Amine oxidase activity of ß-amyloid precursor protein modulates systemic and local catecholamine levels.

The catecholamines dopamine (DA), norepinephrine (NE) and epinephrine (E) are neurotransmitters and hormones that mediate stress responses in tissues and plasma. The expression of ß-amyloid precursor protein (APP) is responsive to stress and is high in tissues rich in catecholamines. We recently reported that APP is a ferroxidase, subsuming, in neurons and other cells, the iron-export activity that ceruloplasmin mediates in glia. Here we report that, like ceruloplasmin, APP also oxidizes synthetic amines and catecholamines catalytically (K(m) NE=0.27 mM), through a site encompassing its ferroxidase motif and selectively inhibited by zinc. Accordingly, APP knockout mice have significantly higher levels of DA, NE and E in brain, plasma and select tissues. Consistent with this, these animals have increased resting heart rate and systolic blood pressure as well as suppressed prolactin and lymphocyte levels. These findings support a role for APP in extracellular catecholaminergic clearance.

Protocol of a Phase II Randomized, Multi-Center, Double-Blind, Placebo-Controlled Trial of S-Adenosyl Methionine in Participants with Mild Cognitive Impairment or Dementia Due to Alzheimer's Disease.

BACKGROUND: S-adenosyl methionine (SAMe) is a pivotal metabolite in multiple pathways required for neuronal homeostasis, several of which are compromised in Alzheimer's disease (AD). Correction of the SAMe deficiency that is characteristic of the AD brain may attenuate or prevent pathological processes driving AD-associated neurodegeneration including aberrant tau hyperphosphorylation and DNA hypomethylation. OBJECTIVES: The primary aim is to test the hypothesis that daily treatment with 400 mg oral SAMe for 180 days will lead to a greater reduction from baseline in plasma levels of p-tau181 compared to placebo in patients with mild cognitive impairment or dementia due to AD. DESIGN, SETTING, PARTICIPANTS: This is a phase II, randomized, multi-center, double-blind, placebo-controlled trial among 60 participants with mild cognitive impairment or dementia due to AD. Participants will be randomized in a 1:1 ratio to receive either SAMe or matching placebo, to be taken as an adjunct to their AD standard of care. MEASUREMENTS AND RESULTS: The primary outcome is change in plasma p-tau181 concentration between baseline and following 180 days of treatment, which will be compared between the active and placebo group. Secondary outcomes are the safety of SAMe administration (incidence of serious adverse events), change from baseline in cognitive performance (as measured by the Repeatable Battery for the Assessment of Neuropsychological Status), and epigenetic changes in DNA methylation. CONCLUSION: Demonstration of effective and safe lowering of plasma p-tau181 with SAMe in this phase II trial would pave the way for an exciting field of translational research and a larger phase III trial.

Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice.

Inhibition of neocortical beta-amyloid (Abeta) accumulation may be essential in an effective therapeutic intervention for Alzheimer's disease (AD). Cu and Zn are enriched in Abeta deposits in AD, which are solubilized by Cu/Zn-selective chelators in vitro. Here we report a 49% decrease in brain Abeta deposition (-375 microg/g wet weight, p = 0.0001) in a blinded study of APP2576 transgenic mice treated orally for 9 weeks with clioquinol, an antibiotic and bioavailable Cu/Zn chelator. This was accompanied by a modest increase in soluble Abeta (1.45% of total cerebral Abeta); APP, synaptophysin, and GFAP levels were unaffected. General health and body weight parameters were significantly more stable in the treated animals. These results support targeting the interactions of Cu and Zn with Abeta as a novel therapy for the prevention and treatment of AD.

Is covalently crosslinked Abeta responsible for synaptotoxicity in Alzheimer's disease?

Alzheimer's disease (AD) is the most common form of dementia in the elderly, and is characterized by the deposition of extracellular amyloid plaques primarily composed of the beta-amyloid peptide (Abeta). While these plaques define the pathology of AD, disease progression has been shown to correlate more closely with the level of synaptotoxicity induced by soluble Abeta oligomers. Recent evidence suggests that these oligomers are covalently crosslinked, possibly due to the interaction of Abeta with redox-active metal ions. These findings offer new avenues for the treatment and prevention of disease, by modulating metal binding or preventing the formation of neurotoxic Abeta oligomers. An understanding of the chemical nature of Abeta is also required to elucidate the synaptotoxic process or processes in AD, which have so far resisted explanation.

Bis(thiosemicarbazone) Metal Complexes as Therapeutics for Neurodegenerative Diseases.

Pathological aggregation of endogenous proteins is a common feature of many neurodegenerative diseases. This is generally accompanied by elevated levels of oxidative stress associated with transition metal dyshomeostasis. As such, strategies targeted toward rectifying metal imbalance are increasingly becoming an attractive therapeutic option. One class of compound showing such therapeutic potential are the bis(thiosemicarbazone) metal complexes. These are small, orally bioavailable compounds capable of crossing the blood brain barrier and capable of delivering bioavailable metal intracellularly. Members of this family of compounds have been shown to successfully treat animal models of several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Here we review the current evidence for the efficacy of bis(thiosemicarbazone) metal complexes in treating these diseases and discuss the implications for future development of these compounds.

Restoration of intestinal function in an MPTP model of Parkinson's Disease.

Patients with Parkinson's disease often experience non-motor symptoms including constipation, which manifest prior to the onset of debilitating motor signs. Understanding the causes of these non-motor deficits and developing disease modifying therapeutic strategies has the potential to prevent disease progression. Specific neuronal subpopulations were reduced within the myenteric plexus of mice 21 days after intoxication by the intraperitoneal administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and was associated with a reduction in stool frequency, indicative of intestinal dysfunction. Oral administration of the divalent copper complex, Cu(II)(atsm), which has been shown to be neuroprotective and restore motor performance to MPTP lesioned mice, improved stool frequency and was correlated with restoration of neuronal subpopulations in the myenteric plexus of MPTP lesioned mice. Restoration of intestinal function was associated with reduced enteric glial cell reactivity and reduction of markers of inflammation. Therapeutics that have been shown to be neuroprotective in the central nervous system, such as Cu(II)(atsm), therefore also provide symptom relief and are disease modifying in the intestinal tract, suggesting that there is a common cause of Parkinson's disease pathogenesis in the enteric nervous system and central nervous system.

The Val-210-Ile pathogenic Creutzfeldt-Jakob disease mutation increases both the helical and aggregation propensities of a sequence corresponding to helix-3 of PrP(C).

A peptide corresponding to the third helical region within the PrP(C) protein, from residues 198 to 218 (helix-3), was synthesised with and without the familial 210-Val to Ile Creutzfeldt-Jakob disease mutation. The NMR structure of PrP(C) predicts no global variation in stability for this mutation, indicating that local sequence rather than global structural factors are involved in the pathological effects of this mutation. 1H NMR analysis of peptides with and without this mutation indicated that it had no significant effect on local helical structure. Temperature denaturation studies monitored by CD showed that the mutation increased the helical content within this region (helical propensity), but did not stabilise the helix toward denaturation (helical stability). Aggregation data indicated that, in addition to increasing helical propensity, this mutation increased the aggregation propensity of this sequence. CD and NMR data indicate that helical interactions, stabilised by the Val-210-Ile mutation, may precede the formation of beta-sheet aggregates in this peptide sequence. Therefore, this pathological mutation probably does not facilitate PrP(C) to PrP(Sc) conversion by directly destabilising the helical structure of PrP(C), but may preferentially stabilise PrP(Sc) by facilitating beta-sheet formation within this sequence region of PrP. In addition, helical interactions between helix-3 in two or more PrP(C) molecules may promote conversion to PrP(Sc).

Helical structure and self-association in a 13 residue neuropeptide Y Y2 receptor agonist: relationship to biological activity.

The solution structure and self-association behaviour of a 13 residue peptide analogue of the C-terminal region of human neuropeptide Y (NPY) have been investigated. NMR analysis of Ac[Leu(28,31)]NPY(24-36), a potent Y2 receptor agonist, shows that it is unstructured in aqueous solution at 5-20 degrees C, but forms a well-defined helix (encompassing residues 25-35) in 40% trifluoroethanol/water at 20 degrees C. Sedimentation experiments show that, in contrast to many peptides in aqueous trifluoroethanol, Ac[Leu(28,31)]NPY(24-36) associates to form a trimer or, more likely, a tetramer in 40% trifluoroethanol, even though it is monomeric in water. This is consistent with the observation of inter-molecular nuclear Overhauser enhancements in trifluoroethanol. Possible models of the associated form that are consistent with the NMR data are described. The relevance of the helical structure observed in trifluoroethanol to the structure of this peptide bound to the NPY Y2 receptor is discussed.

Structure of neurotoxin B-IV from the marine worm Cerebratulus lacteus: a helical hairpin cross-linked by disulphide bonding.

B-IV is a 55-residue, crustacean-selective, neurotoxin secreted by Cerebratulus lacteus, a large marine worm found along the northeastern coast of North America. The 3D structure of this molecule in aqueous solution has been determined by 1H NMR spectroscopy at 600 MHz. The molecule has a well-defined helical hairpin structure, with the branches of the hairpin linked by four disulphide bonds. The disulphide connectivities were established from the NMR data to be 1-8/2-7/3-6/4-5, which differed from those determined previously by chemical means, where 1-7 and 2-8 connectivities were found. Each branch of the hairpin is largely alpha-helical, with the helices in the N and C-terminal branches encompassing residues 11 to 23 and 34 to 49, respectively. The loop connecting the branches of the hairpin contains two inverse gamma-turns centred on residues 24 and 25, a type I beta-turn at residues 28 to 31 and a type II beta-turn at residues 30 to 33. Arg17, -25 and -34, which are important for activity, are all on the same face of the molecule, while Trp30, which is also important for activity, is on the opposite face. Structure comparisons show that the B-IV structure is quite similar to those of Rop (ColE1 repressor of primer) and the heat-stable enterotoxin B from Escherichia coli. These structural similarities are discussed in relation to possible mechanisms of action of B-IV.

Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor.

Two synthetic analogues of murine epidermal growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF. The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF. Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF. [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its 1H chemical shifts suggested that its structure was also very similar to native.

Generating recombinant C-terminal prion protein fragments of exact native sequence.

Transmissibility and distinctive neuropathology are hallmark features of prion diseases differentiating them from other neurodegenerative disorders, with pathogenesis and transmission appearing closely linked to misfolded conformers (PrP(Sc)) of the ubiquitously expressed cellular form of the prion protein (PrP(C)). Given the apparent pathogenic primacy of misfolded PrP, the utilisation of peptides based on the prion protein has formed an integral approach for providing insights into misfolding pathways and pathogenic mechanisms. In parallel with studies employing prion peptides, similar approaches in other neurodegenerative disorders such as Alzheimer Disease, have demonstrated that differential processing of parent proteins and quite minor variations in the primary sequence of cognate peptides generated from the same constitutive processing (such as Aß1-40 versus Aß1-42 produced from γ-secretase activity) can be associated with very different pathogenic consequences. PrP(C) also undergoes constitutive α- or ß-cleavage yielding C1 (residues 112-231 human sequence) or C2 (residues 90-231), respectively, with the full cell biological significance of such processing unresolved; however, it is noteworthy that in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and murine models, the moderately extended C2 fragment predominates in the brain suggesting that the two cleavage events and the consequent C-terminal fragments may differ in their pathogenic significance. Accordingly, studies characterising biologically relevant peptides like C1 and C2, would be most valid if undertaken using peptides completely free of any inherent non-native sequence that arises as a by-product of commonly employed recombinant production techniques. To achieve this aim and thereby facilitate more representative biophysical and neurotoxicity studies, we adapted the combination of high fidelity Taq TA cloning with a SUMO-Hexa-His tag-type approach, incorporating the SUMO protease step. This technique consistently produced sufficient yields (∼10 mg/L) of high purity peptides (>95%) equating to C1 and C2 of exact native primary sequence in the α-helical conformation suitable for biological and biophysical investigations.

La lunga attesa: towards a molecular approach to neuroimaging and therapeutics in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised by the gradual onset of dementia. The pathological hallmarks of the disease are Aß amyloid plaques, neurofibrillary tangles (NFT), synaptic loss and reactive gliosis. Current diagnosis of AD is made by clinical, neuropsychologic, and neuroimaging assessments. Routine structural neuroimaging evaluation with computed tomography (CT) and magnetic resonance imaging (MRI) is based on non-specific features such as atrophy, a late feature in the progression of the disease, hence the crucial importance of developing new approaches for early and specific recognition at the prodromal stages of AD. Functional neuroimaging techniques such as functional magnetic resonance imaging (fMRI), magnetic resonance spectroscopy (MRS), positron emission tomography (PET) and single photon emission computed tomography (SPECT), possibly in conjuction with other related Aß biomarkers in plasma and CSF, could prove to be valuable in the differential diagnosis of AD, as well as in assessing prognosis. With the advent of new therapeutic strategies aimed at reducing the Aß amyloid burden in the brain, there is increasing interest in the development of MRI contrast agents and PET and SPECT radioligands that will permit the assessment of Aß amyloid burden in vivo. - ma dov'è / la lenta processione di stagioni / che fu un'alba infinita e senza strade, / dov'è la lunga attesa e qual è il nome / del vuoto che ci invade. - Eugenio Montale.

Antiviral activity and structural characteristics of the nonglycosylated central subdomain of human respiratory syncytial virus attachment (G) glycoprotein.

Segments of the cystine noose-containing nonglycosylated central subdomain, residues 149-197, of the attachment (G) glycoprotein of human respiratory syncytial virus (HRSV) have been assessed for impact on the cytopathic effect (CPE) of respiratory syncytial virus (RSV). Nalpha-acetyl residues 149-197-amide (G149-197), G149-189, and G149-177 of the A2 strain of HRSV protected 50% of human epithelial HEp-2 cells from the CPE of the A2 strain at concentrations (IC(50)) between 5 and 80 microm. Cystine noose-containing peptides G171-197 and G173-197 did not inhibit the CPE even at concentrations above 150 microm. Systematic C- and N-terminal truncations from G149-189 and G149-177 and alanine substitutions within G154-177 demonstrated that residues 166-170 (EVFNF), within a sequence that is conserved in HRSV strains, were critical for inhibition. Concordantly, G154-177 of bovine RSV and of an antibody escape mutant of HRSV with residues 166-170 of QTLPY and EVSNP, respectively, were not inhibitory. Surprisingly, a variant of G154-177 with an E166A substitution had an IC(50) of 750 nm. NMR analysis demonstrated that G149-177 adopted a well-defined conformation in solution, clustered around F168 and F170. G154-170, particularly EVFNF, may be important in binding of RSV to host cells. These findings constitute a promising platform for the development of antiviral agents for RSV.

Characterization of unique amphipathic antimicrobial peptides from venom of the scorpion Pandinus imperator.

Two novel antimicrobial peptides have been identified and characterized from venom of the African scorpion Pandinus imperator. The peptides, designated pandinin 1 and 2, are alpha-helical polycationic peptides, with pandinin 1 belonging to the group of antibacterial peptides previously described from scorpions, frogs and insects, and pandinin 2 to the group of short magainin-type helical peptides from frogs. Both peptides demonstrated high antimicrobial activity against a range of Gram-positive bacteria (2.4-5.2 microM), but were less active against Gram-negative bacteria (2.4-38.2 microM), and only pandinin 2 affected the yeast Candida albicans. Pandinin 2 also demonstrated strong haemolytic activity (11.1-44.5 microM) against sheep erythrocytes, in contrast with pandinin 1, which was not haemolytic. CD studies and a high-resolution structure of pandinin 2 determined by NMR, showed that the two peptides are both essentially helical, but differ in their overall structure. Pandinin 2 is composed of a single alpha-helix with a predominantly hydrophobic N-terminal sequence, whereas pandinin 1 consists of two distinct alpha-helices separated by a coil region of higher flexibility. This is the first report of magainin-type polycationic antimicrobial peptides in scorpion venom. Their presence brings new insights into the mode of action of scorpion venom and also opens new avenues for the discovery of novel antibiotic molecules from arthropod venoms.

Solution structure of a polypeptide from the N terminus of the HIV protein Nef.

Nef is a 27 kDa myristylated phosphoprotein expressed early in infection by HIV. The N terminus of Nef is thought to play a vital role in the functions of this protein through its interactions with membrane structures. The solution structure of a 25-residue polypeptide corresponding to the N terminus of Nef (Nef1-25) has been investigated by 1H NMR spectroscopy. In aqueous solution at pH 4.8 and 281 K, this peptide underwent conformational averaging, with Pro13 existing in cis and trans conformations in nearly equal proportions. In methanol solution, however, the peptide adopted a well-defined alpha-helical structure from residues 6 to 22, with the N- and C-terminal regions having a less ordered structure. On the basis of a comparison of chemical shifts and NOEs, it appeared that this helical structure was maintained in aqueous trifluoroethanol (50% v/v) and to a lesser extent in a solution of SDS micelles. When the N-acetyl group was replaced by either an N-myristyl or a free ammonium group, there was little effect on the three-dimensional structure of the peptide in methanol; deamidation of the C terminus also had no effect on the structure in methanol. In water, the myristylated peptide aggregated. The similarity between the sequences of Nef1-25 and melittin is reflected in the similar structures of the two molecules, although the N-terminal helix of melittin is more defined. This similarity in structure raises the possibility that Nef1-25 not only interacts with membranes but also may be capable of disrupting them and causing cell lysis. This type of interaction could contribute at least in part to the killing of bystander cells in lymphoid tissues during HIV infection.

Cisplatin binding sites on human albumin.

Reactions of cisplatin (cis-[PtCl2(NH3)2]) with albumin are thought to play an important role in the metabolism of this anticancer drug. They are investigated here via (i) labeling of cisplatin with 15N and use of two-dimensional 1H,15N NMR spectroscopy, (ii) comparison of natural human serum albumin with recombinant human albumin (higher homogeneity and SH content), (iii) chemical modification of Cys, Met, and His residues, (iv) reactions of bound platinum with thiourea, and (v) gel filtration chromatography. In contrast to previous reports, it is shown that the major sulfur-containing binding site involves Met and not Cys-34, and also a N ligand, in the form of an S,N macrochelate. Additional monofunctional adducts involving other Met residues and Cys-34 are also observed. During the later stages of reactions of cisplatin with albumin, release of NH3 occurs due to the strong trans influence of Met sulfur, which weakens the Pt-NH3 bonds, and protein cross-linking is observed. The consequences of these findings for the biological activity of cisplatin-albumin complexes are discussed.

Structural transitions of a GG-platinated DNA duplex induced by pH, temperature and box A of high-mobility-group protein 1.

[1H, 15N] and 1H NMR, and CD spectroscopy are used to show that the duplex d(A-T-A-C-A-T-Pt 7G-Pt7G-T-A-C-A-T-A).d(T-A-T-G-T-A-C-C-A-T-G-T-A-T), where Pt7G is platinated guanine, containing the cis-[Pt(NH3)2]2+ adduct, undergoes reversible temperature-induced (T0.5 310 K) and pH-induced (pKa approximately 4.8) transitions between kinked-duplex and distorted forms, with the latter forms predominating at high temperature and low pH. A related pH-induced structural change was observed for the unplatinated duplex (pKa 4.69, Hill coefficient n = 1.4) but was less cooperative than for the platinated duplex (n = 2). The pH-induced transition is attributed to protonation of cytosine residues and has wider implications, since many reported NMR studies of DNA are carried out near pH 5 to minimize NH-exchange rates. The [Pt(en)]2+ (where en is 1,2-ethanediamine) GG chelate of the same duplex is shown to exist in kinked and distorted forms, and the [1H,15N]-NMR shifts for the kinked form are indicative of the presence of highly stereospecific interactions with the Pt-NH protons. On binding of the duplex platinated with [Pt(NH3)2]2+ to high-mobility-group protein 1 (HMG1) box A, similar changes in shifts of the Pt-NH3 resonances to those induced by raising the temperature or lowering the pH were observed. The specific changes in 1H-NMR chemical shifts of HMG1 box A are consistent with binding of the platinated duplex (intermediate exchange rate on the 1H-NMR time-scale) to the concave face of the protein via helices I and II and the intervening loop.

Identification of platination sites on human serum transferrin using (13)C and (15)N NMR spectroscopy.

Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl(2)] or cis-[PtCl(2)(NH(3))(2)] have been investigated in solution via observation of [(1)H,(15)N] NMR resonances of the Pt complexes, [(1)H,(13)C] resonances of the eCH(3) groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days.

Copper modulation of ion channels of PrP[106-126] mutant prion peptide fragments.

We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106-126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu(2+)-sensitive fast cation channel. To investigate the role of PrP[106-126]'s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu(2+), we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106-126] (AA/SS) and PrP[106-126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the beta-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106-126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common beta sheet-based channel structure. The current-voltage (I-V) relationship of the fast cation channel, which had a reversal potential, E(rev), between -40 and -10 mV, close to the equilibrium potential for K(+) ( E(K) = -35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106-126] channel formation, it is not a binding site for Cu(2+). We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.

Chelation and intercalation: complementary properties in a compound for the treatment of Alzheimer's disease.

Selective application of metal chelators to homogenates of human Alzheimer's disease (AD) brain has led us to propose that the architecture of aggregated beta-amyloid peptide, whether in the form of plaques or soluble oligomers, is determined at least in part by high-affinity binding of transition metals, especially copper and zinc. Of the two metals, copper is implicated in reactive oxygen species generating reactions, while zinc appears to be associated with conformational and antioxidant activity. We tested the copper chelators trientine, penicillamine, and bathophenanthroline for their ability to mobilize brain Abeta as measured against our benchmark compound bathocuproine (BC). All of these agents were effective in solubilizing brain Abeta, although BC was the most consistent across the range of AD brain tissue samples tested. Similarly, all of the copper chelators depleted copper in the high-speed supernatants. BC alone had no significant effect upon zinc levels in the soluble fraction. BC extraction of brain tissue from C100 transgenic mice (which express human Abeta but do not develop amyloid) revealed SDS-resistant dimers as Abeta was mobilized from the sedimentable to the soluble fraction. NMR analysis showed that, in addition to its copper chelating properties, BC interacts with Abeta to form a complex independent of the presence of copper. Such hybrid copper chelating and "chain breaking" properties may form the basis of a rational design for a therapy for Alzheimer's disease.