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.     

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.     

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.     

Adequate prediction for inhibitor affinity of Aβ40 protofibril using the linear interaction energy method

The search for efficient inhibitors targeting Aβ oligomers and fibrils is an important issue in Alzheimer''s disease treatment. As a consequence, an accurate and computationally cheap approach to estimate the binding affinity for many ligands interacting with Aβ peptides is very important. Here, the calculated binding free energies of 30 ligands interacting with 12Aβ_11–40 peptides using the linear interaction energy (LIE) approach are found to be in good correlation with experimental data (R = 0.79). The binding affinities of these complexes are also calculated by using free energy perturbation (FEP) and molecular mechanic/Poisson–Boltzmann surface area (MM/PBSA) methods. The time-consuming FEP method provides results with similar correlation (R = 0.72), whereas MM/PBSA calculations show very low correlation with experimental data (R = 0.27). In all complexes, van der Waals interactions contribute much more than electrostatic interactions. The LIE model, which is much less time-consuming than both the FEP and MM/PBSA methods, opens the door to accurate and rapid affinity prediction of ligands with Aβ peptides and the design of new ligands.

The efficient approach to estimate inhibitors targeting Aβ oligomers and fibrils is an important issue in Alzheimer''s disease treatment.     

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.     

Nanoscopic insights into the surface conformation of neurotoxic amyloid β oligomers

Raman spectroscopy assisted by localized plasmon resonances generating effective hot spots at the gaps between intertwined silver nanowires is herein adopted to unravel characteristic molecular motifs on the surface of Aβ₄₂ misfolded oligomers that are critical in driving intermolecular interactions in neurodegeneration.

Unraveling characteristic structural determinants at the basis of Aβ₄₂ oligomers'' neurotoxicity by a sub-molecular SERS investigation of their surface.     

Geniposide attenuates Aβ25–35-induced neurotoxicity via the TLR4/NF-κB pathway in HT22 cells

Alzheimer''s disease (AD), a neurodegenerative disorder, is marked by the accumulation of amyloid-β (Aβ) and neuroinflammation which promote the development of AD. Geniposide, the main ingredient isolated from Chinese herbal medicine Gardenia jasminoides Ellis, has a variety of pharmacological functions such as anti-apoptosis and anti-inflammatory activity. Hence, we estimated the inflammatory cytotoxicity caused by Aβ_25–35 and the neuroprotective effects of geniposide in HT22 cells. In this research, following incubation with Aβ_25–35 (40 μM, 24 h) in HT22 cells, the methylthiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) release assays showed that the cell survival rate was significantly decreased. In contrast, the reactive oxygen species (ROS) assay indicated that Aβ_25–35 enhanced ROS accumulation and apoptosis showed in both hoechst 33342 staining and annexin V-FITC/PI double staining. And then, immunofluorescence test revealed that Aβ_25–35 promoted p65 to transfer into the nucleus indicating p65 was activated by Aβ_25–35. Moreover, western blot analysis proved that Aβ_25–35 increased the expression of nitric oxide species (iNOS), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-1β (IL-1β). Simultaneously, Aβ_25–35 also promoted the expression of toll-like receptor 4 (TLR4), p-p65 and p-IκB-α accompanied with the increase in the level of beta-secretase 1 (BACE1) and caspase-3 which further supported Aβ_25–35 induced apoptosis and inflammation. Fortunately, this up-regulation was reversed by geniposide. In conclusion, our data suggest that geniposide can alleviate Aβ_25–35-induced inflammatory response to protect neurons, which is possibly involved with the inhibition of the TLR4/NF-κB pathway in HT22 cells. Geniposide may be the latent treatment for AD induced by neuroinflammation and apoptosis.

Alzheimer''s disease (AD), a neurodegenerative disorder, is marked by the accumulation of amyloid-β (Aβ) and neuroinflammation which promote the development of AD.     

Qualitative changes in human γ-secretase underlie familial Alzheimer’s disease

Presenilin (PSEN) pathogenic mutations cause familial Alzheimer’s disease (AD [FAD]) in an autosomal-dominant manner. The extent to which the healthy and diseased alleles influence each other to cause neurodegeneration remains unclear. In this study, we assessed γ-secretase activity in brain samples from 15 nondemented subjects, 22 FAD patients harboring nine different mutations in PSEN1, and 11 sporadic AD (SAD) patients. FAD and control brain samples had similar overall γ-secretase activity levels, and therefore, loss of overall (endopeptidase) γ-secretase function cannot be an essential part of the pathogenic mechanism. In contrast, impaired carboxypeptidase-like activity (γ-secretase dysfunction) is a constant feature in all FAD brains. Significantly, we demonstrate that pharmacological activation of the carboxypeptidase-like γ-secretase activity with γ-secretase modulators alleviates the mutant PSEN pathogenic effects. Most SAD cases display normal endo- and carboxypeptidase-like γ-secretase activities. However and interestingly, a few SAD patient samples display γ-secretase dysfunction, suggesting that γ-secretase may play a role in some SAD cases. In conclusion, our study highlights qualitative shifts in amyloid-β (Aβ) profiles as the common denominator in FAD and supports a model in which the healthy allele contributes with normal Aβ products and the diseased allele generates longer aggregation-prone peptides that act as seeds inducing toxic amyloid conformations.Early-onset familial Alzheimer’s disease (AD [FAD]), starting before age 65, is mainly caused by mutations in the Presenilin 1/2 (PSEN1/2) or the amyloid precursor protein (APP) genes and represents less than 0.1% of the total AD cases (Campion et al., 1999). Although rare, FAD offers a unique model to gain insights into the molecular mechanisms and etiology of sporadic AD (SAD).PSEN is the catalytic subunit of the γ-secretase complex (De Strooper et al., 1998; Wolfe et al., 1999), an intramembrane multimeric protease involved in the processing of many type 1 transmembrane proteins; among them, the Notch receptors and APP have received much attention because of their association with crucial cell signaling events or with AD pathogenesis, respectively (for a review see Jurisch-Yaksi et al. [2013]). Nicastrin (Nct), PSEN enhancer 2 (Pen2), and anterior pharynx defective 1 (APH1) are, together with PSEN, essential components of the protease complex (De Strooper, 2003).More than 150 pathogenic mutations in PSEN1 have been reported so far (http://www.molgen.ua.ac.be/ADMutations); and notably, the vast majority are missense substitutions distributed throughout the primary structure of PSEN1. PSEN/γ-secretase hydrolyzes peptide bonds in a process called regulated intramembrane proteolysis, which allows translation of extracellular signals into the cell. Compelling evidence indicates that γ-secretase cuts membrane proteins sequentially: the first endopeptidase cleavage (ε) releases a soluble intracellular domain (ICD), which may translocate to the nucleus to regulate gene expression while the remaining N-terminal transmembrane domain (TMD) fragment is successively cut by the carboxypeptidase-like activity of γ-secretase (γ-cleavages). Endopeptidase products, either amyloid-β49 (Aβ49) or Aβ48, are then processed along two major product lines: Aβ49 → Aβ46 → Aβ43 → Aβ40 or Aβ48 → Aβ45 → Aβ42 → Aβ38 (Takami et al., 2009). Every γ-cleavage removes a short C-terminal peptide from the TMD, reducing its hydrophobicity and increasing the probability of release. Secretion of an N-terminal fragment into the extracellular/luminal space terminates this sequence (Qi-Takahara et al., 2005; Yagishita et al., 2008; Takami et al., 2009). Importantly, the efficiency of the endopeptidase cleavage determines ICD product levels, which acquires high physiological relevance in the case of the Notch substrate. The carboxypeptidase-like efficiency, the number of cuts per substrate, determines the length of the N-terminal products; the level of efficiency is pathologically very relevant in the case of the APP substrate, as lower efficiency results in the production of longer and more aggregation-prone Aβ peptides (Chávez-Gutiérrez et al., 2012).How mutations in the PSENs cause FAD remains a hotly debated topic in the field. Because FAD is an autosomal-dominant disorder (patients carry both healthy and mutant alleles), a major unknown in the discussion remains the role of the healthy allele and, to a lesser extent, the role of the brain environment on the total (normal + mutant proteases) γ-secretase activity. To what degree do normal and mutant complexes contribute to total γ-secretase activity in the patient brain? Does the healthy allele compensate for the disease allele effects? Despite their relevance, those questions have not been addressed.Only one group, i.e., Potter et al. (2013), have estimated the Aβ production kinetics in the FAD brain by measuring isotope-labeled Aβ peptides in the cerebrospinal fluid of patients (stable isotope-labeled kinetics [SILK]). Feeding the in vivo metabolic labeling patient data into a mathematical model, specifically generated for their approach, they described higher Aβ42 production rates in the central nervous system of PSEN mutation carriers (Potter et al., 2013). Accordingly, Potter et al. (2013) suggest that increments in Aβ42 play a decisive pathogenic role in AD.In contrast, a “revised” loss-of-function (LOF) hypothesis has recently been proposed by Xia et al. (2015). In this view, loss of PSEN/γ-secretase physiological cell signaling function causes neurodegeneration, whereas changes in Aβ peptides are only secondary byproducts that arise from but do not trigger the disease (Xia et al., 2015). The idea is only tenable if FAD-linked PSEN mutations exert a LOF effect on PSEN/γ-secretase and, in addition, a dominant-negative effect on the healthy PSEN allele (normal γ-secretase) in patients, a key part of this hypothesis (Heilig et al., 2013; Xia et al., 2015). However, it should be stressed that γ-secretase haploinsufficiency caused by nonsense, frameshift, and splice site mutations in genes coding for essential subunits of γ-secretase (Nct, Pen2, and PSEN) is pathogenic in nature; such haploinsufficiency causes a chronic inflammatory disease of hair follicles known as familial acne inversa. Most importantly, no clinical association between this disorder and AD has been reported (for a review see Pink et al. [2013]). Furthermore, if FAD-linked PSEN mutations were truly LOF mutations, resulting in “inactive” γ-secretase complexes, homozygous individuals for the disease allele would not be viable because of disturbances in Notch signaling during embryonic development. However, six individuals with homozygous PSEN1 E280A gene mutation have been identified (Kosik et al., 2015).An alternative view to both hypotheses is that pathogenic mutations in PSEN cause disease by qualitative shifts in Aβ profile production (γ-secretase dysfunction; Chávez-Gutiérrez et al., 2012). We have demonstrated that loss of endopeptidase activity is not necessarily observed in γ-secretase complexes containing PSEN1/2 FAD-linked mutations, but reduced carboxypeptidase-like efficiency (γ-secretase dysfunction) is the constant denominator. Furthermore, FAD PSEN mutations may affect the carboxypeptidase-like γ-secretase activity at multiple turnovers, resulting in increased Aβ43 and Aβ42 levels as well as in other longer Aβ peptides, such as Aβ45 and Aβ46 (Quintero-Monzon et al., 2011; Chávez-Gutiérrez et al., 2012; Fernandez et al., 2014). These data support a model in which relative, rather than absolute, changes in Aβ product profiles are at the basis of PSEN/γ-secretase–mediated pathogenicity. However, these findings were based on studies conducted in PSEN1/2-deficient MEFs, which does not fully recapitulate the in vivo heterozygous situation in the FAD patient’s brain.In the current study, we investigated processing of APP by the γ-secretase complex in postmortem human brain samples from FAD and SAD patients and healthy control subjects. Our investigation is the first to directly assess γ-secretase activity in brain material from FAD mutant carriers and to address how the FAD-linked mutant heterozygous situation in patients affects γ-secretase function in brain.     

Islet-Specific T-Cell Responses and Proinflammatory Monocytes Define Subtypes of Autoantibody-Negative Ketosis-Prone Diabetes

OBJECTIVE

Ketosis-prone diabetes (KPD) is characterized by diabetic ketoacidosis (DKA) in patients lacking typical features of type 1 diabetes. A validated classification scheme for KPD includes two autoantibody-negative (“A−”) phenotypic forms: “A−β−” (lean, early onset, lacking β-cell functional reserve) and “A−β+” (obese, late onset, with substantial β-cell functional reserve after the index episode of DKA). Recent longitudinal analysis of a large KPD cohort revealed that the A−β+ phenotype includes two distinct subtypes distinguished by the index DKA episode having a defined precipitant (“provoked,” with progressive β-cell function loss over time) or no precipitant (“unprovoked,” with sustained β-cell functional reserve). These three A− KPD subtypes are characterized by absence of humoral islet autoimmune markers, but a role for cellular islet autoimmunity is unknown.

RESEARCH DESIGN AND METHODS

Islet-specific T-cell responses and the percentage of proinflammatory (CD14+CD16+) blood monocytes were measured in A−β− (n = 7), provoked A−β+ (n = 15), and unprovoked A−β+ (n = 13) KPD patients. Genotyping was performed for type 1 diabetes–associated HLA class II alleles.

RESULTS

Provoked A−β+ and A−β− KPD patients manifested stronger islet-specific T-cell responses (P < 0.03) and higher percentages of proinflammatory CD14+CD16+ monocytes (P < 0.01) than unprovoked A−β+ KPD patients. A significant relationship between type 1 diabetes HLA class II protective alleles and negative T-cell responses was observed.

CONCLUSIONS

Provoked A−β+ KPD and A−β− KPD are associated with a high frequency of cellular islet autoimmunity and proinflammatory monocyte populations. In contrast, unprovoked A−β+ KPD lacks both humoral and cellular islet autoimmunity.Ketosis-prone diabetes (KPD), characterized by presentation with diabetic ketoacidosis (DKA) in patients lacking the typical features of autoimmune type 1 diabetes, is a heterogeneous syndrome (1,2). A validated classification scheme for KPD, based on the presence or absence of β-cell autoantibodies (“A+” or “A−”) and presence or absence of β-cell functional reserve (“β+” or “β−”) (3) includes two autoantibody-negative A− phenotypic forms: “A−β−” (lean, early onset, lacking β-cell functional reserve) and “A−β+” (obese, late onset, with substantial β-cell functional reserve after the index episode of DKA). Long-term longitudinal follow-up of a large cohort of A−β+ KPD patients has revealed that this phenotype comprises two distinct subtypes distinguished by whether the index DKA episode had a defined precipitant (“provoked” A−β+) or no precipitant (“unprovoked” A−β+) (4). Provoked A−β+ KPD patients have progressive loss of β-cell function after initial recovery from the DKA episode, with relapse to insulin dependence, no sex predominance, and an increased frequency of the HLA class II alleles DQB1*0302 and DRB1*04 associated with susceptibility to autoimmune type 1 diabetes. In contrast, unprovoked A−β+ KPD patients have sustained preservation of β-cell function after recovery from the DKA episode, prolonged insulin independence, male predominance, and an increased frequency of the protective allele DQB1*0602 (4). The unique clinical features and natural histories of these two subtypes of A−β+ KPD patients suggest distinctive underlying pathophysiologic processes for each. Although an underlying “occult” autoimmune element is suggested in the provoked A−β+ subtype by progressive β-cell loss and the presence of type 1 diabetes–associated HLA susceptibility alleles, the unprovoked A−β+ subtype could represent a truly nonautoimmune syndrome of KPD.We have previously shown that the T cells of a significant proportion of individuals with an apparent phenotype of type 2 diabetes react strongly to islet antigens, despite lacking β-cell autoantibodies, and that this reactivity is associated with low C-peptide levels, indicating underlying cellular immune damage to β-cells (5,6). This finding expands the range of diabetic phenotypes—including those labeled as having “type 2” diabetes—with a potential pathophysiologic basis in islet autoimmunity. In the current study, we extended these findings to the unique, emerging forms of A− KPD. Specifically, we hypothesized that differences in cellular immune responses might distinguish the three A− KPD subtypes (A−β−, unprovoked A−β+, and provoked A−β+) with regard to a cellular autoimmune etiology. To test this hypothesis, we measured islet-specific T-cell responses using the validated cellular immunoblotting assay and islet autoantibody responses to determine the presence of islet autoimmunity in patients carefully phenotyped for these three KPD subtypes. We further assessed the percentage of proinflammatory (CD14+CD16+) monocytes in the peripheral blood of the three KPD subtypes.In healthy subjects, 90–95% of classical monocytes express high levels of the cell surface marker CD14 (CD14+), without expression of CD16 (CD16−). Inflammation and infection are associated with the emergence of a distinct monocyte population characterized by coexpression of CD14 and CD16 (CD14+CD16+). CD14+CD16+ monocytes secrete high levels of proinflammatory tumor necrosis factor-α and low levels of anti-inflammatory interleukin-10, leading to their designation as proinflammatory monocytes (7).Our results demonstrate that 1) provoked A−β+ KPD, associated with progressive loss of β-cell function, is associated with a high frequency of cellular islet autoimmunity and proinflammatory monocyte populations; 2) unprovoked A− KPD is a distinct syndrome of reversible β-cell dysfunction lacking evidence of humoral or cellular islet autoimmunity; and 3) a substantial proportion of A−β− KPD patients, who resemble patients with type 1 diabetes but lack autoantibodies, have evidence of cellular islet autoimmunity.     

Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of Aβ amyloidosis

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for Alzheimer’s disease (AD). The influence of apoE on amyloid β (Aβ) accumulation may be the major mechanism by which apoE affects AD. ApoE interacts with Aβ and facilitates Aβ fibrillogenesis in vitro. In addition, apoE is one of the protein components in plaques. We hypothesized that certain anti-apoE antibodies, similar to certain anti-Aβ antibodies, may have antiamyloidogenic effects by binding to apoE in the plaques and activating microglia-mediated amyloid clearance. To test this hypothesis, we developed several monoclonal anti-apoE antibodies. Among them, we administered HJ6.3 antibody intraperitoneally to 4-mo-old male APPswe/PS1ΔE9 mice weekly for 14 wk. HJ6.3 dramatically decreased amyloid deposition by 60–80% and significantly reduced insoluble Aβ40 and Aβ42 levels. Short-term treatment with HJ6.3 resulted in strong changes in microglial responses around Aβ plaques. Collectively, these results suggest that anti-apoE immunization may represent a novel AD therapeutic strategy and that other proteins involved in Aβ binding and aggregation might also be a target for immunotherapy. Our data also have important broader implications for other amyloidosis. Immunotherapy to proteins tightly associated with misfolded proteins might open up a new treatment option for many protein misfolding diseases.Accumulation of amyloid β (Aβ) peptide in the brain is hypothesized to initiate pathogenic cascades that lead to Alzheimer’s disease (AD; Golde et al., 2011; Holtzman et al., 2011). Therapeutic strategies to inhibit Aβ accumulation, such as Aβ immunotherapy, are currently being tested as disease-modifying therapies (Golde et al., 2011; Holtzman et al., 2011). Sequential proteolytic cleavages of Aβ precursor protein (APP) by β- and γ-secretase produce the Aβ peptide. Although rare, early-onset AD mutations in the APP, presenilin 1 (PS1), and PS2 genes have supported the critical role of Aβ in AD pathogenesis, the ε4 allele of apolipoprotein E (APOE) gene is the strongest genetic risk factor for the common late-onset form of AD, and the ε2 allele is protective (Kim et al., 2009a). Among several mechanisms proposed to explain the effects of apoE on AD pathogenesis, prevailing evidence suggests that apoE’s effect on Aβ accumulation may be the major mechanism (Kim et al., 2009a). ApoE appears to affect Aβ accumulation by modulating both Aβ aggregation and clearance (Ma et al., 1994; Wisniewski et al., 1994; Castano et al., 1995; Sadowski et al., 2006; Deane et al., 2008; Jiang et al., 2008; Castellano et al., 2011). Along with other amyloid-associated proteins, apoE is found in amyloid plaques (Namba et al., 1991; Wisniewski and Frangione, 1992). In this study, we hypothesized that after peripheral administration of monoclonal anti-apoE antibodies, the small fraction of anti-apoE antibodies that enter the brain would bind to apoE in plaques and decrease Aβ accumulation by activating microglia-mediated clearance of Aβ aggregates, similar to what has been described for certain anti-Aβ antibodies (Bard et al., 2000; Brody and Holtzman, 2008).     

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.     

Potential role of orexin and sleep modulation in the pathogenesis of Alzheimer’s disease

Age-related aggregation of amyloid-β (Aβ) is an upstream pathological event in Alzheimer’s disease (AD) pathogenesis, and it disrupts the sleep–wake cycle. The amount of sleep declines with aging and to a greater extent in AD. Poor sleep quality and insufficient amounts of sleep have been noted in humans with preclinical evidence of AD. However, how the amount and quality of sleep affects Aβ aggregation is not yet well understood. Orexins (hypocretins) initiate and maintain wakefulness, and loss of orexin-producing neurons causes narcolepsy. We tried to determine whether orexin release or secondary changes in sleep via orexin modulation affect Aβ pathology. Amyloid precursor protein (APP)/Presenilin 1 (PS1) transgenic mice, in which the orexin gene is knocked out, showed a marked decrease in the amount of Aβ pathology in the brain with an increase in sleep time. Focal overexpression of orexin in the hippocampus in APP/PS1 mice did not alter the total amount of sleep/wakefulness and the amount of Aβ pathology. In contrast, sleep deprivation or increasing wakefulness by rescue of orexinergic neurons in APP/PS1 mice lacking orexin increased the amount of Aβ pathology in the brain. Collectively, modulation of orexin and its effects on sleep appear to modulate Aβ pathology in the brain.Age-related aggregation of amyloid-β (Aβ) is an upstream pathological event in Alzheimer’s disease (AD) pathogenesis (Holtzman et al., 2011; Sperling et al., 2011). As the accumulation and aggregation of Aβ in the brain is known to develop ∼10–15 yr before the initial symptoms of AD, understanding the factors that lead to Aβ aggregation are likely to be important in delaying the onset of this pathology and delaying/preventing AD (Holtzman et al., 2011; Sperling et al., 2011). Diverse lines of in vitro and in vivo studies have shown that synaptic activity and specifically synaptic vesicle release is coupled with presynaptic Aβ release (Kamenetz et al., 2003; Cirrito et al., 2008; Bero et al., 2011; Roh et al., 2012). In addition, sleep plays a role in the regulation of synaptic weight in the brain such that sleep appears to downscale the slow wave activity of the brain caused by accumulated load of synaptic potentiation during wakefulness (Vyazovskiy et al., 2009). Poor sleep quality and insufficient amounts of sleep have been noted in humans with preclinical evidence of AD (Roh et al., 2012; Ju and Holtzman, 2013; Ju et al., 2014). These observations suggest that understanding how integrated synaptic and network activity as measured by the sleep–wake cycle regulates Aβ may provide novel insights into AD pathogenesis. However, how the amount and quality of sleep affect Aβ aggregation is not yet well understood (Ju et al., 2014). Orexins (hypocretins) initiate and maintain wakefulness, and loss of orexin-producing neurons causes narcolepsy (de Lecea et al., 1998; Chemelli et al., 1999).Levels of soluble Aβ in the extracellular interstitial fluid (ISF) of the hippocampus in mice are dynamically and positively associated with minutes awake per hour and negatively associated with time asleep (Kang et al., 2009). In addition, the sleep–wake cycle also affects the Aβ pathology in the brain. A study in mice showed that intracerebral administration of orexin can acutely increase both wakefulness and Aβ levels and systemic treatment with an orexin receptor antagonist decreased Aβ deposition in amyloid precursor protein (APP) transgenic mouse models (Kang et al., 2009). Pharmacological experiments suggest that orexin and the sleep–wake cycle appear to be related to regulation of Aβ levels. We sought to determine for the first time whether genetic manipulation of orexin has similar effects as pharmacological manipulation and, importantly, whether orexin is influencing Aβ levels and Aβ pathology directly via orexin signaling or indirectly via its effects on the sleep–wake cycle.     

In vitro and in silico determination of glutaminyl cyclase inhibitors

Alzheimer''s disease (AD) is the most common form of neurodegenerative disease currently. It is widely accepted that AD is characterized by the self-assembly of amyloid beta (Aβ) peptides. The human glutaminyl cyclase (hQC) enzyme is characterized by association with Aβ peptide generation. The development of hQC inhibitors could prevent the self-aggregation of Aβ peptides, resulting in impeding AD. Utilizing structural knowledge of the hQC substrates and known hQC inhibitors, new heterocyclic and peptidomimetic derivatives were synthesized and were able to inhibit the hQC enzyme. The inhibiting abilities of these compounds were evaluated using a fluorometric assay. The binding mechanism at the atomic level was estimated using molecular docking, free energy perturbation, and quantum chemical calculation methods. The predicted log(BBB) and human intestinal absorption values indicated that these compounds are able to permeate the blood–brain barrier and be well-absorbed through the gastrointestinal tract. Overall, 5,6-dimethoxy-N-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1H-benzo[d]imidazol-2-amine (1_2) was indicated as a potential drug for AD treatment.

Rational design of new hQC inhibitors.     

Fullerenemalonates inhibit amyloid beta aggregation,in vitro and in silico evaluation

The onset of Alzheimer''s disease (AD) is associated with the presence of neurofibrillary pathology such as amyloid β (Aβ) plaques. Different therapeutic strategies have focused on the inhibition of Aβ aggregate formation; these pathological structures lead to neuronal disorder and cognitive impairment. Fullerene C₆₀ has demonstrated the ability to interact and prevent Aβ fibril development; however, its low solubility and toxicity to cells remain significant problems. In this study, we synthesized, characterized and compared diethyl fullerenemalonates and the corresponding sodium salts, adducts of C₆₀ bearing 1 to 3 diethyl malonyl and disodium malonyl substituents to evaluate the potential inhibitory effect on the aggregation of Aβ₄₂ and their biocompatibility. The dose-dependent inhibitory effect of fullerenes on Aβ₄₂ aggregation was studied using a thioflavin T fluorescent assay, and the IC₅₀ value demonstrated a low range of fullerene concentration for inhibition, as confirmed by electron microscopy. The exposure of neuroblastoma to fullerenemalonates showed low toxicity, primarily in the presence of the sodium salt-adducts. An isomeric mixture of bisadducts, trisadducts and a C₃-symetrical trisadduct demonstrated the highest efficacy among the tests. In silico calculations were performed to complement the experimental data, obtaining a deeper understanding of the Aβ inhibitory mechanism; indicating that C₃-symetrical trisadduct interacts mainly with 1D to 16K residues of Aβ₄₂ peptide. These data suggest that fullerenemalonates require specific substituents designed as sodium salt molecules to inhibit Aβ fibrillization and perform with low toxicity. These are promising molecules for developing future therapies involving Aβ aggregates in diseases such as AD and other types of dementia.

Synthesis of new non toxic nanomaterials, with high anti-amyloid fibrils formation effect, in vitro and in silico.     

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.     

Neuroprotection by cyclodextrin in cell and mouse models of Alzheimer disease

There is extensive evidence that cholesterol and membrane lipids play a key role in Alzheimer disease (AD) pathogenesis. Cyclodextrins (CD) are cyclic oligosaccharide compounds widely used to bind cholesterol. Because CD exerts significant beneficial effects in Niemann-Pick type C disease, which shares neuropathological features with AD, we examined the effects of hydroxypropyl-β-CD (HP-β-CD) in cell and mouse models of AD. Cell membrane cholesterol accumulation was detected in N2a cells overexpressing Swedish mutant APP (SwN2a), and the level of membrane cholesterol was reduced by HP-β-CD treatment. HP-β-CD dramatically lowered the levels of Aβ42 in SwN2a cells, and the effects were persistent for 24 h after withdrawal. 4 mo of subcutaneous HP-β-CD administration significantly improved spatial learning and memory deficits in Tg19959 mice, diminished Aβ plaque deposition, and reduced tau immunoreactive dystrophic neurites. HP-β-CD lowered levels of Aβ42 in part by reducing β cleavage of the APP protein, and it also up-regulated the expression of genes involved in cholesterol transport and Aβ clearance. This is the first study to show neuroprotective effects of HP-β-CD in a transgenic mouse model of AD, both by reducing Aβ production and enhancing clearance mechanisms, which suggests a novel therapeutic strategy for AD.There is extensive evidence implicating cholesterol in the pathogenesis of AD. Epidemiological studies have shown that hypercholesterolemia is an important risk factor for AD (Kivipelto et al., 2001; Puglielli et al., 2003). Furthermore, higher cholesterol levels are observed in the brains of AD patients (Xiong et al., 2008). Cholesterol is a major component of the lipid bilayer membrane, where β- and γ-secretases are located. β-C-terminal fragments (β-CTFs), which are the products of β cleavage of the amyloid precursor protein (APP), directly bind cholesterol (Barrett et al., 2012). Increased cellular cholesterol increases the association of APP with β- and γ-secretases in cholesterol-rich lipid rafts, which stimulates cleavage of APP into Aβ (Lee et al., 1998; Ehehalt et al., 2003; Xiong et al., 2008; Kodam et al., 2010; Vetrivel and Thinakaran, 2010).Cholesterol homeostasis is tightly regulated by feedback mechanisms: sterol regulatory element binding proteins (SREBPs) and liver X receptors (LXRs) regulate the expression of genes that control the uptake, synthesis, and export of cholesterol. SREBPs regulate the expression of genes encoding HMG-CoA reductase and HMG-CoA synthase, which play essential roles in cholesterol synthesis (Brown and Goldstein, 1997; Bełtowski, 2008; Sato, 2010). LXRs activate genes encoding the ATP-binding cassette subfamily A/G transporters (ABCA/Gs), which control cholesterol efflux across the plasma membrane to apolipoprotein acceptors, initiating the reverse cholesterol transport pathway (Schmitz and Drobnik, 2002; Jessup et al., 2006). Studies in mouse models of AD indicate that ABCA1 plays an important role in the induction of ApoE lipidation, in facilitating Aβ transport to ApoE-containing carriers for clearance, and in suppression of Aβ generation. Disruption of ABCA1 leads to increased insoluble Aβ and parenchymal amyloid plaques, with no change in APP processing (Hirsch-Reinshagen et al., 2005; Koldamova et al., 2005a; Wahrle et al., 2005; Kim et al., 2007; Cramer et al., 2012). Activation of LXRs produces behavioral improvement and removal of fibrillar Aβ in transgenic mouse models expressing mutant APP (Koldamova et al., 2005b; Terwel et al., 2011).Cyclodextrins (CDs) are a family of cyclic polysaccharide compounds that can extract cholesterol from cultured cells (Zidovetzki and Levitan, 2007; Coskun and Simons, 2010; Di Paolo and Kim, 2011). In transgenic mouse models of the lipid storage disease Niemann-Pick type C1 (NPC1), subcutaneous administration of CDs improved cholesterol trafficking, corrected abnormal cholesterol metabolism, and prevented neurodegenerative changes in NPC1^−/− mice (Liu et al., 2008, 2010; Zhang et al., 2009; Ramirez et al., 2010; Aqul et al., 2011). Notably, intriguing parallels exist between NPC1 and AD, including neurofibrillary tangles and prominent lysosomal system dysfunction (Nixon, 2004; Liu et al., 2010). A recent study showed that CDs reversed attenuated behavioral and pathological abnormalities in a mouse model generated by crossing NPC1^+/− mice with mutant APP transgenic mice (Maulik et al., 2012). It is therefore plausible that CDs may also benefit AD (Aqul et al., 2011). In vitro studies showed that CDs reduce membrane cholesterol levels and reduce both BACE and γ-secretase activities (Hartmann et al., 2007), leading to additive effects in reducing amyloid-β production (Simons et al., 1998).In this study, we examined the hydroxypropyl form of β-CD (HP-β-CD) in Tg19959 mice, which overexpress human APP with the Swedish and Indiana familial AD mutations. These mice robustly develop Aβ pathology and memory deficits at 3–4 mo of age (Yao et al., 2010). We injected the mice subcutaneously with HP-β-CD starting at 7 d of age, twice weekly until 4 mo of age. Treatment with HP-β-CD significantly reduced Aβ burden and phosphorylated tau pathology, and improved memory in the Morris water maze. HP-β-CD activated expression of genes, including ABCA1, suggesting that HP-β-CD increases cholesterol transport and enhances Aβ clearance.     

Stewartiacids A–N,C-23 carboxylated triterpenoids from Chinese Stewartia and their inhibitory effects against ATP-citrate lyase and NF-κB

Fourteen previously undescribed naturally occurring C-23 carboxylated triterpenoids, stewartiacids A–N (1–14), were isolated and characterized from the twigs and leaves of the ornamental and medicinal plant Stewartia sinensis (Chinese Stewartia), a ‘vulnerable’ species endemic to China. The new structures were elucidated on the basis of spectroscopic data, single crystal X-ray diffraction, and electronic circular dichroism (ECD) analyses. Stewartiacids A (1) and B (2) are isoursenol derivatives. Stewartiacid C (3) is a 12-oxo-γ-amyrin analogue. Both isoursenol and γ-amyrin derivatives are quite rare in nature. Stewartiacids D (4) and E (5) are 13,27-cycloursane-type compounds. Stewartiacids K (11) and L (12) are ursane-type triterpene and phenylpropanol adducts built through a 1,4-dioxane ring, which are also seldom reported in the literature. The rest are common C-23 carboxylated ursane-type (6–10) and oleanane-type (13, 14) pentacyclic triterpenoids. Stewartiacids G (7), K (11), and L (12) showed moderate inhibitory effects against ATP-citrate lyase (ACL), with IC₅₀ values of 12.5, 2.8, and 10.6 μM, respectively. Stewartiacid K (11) also exhibited moderate inhibition (IC₅₀: 16.8 μM) of NF-κB.

Fourteen new C-23 carboxylated triterpenoids (stewartiacids A–N, 1–14) were obtained from the ‘vulnerable’ plant Stewartia sinensis. 11 and 12 showed inhibitory effects against ACL.     

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     

Effect of C-terminus amidation of Aβ39–42 fragment derived peptides as potential inhibitors of Aβ aggregation

The C-terminus fragment (Val-Val-Ile-Ala) of amyloid-β is reported to inhibit the aggregation of the parent peptide. In an attempt to investigate the effect of sequential amino-acid scan and C-terminus amidation on the biological profile of the lead sequence, a series of tetrapeptides were synthesized using MW-SPPS. Peptide D-Phe-Val-Ile-Ala-NH₂ (12c) exhibited high protection against β-amyloid-mediated-neurotoxicity by inhibiting Aβ aggregation in the MTT cell viability and ThT-fluorescence assay. Circular dichroism studies illustrate the inability of Aβ₄₂ to form β-sheet in the presence of 12c, further confirmed by the absence of Aβ₄₂ fibrils in electron microscopy experiments. The peptide exhibits enhanced BBB permeation, no cytotoxicity along with prolonged proteolytic stability. In silico studies show that the peptide interacts with the key amino acids in Aβ, which potentiate its fibrillation, thereby arresting aggregation propensity. This structural class of designed scaffolds provides impetus towards the rational development of peptide-based-therapeutics for Alzheimer''s disease (AD).

Amidated C-terminal fragment, Aβ_39–42 derived non-cytotoxic β-sheet breaker peptides exhibit excellent potency, enhanced bioavailability and improved proteolytic stability.

First reported by Alois Alzheimer in 1906, Alzheimer''s Disease (AD) is a progressive, neurodegenerative disorder with an irreversible decline in memory and cognition.¹ It is commonly seen in elderly populations and is marked by two major histopathological hallmarks, amyloid-β (Aβ) plaques and neurofibrillary tangles (NFT).² The number of patients suffering from AD has been increasing at an alarming rate. It is estimated that around 60 million patients will be suffering from AD by the end of 2020 and half of this patient population would require the care equivalent to that of a nursing home.³ Even after a century of its discovery, there has been no treatment that targets the pathophysiology of AD.⁴ The current treatment regimen includes acetylcholine-esterase inhibitors (AChEI) comprising of donepezil, rivastigmine and galantamine as well as N-methyl-d-aspartate (NMDA)-receptor antagonist, memantine. These provide only symptomatic relief to the patient. Most of the therapeutics that are currently being tested in clinical trials couldn''t proceed beyond phase II and phase III clinical trials due to lower efficacy in elderly patients, multiple side effects and limitations in their pharmacokinetic and pharmacodynamic profiles.^5–9 This has created challenges on the societal and economic upfront.Literature analysis reveals that the soluble oligomeric Aβ species is the culprit for neurotoxicity. It interrupts normal physiological functioning of the human brain. The central hydrophobic fragment Aβ_16–22 (KLVFFAE) and the C-terminus region fragment Aβ_31–42 (IIGLMVGGVVIA) are responsible for controlling the aggregation kinetics of the monomeric species, wherein the later still remains relatively less explored.^8,9 Our group is focused on development of peptidomimetic analogues for preventing Aβ aggregation. Studies on a complete peptide scan on the C-terminus region regions has already been published previously.^10–12 In an attempt to enhance the biological efficacy of the previously designed scaffolds,¹² we rationalized the use of sequential amino acid scan by modifying/replacing individual residues, as well as amide protection of the C-terminus on the lead tetrapeptide sequence (Val-Val-Ile-Ala) to enhance the proteolytic stability of the peptides.C-terminus amidated peptides were synthesized by microwave-assisted Fmoc-solid phase peptide synthesis protocol. Scheme 1 shows the general route for the synthesis of peptides employing Rink amide resin (detailed methodology is mentioned in the ESI, Section 1^†). These peptides were characterized using by analytical HPLC, ¹H and ¹³C NMR, APCI/ESI-MS and HRMS.Open in a separate windowScheme 1Synthesis of tetrapeptide 12c using MW-assisted Fmoc-solid phase peptide synthesis protocol employing Rink amide resin. Reaction conditions: (i) 20% piperidine in DMF (7 mL), MW (40 W), 60 °C, 2 cycles – 1.5 & 3 min; (ii) 2, 4, 6, 8 (4 equiv.), TBTU (4 equiv.), HoBt (4 equiv.), DIPEA (5 equiv.), DMF (3.5 mL), MW (40 W), 60 °C, 13.5 min; (iii) TFA : TIPS : H₂O (95 : 2.5 : 2.5), rt, 2.5 h; reaction monitoring was done by: UV measurement: Fmoc deprotection-dibenzofulvene adduct, Kaiser test: 1° amines; acetaldehyde test: 2° amines.Aggregation of Aβ₄₂ results in accumulation of toxic species, which interact with neurons and hinder their functioning, leading to loss of memory and cognition. Inhibiting the process of Aβ₄₂ aggregation would alleviate the neurotoxicity imparted in PC-12 cells; this was evaluated by MTT cell viability assay.¹³ Viability of untreated cells is considered 100%. Upon treatment with 2 μM of Aβ₄₂, only 74% cells were found to be viable. Out of the total tested peptides, seven tetrapeptides 12a, 12c, 12f, 13e, 14b, 15b and 15f showed complete inhibition of Aβ₄₂-induced toxicity by restoring the cell viability to 100%, at respective concentrations. Results for cell viability assay along with Thioflavin-T fluorescence assay for all the synthesized tetrapeptides has been summarized in