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1.  Isotope-edited FTIR reveals distinct aggregation and structural behaviors of unmodified and pyroglutamylated amyloid β peptides 
Amyloid β peptide (Aβ) is causatively associated with Alzheimer’s disease (AD), and N-terminally truncated and pyroglutamylated Aβ peptides (AβpE) exert hypertoxic effect by an unknown mechanism. Recent evidence has identified the prefibrillar oligomers of Aβ, not the fibrils, as the prevalent cytotoxic species. Structural characterization of Aβ and AβpE oligomers is therefore important for better understanding of their toxic effect. Here we have used isotope-edited Fourier transform infrared (FTIR) spectroscopy to identify the conformational changes in Aβ1-42 and AβpE3-42 upon aggregation, individually and in 1:1 molar combination. During the first two hours of exposure to aqueous buffer, the peptides undergo transition from mostly α-helical to mostly β-sheet structure. Data on peptides 13C,15N-labeled at K16L17V18 or V36G37G38V39 allowed construction of structural models for the monomer and early oligomers. The peptide monomer comprises a β-hairpin that involves residues upstream of the K16L17V18 sequence and an N-terminal α-helix. The oligomers form by non-H-bonding interactions between the β-strands of neighboring β-hairpins, in lateral or staggered manner, with the strands running parallel or antiparallel. Relative α-helical and β-sheet propensities of Aβ1-42 and AβpE3-42 depend on the ionic strength of the buffer, emphasizing the importance of ionic interactions in Aβ peptide structure and aggregation. It is inferred that N-terminal modification of AβpE3-42 affects the helix stability and thereby modulates β-sheet oligomer formation. The data thus provide new insight into the molecular mechanism of Aβ oligomerization by emphasizing the role of the N-terminal transient α-helical structure and by identifying structural constraints for molecular organization of the oligomers.
PMCID: PMC4668202  PMID: 26214017
2.  Activity and Architecture of Pyroglutamate-Modified Amyloid-β (AβpE3-42) Pores 
The Journal of Physical Chemistry. B  2014;118(26):7335-7344.
Among the family of Aβ peptides, pyroglutamate-modified Aβ (AβpE) peptides are particularly associated with cytotoxicity in Alzheimer’s disease (AD). They represent the dominant fraction of Aβ oligomers in the brains of AD patients, but their accumulation in the brains of elderly individuals with normal cognition is significantly lower. Accumulation of AβpE plaques precedes the formation of plaques of full-length Aβ (Aβ1-40/42). Most of these properties appear to be associated with the higher hydrophobicity of AβpE as well as an increased resistance to enzymatic degradation. However, the important question of whether AβpE peptides induce pore activity in lipid membranes and their potential toxicity compared with other Aβ pores is still open. Here we examine the activity of AβpE pores in anionic membranes using planar bilayer electrical recording and provide their structures using molecular dynamics simulations. We find that AβpE pores spontaneously induce ionic current across the membrane and have some similar properties to the other previously studied pores of the Aβ family. However, there are also some significant differences. The onset of AβpE3-42 pore activity is generally delayed compared with Aβ1-42 pores. However, once formed, AβpE3-42 pores produce increased ion permeability of the membrane, as indicated by a greater occurrence of higher conductance electrical events. Structurally, the lactam ring of AβpE peptides induces a change in the conformation of the N-terminal strands of the AβpE3-42 pores. While the N-termini of wild-type Aβ1–42 peptides normally reside in the bulk water region, the N-termini of AβpE3-42 peptides tend to reside in the hydrophobic lipid core. These studies provide a first step to an understanding of the enhanced toxicity attributed to AβpE peptides.
PMCID: PMC4096221  PMID: 24922585
3.  N-truncated Abeta starting with position four: early intraneuronal accumulation and rescue of toxicity using NT4X-167, a novel monoclonal antibody 
The amyloid hypothesis in Alzheimer disease (AD) considers amyloid β peptide (Aβ) deposition causative in triggering down-stream events like neurofibrillary tangles, cell loss, vascular damage and memory decline. In the past years N-truncated Aβ peptides especially N-truncated pyroglutamate AβpE3-42 have been extensively studied. Together with full-length Aβ1–42 and Aβ1–40, N-truncated AβpE3-42 and Aβ4–42 are major variants in AD brain. Although Aβ4–42 has been known for a much longer time, there is a lack of studies addressing the question whether AβpE3-42 or Aβ4–42 may precede the other in Alzheimer’s disease pathology.
Using different Aβ antibodies specific for the different N-termini of N-truncated Aβ, we discovered that Aβ4-x preceded AβpE3-x intraneuronal accumulation in a transgenic mouse model for AD prior to plaque formation. The novel Aβ4-x immunoreactive antibody NT4X-167 detected high molecular weight aggregates derived from N-truncated Aβ species. While NT4X-167 significantly rescued Aβ4–42 toxicity in vitro no beneficial effect was observed against Aβ1–42 or AβpE3-42 toxicity. Phenylalanine at position four of Aβ was imperative for antibody binding, because its replacement with alanine or proline completely prevented binding. Although amyloid plaques were observed using NT4X-167 in 5XFAD transgenic mice, it barely reacted with plaques in the brain of sporadic AD patients and familial cases with the Arctic, Swedish and the presenilin-1 PS1Δ9 mutation. A consistent staining was observed in blood vessels in all AD cases with cerebral amyloid angiopathy. There was no cross-reactivity with other aggregates typical for other common neurodegenerative diseases showing that NT4X-167 staining is specific for AD.
Aβ4-x precedes AβpE3-x in the well accepted 5XFAD AD mouse model underlining the significance of N-truncated species in AD pathology. NT4X-167 therefore is the first antibody reacting with Aβ4-x and represents a novel tool in Alzheimer research.
PMCID: PMC3893517  PMID: 24252153
Pyroglutamate Abeta; Abeta oligomer; Toxicity; Arctic; Swedish; Presenilin-1; 5XFAD; Transgenic mouse model; Familial Alzheimer’s disease; Sporadic Alzheimer’s disease; Abeta 4-40; Abeta 4–42
4.  Pyroglutamate Abeta pathology in APP/PS1KI mice, sporadic and familial Alzheimer’s disease cases 
Journal of Neural Transmission  2009;117(1):85-96.
The presence of AβpE3 (N-terminal truncated Aβ starting with pyroglutamate) in Alzheimer’s disease (AD) has received considerable attention since the discovery that this peptide represents a dominant fraction of Aβ peptides in senile plaques of AD brains. This was later confirmed by other reports investigating AD and Down’s syndrome postmortem brain tissue. Importantly, AβpE3 has a higher aggregation propensity, and stability, and shows an increased toxicity compared to full-length Aβ. We have recently shown that intraneuronal accumulation of AβpE3 peptides induces a severe neuron loss and an associated neurological phenotype in the TBA2 mouse model for AD. Given the increasing interest in AβpE3, we have generated two novel monoclonal antibodies which were characterized as highly specific for AβpE3 peptides and herein used to analyze plaque deposition in APP/PS1KI mice, an AD model with severe neuron loss and learning deficits. This was compared with the plaque pattern present in brain tissue from sporadic and familial AD cases. Abundant plaques positive for AβpE3 were present in patients with sporadic AD and familial AD including those carrying mutations in APP (arctic and Swedish) and PS1. Interestingly, in APP/PS1KI mice we observed a continuous increase in AβpE3 plaque load with increasing age, while the density for Aβ1-x plaques declined with aging. We therefore assume that, in particular, the peptides starting with position 1 of Aβ are N-truncated as disease progresses, and that, AβpE3 positive plaques are resistant to age-dependent degradation likely due to their high stability and propensity to aggregate.
PMCID: PMC2789212  PMID: 19823761
Transgenic mice; Arctic mutation; Swedish mutation; Presenilin-1 mutation; Sporadic Alzheimer’s disease; Pyroglutamate Abeta; Biacore; Antibody specificity
5.  Intracellular accumulation of aggregated pyroglutamate amyloid beta: convergence of aging and Aβ pathology at the lysosome 
Age  2012;35(3):673-687.
Deposition of aggregated amyloid beta (Aβ) is a major hallmark of Alzheimer’s disease (AD)—a common age-related neurodegenerative disorder. Typically, Aβ is generated as a peptide of varying lengths. However, a major fraction of Aβ peptides in the brains of AD patients has undergone posttranslational modifications, which often radically change the properties of the peptides. Aβ3(pE)-42 is an N-truncated, pyroglutamate-modified variant that is abundantly present in AD brain and was suggested to play a role early in the pathogenesis. Here we show that intracellular accumulation of oligomeric aggregates of Aβ3(pE)-42 results in loss of lysosomal integrity. Using a novel antibody specific for aggregates of AβpE3, we show that in postmortem human brain tissue, aggregated AβpE3 is predominantly found in the lysosomes of both neurons and glial cells. Our data further demonstrate that AβpE3 is relatively resistant to lysosomal degradation, which may explain its accumulation in the lysosomes. The intracellular AβpE3 aggregates increase in an age-dependent manner. The results presented in this study support a model where Aβ pathology and aging converge, leading to accumulation of the degradation-resistant pE-modified Aβ in the lysosomes, lysosomal dysfunction, and neurodegeneration.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-012-9403-0) contains supplementary material, which is available to authorized users.
PMCID: PMC3636379  PMID: 22477259
Alzheimer’s disease; Pyroglutamate amyloid beta; Lysosomal pathology
6.  Role of the Fast Kinetics of Pyroglutamate-Modified Amyloid-β Oligomers in Membrane Binding and Membrane Permeability 
Biochemistry  2014;53(28):4704-4714.
Membrane permeability to ions and small molecules is believed to be a critical step in the pathology of Alzheimer’s disease (AD). Interactions of oligomers formed by amyloid-β (Aβ) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aβs, pyroglutamate (pE)-modified Aβ peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aβ1–40/42 peptides, these have not been sufficiently characterized for the more abundant AβpE3–42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AβpE3–42 and Aβ1–42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AβpE3–42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AβpE3–42, and the lower concentrations are attributed to weaker interactions with zwitterionic lipid headgroups. While adsorbed oligomers produced little or no significant membrane structural damage, increased membrane permeabilization to ionic species is understood in terms of enlarged membrane-inserted oligomers. Membrane-inserted AβpE3–42 oligomers were also found to modify the mechanical properties of the membrane. Taken together, our results suggest that membrane-inserted oligomers are the primary species responsible for membrane permeability.
PMCID: PMC4215883  PMID: 24950761
7.  Pyroglutamate-Aβ 3 and 11 colocalize in amyloid plaques in Alzheimer’s disease cerebral cortex with pyroglutamate-Aβ 11 forming the central core 
Neuroscience Letters  2011;505(2):109-112.
N-terminal truncated amyloid beta (Aβ) derivatives, especially the forms having pyroglutamate at the 3 position (AβpE3) or at the 11 position (AβpE11) have become the topic of considerable study. AβpE3 is known to make up a substantial portion of the Aβ species in senile plaques while AβpE11 has received less attention. We have generated very specific polyclonal antibodies against both species. Each antibody recognizes only the antigen against which it was generated on Western blots and neither recognizes full length Aβ. Both anti-AβpE3 and anti-AβpE11 stain senile plaques specifically in Alzheimer’s disease cerebral cortex and colocalize with Aβ, as shown by confocal microscopy. In a majority of plaques examined, AβpE11 was observed to be the dominant form in the innermost core. These data suggest that AβpE11 may serve as a generating site for senile plaque formation.
PMCID: PMC3253715  PMID: 22001577
8.  Neurosteroids block the increase in intracellular calcium level induced by Alzheimer’s β-amyloid protein in long-term cultured rat hippocampal neurons 
The neurotoxicity of β-amyloid protein (AβP) is implicated in the etiology of Alzheimer’s disease. We previously have demonstrated that AβP forms Ca2+-permeable pores on neuronal membranes, causes a marked increase in intracellular calcium level, and leads to neuronal death. Here, we investigated in detail the features of AβP-induced changes in intracellular Ca2+ level in primary cultured rat hippocampal neurons using a multisite Ca2+-imaging system with fura-2 as a fluorescent probe. Only a small fraction of short-term cultured hippocampal neurons (ca 1 week in vitro) exhibited changes in intracellular Ca2+ level after AβP exposure. However, AβP caused an acute increase in intracellular Ca2+ level in long-term cultured neurons (ca 1 month in vitro). The responses to AβP were highly heterogeneous, and immunohistochemical analysis using an antibody to AβP revealed that AβP is deposited on some but not all neurons. Considering that the disruption of Ca2+ homeostasis is the primary event in AβP neurotoxicity, substances that protect neurons from an AβP-induced intracellular Ca2+ level increase may be candidates as therapeutic drugs for Alzheimer’s disease. In line with the search for such protective substances, we found that the preadministration of neurosteroids including dehydroepiandrosterone, dehydroepiandrosterone sulfate, and pregnenolone significantly inhibits the increase in intracellular calcium level induced by AβP. Our results suggest the possible significance of neurosteroids, whose levels are reduced in the elderly, in preventing AβP neurotoxicity.
PMCID: PMC2515900  PMID: 18728806
neurotoxicity; pore; calcium homeostasis; channel; aging
9.  Amyloid-beta peptide Aβp3-42 affects early aggregation of full-length Aβ1-42 
Peptides  2009;30(5):849-854.
The major amyloid beta (Aβ) peptides found in the brain of familial and late onset Alzheimer’s disease include the full-length Aβ1-42 and N-terminally truncated, pyroglutamylated peptides Aβp3-42 and Aβp11-42. The biophysical properties of Aβ1-42 have been extensively studied, yet little is known about the other modified peptides. We investigated the aggregation kinetics of brain-specific Aβ peptides to better understand their potential roles in plaque formation. Synthetic peptides were analyzed individually and in mixtures representing various ratios found in the brain. Spectrofluorometric analyses using Thioflavin-T showed that the aggregation of Aβ1-42 was faster compared to Aβp3-42; however, Aβp11-42 displayed similar kinetics. Surprisingly, mixtures of full-length Aβ1-42 and Aβp3-42 showed an initial delay in beta-sheet formation from both equimolar and non-equimolar samples. Electron microscopy of peptides individually and in mixtures further supported fluorescence data. These results indicate that Aβ-Aβ peptide interactions involving different forms may play a critical role in senile plaque formation and maintenance of the soluble Aβ pool in the brain.
PMCID: PMC2752682  PMID: 19428760
Alzheimer’s disease; Amyloid-beta peptides; Aggregation; Pyroglutamate
10.  Choline Modulation of the Aβ P1-40 Channel Reconstituted into a Model Lipid Membrane 
Nicotinic acetylcholine receptors (AChRs), implicated in memory and learning, in subjects affected by Alzheimer's disease result altered. Stimulation of α7-nAChRs inhibits amyloid plaques and increases ACh release. β-amyloid peptide (AβP) forms ion channels in the cell and model phospholipid membranes that are retained responsible in Alzheimer disease. We tested if choline, precursor of ACh, could affect the AβP1-40 channels in oxidized cholesterol (OxCh) and in palmitoyl-oleoyl-phosphatidylcholine (POPC):Ch lipid bilayers. Choline concentrations of 5 × 10−11 M–1.5 × 10−8 M added to the cis- or trans-side of membrane quickly increased AβP1-40 ion channel frequency (events/min) and ion conductance in OxCh membranes, but not in POPC:Ch membranes. Circular Dichroism (CD) spectroscopy shows that after 24 and 48 hours of incubation with AβP1-40, choline stabilizes the random coil conformation of the peptide, making it less prone to fibrillate. These actions seem to be specific in that ACh is ineffective either in solution or on AβP1-40 channel incorporated into PLMs.
PMCID: PMC3022179  PMID: 21253469
11.  Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein 
Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's β-amyloid protein (AβP) play crucial roles in the pathogenesis of Alzheimer's disease (AD). Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”), and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.
PMCID: PMC3087492  PMID: 21547225
12.  From BACE1 Inhibitor to Multifunctionality of Tryptoline and Tryptamine Triazole Derivatives for Alzheimer’s Disease 
Molecules (Basel, Switzerland)  2012;17(7):8312-8333.
Efforts to discover new drugs for Alzheimer’s disease emphasizing multiple targets was conducted seeking to inhibit amyloid oligomer formation and to prevent radical formation. The tryptoline and tryptamine cores of BACE1 inhibitors previously identified by virtual screening were modified in silico for additional modes of action. These core structures were readily linked to different side chains using 1,2,3-triazole rings as bridges by copper catalyzed azide-alkyne cycloaddition reactions. Three compounds among the sixteen designed compounds exerted multifunctional activities including β-secretase inhibitory action, anti-amyloid aggregation, metal chelating and antioxidant effects at micromolar levels. The neuroprotective effects of the multifunctional compounds 6h, 12c and 12h on Aβ1–42 induced neuronal cell death at 1 μM were significantly greater than those of the potent single target compound, BACE1 inhibitor IV and were comparable to curcumin. The observed synergistic effect resulting from the reduction of the Aβ1–42 neurotoxicity cascade substantiates the validity of our multifunctional strategy in drug discovery for Alzheimer’s disease.
PMCID: PMC3618987  PMID: 22781443
multifunction drugs; BACE1 inhibitor; anti-amyloid aggregation; chelator; antioxidant; neuroprotection
13.  A Chemical Analog of Curcumin as an Improved Inhibitor of Amyloid Abeta Oligomerization 
PLoS ONE  2012;7(3):e31869.
Amyloid-like plaques are characteristic lesions defining the neuropathology of Alzheimer's disease (AD). The size and density of these plaques are closely associated with cognitive decline. To combat this disease, the few therapies that are available rely on drugs that increase neurotransmission; however, this approach has had limited success as it has simply slowed an imminent decline and failed to target the root cause of AD. Amyloid-like deposits result from aggregation of the Aβ peptide, and thus, reducing amyloid burden by preventing Aβ aggregation represents an attractive approach to improve the therapeutic arsenal for AD. Recent studies have shown that the natural product curcumin is capable of crossing the blood-brain barrier in the CNS in sufficient quantities so as to reduce amyloid plaque burden. Based upon this bioactivity, we hypothesized that curcumin presents molecular features that make it an excellent lead compound for the development of more effective inhibitors of Aβ aggregation. To explore this hypothesis, we screened a library of curcumin analogs and identified structural features that contribute to the anti-oligomerization activity of curcumin and its analogs. First, at least one enone group in the spacer between aryl rings is necessary for measureable anti-Aβ aggregation activity. Second, an unsaturated carbon spacer between aryl rings is essential for inhibitory activity, as none of the saturated carbon spacers showed any margin of improvement over that of native curcumin. Third, methoxyl and hydroxyl substitutions in the meta- and para-positions on the aryl rings appear necessary for some measure of improved inhibitory activity. The best lead inhibitors have either their meta- and para-substituted methoxyl and hydroxyl groups reversed from that of curcumin or methoxyl or hydroxyl groups placed in both positions. The simple substitution of the para-hydroxy group on curcumin with a methoxy substitution improved inhibitor function by 6-7-fold over that measured for curcumin.
PMCID: PMC3307704  PMID: 22442659
14.  Structural Conversion of Aβ17–42 Peptides from Disordered Oligomers to U-Shape Protofilaments via Multiple Kinetic Pathways 
PLoS Computational Biology  2015;11(5):e1004258.
Discovering the mechanisms by which proteins aggregate into fibrils is an essential first step in understanding the molecular level processes underlying neurodegenerative diseases such as Alzheimer’s and Parkinson's. The goal of this work is to provide insights into the structural changes that characterize the kinetic pathways by which amyloid-β peptides convert from monomers to oligomers to fibrils. By applying discontinuous molecular dynamics simulations to PRIME20, a force field designed to capture the chemical and physical aspects of protein aggregation, we have been able to trace out the entire aggregation process for a system containing 8 Aβ17–42 peptides. We uncovered two fibrillization mechanisms that govern the structural conversion of Aβ17–42 peptides from disordered oligomers into protofilaments. The first mechanism is monomeric conversion templated by a U-shape oligomeric nucleus into U-shape protofilament. The second mechanism involves a long-lived and on-pathway metastable oligomer with S-shape chains, having a C-terminal turn, en route to the final U-shape protofilament. Oligomers with this C-terminal turn have been regarded in recent experiments as a major contributing element to cell toxicity in Alzheimer’s disease. The internal structures of the U-shape protofilaments from our PRIME20/DMD simulation agree well with those from solid state NMR experiments. The approach presented here offers a simple molecular-level framework to describe protein aggregation in general and to visualize the kinetic evolution of a putative toxic element in Alzheimer’s disease in particular.
Author Summary
Understanding the mechanisms of protein folding and aggregation is of fundamental importance in elucidating the biological function of proteins and their complex. Many advances have been made in our ability to describe protein folding based both on ideas from biophysics and improvements in supercomputing power, yet realistic simulations of the entire kinetic process of protein aggregation including fibril formation still remain challenging tasks in biophysics and computational biology. This work describes a breakthrough in our ability to simulate the aggregation of proteins on a molecular level and the emergence of the toxic species responsible for the cause of neuro-degenerative diseases such as Alzheimer’s disease. Based on this work, one can now trace the entire aggregation process starting from disordered monomers to meta-stable oligomers to protofilament and then amyloid fibril. This is a significant advance over the current state of the art in both biophysics and computational biology in uncovering the fundamental mechanisms behind the amyloid fibril formation for aggregation-prone proteins.
PMCID: PMC4425657  PMID: 25955249
Neurodegenerative diseases result in the loss of functional neurons and synapses. Although future stem cell therapies offer some hope, current treatments for most of these diseases are less than adequate and our best hope is to prevent these devastating diseases. Neuroprotective approaches work best prior to the initiation of damage, suggesting that some safe and effective prophylaxis would be highly desirable. Curcumin has an outstanding safety profile and a number of pleiotropic actions with potential for neuroprotective efficacy, including anti-inflammatory, antioxidant, and anti-protein-aggregate activities. These can be achieved at sub-micromolar levels. Curcumin’s dose–response curves are strongly dose dependent and often biphasic so that in vitro data need to be cautiously interpreted; many effects might not be achievable in target tissues in vivo with oral dosing. However, despite concerns about poor oral bioavailability, curcumin has at least 10 known neuroprotective actions and many of these might be realized in vivo. Indeed, accumulating cell culture and animal model data show that dietary curcumin is a strong candidate for use in the prevention or treatment of major disabling age-related neurodegenerative diseases like Alzheimer’s, Parkinson’s, and stroke. Promising results have already led to ongoing pilot clinical trials.
PMCID: PMC2527619  PMID: 17569212
16.  Discovery of 5-(4-Hydroxyphenyl)-3-oxo-pentanoic Acid [2-(5-Methoxy-1H-indol-3-yl)-ethyl]-amide as a Neuroprotectant for Alzheimer’s Disease by Hybridization of Curcumin and Melatonin 
ACS Chemical Neuroscience  2014;5(8):690-699.
In our effort to develop effective neuroprotectants as potential treatments for Alzheimer’s disease (AD), hybrid compounds of curcumin and melatonin, two natural products that have been extensively studied in various AD models, were designed, synthesized, and biologically characterized. A lead hybrid compound (7) was discovered to show significant neuroprotection with nanomolar potency (EC50 = 27.60 ± 9.4 nM) in MC65 cells, a cellular AD model. Multiple in vitro assay results established that 7 exhibited moderate inhibitory effects on the production of amyloid-β oligomers (AβOs) in MC65 cells, but not on the aggregation of Aβ species. It also exhibited significant antioxidative properties. Further mechanistic studies demonstrated that 7’s antioxidant effects correlate well with its neuroprotective potency for MC65 cells, and these effects might be due to its interference with the interactions of AβOs within the mitochondria of MC65 cells. Furthermore, 7 was confirmed to cross the blood-brain barrier (BBB) and deliver a sufficient amount to brain tissue after oral administration. Collectively, these results strongly support the hybridization approach as an efficient strategy to help identify novel scaffolds with a desired pharmacology, and strongly encourage further optimization of 7 to develop more potent neuroprotectants for AD.
PMCID: PMC4140595  PMID: 24825313
Neuroprotectants; hybridization; Alzheimer’s disease; curcumin; melatonin
17.  In Vitro and In Vivo Neurotoxicity of Prion Protein Oligomers 
PLoS Pathogens  2007;3(8):e125.
The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers.
Author Summary
Prion diseases are transmissible neurodegenerative diseases caused by an infectious agent thought to be composed mainly of a host protein, the prion protein (PrP). The mechanisms of neurodegeneration prevailing in these diseases are not well understood. In the present study, we demonstrate that small PrP aggregates, called oligomers, cause the death of neurons in culture and after injection in vivo. On the contrary, larger PrP aggregates, visualized as fibrils by electron microscopy, do not cause the death of cultured neurons and are much less toxic than PrP oligomers in vivo. We propose that the PrP oligomers exert their toxicity by disturbing neuronal membranes, as well as by an excessive intracellular concentration leading to the generation of death signals (also called apoptotic signals) by the cell. Moreover, the use of antibodies recognizing a certain portion of the PrP oligomers could prevent neuronal death. This study assigns prion diseases to the same group of diseases as Alzheimer disease, in which protein oligomers constitute the major trigger of the neurodegenerative process, and suggests new possible neuroprotective approaches for therapeutic strategies.
PMCID: PMC1959381  PMID: 17784787
18.  Complete Phenotypic Recovery of an Alzheimer's Disease Model by a Quinone-Tryptophan Hybrid Aggregation Inhibitor 
PLoS ONE  2010;5(6):e11101.
The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated β-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Aβ oligomerization and fibrillization, as well as the cytotoxic effect of Aβ oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Aβ while immuno-staining of the 3rd instar larval brains showed a significant reduction in Aβ accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Aβ. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease.
PMCID: PMC2885425  PMID: 20559435
19.  Telomerase: A Target for Therapeutic Effects of Curcumin and a Curcumin Derivative in Aβ1-42 Insult In Vitro 
PLoS ONE  2014;9(7):e101251.
This study was designed to investigate whether telomerase was involved in the neuroprotective effect of curcumin and Cur1. Alzheimer's disease is a consequence of an imbalance between the generation and clearance of amyloid-beta peptide in the brain. In this study, we used Aβ1-42 (10 µg/ml) to establish a damaged cell model, and curcumin and Cur1 were used in treatment groups. We measured cell survival and cell growth, intracellular oxidative stress and hTERT expression. After RNA interference, the effects of curcumin and Cur1 on cells were verified. Exposure to Aβ1–42 resulted in significant oxidative stress and cell toxicity, and the expression of hTERT was significantly decreased. Curcumin and Cur1 both protected SK-N-SH cells from Aβ1–42 and up-regulated the expression of hTERT. Furthermore, Cur1 demonstrated stronger protective effects than curcumin. However, when telomerase was inhibited by TERT siRNA, the neuroprotection by curcumin and Cur1 were ceased. Our study indicated that the neuroprotective effects of curcumin and Cur1 depend on telomerase, and thus telomerase may be a target for therapeutic effects of curcumin and Cur1.
PMCID: PMC4077729  PMID: 24983737
20.  Flavonoids as Therapeutic Compounds Targeting Key Proteins Involved in Alzheimer’s Disease 
ACS Chemical Neuroscience  2013;5(2):83-92.
Alzheimer’s disease is characterized by pathological aggregation of protein tau and amyloid-β peptides, both of which are considered to be toxic to neurons. Naturally occurring dietary flavonoids have received considerable attention as alternative candidates for Alzheimer’s therapy taking into account their antiamyloidogenic, antioxidative, and anti-inflammatory properties. Experimental evidence supports the hypothesis that certain flavonoids may protect against Alzheimer’s disease in part by interfering with the generation and assembly of amyloid-β peptides into neurotoxic oligomeric aggregates and also by reducing tau aggregation. Several mechanisms have been proposed for the ability of flavonoids to prevent the onset or to slow the progression of the disease. Some mechanisms include their interaction with important signaling pathways in the brain like the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways that regulate prosurvival transcription factors and gene expression. Other processes include the disruption of amyloid-β aggregation and alterations in amyloid precursor protein processing through the inhibition of β-secretase and/or activation of α-secretase, and inhibiting cyclin-dependent kinase-5 and glycogen synthase kinase-3β activation, preventing abnormal tau phosphorylation. The interaction of flavonoids with different signaling pathways put forward their therapeutic potential to prevent the onset and progression of Alzheimer’s disease and to promote cognitive performance. Nevertheless, further studies are needed to give additional insight into the specific mechanisms by which flavonoids exert their potential neuroprotective actions in the brain of Alzheimer’s disease patients.
PMCID: PMC3930994  PMID: 24328060
Flavonoids; Alzheimer’s disease; amyloid precursor protein; amyloid beta; BACE-1; tau; signaling
21.  Systematic In Vivo Analysis of the Intrinsic Determinants of Amyloid β Pathogenicity 
PLoS Biology  2007;5(11):e290.
Protein aggregation into amyloid fibrils and protofibrillar aggregates is associated with a number of the most common neurodegenerative diseases. We have established, using a computational approach, that knowledge of the primary sequences of proteins is sufficient to predict their in vitro aggregation propensities. Here we demonstrate, using rational mutagenesis of the Aβ42 peptide based on such computational predictions of aggregation propensity, the existence of a strong correlation between the propensity of Aβ42 to form protofibrils and its effect on neuronal dysfunction and degeneration in a Drosophila model of Alzheimer disease. Our findings provide a quantitative description of the molecular basis for the pathogenicity of Aβ and link directly and systematically the intrinsic properties of biomolecules, predicted in silico and confirmed in vitro, to pathogenic events taking place in a living organism.
Author Summary
A wide range of diseases, including diabetes and common brain diseases of old age, are characterised by the deposition of protein in the affected tissues. Alzheimer disease, the most common neurodegenerative disorder, is caused by the aggregation and deposition of a peptide called Aβ in the brain. We have previously developed a computational procedure that predicts a particular peptide or protein's speed of aggregation in the test tube. Our goal was to test whether the speed of aggregate formation that we observe in the test tube is directly linked to the brain toxicity that we see in our fruit fly model of Alzheimer disease. We made flies that produce each of 17 variant forms of Aβ and show that the toxicity of each variant is closely linked to the tendency of each variant to form small soluble aggregates. Our computational procedure has previously been shown to be applicable to a wide range of different proteins and diseases, and so this demonstration that it can predict toxicity in an animal model system has implications for many areas of disease-related research.
A systematic analysis of Alzheimer disease amyloid β peptide variants inDrosophila brain demonstrates that their predicted propensity to form protofibrillar aggregates correlates best with toxicity.
PMCID: PMC2043051  PMID: 17973577
22.  Atomic and Dynamic Insights into the Beneficial Effect of the 1,4-Naphthoquinon-2-yl-l-tryptophan Inhibitor on Alzheimer’s Aβ1–42 Dimer in Terms of Aggregation and Toxicity 
ACS Chemical Neuroscience  2013;5(2):148-159.
Aggregation of the amyloid β protein (Aβ) peptide with 40 or 42 residues is one key feature in Alzheimer’s disease (AD). The 1,4-naphthoquinon-2-yl-l-tryptophan (NQTrp) molecule was reported to alter Aβ self-assembly and reduce toxicity. Though nuclear magnetic resonance experiments and various simulations provided atomic information about the interaction of NQTrp with Aβ peptides spanning the regions of residues 12–28 and 17–42, none of these studies were conducted on the full-length Aβ1–42 peptide. To this end, we performed extensive atomistic replica exchange molecular dynamics simulations of Aβ1–42 dimer with two NQTrp molecules in explicit solvent, by using a force field known to fold diverse proteins correctly. The interactions between NQTrp and Aβ1–42, which change the Aβ interface by reducing most of the intermolecular contacts, are found to be very dynamic and multiple, leading to many transient binding sites. The most favorable binding residues are Arg5, Asp7, Tyr10, His13, Lys16, Lys18, Phe19/Phe20, and Leu34/Met35, providing therefore a completely different picture from in vitro and in silico experiments with NQTrp with shorter Aβ fragments. Importantly, the 10 hot residues that we identified explain the beneficial effect of NQTrp in reducing both the level of Aβ1–42 aggregation and toxicity. Our results also indicate that there is room to design more efficient drugs targeting Aβ1–42 dimer against AD.
PMCID: PMC3930991  PMID: 24246047
Alzheimer’s disease; amyloid β dimer; NQTrp; replica exchange molecular dynamics simulation
23.  Nitrosative stress-induced Parkinsonian Lewy-like aggregates prevented through polyphenolic phytochemical analog intervention 
Nitrosative stress has recently been demonstrated as a causal in a select sporadic variant of Parkinson’s (PD) and Alzheimer’s (AD) diseases. Specifically, elevated levels of NO disrupt the redox activity of protein disulfide isomerase, a key endoplasmic reticulum-resident chaperone by S-nitroso modification of its redox-active cysteines. This leads to accumulation of misfolded AD- and PD-specific protein debris. We have recently demonstrated in vitro that polyphenolic phytochemicals, curcumin and masoprocol, can rescue S-nitroso-PDI formation by scavenging NOx. In this study, using dopaminergic SHSY-5Y cells, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1 (a known constituent of PD Lewy neurites) as a function of rotenone-induced nitrosative stress. Importantly, we demonstrate a marked decrease in synphilin-1 aggregation when the cell line is previously incubated with 3,5-bis(2-flurobenzylidene) piperidin-4-one (EF-24), a curcumin analogue, prior to rotenone insult. Furthermore, our data also reveal that rotenone attenuates PDI expression in the same cell line, a phenomenon that can be mitigated through EF-24 intervention. Together, these results suggest that EF-24 can exert neuroprotective effects by ameliorating nitrosative stress-linked damage to PDI and the associated onset of PD and AD. Essentially, EF-24 can serve as a scaffold for the design and development of PD and AD specific prophylactics.
PMCID: PMC3169436  PMID: 21130735
protein disulfide isomerase; nitrosative stress; neurodegenerative disorders; synphilin-1; EF-24; Lewy body
24.  Amyloid β-protein oligomers and Alzheimer’s disease 
The oligomer cascade hypothesis, which states that oligomers are the initiating pathologic agents in Alzheimer’s disease, has all but supplanted the amyloid cascade hypothesis, which suggested that fibers were the key etiologic agents in Alzheimer’s disease. We review here the results of in vivo, in vitro and in silico studies of amyloid β-protein oligomers, and discuss important caveats that should be considered in the evaluation of these results. This article is divided into four sections that mirror the main approaches used in the field to better understand oligomers: (1) attempts to locate and examine oligomers in vivo in situ; that is, without removing these species from their environment; (2) studies involving oligomers extracted from human or animal tissues and the subsequent characterization of their properties ex vivo; (3) studies of oligomers that have been produced synthetically and studied using a reductionist approach in relatively simple in vitro biophysical systems; and (4) computational studies of oligomers in silico. These multiple orthogonal approaches have revealed much about the molecular and cell biology of amyloid β-protein. However, as informative as these approaches have been, the amyloid β-protein oligomer system remains enigmatic.
PMCID: PMC3978746  PMID: 24289820
25.  Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta 
eLife  2013;2:e00857.
Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer’s, Parkinson’s, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer’s disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind to Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers.
eLife digest
Alzheimer’s disease is the most common form of dementia, estimated to affect roughly five million people in the United States, and its incidence is steadily increasing as the population ages. A pathological hallmark of Alzheimer’s disease is the presence in the brain of aggregates of two proteins: tangles of a protein called tau; and fibers and smaller units (oligomers) of a peptide called amyloid beta.
Many attempts have been made to screen libraries of natural and synthetic compounds to identify substances that might prevent the aggregation and toxicity of amyloid. Such studies revealed that polyphenols found in green tea and in the spice turmeric can inhibit the formation of amyloid fibrils. Moreover, a number of dyes reduce the toxic effects of amyloid on cells, although significant side effects prevent these from being used as drugs.
Structure-based drug design, in which the structure of a target protein is used to help identify compounds that will interact with it, has been used to generate therapeutic agents for a number of diseases. Here, Jiang et al. report the first application of this technique in the hunt for compounds that inhibit the cytotoxicity of amyloid beta. Using the known atomic structure of the protein in complex with a dye, Jiang et al. performed a computational screen of 18,000 compounds in search of those that are likely to bind effectively.
The compounds that showed the strongest predicted binding were then tested for their ability to interfere with the aggregation of amyloid beta and to protect cells grown in culture from its toxic effects. Compounds that reduced toxicity did not reduce the abundance of protein aggregates, but they appear to increase the stability of fibrils. This is consistent with other evidence suggesting that small, soluble forms (oligomers) of amyloid beta that break free from the fibrils may be the toxic agent in Alzheimer’s disease, rather than the fibrils themselves.
In addition to uncovering compounds with therapeutic potential in Alzheimer’s disease, this work presents a new approach for identifying proteins that bind to amyloid fibrils. Given that amyloid accumulation is a feature of many other diseases, including Parkinson’s disease, Huntington’s disease and type 2 diabetes, the approach could have broad therapeutic applications.
PMCID: PMC3713518  PMID: 23878726
amyloid fiber; computational biology; drug discovery; Alzheimer's disease; ligand docking; Other

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