Keyla Perez

Rapid Restoration of Cognition in Alzheimer's Transgenic Mice with 8-Hydroxy Quinoline Analogs Is Associated with Decreased Interstitial Aβ

As a disease-modifying approach for Alzheimers disease (AD), clioquinol (CQ) targets beta-amyloid (Abeta) reactions with synaptic Zn and Cu yet promotes metal uptake. Here we characterize the second-generation 8-hydroxy quinoline analog PBT2, which also targets metal-induced aggregation of Abeta, but is more effective as a Zn/Cu ionophore and has greater blood-brain barrier permeability. Given orally to two types of amyloid-bearing transgenic mouse models of AD, PBT2 outperformed CQ by markedly decreasing soluble interstitial brain Abeta within hours and improving cognitive performance to exceed that of normal littermate controls within days. Nontransgenic mice were unaffected by PBT2. The current data demonstrate that ionophore activity, inhibition of in vitro metal-mediated Abeta reactions, and blood-brain barrier permeability are indices that predict a potential disease-modifying drug for AD. The speed of recovery of the animals underscores the acutely reversible nature of the cognitive deficits associated with transgenic models of AD.

Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease.

Alzheimers Disease (AD) is complicated by pro-oxidant intraneuronal Fe(2+) elevation as well as extracellular Zn(2+) accumulation within amyloid plaque. We found that the AD β-amyloid protein precursor (APP) possesses ferroxidase activity mediated by a conserved H-ferritin-like active site, which is inhibited specifically by Zn(2+). Like ceruloplasmin, APP catalytically oxidizes Fe(2+), loads Fe(3+) into transferrin, and has a major interaction with ferroportin in HEK293T cells (that lack ceruloplasmin) and in human cortical tissue. Ablation of APP in HEK293T cells and primary neurons induces marked iron retention, whereas increasing APP695 promotes iron export. Unlike normal mice, APP(-/-) mice are vulnerable to dietary iron exposure, which causes Fe(2+) accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD postmortem neocortex is inhibited by endogenous Zn(2+), which we demonstrate can originate from Zn(2+)-laden amyloid aggregates and correlates with Aβ burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD.

Amyloid-β Peptide (Aβ) Neurotoxicity Is Modulated by the Rate of Peptide Aggregation: Aβ Dimers and Trimers Correlate with Neurotoxicity

Alzheimers disease is an age-related neurodegenerative disorder with its toxicity linked to the generation of amyloid-β peptide (Aβ). Within the Aβ sequence, there is a systemic repeat of a GxxxG motif, which theoretical studies have suggested may be involved in both peptide aggregation and membrane perturbation, processes that have been implicated in Aβ toxicity. We synthesized modified Aβ peptides, substituting glycine for leucine residues within the GxxxG repeat motif (GSL peptides). These GSL peptides undergo β-sheet and fibril formation at an increased rate compared with wild-type Aβ. The accelerated rate of amyloid fibril formation resulted in a decrease in the presence of small soluble oligomers such as dimeric and trimeric forms of Aβ in solution, as detected by mass spectrometry. This reduction in the presence of small soluble oligomers resulted in reduced binding to lipid membranes and attenuated toxicity for the GSL peptides. The potential role that dimer and trimer species binding to lipid plays in Aβ toxicity was further highlighted when it was observed that annexin V, a protein that inhibits Aβ toxicity, specifically inhibited Aβ dimers from binding to lipid membranes.

Increasing Cu bioavailability inhibits Aβ oligomers and tau phosphorylation

Cognitive decline in Alzheimers disease (AD) involves pathological accumulation of synaptotoxic amyloid-β (Aβ) oligomers and hyperphosphorylated tau. Because recent evidence indicates that glycogen synthase kinase 3β (GSK3β) activity regulates these neurotoxic pathways, we developed an AD therapeutic strategy to target GSK3β. The strategy involves the use of copper-bis(thiosemicarbazonoto) complexes to increase intracellular copper bioavailability and inhibit GSK3β through activation of an Akt signaling pathway. Our lead compound CuII(gtsm) significantly inhibited GSK3β in the brains of APP/PS1 transgenic AD model mice. CuII(gtsm) also decreased the abundance of Aβ trimers and phosphorylated tau, and restored performance of AD mice in the Y-maze test to levels expected for cognitively normal animals. Improvement in the Y-maze correlated directly with decreased Aβ trimer levels. This study demonstrates that increasing intracellular copper bioavailability can restore cognitive function by inhibiting the accumulation of neurotoxic Aβ trimers and phosphorylated tau.

Inhibition of γ-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes

Alzheimers disease (AD) is the most common form of dementia and is associated with the deposition of the 39‐ to 43‐amino acid β‐amyloid peptide (Aβ) in the brain. C‐terminal fragments (CTFs) of amyloid precursor protein (APP) can accumulate in endosomally derived multivesicular bodies (MVBs). These intracellular structures contain intraluminal vesicles that are released from the cell as exosomes when the MVB fuses with the plasma membrane. Here we have investigated the role of exosomes in the processing of APP and show that these vesicles contain APP‐ CTFs, as well as Aβ. In addition, inhibition of γ‐secre‐ tase results in a significant increase in the amount of α‐ and β‐secretase cleavage, further increasing the amount of APP‐CTFs contained within these exosomes. We identify several key members of the secretase family of proteases (BACE, PS1, PS2, and ADAM10) to be localized in exosomes, suggesting they may be a previously unidentified site of APP cleavage. These results provide further evidence for a novel pathway in which APP fragments are released from cells and have implications for the analysis of APP processing and diagnostics for Alzheimers disease.—Sharples, R. A., Vella, L. J., Nisbet, R. M., Naylor, R., Perez, K., Barnham, K. J., Masters, C. L., Hill, A. F. Inhibition of γ‐secretase causes increased secretion of amyloid precursor protein C‐terminal fragments in association with exosomes. FASEB J. 22, 1469–1478 (2008)

In Vitro Characterization of Pittsburgh Compound-B Binding to Lewy Bodies

Dementia with Lewy bodies (DLB) is pathologically characterized by the presence of α-synuclein-containing Lewy bodies within the neocortical, limbic, and paralimbic regions. Like Alzheimers disease (AD), Aβ plaques are also present in most DLB cases. The contribution of Aβ to the development of DLB is unclear. [11C]-Pittsburgh compound B ([11C]-PIB) is a thioflavin-T derivative that has allowed in vivo Aβ burden to be quantified using positron emission tomography (PET). [11C]-PIB PET studies have shown similar high cortical [11C]-PIB binding in AD and DLB subjects. To establish the potential binding of PIB to α-synuclein in DLB patients, we characterized the in vitro binding of PIB to recombinant human α-synuclein and DLB brain homogenates. Analysis of the in vitro binding studies indicated that [3H]-PIB binds to α-synuclein fibrils but with lower affinity than that demonstrated/reported for Aβ1–42 fibrils. Furthermore, [3H]-PIB was observed to bind to Aβ plaque-containing DLB brain homogenates but failed to bind to DLB homogenates that were Aβ plaque-free (“pure DLB”). Positive PIB fluorescence staining of DLB brain sections colocalized with immunoreactive Aβ plaques but failed to stain Lewy bodies. Moreover, image quantification analysis suggested that given the small size and low density of Lewy bodies within the brains of DLB subjects, any contribution of Lewy bodies to the [11C]-PIB PET signal would be negligible. These studies indicate that PIB retention observed within the cortical gray matter regions of DLB subjects in [11C]-PIB PET studies is largely attributable to PIB binding to Aβ plaques and not Lewy bodies.

Copper-mediated Amyloid-β Toxicity Is Associated with an Intermolecular Histidine Bridge

Amyloid-β peptide (Aβ) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic Aβ-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/peptide ratios of >0.6:1 by EPR spectroscopy. The toxicity of the Aβ-Cu2+ complex to cultured primary cortical neurons was attenuated when either the π -or τ-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl-1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. 31P magic angle spinning solid-state NMR showed that Aβ and Aβ-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the Aβ toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.

Platinum-based inhibitors of amyloid-β as therapeutic agents for Alzheimer's disease

Amelyoid-β peptide (Aβ) is a major causative agent responsible for Alzheimers disease (AD). Aβ contains a high affinity metal binding site that modulates peptide aggregation and toxicity. Therefore, identifying molecules targeting this site represents a valid therapeutic strategy. To test this hypothesis, a range of L-PtCl2 (L = 1,10-phenanthroline derivatives) complexes were examined and shown to bind to Aβ, inhibit neurotoxicity and rescue Aβ-induced synaptotoxicity in mouse hippocampal slices. Coordination of the complexes to Aβ altered the chemical properties of the peptide inhibiting amyloid formation and the generation of reactive oxygen species. In comparison, the classic anticancer drug cisplatin did not affect any of the biochemical and cellular effects of Aβ. This implies that the planar aromatic 1,10-phenanthroline ligands L confer some specificity for Aβ onto the platinum complexes. The potent effect of the L-PtCl2 complexes identifies this class of compounds as therapeutic agents for AD.

Formation of dopamine-mediated α-synuclein-soluble oligomers requires methionine oxidation

alpha-Synuclein is the major component of the intracellular Lewy body inclusions present in Parkinson disease (PD) neurons. PD involves the loss of dopaminergic neurons in the substantia nigra and the subsequent depletion of dopamine (DA) in the striatum. DA can inhibit alpha-synuclein fibrillization in vitro and promote alpha-synuclein aggregation into soluble oligomers. We have studied the mechanism by which DA mediates alpha-synuclein aggregation into soluble oligomers. Reacting alpha-synuclein with DA increased the mass of alpha-synuclein by 64 Da. NMR showed that all four methionine residues were oxidized by DA, consistent with the addition of 64 Da. Substituting all four methionines to alanine significantly reduced the formation of DA-mediated soluble oligomers. The (125)YEMPS(129) motif in alpha-synuclein can modulate DA inhibition of alpha-synuclein fibrillization. However, alpha-synuclein ending before the (125)YEMPS(129) motif (residues 1-124) could still form soluble oligomers. The addition of exogenous synthetic YEMPS peptide inhibited the formation of soluble oligomers and resulted in the YEMPS peptide being oxidized. Therefore, the (125)YEMPS(129) acts as an antioxidant rather than interacting directly with DA. Our study defines methionine oxidation as the dominant mechanism by which DA generates soluble alpha-synuclein oligomers and highlights the potential role for oxidative stress in modulating alpha-synuclein aggregation.

The Caenorhabditis elegans Aβ1–42 Model of Alzheimer Disease Predominantly Expresses Aβ3–42

Transgenic expression of human amyloid β (Aβ) peptide in body wall muscle cells of Caenorhabditis elegans has been used to better understand aspects of Alzheimer disease (AD). In human aging and AD, Aβ undergoes post-translational changes including covalent modifications, truncations, and oligomerization. Amino truncated Aβ is increasingly recognized as potentially contributing to AD pathogenesis. Here we describe surface-enhanced laser desorption ionization-time of flight mass spectrometry mass spectrometry of Aβ peptide in established transgenic C. elegans lines. Surprisingly, the Aβ being expressed is not full-length 1–42 (amino acids) as expected but rather a 3–42 truncation product. In vitro analysis demonstrates that Aβ3–42 self-aggregates like Aβ1–42, but more rapidly, and forms fibrillar structures. Similarly, Aβ3–42 is also the more potent initiator of Aβ1–40 aggregation. Seeded aggregation via Aβ3–42 is further enhanced via co-incubation with the transition metal Cu(II). Although unexpected, the C. elegans model of Aβ expression can now be co-opted to study the proteotoxic effects and processing of Aβ3–42.