Impact of sphingosine and acetylsphingosines on the aggregation and toxicity of metal-free and metal-treated amyloid-β

Pathophysiological shifts in the cerebral levels of sphingolipids in Alzheimer''s disease (AD) patients suggest a link between sphingolipid metabolism and the disease pathology. Sphingosine (SP), a structural backbone of sphingolipids, is an amphiphilic molecule that is able to undergo aggregation into micelles and micellar aggregates. Considering its structural properties and cellular localization, we hypothesized that SP potentially interacts with amyloid-β (Aβ) and metal ions that are found as pathological components in AD-affected brains, with manifesting its reactivity towards metal-free Aβ and metal-bound Aβ (metal–Aβ). Herein, we report, for the first time, that SP is capable of interacting with both Aβ and metal ions and consequently affects the aggregation of metal-free Aβ and metal–Aβ. Moreover, incubation of SP with Aβ in the absence and presence of metal ions results in the aggravation of toxicity induced by metal-free Aβ and metal–Aβ in living cells. As the simplest acyl derivatives of SP, N-acetylsphingosine and 3-O-acetylsphingosine also influence metal-free Aβ and metal–Aβ aggregation to different degrees, compared to SP. Such slight structural modifications of SP neutralize its ability to exacerbate the cytotoxicity triggered by metal-free Aβ and metal–Aβ. Notably, the reactivity of SP and the acetylsphingosines towards metal-free Aβ and metal–Aβ is determined to be dependent on their formation of micelles and micellar aggregates. Our overall studies demonstrate that SP and its derivatives could directly interact with pathological factors in AD and modify their pathogenic properties at concentrations below and above critical aggregation concentrations.

The reactivity of sphingosine and acetylsphingosines towards both metal-free and metal-treated amyloid-β is demonstrated showing a correlation of their micellization properties.     

An explicitly designed paratope of amyloid-β prevents neuronal apoptosis in vitro and hippocampal damage in rat brain

Synthetic antibodies hold great promise in combating diseases, diagnosis, and a wide range of biomedical applications. However, designing a therapeutically amenable, synthetic antibody that can arrest the aggregation of amyloid-β (Aβ) remains challenging. Here, we report a flexible, hairpin-like synthetic paratope (SP1, ∼2 kDa), which prevents the aggregation of Aβ monomers and reverses the preformed amyloid fibril to a non-toxic species. Structural and biophysical studies further allowed dissecting the mode and affinity of molecular recognition events between SP1 and Aβ. Subsequently, SP1 reduces Aβ-induced neurotoxicity, neuronal apoptosis, and ROS-mediated oxidative damage in human neuroblastoma cells (SH-SY5Y). The non-toxic nature of SP1 and its ability to ameliorate hippocampal neurodegeneration in a rat model of AD demonstrate its therapeutic potential. This paratope engineering module could readily implement discoveries of cost-effective molecular probes to nurture the basic principles of protein misfolding, thus combating related diseases.

Herein, the therapeutic potentials of an explicitly designed peptide probe are systematically illuminated in vitro and in vivo against Aβ aggregation. The probe demonstrates remarkable potency for attenuating neurotoxicity and hippocampal damage.     

Amyloid-β peptide 37, 38 and 40 individually and cooperatively inhibit amyloid-β 42 aggregation

The pathology of Alzheimer''s disease is connected to the aggregation of β-amyloid (Aβ) peptide, which in vivo exists as a number of length-variants. Truncations and extensions are found at both the N- and C-termini, relative to the most commonly studied 40- and 42-residue alloforms. Here, we investigate the aggregation of two physiologically abundant alloforms, Aβ₃₇ and Aβ₃₈, as pure peptides and in mixtures with Aβ₄₀ and Aβ₄₂. A variety of molar ratios were applied in quaternary mixtures to investigate whether a certain ratio is maximally inhibiting of the more toxic alloform Aβ₄₂. Through kinetic analysis, we show that both Aβ₃₇ and Aβ₃₈ self-assemble through an autocatalytic secondary nucleation reaction to form fibrillar β-sheet-rich aggregates, albeit on a longer timescale than Aβ₄₀ or Aβ₄₂. Additionally, we show that the shorter alloforms co-aggregate with Aβ₄₀, affecting both the kinetics of aggregation and the resulting fibrillar ultrastructure. In contrast, neither Aβ₃₇ nor Aβ₃₈ forms co-aggregates with Aβ₄₂; however, both short alloforms reduce the rate of Aβ₄₂ aggregation in a concentration-dependent manner. Finally, we show that the aggregation of Aβ₄₂ is more significantly impeded by a combination of Aβ₃₇, Aβ₃₈, and Aβ₄₀ than by any of these alloforms independently. These results demonstrate that the aggregation of any given Aβ alloform is significantly perturbed by the presence of other alloforms, particularly in heterogeneous mixtures, such as is found in the extracellular fluid of the brain.

The pathology of Alzheimer''s disease is connected to the aggregation of β-amyloid (Aβ) peptide, which in vivo exists as a number of length-variants. This study identifies the Aβ37/38/40 ratio that is maximally inhibitory to Aβ₄₂ aggregation.     

Concurrent suppression of Aβ aggregation and NLRP3 inflammasome activation for treating Alzheimer's disease

Alzheimer''s disease (AD) is a neurodegenerative illness accompanied by severe memory loss, cognitive disorders and impaired behavioral ability. Amyloid β-peptide (Aβ) aggregation and nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome play crucial roles in the pathogenesis of AD. Aβ plaques not only induce oxidative stress and impair neurons, but also activate the NLRP3 inflammasome, which releases inflammatory cytokine IL-1β to trigger neuroinflammation. A bifunctional molecule, 2-[2-(benzo[d]thiazol-2-yl)phenylamino]benzoic acid (BPBA), with both Aβ-targeting and inflammasome-inhibiting capabilities was designed and synthesized. BPBA inhibited self- and Cu²⁺- or Zn²⁺-induced Aβ aggregation, disaggregated the already formed Aβ aggregates, and reduced the neurotoxicity of Aβ aggregates; it also inhibited the activation of the NLRP3 inflammasome and reduced the release of IL-1β in vitro and vivo. Moreover, BPBA decreased the production of reactive oxygen species (ROS) and alleviated Aβ-induced paralysis in transgenic C. elegans with the human Aβ₄₂ gene. BPBA exerts an anti-AD effect mainly through dissolving Aβ aggregates and inhibiting NLRP3 inflammasome activation synergistically.

Bifunctional molecule BPBA inhibits Aβ aggregation and NLRP3 inflammasome activation, thereby decreasing ROS and IL-1β in vitro and vivo; it synergistically prevents Alzheimer''s disease via alleviating Aβ neurotoxicity and reducing neuroinflammation.     

Differentiating Aβ40 and Aβ42 in amyloid plaques with a small molecule fluorescence probe

Differentiating amyloid beta (Aβ) subspecies Aβ40 and Aβ42 has long been considered an impossible mission with small-molecule probes. In this report, based on recently published structures of Aβ fibrils, we designed iminocoumarin–thiazole (ICT) fluorescence probes to differentiate Aβ40 and Aβ42, among which Aβ42 has much higher neurotoxicity. We demonstrated that ICTAD-1 robustly responds to Aβ fibrils, evidenced by turn-on fluorescence intensity and red-shifting of emission peaks. Remarkably, ICTAD-1 showed different spectra towards Aβ40 and Aβ42 fibrils. In vitro results demonstrated that ICTAD-1 could be used to differentiate Aβ40/42 in solutions. Moreover, our data revealed that ICTAD-1 could be used to separate Aβ40/42 components in plaques of AD mouse brain slides. In addition, two-photon imaging suggested that ICTAD-1 was able to cross the BBB and label plaques in vivo. Interestingly, we observed that ICTAD-1 was specific toward plaques, but not cerebral amyloid angiopathy (CAA) on brain blood vessels. Given Aβ40 and Aβ42 species have significant differences of neurotoxicity, we believe that ICTAD-1 can be used as an important tool for basic studies and has the potential to provide a better diagnosis in the future.

A small molecule fluorescence probe ICTAD-1 was rationally designed for differentiating Aβ40 and Aβ42 in solutions and in Aβ plaques.     

Multifunctional peptide-assembled micelles for simultaneously reducing amyloid-β and reactive oxygen species

The excessive production and deposition of amyloid-β (Aβ) is one of the most important etiologies of Alzheimer''s disease (AD). The interaction between Aβ and metal ions produces aberrant reactive oxygen species (ROS), which induce oxidative stress and accelerate the progression of AD. To reduce Aβ plaques and ROS to maintain their homeostasis is an emerging and ingenious strategy for effective treatment of AD. Herein, we report the rational design of multifunctional micelles (MPGLT) based on a polymer-grafted peptide to simultaneously clear Aβ and ROS for AD therapy. The MPGLT integrating three functional peptides as a ROS scavenger (tk-GSH), β-sheet breaker (LP) and an autophagy activator (TK) respectively, could capture and degrade Aβ. Meanwhile, the tk-GSH on the surface of MPGLT effectively scavenges the intracellular ROS. Consequently, MPGLT reduced the cytotoxicity of Aβ and ROS. In vivo animal studies using an AD mouse model further showed that MPGLT could transport across the blood–brain barrier for decreasing the Aβ plaque and eliminating ROS in vivo. This peptide micelle-based synergistic strategy may provide novel insight for AD therapy.

Multifunctional micelles based on a peptide–polymer for simultaneously targeting Aβ degradation and ROS scavenging for AD therapy.     

Metal-chelating benzothiazole multifunctional compounds for the modulation and 64Cu PET imaging of Aβ aggregation

While Alzheimer''s Disease (AD) is the most common neurodegenerative disease, there is still a dearth of efficient therapeutic and diagnostic agents for this disorder. Reported herein are a series of new multifunctional compounds (MFCs) with appreciable affinity for amyloid aggregates that can be potentially used for both the modulation of Aβ aggregation and its toxicity, as well as positron emission tomography (PET) imaging of Aβ aggregates. Firstly, among the six compounds tested HYR-16 is shown to be capable to reroute the toxic Cu-mediated Aβ oligomerization into the formation of less toxic amyloid fibrils. In addition, HYR-16 can also alleviate the formation of reactive oxygen species (ROS) caused by Cu²⁺ ions through Fenton-like reactions. Secondly, these MFCs can be easily converted to PET imaging agents by pre-chelation with the ⁶⁴Cu radioisotope, and the Cu complexes of HYR-4 and HYR-17 exhibit good fluorescent staining and radiolabeling of amyloid plaques both in vitro and ex vivo. Importantly, the ⁶⁴Cu-labeled HYR-17 is shown to have a significant brain uptake of up to 0.99 ± 0.04 %ID per g. Overall, by evaluating the various properties of these MFCs valuable structure–activity relationships were obtained that should aid the design of improved therapeutic and diagnostic agents for AD.

A series of multifunctional compounds and their ⁶⁴Cu complexes exhibit good affinity for Aβ aggregates and can also control Aβ toxicity.     

Modification of amyloid-beta peptide aggregation via photoactivation of strained Ru(ii) polypyridyl complexes

Alzheimer''s disease (AD) is a chronic neurodegenerative disorder characterized by progressive and irreversible damage to the brain. One of the hallmarks of the disease is the presence of both soluble and insoluble aggregates of the amyloid beta (Aβ) peptide in the brain, and these aggregates are considered central to disease progression. Thus, the development of small molecules capable of modulating Aβ peptide aggregation may provide critical insight into the pathophysiology of AD. In this work we investigate how photoactivation of three distorted Ru(ii) polypyridyl complexes (Ru1–3) alters the aggregation profile of the Aβ peptide. Photoactivation of Ru1–3 results in the loss of a 6,6′-dimethyl-2,2′-bipyridyl (6,6′-dmb) ligand, affording cis-exchangeable coordination sites for binding to the Aβ peptide. Both Ru1 and Ru2 contain an extended planar imidazo[4,5-f][1,10]phenanthroline ligand, as compared to a 2,2′-bipyridine ligand for Ru3, and we show that the presence of the phenanthroline ligand promotes covalent binding to Aβ peptide His residues, and in addition, leads to a pronounced effect on peptide aggregation immediately after photoactivation. Interestingly, all three complexes resulted in a similar aggregate size distribution at 24 h, forming insoluble amorphous aggregates as compared to significant fibril formation for peptide alone. Photoactivation of Ru1–3 in the presence of pre-formed Aβ_1–42 fibrils results in a change to amorphous aggregate morphology, with Ru1 and Ru2 forming large amorphous aggregates immediately after activation. Our results show that photoactivation of Ru1–3 in the presence of either monomeric or fibrillar Aβ_1–42 results in the formation of large amorphous aggregates as a common endpoint, with Ru complexes incorporating the extended phenanthroline ligand accelerating this process and thereby limiting the formation of oligomeric species in the initial stages of the aggregation process that are reported to show considerable toxicity.

Photoactivation of a series of Ru(ii) polypyridyl complexes leads to ligand exchange and modulation of amyloid-beta peptide aggregation of relevance to Alzheimer''s disease.     

Small molecule-mediated co-assembly of amyloid-β oligomers reduces neurotoxicity through promoting non-fibrillar aggregation

Amyloid-β (Aβ) oligomers, particularly low molecular weight (LMW) oligomers, rather than fibrils, contribute very significantly to the onset and progression of Alzheimer''s Disease (AD). However, due to the inherent heterogeneity and metastability of oligomers, most of the conventional anti-oligomer therapies have indirectly modulated oligomers'' toxicity through manipulating Aβ self-assembly to reduce oligomer levels, which are prone to suffering from the risk of regenerating toxic oligomers from the products of modulation. To circumvent this disadvantage, we demonstrate, for the first time, rational design of rigid pincer-like scaffold-based small molecules with blood–brain barrier permeability that specifically co-assemble with LMW Aβ oligomers through directly binding to the exposed hydrophobic regions of oligomers to form non-fibrillar, degradable, non-toxic co-aggregates. As a proof of concept, treatment with a europium complex (EC) in such a structural mode can rescue Aβ-mediated dysfunction in C. elegans models of AD in vivo. This small molecule-mediated oligomer co-assembly strategy offers an efficient approach for AD treatment.

A rational design of pincer-like scaffold-based small molecule with blood-brain barrier permeability that can specifically co-assemble with low molecular weight Aβ oligomers to form non-fibrillar, degradable, non-toxic co-aggregates.     

A rationally designed small molecule for identifying an in vivo link between metal–amyloid-β complexes and the pathogenesis of Alzheimer's disease

Multiple factors, including amyloid-β (Aβ), metals, and reactive oxygen species (ROS), are involved in the development of Alzheimer''s disease (AD). Metal ions can interact with Aβ species generating toxic oligomers and ROS in vitro; however, the involvement of metal–Aβ complexes in AD pathology in vivo remains unclear. To solve this uncertainty, we have developed a chemical tool (L2-b) that specifically targets metal–Aβ complexes and modulates their reactivity (i.e., metal–Aβ aggregation, toxic oligomer formation, and ROS production). Through the studies presented herein, we demonstrate that L2-b is able to specifically interact with metal–Aβ complexes over metal-free Aβ analogues, redirect metal–Aβ aggregation into off-pathway, nontoxic less structured Aβ aggregates, and diminish metal–Aβ-induced ROS production, overall mitigating metal–Aβ-triggered toxicity, confirmed by multidisciplinary approaches. L2-b is also verified to enter the brain in vivo with relative metabolic stability. Most importantly, upon treatment of 5XFAD AD mice with L2-b, (i) metal–Aβ complexes are targeted and modulated in the brain; (ii) amyloid pathology is reduced; and (iii) cognition deficits are significantly improved. To the best of our knowledge, by employing an in vivo chemical tool specifically prepared for investigating metal–Aβ complexes, we report for the first time experimental evidence that metal–Aβ complexes are related directly to AD pathogenesis.     

Beneficial Effects of Cyclic Ether 2-Butoxytetrahydrofuran from Sea Cucumber Holothuria scabra against Aβ Aggregate Toxicity in Transgenic Caenorhabditis elegans and Potential Chemical Interaction

The pathological finding of amyloid-β (Aβ) aggregates is thought to be a leading cause of untreated Alzheimer’s disease (AD). In this study, we isolated 2-butoxytetrahydrofuran (2-BTHF), a small cyclic ether, from Holothuria scabra and demonstrated its therapeutic potential against AD through the attenuation of Aβ aggregation in a transgenic Caenorhabditis elegans model. Our results revealed that amongst the five H. scabra isolated compounds, 2-BTHF was shown to be the most effective in suppressing worm paralysis caused by Aβ toxicity and in expressing strong neuroprotection in CL4176 and CL2355 strains, respectively. An immunoblot analysis showed that CL4176 and CL2006 treated with 2-BTHF showed no effect on the level of Aβ monomers but significantly reduced the toxic oligomeric form and the amount of 1,4-bis(3-carboxy-hydroxy-phenylethenyl)-benzene (X-34)-positive fibril deposits. This concurrently occurred with a reduction of reactive oxygen species (ROS) in the treated CL4176 worms. Mechanistically, heat shock factor 1 (HSF-1) (at residues histidine 63 (HIS63) and glutamine 72 (GLN72)) was shown to be 2-BTHF’s potential target that might contribute to an increased expression of autophagy-related genes required for the breakdown of the Aβ aggregate, thus attenuating its toxicity. In conclusion, 2-BTHF from H. scabra could protect C. elegans from Aβ toxicity by suppressing its aggregation via an HSF-1-regulated autophagic pathway and has been implicated as a potential drug for AD.     

Antidementia Effects of Alternanthera philoxeroides in Ovariectomized Mice Supported by NMR-Based Metabolomic Analysis

The crude ethanol extract of the whole plant of Alternanthera philoxeroides (Mart.) Griseb was investigated for its potential as antidementia, induced by estrogen deprivation, based on in vitro antioxidant activity, β-amyloid aggregation inhibition and cholinesterase inhibitory activity, as well as in vivo Morris water maze task (MWMT), novel object recognition task (NORT), and Y-maze task. To better understand the effect of the extract, oxidative stress-induced brain membrane damage through lipid peroxidation in the whole brain was also investigated. Additionally, expressions of neuroinflammatory cytokines (IL-1β, IL-6 and TNF-α) and estrogen receptor-mediated facilitation genes such as PI3K and AKT mRNA in the hippocampus and frontal cortex were also evaluated. These effects were confirmed by the determination of its serum metabolites by NMR metabolomic analysis. Both the crude extract of A. philoxeroides and its flavone constituents were found to inhibit β-amyloid (Aβ) aggregation.     

A fluorescent molecular imaging probe with selectivity for soluble tau aggregated protein

Soluble forms of aggregated tau misfolded protein, generally termed oligomers, are considered to be the most toxic species of the different assembly states that are the pathological components of neurodegenerative disorders. Therefore, a critical biomedical need exists for imaging probes that can identify and quantify them. We have designed and synthesized a novel fluorescent probe, pTP-TFE for which binding and selectivity profiles towards aggregated tau and Aβ proteins were assessed. Our results have shown pTP-TFE to be selective for early forms of soluble tau aggregates, with high affinity of dissociation constants (K_d) = 66 nM, and tenfold selectivity over mature tau fibrils. Furthermore, we found that pTP-TFE is selective for tau over Aβ aggregates and had good cell permeability. This selectivity of pTP-TFE towards early forms of aggregated tau protein ex vivo was also supported with studies on human brain tissue containing tau and Aβ pathology. To the best of our knowledge, this is the first fluorescent molecule to be reported to have this form of selectivity profile, which suggests that pTP-TFE is a unique probe candidate for imaging-based detection of early stages of Alzheimer''s disease and other tauopathies.

pTP-TFE imaging probe can distinguish soluble tau aggregated proteins from other aggregated proteins enabling earlier detection of neurodegenerative diseases.     

Monitoring phagocytic uptake of amyloid β into glial cell lysosomes in real time

Phagocytosis by glial cells is essential to regulate brain function during health and disease. Therapies for Alzheimer''s disease (AD) have primarily focused on targeting antibodies to amyloid β (Aβ) or inhibitng enzymes that make it, and while removal of Aβ by phagocytosis is protective early in AD it remains poorly understood. Impaired phagocytic function of glial cells during later stages of AD likely contributes to worsened disease outcome, but the underlying mechanisms of how this occurs remain unknown. We have developed a human Aβ_1–42 analogue (Aβ^pH) that exhibits green fluorescence upon internalization into the acidic organelles of cells but is non-fluorescent at physiological pH. This allowed us to image, for the first time, glial uptake of Aβ^pH in real time in live animals. We find that microglia phagocytose more Aβ^pH than astrocytes in culture, in brain slices and in vivo. Aβ^pH can be used to investigate the phagocytic mechanisms responsible for removing Aβ from the extracellular space, and thus could become a useful tool to study Aβ clearance at different stages of AD.

Glial cell phagocytosis of pH-dependent amyloid-β, Aβ^pH, in live and fixed cultures, brain tissue sections, retina, cortex and in live animals useful for studying function in health and disease.     

Nanomaterials for Modulating the Aggregation of β-Amyloid Peptides

The aberrant aggregation of amyloid-β (Aβ) peptides in the brain has been recognized as the major hallmark of Alzheimer’s disease (AD). Thus, the inhibition and dissociation of Aβ aggregation are believed to be effective therapeutic strategiesforthe prevention and treatment of AD. When integrated with traditional agents and biomolecules, nanomaterials can overcome their intrinsic shortcomings and boost their efficiency via synergistic effects. This article provides an overview of recent efforts to utilize nanomaterials with superior properties to propose effective platforms for AD treatment. The underlying mechanismsthat are involved in modulating Aβ aggregation are discussed. The summary of nanomaterials-based modulation of Aβ aggregation may help researchers to understand the critical roles in therapeutic agents and provide new insight into the exploration of more promising anti-amyloid agents and tactics in AD theranostics.     

Intermediates involved in serotonin oxidation catalyzed by Cu bound Aβ peptides

The degradation of neurotransmitters is a hallmark feature of Alzheimer''s disease (AD). Copper bound Aβ peptides, invoked to be involved in the pathology of AD, are found to catalyze the oxidation of serotonin (5-HT) by H₂O₂. A combination of EPR and resonance Raman spectroscopy reveals the formation of a Cu(ii)–OOH species and a dimeric, EPR silent, Cu₂O₂ bis-μ-oxo species under the reaction conditions. The Cu(ii)–OOH species, which can be selectively formed in the presence of excess H₂O₂, is the reactive intermediate responsible for 5-HT oxidation. H₂O₂ produced by the reaction of O₂ with reduced Cu(i)–Aβ species can also oxidize 5-HT. Both these pathways are physiologically relevant and may be involved in the observed decay of neurotransmitters as observed in AD patients.

The mononuclear copper hydroperoxo species (Cu(ii)–OOH) of Cu–Aβ is the active oxidant responsible for serotonin oxidation by Cu–Aβ in the presence of physiologically relevant oxidants like O₂ and H₂O₂, which can potentially cause oxidative degradation of neurotransmitters, a marker of Alzheimer''s disease.     

Isoorientin Inhibits Amyloid β25–35-Induced Neuronal Inflammation in BV2 Cells by Blocking the NF-κB Signaling Pathway

Alzheimer’s disease (AD) is a severe neurodegenerative disorder. AD is pathologically characterized by the formation of intracellular neurofibrillary tangles, and extracellular amyloid plaques which were comprised of amyloid-beta (Aβ) peptides. Aβ induces neurodegeneration by activating microglia, which triggers neurotoxicity by releasing various inflammatory mediators and reactive oxygen species (ROS). Nuclear factor-kappa B (NF-κB) is expressed in human tissues including the brain and plays an important role in Aβ-mediated neuronal inflammation. Thus, the identification of molecules that inhibit the NF-κB pathway is considered an attractive strategy for the treatment and prevention of AD. Isoorientin (3′,4′,5,7-Tetrahydroxy-6-C-glucopyranosyl flavone; ISO), which can be extracted from several plant species, such as Philostachys and Patrinia is known to have various pharmacological activities such as anticancer, antioxidant, and antibacterial activity. However, the effect of ISO on Aβ-mediated inflammation and apoptosis in the brain has yet to be elucidated. In the present study, we investigated whether ISO regulated Aβ-induced neuroinflammation in microglial cells and further explored the underlying mechanisms. Our results showed that ISO inhibited the expression of iNOS and COX-2 induced by Aβ_25–35. And, it inhibited the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, ISO reduced the ROS production in Aβ_25–35-induced BV2 cells and inhibited NF-κB activation. Furthermore, ISO blocked Aβ_25–35-induced apoptosis of BV2 cells. Based on these findings, we suggest that ISO represents a promising therapeutic drug candidate for the treatment and prevention of AD.     

Modulation of γ-Secretase Activity by a Carborane-Based Flurbiprofen Analogue

All over the world, societies are facing rapidly aging populations combined with a growing number of patients suffering from Alzheimer’s disease (AD). One focus in pharmaceutical research to address this issue is on the reduction of the longer amyloid-β (Aβ) fragments in the brain by modulation of γ-secretase, a membrane-bound protease. R-Flurbiprofen (tarenflurbil) was studied in this regard but failed to show significant improvement in AD patients in a phase 3 clinical trial. This was mainly attributed to its low ability to cross the blood–brain barrier (BBB). Here, we present the synthesis and in vitro evaluation of a racemic meta-carborane analogue of flurbiprofen. By introducing the carborane moiety, the hydrophobicity could be shifted into a more favourable range for the penetration of the blood–brain barrier, evident by a logD_7.4 value of 2.0. Furthermore, our analogue retained γ-secretase modulator activity in comparison to racemic flurbiprofen in a cell-based assay. These findings demonstrate the potential of carboranes as phenyl mimetics also in AD research.     

3D-visualization of amyloid-β oligomer interactions with lipid membranes by cryo-electron tomography

Amyloid-β (Aβ) assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimer''s disease. However, molecular mechanisms of Aβ cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various Aβ assembly types and their interaction with liposomes. Aβ oligomers and curvilinear protofibrils bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Aβ oligomers concentrate at the interface of vesicles and form a network of Aβ-linked liposomes, while crucially, monomeric and fibrillar Aβ have relatively little impact on the membrane. Changes to lipid membrane composition highlight a significant role for GM1-ganglioside in promoting Aβ-membrane interactions. The different effects of Aβ assembly forms observed align with the highlighted cytotoxicity reported for Aβ oligomers. The wide-scale incorporation of Aβ oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.

Cryo-electron tomography 3D imaging of amyloid-β oligomers carpeting the surface of lipid bilayers in near native conditions.     

From Combinations to Single-Molecule Polypharmacology—Cromolyn-Ibuprofen Conjugates for Alzheimer’s Disease

Despite Alzheimer’s disease (AD) incidence being projected to increase worldwide, the drugs currently on the market can only mitigate symptoms. Considering the failures of the classical paradigm “one target-one drug-one disease” in delivering effective medications for AD, polypharmacology appears to be a most viable therapeutic strategy. Polypharmacology can involve combinations of multiple drugs and/or single chemical entities modulating multiple targets. Taking inspiration from an ongoing clinical trial, this work aims to convert a promising cromolyn–ibuprofen drug combination into single-molecule “codrugs.” Such codrugs should be able to similarly modulate neuroinflammatory and amyloid pathways, while showing peculiar pros and cons. By exploiting a linking strategy, we designed and synthesized a small set of cromolyn–ibuprofen conjugates (4–6). Preliminary plasma stability and neurotoxicity assays allowed us to select diamide 5 and ethanolamide 6 as promising compounds for further studies. We investigated their immunomodulatory profile in immortalized microglia cells, in vitro anti-aggregating activity towards Aβ₄₂-amyloid self-aggregation, and their cellular neuroprotective effect against Aβ₄₂-induced neurotoxicity. The fact that 6 effectively reduced Aβ-induced neuronal death, prompted its investigation into an in vivo model. Notably, 6 was demonstrated to significantly increase the longevity of Aβ₄₂-expressing Drosophila and to improve fly locomotor performance.