Discovery of an Orally Bioavailable Benzofuran Analogue That Serves as a ß-Amyloid Aggregation Inhibitor for the Potential Treatment of Alzheimer's Disease.

We developed an orally active and blood-brain-barrier-permeable benzofuran analogue (8, MDR-1339) with potent antiaggregation activity. Compound 8 restored cellular viability from Aß-induced cytotoxicity but also improved the learning and memory function of AD model mice by reducing the Aß aggregates in the brains. Given the high bioavailability and brain permeability demonstrated in our pharmacokinetic studies, 8 will provide a novel scaffold for an Aß-aggregation inhibitor that may offer an alternative treatment for AD.

Discovery of Potent Human Glutaminyl Cyclase Inhibitors as Anti-Alzheimer's Agents Based on Rational Design.

Glutaminyl cyclase (QC) has been implicated in the formation of toxic amyloid plaques by generating the N-terminal pyroglutamate of ß-amyloid peptides (pGlu-Aß) and thus may participate in the pathogenesis of Alzheimer's disease (AD). We designed a library of glutamyl cyclase (QC) inhibitors based on the proposed binding mode of the preferred substrate, Aß3E-42. An in vitro structure-activity relationship study identified several excellent QC inhibitors demonstrating 5- to 40-fold increases in potency compared to a known QC inhibitor. When tested in mouse models of AD, compound 212 significantly reduced the brain concentrations of pyroform Aß and total Aß and restored cognitive functions. This potent Aß-lowering effect was achieved by incorporating an additional binding region into our previously established pharmacophoric model, resulting in strong interactions with the carboxylate group of Glu327 in the QC binding site. Our study offers useful insights in designing novel QC inhibitors as a potential treatment option for AD.

PiB-PET Imaging-Based Serum Proteome Profiles Predict Mild Cognitive Impairment and Alzheimer's Disease.

Development of a simple, non-invasive early diagnosis platform of Alzheimer's disease (AD) using blood is urgently required. Recently, PiB-PET imaging has been shown to be powerful to quantify amyloid-ß plaque loads leading to pathophysiological alterations in AD brains. Thus, there has been a need for serum biomarkers reflecting PiB-PET imaging data as an early diagnosis platform of AD. Here, using LC-MS/MS analysis coupled with isobaric tagging, we performed comprehensive proteome profiling of serum samples from cognitively normal controls, mild cognitive impairment (MCI), and AD patients, who were selected using PiB-PET imaging. Comparative analysis of the proteomes revealed 79 and 72 differentially expressed proteins in MCI and AD, respectively, compared to controls. Integrated analysis of these proteins with genomic and proteomic data of AD brain tissues, together with network analysis, identified three biomarker candidates representing the altered proteolysis-related process in MCI or AD: proprotein convertase subtilisin/kexin type 9 (PCSK9), coagulation factor XIII, A1 polypeptide (F13A1), and dermcidin (DCD). In independent serum samples of MCI and AD, we confirmed the elevation of the candidates using western blotting and ELISA. Our results suggest that these biomarker candidates can serve as a potential non-invasive early diagnosis platform reflecting PiB-PET imaging for MCI and AD.

The novel RAGE interactor PRAK is associated with autophagy signaling in Alzheimer's disease pathogenesis.

BACKGROUND: The receptor for advanced glycation end products (RAGE) has been found to interact with amyloid ß (Aß). Although RAGE does not have any kinase motifs in its cytosolic domain, the interaction between RAGE and Aß triggers multiple cellular signaling involved in Alzheimer's disease (AD). However, the mechanism of signal transduction by RAGE remains still unknown. Therefore, identifying binding proteins of RAGE may provide novel therapeutic targets for AD. RESULTS: In this study, we identified p38-regulated/activated protein kinase (PRAK) as a novel RAGE interacting molecule. To investigate the effect of Aß on PRAK mediated RAGE signaling pathway, we treated SH-SY5Y cells with monomeric form of Aß. We demonstrated that Aß significantly increased the phosphorylation of PRAK as well as the interaction between PRAK and RAGE. We showed that knockdown of PRAK rescued mTORC1 inactivation induced by Aß treatment and decreased the formation of Aß-induced autophagosome. CONCLUSIONS: We provide evidence that PRAK plays a critical role in AD pathology as a key interactor of RAGE. Thus, our data suggest that PRAK might be a potential therapeutic target of AD involved in RAGE-mediated cell signaling induced by Aß.

3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis.

Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.

Identification of disulfide cross-linked tau dimer responsible for tau propagation.

Recent evidence suggests that tau aggregates are not only neurotoxic, but also propagate in neurons acting as a seed for native tau aggregation. Prion-like tau transmission is now considered as an important pathogenic mechanism driving the progression of tau pathology in the brain. However, prion-like tau species have not been clearly characterized. To identify infectious tau conformers, here we prepared diverse tau aggregates and evaluated the effect on inducing intracellular tau-aggregation. Among tested, tau dimer containing P301L-mutation is identified as the most infectious form to induce tau pathology. Biochemical analysis reveals that P301L-tau dimer is covalently cross-linked with a disulfide bond. The relatively small and covalently cross-linked tau dimer induced tau pathology efficiently in primary neurons and also in tau-transgenic mice. So far, the importance of tau disulfide cross-linking has been overlooked in the study of tau pathology. Here our results suggested that tau disulfide cross-linking might play critical role in tau propagation by producing structurally stable and small tau conformers.

Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease.

Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aß-treated cells. Indeed, Aß bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aß-induced impairment in ATP production and ATPase activity. These results indicate that Aß-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aß and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A.

Tomoregulin (TMEFF2) Binds Alzheimer's Disease Amyloid-ß (Aß) Oligomer and AßPP and Protects Neurons from Aß-Induced Toxicity.

Amyloid-ß (Aß) protein causes neurotoxicity and its abnormal aggregation into amyloid is a pathological hallmark of Alzheimer's disease (AD). Cellular proteins able to interact with Aß or its precursor, AßPP (amyloid-ß protein precursor), may regulate Aß production and neurotoxicity. We identified a brain-enriched type I transmembrane protein, tomoregulin (TR), that directly binds Aß and Aß oligomers (AßO). TR co-immunoprecipitated with Aß and AßO in cultured cells and co-localized with amyloid plaques and intraneuronal Aß in the 5xFAD AD mouse model. TR was also enriched in astrocytic processes reactive to amyloid plaques. Surface plasmon resonance spectroscopy studies showed that the extracellular domain of TR binds to AßO with a high affinity (KD = 76.8 nM). Electron paramagnetic resonance spectroscopy also demonstrated a physical interaction between spin-labeled Aß and the TR extracellular domain in solution. Furthermore, TR also interacted with AßPP and enhanced its cleavage by α-secretase. Both cellular expression of TR and application of recombinant TR extracellular domain protected N2a neurons from AßO-induced neuronal death. These data provide first evidence that neuronal and astrocytic expression of TR is intimately related to Aß metabolism and toxicity, and could be neuroprotective through its direct interaction with Aß and AßPP.

6-Phenoxy-2-phenylbenzoxazoles, novel inhibitors of receptor for advanced glycation end products (RAGE).

Receptor for advanced glycation end products (RAGE) is known to be involved in the transportation of amyloid ß (Aß) peptides and causes the accumulation of Aß in the brain. Moreover, recent studies suggest that the interactions between RAGE and Aß peptides may be the culprit behind Alzheimer's disease (AD). Inhibitors of the RAGE-Aß interactions would not only prevent the accumulation of toxic Aß in the brain, and but also block the progress of AD, therefore, have the potential to provide a 'disease-modifying therapy'. In this study, we have developed a series of 6-phenoxy-2-phenylbenzoxazole analogs as novel inhibitors of RAGE. Among these derivatives, we found several effective inhibitors that block the RAGE-Aß interactions without causing significant cellular toxicity. Further testing showed that compound 48 suppressed Aß induced toxicity in mouse hippocampal neuronal cells and reduced Aß levels in the brains of a transgenic mouse model of AD after oral administration.

Synthesis and Evaluation of a Novel Deguelin Derivative, L80, which Disrupts ATP Binding to the C-terminal Domain of Heat Shock Protein 90.

The clinical benefit of current anticancer regimens for lung cancer therapy is still limited due to moderate efficacy, drug resistance, and recurrence. Therefore, the development of effective anticancer drugs for first-line therapy and for optimal second-line treatment is necessary. Because the 90-kDa molecular chaperone heat shock protein (Hsp90) contributes to the maturation of numerous mutated or overexpressed oncogenic proteins, targeting Hsp90 may offer an effective anticancer therapy. Here, we investigated antitumor activities and toxicity of a novel deguelin-derived C-terminal Hsp90 inhibitor, designated L80. L80 displayed significant inhibitory effects on the viability, colony formation, angiogenesis-stimulating activity, migration, and invasion of a panel of non-small cell lung cancer cell lines and their sublines with acquired resistance to paclitaxel with minimal toxicity to normal lung epithelial cells, hippocampal cells, vascular endothelial cells, and ocular cells. Biochemical analyses and molecular docking simulation revealed that L80 disrupted Hsp90 function by binding to the C-terminal ATP-binding pocket of Hsp90, leading to the disruption of the interaction between hypoxia-inducible factor (HIF)-1α and Hsp90, downregulation of HIF-1α and its target genes, including vascular endothelial growth factor (VEGF) and insulin-like growth factor 2 (IGF2), and decreased the expression of various Hsp90 client proteins. Consistent with these in vitro findings, L80 exhibited significant antitumor and antiangiogenic activities in H1299 xenograft tumors. These results suggest that L80 represents a novel C-terminal Hsp90 inhibitor with effective anticancer activities with minimal toxicities.

Two ß-strands of RAGE participate in the recognition and transport of amyloid-ß peptide across the blood brain barrier.

Amyloid-ß (Aß) peptide is central to the development of brain pathology in Alzheimer disease (AD) patients. Association with receptors for advanced glycation end-products (RAGE) enables the transport of Aß peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two ß-strands of RAGE participate in the interaction with Aß peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth ß-strands are required for interaction with Aß peptide. Site-directed mutagenesis of amino acids located in the third and eighth ß-strands abolish the interaction of RAGE with Aß peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aß peptide treatment, while a RAGE mutant defective in Aß binding does not. Furthermore, use of peptide for the third ß-strand or a RAGE monoclonal antibody that targets the RAGE-Aß interaction interface inhibited transport of the Aß peptide across the blood brain barrier in a mice model. These results provide information crucial to the development of RAGE-derived therapeutic reagents for Alzheimer disease.

O-linked ß-N-acetylglucosaminidase inhibitor attenuates ß-amyloid plaque and rescues memory impairment.

Deposition of ß-amyloid (Aß) as senile plaques and disrupted glucose metabolism are two main characteristics of Alzheimer's disease (AD). It is unknown, however, how these two processes are related in AD. Here we examined the relationship between O-GlcNAcylation, which is a glucose level-dependent post-translational modification that adds O-linked ß-N-acetylglucosamine (O-GlcNAc) to proteins, and Aß production in a mouse model of AD carrying 5XFAD genes. We found that 1,2-dideoxy-2'-propyl-α-d-glucopyranoso-[2,1-D]-Δ2'-thiazoline (NButGT), a specific inhibitor of O-GlcNAcase, reduces Aß production by lowering γ-secretase activity both in vitro and in vivo. We also found that O-GlcNAcylation takes place at the S708 residue of nicastrin, which is a component of γ-secretase. Moreover, NButGT attenuated the accumulation of Aß, neuroinflammation, and memory impairment in the 5XFAD mice. This is the first study to show the relationship between Aß generation and O-GlcNAcylation in vivo. These results suggest that O-GlcNAcylation may be a suitable therapeutic target for the treatment of AD.

Ligand-based design, synthesis, and biological evaluation of 2-aminopyrimidines, a novel series of receptor for advanced glycation end products (RAGE) inhibitors.

Using the approach of ligand-based drug design, we discovered a novel series of 4,6-disubstituted 2-aminopyrimidines as RAGE inhibitors. In transgenic mouse models of AD, one of the 4,6-bis(4-chlorophenyl)pyrimidine analogs, 59, significantly lowered the concentration of toxic soluble Aß in the brain and improved cognitive function. SPR analysis confirmed the direct binding of 59 with RAGE, which should contribute to its biological activities via inhibition of the RAGE-Aß interaction. We also predicted the binding mode of the 4,6-bis(4-chlorophenyl)pyrimidine analogs to the RAGE V-domain through flexible docking study.

Aß1₋42-RAGE interaction disrupts tight junctions of the blood-brain barrier via Ca²âº-calcineurin signaling.

The blood-brain barrier (BBB), which is formed by adherens and tight junctions (TJs) of endothelial cells, maintains homeostasis of the brain. Disrupted intracellular Ca²âº homeostasis and breakdown of the BBB have been implicated in the pathogenesis of Alzheimer's disease (AD). The receptor for advanced glycation end products (RAGE) is known to interact with amyloid ß-peptide (Aß) and mediate Aß transport across the BBB, contributing to the deposition of Aß in the brain. However, molecular mechanisms underlying Aß-RAGE interaction-induced alterations in the BBB have not been identified. We found that Aß1₋42 induces enhanced permeability, disruption of zonula occludin-1 (ZO-1) expression in the plasma membrane, and increased intracellular calcium and matrix metalloproteinase (MMP) secretion in cultured endothelial cells. Neutralizing antibodies against RAGE and inhibitors of calcineurin and MMPs prevented Aß1₋42-induced changes in ZO-1, suggesting that Aß-RAGE interactions alter TJ proteins through the Ca²âº-calcineurin pathway. Consistent with these in vitro findings, we found disrupted microvessels near Aß plaque-deposited areas, elevated RAGE expression, and enhanced MMP secretion in microvessels of the brains of 5XFAD mice, an animal model for AD. We have identified a potential molecular pathway underlying Aß-RAGE interaction-induced breakage of BBB integrity. This pathway might play an important role in the pathogenesis of AD.

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aß peptide.

BACKGROUND: The deposition and oligomerization of amyloid ß (Aß) peptide plays a key role in the pathogenesis of Alzheimer's disease (AD). Aß peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by ß and γ secretases. Several lines of evidence point to the soluble Aß oligomer (AßO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AßO species. METHODOLOGY/PRINCIPAL FINDINGS: To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aß peptide to investigate the binding and influence of fluorene compounds on AßO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AßO toxicity and a route to directly observe the binding of the fluorene to the AßO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AßO toxicity, scavenge free radicals and diminish the formation of intracellular AßO species. CONCLUSIONS: Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aß peptide toxicity, because they possess both antioxidant properties and the ability to disrupt AßO species.

Impaired short-term plasticity in mossy fiber synapses caused by mitochondrial dysfunction of dentate granule cells is the earliest synaptic deficit in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) in the early stages is characterized by memory impairment, which may be attributable to synaptic dysfunction. Oxidative stress, mitochondrial dysfunction, and Ca²âº dysregulation are key factors in the pathogenesis of AD, but the causal relationship between these factors and synaptic dysfunction is not clearly understood. We found that in the hippocampus of an AD mouse model (Tg2576), mitochondrial Ca²âº handling in dentate granule cells was impaired as early as the second postnatal month, and this Ca²âº dysregulation caused an impairment of post-tetanic potentiation in mossy fiber-CA3 synapses. The alteration of cellular Ca²âº clearance in Tg2576 mice is region-specific within hippocampus because in another region, CA1 pyramidal neuron, no significant difference in Ca²âº clearance was detected between wild-type and Tg2576 mice at this early stage. Impairment of mitochondrial Ca²âº uptake was associated with increased mitochondrial reactive oxygen species and depolarization of mitochondrial membrane potential. Mitochondrial dysfunctions in dentate granule cells and impairment of post-tetanic potentiation in mossy fiber-CA3 synapses were fully restored when brain slices obtained from Tg2576 were pretreated with antioxidant, suggesting that mitochondrial oxidative stress initiates other dysfunctions. Reversibility of early dysfunctions by antioxidants at the preclinical stage of AD highlights the importance of early diagnosis and antioxidant therapy to delay or prevent the disease processes.

Mitochondria-specific accumulation of amyloid ß induces mitochondrial dysfunction leading to apoptotic cell death.

Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid ß (Aß) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aß impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aß accumulation has been detected in the AD brain. However, the underlying mechanism of how Aß affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aß accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aß(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aß(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aß(1-42) in HT22 cells using Aß(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aß(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aß(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aß(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aß(1-42) accumulation, which mimics the apoptosis process in exogenous Aß(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aß(1-42) accumulation is the necessary and sufficient condition for Aß-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aß-mediated signaling alterations.

Astrocyte-originated ATP protects Aß(1-42)-induced impairment of synaptic plasticity.

Activated microglia and reactive astrocytes are commonly found in and around the senile plaque, which is the central pathological hallmark of Alzheimer's disease. Astrocytes respond to neuronal activity through the release of gliotransmitters such as glutamate, D-serine, and ATP. However, it is largely unknown whether and how gliotransmitters affect neuronal functions. In this study, we explored the effect of a gliotransmitter, ATP, on neurons damaged by ß-amyloid peptide (Aß). We found that Aß(1-42) (Aß42) increased the release of ATP in cultures of primary astrocytes and U373 astrocyte cell line. We also found that exogenous ATP protected Aß42-mediated reduction in synaptic molecules, such as NMDA receptor 2A and PSD-95, through P2 purinergic receptors and prevented Aß42-induced spine reduction in cultured primary hippocampal neurons. Moreover, ATP prevented Aß42-induced impairment of long-term potentiation in acute hippocampal slices. Our findings suggest that Aß-induced release of gliotransmitter ATP plays a protective role against Aß42-mediated disruption of synaptic plasticity.

Intracellular amyloid-ß accumulation in calcium-binding protein-deficient neurons leads to amyloid-ß plaque formation in animal model of Alzheimer's disease.

One of the major hallmarks of Alzheimer's disease (AD) is the extracellular deposition of amyloid-ß (Aß) as senile plaques in specific brain regions. Clearly, an understanding of the cellular processes underlying Aß deposition is a crucial issue in the field of AD research. Recent studies have found that accumulation of intraneuronal Aß (iAß) is associated with synaptic deficits, neuronal death, and cognitive dysfunction in AD patients. In this study, we found that Aß deposits had several shapes and sizes, and that iAß occurred before the formation of extracellular amyloid plaques in the subiculum of 5XFAD mice, an animal model of AD. We also observed pyroglutamate-modified Aß (N3pE-Aß), which has been suggested to be a seeding molecule for senile plaques, inside the Aß plaques only after iAß accumulation, which argues against its seeding role. In addition, we found that iAß accumulates in calcium-binding protein (CBP)-free neurons, induces neuronal death, and then develops into senile plaques in 2-4-month-old 5XFAD mice. These findings suggest that N3pE-Aß-independent accumulation of Aß in CBP-free neurons might be an early process that triggers neuronal damage and senile plaque formation in AD patients. Our results provide new insights into several long-standing gaps in AD research, namely how Aß plaques are formed, what happens to iAß and how Aß causes selective neuronal loss in AD patients.

Human serum transthyretin levels correlate inversely with Alzheimer's disease.

Alzheimer's disease (AD) is the fastest growing neurodegenerative disease in the elderly population, and the search for therapeutic targets and diagnostic AD biomarkers is an exigent issue. Because amyloid-ß (Aß) aggregation constitutes the epicenter of AD pathology, Aß-binding proteins that regulate Aß aggregation, such as transthyretin (TTR), have attracted much attention. TTR binds to Aß, prevents its aggregation, and consequently inhibits Aß-induced cellular toxicity. Decreased TTR levels in cerebrospinal fluid (CSF) from AD patients suggest that TTR is a biomarker of AD. But, studies on TTR as a biomarker have focused on CSF; no study has evaluated peripheral levels of TTR in AD. Here, we examined the relationship between serum TTR levels and AD. We measured TTR levels in serum samples from 90 nondemented controls and 111 AD patients and observed significantly lower serum TTR levels in AD (p < 0.001). Notably, females in the control group had lower serum TTR levels compared with male in the control (p = 0.006), while no difference in gender was noted in the AD group. There were no age-related changes in serum TTR levels. Thus, this study demonstrates a clear negative correlation between serum TTR levels and AD, suggesting that TTR is not only involved in AD pathological process but also suggested as possible peripheral biomarker for AD diagnosis in serum level.