Margaret M. Condron

Amyloid β-Protein Fibrillogenesis STRUCTURE AND BIOLOGICAL ACTIVITY OF PROTOFIBRILLAR INTERMEDIATES

Alzheimer’s disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid β-protein (Aβ). Fibrils, both in vitro andin vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control Aβ production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of Aβ fibril formation, the amyloid protofibril (Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) J. Biol. Chem. 272, 22364–22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight Aβ (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer’s disease and for our understanding of fibril assembly are discussed.

Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate.

Fibrillogenesis of the amyloid β-protein (Aβ) is a seminal pathogenetic event in Alzheimer’s disease. Inhibiting fibrillogenesis is thus one approach toward disease therapy. Rational design of fibrillogenesis inhibitors requires elucidation of the stages and kinetics of Aβ fibrillogenesis. We report results of studies designed to examine the initial stages of Aβ oligomerization. Size exclusion chromatography, quasielastic light scattering spectroscopy, and electron microscopy were used to characterize fibrillogenesis intermediates. After dissolution in 0.1 m Tris-HCl, pH 7.4, and removal of pre-existent seeds, Aβ chromatographed almost exclusively as a single peak. The molecules composing the peak had average hydrodynamic radii of 1.8 ± 0.2 nm, consistent with the predicted size of dimeric Aβ. Over time, an additional peak, with a molecular weight >100,000, appeared. This peak contained predominantly curved fibrils, 6–8 nm in diameter and <200 nm in length, which we have termed “protofibrils.” The kinetics of protofibril formation and disappearance are consistent with protofibrils being intermediates in the evolution of amyloid fibers. Protofibrils appeared during the polymerization of Aβ-(1–40), Aβ-(1–42), and Aβ-(1–40)-Gln22, peptides associated with both sporadic and inherited forms of Alzheimer’s disease, suggesting that protofibril formation may be a general phenomenon in Aβ fibrillogenesis. If so, protofibrils could be attractive targets for fibrillogenesis inhibitors.

Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinsons disease and Alzheimers disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimers disease.

Structure–neurotoxicity relationships of amyloid β-protein oligomers

Amyloid β-protein (Aβ) oligomers may be the proximate neurotoxins in Alzheimers disease (AD). “Oligomer” is an ill-defined term because many kinds have been reported and they often exist in rapid equilibrium with monomers and higher-order assemblies. We report here results of studies in which specific oligomers have been stabilized structurally, fractionated in pure form, and then studied by using a combination of CD spectroscopy, Thioflavin T fluorescence, EM, atomic force microscopy (AFM), and neurotoxicity assays. Aβ monomers were largely unstructured, but oligomers exhibited order-dependent increases in β-sheet content. EM and AFM data suggest that dimerization and subsequent monomer addition are processes in which significant and asymmetric monomer conformational changes occur. Oligomer secondary structure and order correlated directly with fibril nucleation activity. Neurotoxic activity increased disproportionately (order dependence >1) with oligomer order. The structure–activity correlations reported here significantly extend our understanding of the conformational dynamics, structure, and relative toxicity of pure Aβ oligomers of specific order.

Presenilin‐dependent γ‐secretase processing of β‐amyloid precursor protein at a site corresponding to the S3 cleavage of Notch

The presenilin (PS)‐dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar γ‐secretase cleavage of the β‐amyloid precursor protein (βAPP) results in the secretion of amyloid β‐peptide (Aβ). However, little is known about the corresponding C‐terminal cleavage product (CTFγ). We have now identified CTFγ in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFγ is facilitated by PSs, since a dominant‐negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, γ‐secretase inhibitors, including one that is known to bind to PS, also block CTFγ generation. Sequence analysis revealed that CTFγ is produced by a novel γ‐secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.

Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nigriscans

Munumbicins A, B, C and D are newly described antibiotics with a wide spectrum of activity against many human as well as plant pathogenic fungi and bacteria, and a Plasmodium sp. These compounds were obtained from Streptomyces NRRL 3052, which is endophytic in the medicinal plant snakevine (Kennedia nigriscans), native to the Northern Territory of Australia. This endophyte was cultured, the broth was extracted with an organic solvent and the contents of the residue were purified by bioassay-guided HPLC. The major components were four functionalized peptides with masses of 1269.6, 1298.5, 1312.5 and 1326.5 Da. Numerous other related compounds possessing bioactivity, with differing masses, were also present in the culture broth extract in lower quantities. With few exceptions, the peptide portion of each component contained only the common amino acids threonine, aspartic acid (or asparagine), glutamic acid (or glutamine), valine and proline, in varying ratios. The munumbicins possessed widely differing biological activities depending upon the target organism. For instance, munumbicin B had an MIC of 2.5 microg x ml(-1) against a methicillin-resistant strain of Staphylococcus aureus, whereas munumbicin A was not active against this organism. In general, the munumbicins demonstrated activity against Gram-positive bacteria such as Bacillus anthracis and multidrug-resistant Mycobacterium tuberculosis. However, the most impressive biological activity of any of the munumbicins was that of munumbicin D against the malarial parasite Plasmodium falciparum, having an IC(50) of 4.5+/-0.07 ng x ml(-1). This report also describes the potential of the munumbicins in medicine and agriculture.

Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina

A unique lipopeptide antimycotic, termed cryptocandin, is described from Cryptosporiopsis cf. quercina, an endophytic fungus. Cryptocandin, with a molecular mass of 1079 Da, contains equimolar amounts of 3,4-dihydroxyhomotyrosine, 4-hydroxyproline, threonine, glutamine, 3-hydroxy-4-hydroxymethylproline, 4,5-dihydroxyornithine and palmitic acid. Cryptocandin is chemically related to well-known antimycotics, the echinocandins and pneumocandins, which are produced by such fungi as Zalerion arboricola, Pezicula spp. and Aspergillus spp. Cryptocandin has minimal inhibitory concentration values of 0.03-0.07 microgram ml-1 against isolates of Candida albicans, Trichophyton mentagrophytes and Trichophyton rubrum. Cryptocandin is also active against a number of plant-pathogenic fungi including Sclerotinia sclerotiorum and Botrytis cinerea.

Effects of Grape Seed-derived Polyphenols on Amyloid β-Protein Self-assembly and Cytotoxicity

Epidemiological evidence suggests that moderate consumption of red wine reduces the incidence of Alzheimer disease (AD). To study the protective effects of red wine, experiments recently were executed in the Tg2576 mouse model of AD. These studies showed that a commercially available grape seed polyphenolic extract, MegaNatural-AZ (MN), significantly attenuated AD-type cognitive deterioration and reduced cerebral amyloid deposition (Wang, J., Ho, L., Zhao, W., Ono, K., Rosensweig, C., Chen, L., Humala, N., Teplow, D. B., and Pasinetti, G. M. (2008) J. Neurosci. 28, 6388–6392). To elucidate the mechanistic bases for these observations, here we used CD spectroscopy, photo-induced cross-linking of unmodified proteins, thioflavin T fluorescence, size exclusion chromatography, and electron microscopy to examine the effects of MN on the assembly of the two predominant disease-related amyloid β-protein alloforms, Aβ40 and Aβ42. We also examined the effects of MN on Aβ-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide metabolism and lactate dehydrogenase activity in Aβ-treated, differentiated pheochromocytoma (PC12) cells. Initial studies revealed that MN blocked Aβ fibril formation. Subsequent evaluation of the assembly stage specificity of the effect showed that MN was able to inhibit protofibril formation, pre-protofibrillar oligomerization, and initial coil → α-helix/β-sheet secondary structure transitions. Importantly, MN had protective effects in assays of cytotoxicity in which MN was mixed with Aβ prior to peptide assembly or following assembly and just prior to peptide addition to cells. These data suggest that MN is worthy of consideration as a therapeutic agent for AD.

C-terminal peptides coassemble into Aβ42 oligomers and protect neurons against Aβ42-induced neurotoxicity

Alzheimers disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of Aβ42 are believed to be the main cause of AD; therefore, disruption of Aβ oligomerization is a promising approach for developing therapeutics for AD. Formation of Aβ42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of Aβ42 may get incorporated into oligomers of Aβ42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using Aβ fragments with the general formula Aβ(x−42) (x = 28–39). A cell viability screen identified Aβ(31–42) as the most potent inhibitor. In addition, the shortest peptide, Aβ(39–42), also had high activity. Both Aβ(31–42) and Aβ(39–42) inhibited Aβ-induced cell death and rescued disruption of synaptic activity by Aβ42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of Aβ42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with Aβ42 to form heterooligomers. Thus, Aβ(31–42) and Aβ(39–42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure–activity approach.

A γ-secretase-like intramembrane cleavage of TNFα by the GxGD aspartyl protease SPPL2b

γ-secretase and signal peptide peptidase (SPP) are unusual GxGD aspartyl proteases, which mediate intramembrane proteolysis. In addition to SPP, a family of SPP-like proteins (SPPLs) of unknown function has been identified. We demonstrate that SPPL2b utilizes multiple intramembrane cleavages to liberate the intracellular domain of tumor necrosis factor α (TNFα) into the cytosol and the carboxy-terminal counterpart into the extracellular space. These findings suggest common principles for regulated intramembrane proteolysis by GxGD aspartyl proteases.