefer to bind to the N-terminal β-strand of the D23N mutant trimer, while they mostly bind to the N-terminal loop region of the WT. It is important to take into account the difference in the binding of ligands to mutant and wild type Aβ peptides in designing efficient inhibitors for various types of AD.

Amyloid beta peptide oligomers are believed to play key roles in Alzheimer''s disease pathogenesis. D23N mutation significantly changes their structure and how they bind potential inhibitors.     

Synthetic and biochemical studies on the effect of persulfidation on disulfide dimer models of amyloid β42 at position 35 in Alzheimer's etiology

Protein persulfidation plays a role in redox signaling as an anti-oxidant. Dimers of amyloid β42 (Aβ42), which induces oxidative stress-associated neurotoxicity as a causative agent of Alzheimer''s disease (AD), are minimum units of oligomers in AD pathology. Met35 can be susceptible to persulfidation through its substitution to homoCys residue under the condition of oxidative stress. In order to verify whether persulfidation has an effect in AD, herein we report a chemical approach by synthesizing disulfide dimers of Aβ42 and their evaluation of biochemical properties. A homoCys-disulfide dimer model at position 35 of Aβ42 formed a partial β-sheet structure, but its neurotoxicity was much weaker than that of the corresponding monomer. In contrast, the congener with an alkyl linker generated β-sheet-rich 8–16-mer oligomers with potent neurotoxicity. The length of protofibrils generated from the homoCys-disulfide dimer model was shorter than that of its congener with an alkyl linker. Therefore, the current data do not support the involvement of Aβ42 persulfidation in Alzheimer''s disease.

Our data do not support the Aβ42 persulfidation hypothesis in Alzheimer''s etiology because the neurotoxicity of the homoCys-disulfide-Aβ42 dimer was very weak.     

Anti-Abeta42- and anti-Abeta40-specific mAbs attenuate amyloid deposition in an Alzheimer disease mouse model

Accumulation and aggregation of amyloid β peptide 1–42 (Aβ₄₂) in the brain has been hypothesized as triggering a pathological cascade that causes Alzheimer disease (AD). To determine whether selective targeting of Aβ₄₂ versus Aβ₄₀ or total Aβ is an effective way to prevent or treat AD, we compared the effects of passive immunization with an anti-Aβ₄₂ mAb, an anti-Aβ₄₀ mAb, and multiple Aβ_1–16 mAbs. We established in vivo binding selectivity of the anti-Aβ₄₂ and anti-Aβ₄₀ mAbs using novel TgBRI-Aβ mice. We then conducted a prevention study in which the anti-Aβ mAbs were administered to young Tg2576 mice, which have no significant Aβ deposition, and therapeutic studies in which mAbs were administered to Tg2576 or CRND8 mice with modest levels of preexisting Aβ deposits. Anti-Aβ₄₂, anti-Aβ₄₀, and anti-Aβ_1–16 mAbs attenuated plaque deposition in the prevention study. In contrast, anti-Aβ₄₂ and anti-Aβ₄₀ mAbs were less effective in attenuating Aβ deposition in the therapeutic studies and were not effective in clearing diffuse plaques following direct injection into the cortex. These data suggest that selective targeting of Aβ₄₂ or Aβ₄₀ may be an effective strategy to prevent amyloid deposition, but may have limited benefit in a therapeutic setting.     

N-Amino peptide scanning reveals inhibitors of Aβ42 aggregation

The aggregation of amyloids into toxic oligomers is believed to be a key pathogenic event in the onset of Alzheimer''s disease. Peptidomimetic modulators capable of destabilizing the propagation of an extended network of β-sheet fibrils represent a potential intervention strategy. Modifications to amyloid-beta (Aβ) peptides derived from the core domain have afforded inhibitors capable of both antagonizing aggregation and reducing amyloid toxicity. Previous work from our laboratory has shown that peptide backbone amination stabilizes β-sheet-like conformations and precludes β-strand aggregation. Here, we report the synthesis of N-aminated hexapeptides capable of inhibiting the fibrillization of full-length Aβ₄₂. A key feature of our design is N-amino substituents at alternating backbone amides within the aggregation-prone Aβ_16–21 sequence. This strategy allows for maintenance of an intact hydrogen-bonding backbone edge as well as side chain moieties important for favorable hydrophobic interactions. An N-amino scan of Aβ_16–21 resulted in the identification of peptidomimetics that block Aβ₄₂ fibrilization in several biophysical assays.

Structure-based design of backbone-aminated peptides affords novel β-strand mimics that inhibit amyloid-beta fibrillogenesis.     

USP25 inhibition ameliorates Alzheimer’s pathology through the regulation of APP processing and Aβ generation

Down syndrome (DS), or trisomy 21, is one of the critical risk factors for early-onset Alzheimer’s disease (AD), implicating key roles for chromosome 21–encoded genes in the pathogenesis of AD. We previously identified a role for the deubiquitinase USP25, encoded on chromosome 21, in regulating microglial homeostasis in the AD brain; however, whether USP25 affects amyloid pathology remains unknown. Here, by crossing 5×FAD AD and Dp16 DS mice, we observed that trisomy 21 exacerbated amyloid pathology in the 5×FAD brain. Moreover, bacterial artificial chromosome (BAC) transgene–mediated USP25 overexpression increased amyloid deposition in the 5×FAD mouse brain, whereas genetic deletion of Usp25 reduced amyloid deposition. Furthermore, our results demonstrate that USP25 promoted β cleavage of APP and Aβ generation by reducing the ubiquitination and lysosomal degradation of both APP and BACE1. Importantly, pharmacological inhibition of USP25 ameliorated amyloid pathology in the 5×FAD mouse brain. In summary, we identified the DS-related gene USP25 as a critical regulator of AD pathology, and our data suggest that USP25 serves as a potential pharmacological target for AD drug development.     

Angiotensin-converting enzyme overexpression in myelomonocytes prevents Alzheimer’s-like cognitive decline

Cognitive decline in patients with Alzheimer’s disease (AD) is associated with elevated brain levels of amyloid β protein (Aβ), particularly neurotoxic Aβ_1–42. Angiotensin-converting enzyme (ACE) can degrade Aβ_1–42, and ACE overexpression in myelomonocytic cells enhances their immune function. To examine the effect of targeted ACE overexpression on AD, we crossed ACE^10/10 mice, which overexpress ACE in myelomonocytes using the c-fms promoter, with the transgenic APP_SWE/PS1_ΔE9 mouse model of AD (AD⁺). Evaluation of brain tissue from these AD⁺ACE^10/10 mice at 7 and 13 months revealed that levels of both soluble and insoluble brain Aβ_1–42 were reduced compared with those in AD⁺ mice. Furthermore, both plaque burden and astrogliosis were drastically reduced. Administration of the ACE inhibitor ramipril increased Aβ levels in AD⁺ACE^10/10 mice compared with the levels induced by the ACE-independent vasodilator hydralazine. Overall, AD⁺ACE^10/10 mice had less brain-infiltrating cells, consistent with reduced AD-associated pathology, though ACE-overexpressing macrophages were abundant around and engulfing Aβ plaques. At 11 and 12 months of age, the AD⁺ACE^10/WT and AD⁺ACE^10/10 mice were virtually equivalent to non-AD mice in cognitive ability, as assessed by maze-based behavioral tests. Our data demonstrate that an enhanced immune response, coupled with increased myelomonocytic expression of catalytically active ACE, prevents cognitive decline in a murine model of AD.     

Retracted Article: Berberine alleviates amyloid beta-induced injury in Alzheimer's disease by miR-107/ZNF217

Berberine plays a neuroprotective role in neurodegenerative disorders, including Alzheimer''s disease (AD). However, the underlying mechanism by which berberine inhibits AD progression remains largely unclear. The AD model was established using PC12 cells after treatment of amyloid beta (Aβ)_25-35. Cells were transfected with microRNA (miRNA)-107 mimic, inhibitor, zinc finger protein 217 (ZNF217) overexpression or corresponding negative controls. Cell viability, apoptosis and inflammatory cytokine secretion were measured by MTT, flow cytometry or enzyme linked immunosorbent assay, respectively. The expressions of miR-107, ZNF217 and phosphorylated tau (p-Tau) were detected by quantitative real-time polymerase chain reaction or Western blot. The association between miR-107 and ZNF217 was explored by luciferase reporter assay and RNA immunoprecipitation. Berberine attenuated Aβ_25-35-induced viability suppression in PC12 cells. Moreover, berberine inhibited the Aβ_25-35-induced increase of inflammatory cytokine expression, apoptosis and p-Tau level in PC12 cells. miR-107 expression was reduced in Aβ_25-35-treated PC12 cells and its overexpression alleviated Aβ_25-35-induced injury, which was further weakened by combination with berberine. ZNF217 was a target of miR-107 and its addition reversed miR-107-mediated inhibition of inflammatory injury, apoptosis and phosphorylation of tau. Besides, ZNF217 protein level was decreased by berberine via regulating miR-107 in Aβ_25-35-treated PC12 cells. Berberine protected against Aβ_25-35-induced inflammatory injury, apoptosis and phosphorylation of tau by regulating miR-107 and ZNF217, indicating berberine as a promising neuroprotective agent for therapeutics of AD.

Berberine plays a neuroprotective role in neurodegenerative disorders, including Alzheimer''s disease (AD).     

Disassembly of Shank and Homer Synaptic Clusters Is Driven by Soluble β-Amyloid1-40 through Divergent NMDAR-Dependent Signalling Pathways

Disruption of the postsynaptic density (PSD), a network of scaffold proteins located in dendritic spines, is thought to be responsible for synaptic dysfunction and loss in early-stage Alzheimer's disease (AD). Extending our previous demonstration that derangement of the PSD by soluble amyloid-β (Aβ) involves proteasomal degradation of PSD-95, a protein important for ionotropic glutamate receptor trafficking, we now show that Aβ also disrupts two other scaffold proteins, Homer1b and Shank1, that couple PSD-95 with ionotropic and metabotropic glutamate receptors. Treatment of fronto-cortical neurons with soluble Aβ results in rapid (within 1 h) and significant thinning of the PSD, decreased synaptic levels of Homer1b and Shank1, and reduced synaptic mGluR1 levels. We show that de novo protein synthesis is required for the declustering effects of Aβ on Homer1b (but not Shank1) and that, in contrast to PSD-95, Aβ-induced Homer1b and Shank1 cluster disassembly does not depend on proteasome activity. The regulation of Homer1b and Shank1 by Aβ diverges in two other respects: i) whereas the activity of both NMDAR and VDCC is required for Aβ-induced declustering of Homer1b, Aβ-induced declustering of Shank1 only requires NMDAR activity; and ii) whereas the effects of Aβ on Homer1b involve engagement of the PI-3K pathway and calcineurin phosphatase (PP2B) activity, those on Shank1 involve activation of the ERK pathway. In summary, soluble Aβ recruits discrete signalling pathways to rapidly reduce the synaptic localization of major components of the PSD and to regulate the availability of mGluR1 in the synapse.     

Development of an MRI contrast agent for both detection and inhibition of the amyloid-β fibrillation process

A curcumin derivative conjugated with Gd-DO3A (Gd-DO3A-Comp.B) was synthesised as an MRI contrast agent for detecting the amyloid-β (Aβ) fibrillation process. Gd-DO3A-Comp.B inhibited Aβ aggregation significantly and detected the fibril growth at 20 μM of Aβ with 10 μM of probe concentration by T₁-weighted MR imaging.

A curcumin derivative conjugated with Gd-DO3A (Gd-DO3A-Comp.B) was developed to significantly inhibit the amyloid-β (Aβ) aggregation and detect the fibril growth by T₁-weighted MR imaging.

A significant increase of Alzheimer''s disease (AD) patients urges the development of therapeutic and diagnostic technology.¹ As with the therapeutic development, diagnostic technology also faces several obstacles. To date, the definite diagnosis of AD relies on the histopathological data of post-mortem.^2,3 The non-invasive imaging technology targeting AD biomarkers such as amyloid β (Aβ) could provide phenotypical diagnostics, although the development of Aβ probes still remains challenging. Several contrast agents for single photon emission computed tomography (SPECT) and positron emission tomography (PET) such as Florbetapir-¹⁸F and Pittsburgh compound-B ([¹¹C]PiB) were developed as efficient tracers in mild cognitive impairment patients.^4,5 However, PET- and SPECT-based diagnostics require injection of radioactive probes, which cannot be measured frequently due to radiation exposure and limited availability of facilities. They also provide limited information on the anatomic profile of biomarkers due to their low spatial resolution and imprecise microscopic localization.⁶ In contrast, magnetic resonance imaging (MRI) contrast agents could quantify the Aβ accumulation in the anatomic brain image.⁷Several reported MRI contrast agents using gadolinium (Gd) complexes demonstrate potential use of Aβ detection. A clinically approved contrast agent, Gd(iii) diethylenetriaminepentaacetic acid (Gd-DTPA) complex accumulates in brain after opening the blood–brain barrier (BBB) by using mannitol and detects Aβ deposits in the mice AD-model.⁸ To improve the selectivity, Gd complexes were conjugated with compounds binding to Aβ such as Pittsburgh compound B (Gd-DO3A-PiB) which also serves as an approach for increasing MRI sensitivity.^9,10 An α,β-unsaturated ketone compound curcumin has been widely reported as an Aβ probe due to its ability to bind the hydrophobic site of Aβ.^11,12 Allen et al. firstly reported the direct conjugation of curcumin with Gd-DTPA which binds to Aβ with four times higher relaxivity than free Gd-DTPA.¹³ Furthermore, a polymalic acid-based nanoparticle covalently linked with curcumin and Gd-DOTA could also detect Aβ in human brain specimen by MRI.¹⁴ These previous studies demonstrate that the curcumin structure has significant potential for the development of MRI contrast agents for AD diagnosis.Previously, we reported a curcumin derivative, compound B, possesses 100-times stronger inhibitory activity of Aβ aggregation than curcumin on the basis of thioflavin T (ThT) competitive binding assay.^15,16 According to this result, we designed curcumin-based Gd probes for the detection and inhibition of Aβ (Fig. 1A–C). We hypothesized that these probes could accelerate proton longitudinal relaxation depending on the fibrillation stage of Aβ, because molecular tumbling rate of the Gd complexes becomes slower (Fig. 1A).¹⁷ As a result, the probes permit the detection of Aβ by longitudinal relaxation time (T₁)-weighted imaging. This mechanism could also be utilized to estimate the inhibitory activity of the probes by T₁-based analysis (Fig. 1B). The curcumin and compound B were directly conjugated with the macrocyclic DO3A ligand through the propylamine linker to obtain Gd-DO3A-Cur and Gd-DO3A-Comp.B, respectively (Fig. 1C).Open in a separate windowFig. 1(A) A probe concept that produces T₁ in a dependent manner of Aβ fibrillation process. (B) Inhibitor-based probes that cause moderate T₁ decreases due to inhibitory activity of fibrillation. (C) The chemical structures of the synthesized Gd probes for Aβ detection and inhibition.Gd-DO3A-Cur and Gd-DO3A-Comp.B were synthesized according to Scheme 1 (detail in Scheme S1, ESI^†). The compound 5a and 5b, which have asymmetric curcumin derivatives containing carboxylic acid group, were synthesized by three step reactions. Amide bond formation with DO3A(tBu)₃-propylamine ligand¹⁸ by condensation reaction afforded compound 7a and 7b. The tert-butyl groups were deprotected by trifluoroacetic acid producing compound 8a and 8b. The complexation was performed with GdCl₃·6H₂O by adjusting the reaction pH to 7, giving 43 and 41% yields of Gd-DO3A-Cur and Gd-DO3A-Comp.B, respectively. The T₁ relaxivities (r₁) of the curcumin-based Gd probes were estimated by T₁ measurement using a 1 tesla NMR relaxometry (Fig. S1, ESI^†). For the comparison, we synthesized Gd-DO3A-Chal which is a reported probe for Aβ.¹⁹ The r₁ of Gd-DO3A-Comp.B, Gd-DO3A-Cur, and Gd-DO3A-Chal were 7.1, 6.1 and 5.3 mM⁻¹ s⁻¹, respectively. These r₁ values are higher than that of clinically approved Gd-DOTA (3.9 mM⁻¹ s⁻¹).²⁰ The molecular weight of Gd-DO3A-Comp.B and Gd-DO3A-Cur is almost two times larger than that of Gd-DOTA. Because the r₁ increases approximately linearly with molecular weight in low magnetic field,¹⁷ the high r₁ values of Gd-DO3A-Comp.B and Gd-DO3A-Cur might be mainly attributed to their high rotational correlation time, rather than the high number of coordinated water molecules. The r₁ of Gd-DO3A-Chal was comparable to the value reported previously.¹⁹Open in a separate windowScheme 1Synthetic scheme of Gd-DO3A-Cur and Gd-DO3A-Comp.B. (a) B(OH)₃, morpholine, DMF, 100 °C, 10 min. (b) 3a/3b, B(OH)₃, morpholine, DMF, 100 °C, 10 min. (c) TFA, DCM. (d) DO3A(tBu)₃-propylamine ligand, PyBOP, HOBt, Et₃N, DMF. (e) 7a/7b, TFA, DCM. (f) GdCl₃·6H₂O, NaOH, H₂O.We evaluated the inhibitory effect of three probes toward Aβ aggregation by Congo red assay.²¹ After 24 h incubation of 20 μM Aβ with 10 μM probe, Gd-DO3A-Comp.B showed the lowest fluorescence intensity, indicating the strongest inhibitory activity followed by Gd-DO3A-Cur (Fig. 2A). As the comparison, the reported MRI agents, Gd-DO3A-Chal showed slight inhibitory activity. The inhibitory effect was further evaluated by transmission electron microscopy (TEM) with negative staining (Fig. 2B). In the absence of the probes, Aβ formed huge and massive fibril similar to the typical morphology of Aβ fibril.²² The TEM images of Aβ with Gd-DO3A-Comp.B showed the presence of white spheres below 10 nm, demonstrating that Gd-DO3A-Comp.B strongly inhibits Aβ aggregation. In fact, the fibril growth stopped at a stage of oligomer formation. Lower inhibitory activity of Gd-DO3A-Cur was also found to provide a shortened worm-like fibril, which is the typical morphology of Aβ exposed to curcumin.²³ In contrast, the small amount of white spheres and partial fibril disruption were found in the image of Aβ with Gd-DO3A-Chal. In comparison with a reported Gd-DTPA-curcumin possessing inhibitory activity starting at 50 μM, Gd-DO3A-Comp.B possessed stronger inhibition of Aβ aggregation at 10 μM.²⁴ The MTT assay using Neuro 2a cells showed that IC₅₀ of Gd-DO3A-Cur and Gd-DO3A-Comp.B. were more than 500 μM, indicating that these compounds did not possess significant cytotoxicity (Fig. S2, ESI^†).Open in a separate windowFig. 2Inhibitory effect of the Gd probes toward Aβ aggregation measured by Congo red assay (A) and negative staining TEM images (B). The Gd probes were co-incubated with monomeric Aβ for 24 h in PBS at pH 7.4. [Gd] = 10 μM, [Aβ] = 20 μM. Scale bars = 100 nm.To detect fibrillation process by NMR relaxometry, we measured T₁ of the probe mixture with Aβ which were pre-incubated for 1, 3, 6, 12, and 24 h to make it form the fibrils of different growth stages (Fig. 3A and B). The T₁ of Gd-DO3A-Comp.B solution decreased with pre-incubation time of Aβ, demonstrating that the Gd-DO3A-Comp.B can detect Aβ fibril depending on the growth stage (Fig. 3B). Lower T₁ involved with Aβ growth could be caused by the reduction in tumbling rate of the Gd complex.²⁵ We also co-incubated the probes with the Aβ monomer and monitored T₁ changes over the incubation time (Fig. 3A, B and S3, ESI^†). Interestingly, the Gd-DO3A-Comp.B did not cause significant T₁ decreases even after 24 hours co-incubation with Aβ monomers, demonstrating that Gd-DO3A-Comp.B has a strong inhibitory effect on fibril formation and the inhibition can be monitored by T₁ measurement (Fig. 3B). The inhibitory effect was consistent with the results of Congo red assay and TEM (Fig. 2). On the other hand, the time-dependent increases of T₁ were observed in Gd-DO3A-Chal and Gd-DO3A-Cur. This might be because these two probes were buried in the hydrophobic pocket as Aβ fibril grew up and fewer water molecules permitted access to the Gd ions. It is also possible that these probes have lower binding affinity, especially for matured fibril, and require higher concentrations to produce significant T₁ changes.²⁶ These probe did not produce the significant ΔT₁ between monomer and fibril samples (Fig. 3B and S3, ESI^†), although they showed little inhibition in Congo red assay and TEM (Fig. 2).Open in a separate windowFig. 3(A) Experimental design of T₁-based detection of Aβ fibrillation and inhibition by using the Gd probes. (B) T₁ changes of the Gd probe solutions with pre-incubated fibrils and monomers in PBS at pH 7.4 (mean ± SEM, n = 3). [Gd] = 10 μM, [Aβ] = 20 μM.The feasibility of the Gd probes was further evaluated by in vitro MRI measurement using a 1 tesla scanner. The T₁-weighted images showed that Gd-DO3A-Comp.B produced slight T₁ signal increases with Aβ monomers for 2 and 24 h (Fig. 4A and B). More significant signal increases were observed in the Gd-DO3A-Comp.B with Aβ fibril pre-incubated for 24 h (Fig. 4C). In contrast, Gd-DO3A-Chal and Gd-DO3A-Cur did not show significant signal changes in the presence of Aβ monomers or fibrils (Fig. 4A–C). These results were mostly consistent with the T₁ profile measured by NMR (Fig. 3). Compared to the previously reported Gd-DO3A-Chal that required 100 μM of the probe concentration to detect the equimolar Aβ,¹⁹ Gd-DO3A-Comp.B could detect five-times lower concentration of Aβ (20 μM) with ten-times lower probe concentration (10 μM). Therefore, Gd-DO3A-Comp.B could be promising to further develop highly sensitive diagnostic MRI contrast agents of AD.Open in a separate windowFig. 4 T ₁-weighted images of the Gd probe solutions in the presence of monomeric Aβ at 2 h incubation (A), monomeric Aβ at 24 h incubation (B), and Aβ fibrils pre-incubated for 24 h (C). Incubation was conducted in PBS at pH 7.4.In conclusion, we synthesized the curcumin-based Gd probes which enabled the detection and inhibition of Aβ fibril formation. Gd-DO3A-Comp.B allowed for the highly sensitive detection of Aβ fibril by the T₁ measurement. Moreover, the inhibitory activity could be estimated by T₁ measurement, because Gd-DO3A-Comp.B decreased T₁ depending on the growth stage of Aβ fibril formation. Such unique modality would be useful not only for the diagnostics but also for the direct evaluation of the therapeutic efficacy in vivo. For the future application, it would be important to combine with BBB penetration methods targeting the brain such as transient opening of the BBB using focused ultrasound or mannitol injection.^27,28     

Preparation and photo-induced activities of water-soluble amyloid β-C60 complexes

We have shown that fullerene (C₆₀) becomes soluble in water by mixing fullerene and amyloid β peptide (Aβ40) whose fibril structures are considered to be associated with Alzheimer''s disease. The water-solubility of fullerene arises from the generation of a nanosized complex between fullerene and the monomer species of Aβ40 (Aβ40-C₆₀). The prepared Aβ40-C₆₀ exhibits photo-induced activity with visible light to induce the inhibition of Aβ40 fibrillation and the cytotoxicity for cultured HeLa cells. The observed photo-induced phenomena result from the generation of singlet oxygen via photoexcitation, inducing oxidative damage to Aβ40 and HeLa cells. The oxidized Aβ40 following photoexcitation of Aβ40-C₆₀ was confirmed by mass spectrometry.

We have shown that fullerene (C₆₀) becomes soluble in water by mixing fullerene and amyloid β peptide (Aβ40) whose fibril structures are considered to be associated with Alzheimer''s disease.