Denaturation improves the abilities of some globular proteins to inhibit Aβ fibrillogenesis

Poster Prrsem~tion:

Alzheimer’,s

Diseme

and Related

Dementias

Sll

II

Thir depressed transmission is nomnhred if infected slices are incubated in the presence of high Mg (which, as indicated above, reduce the secretion of AP from infected cells). We are testing if secretion of AP is responsible for these observation by exprasing various different APP constructs that are known to affect APP processing in specific manners. These results uggest that therapeutic perturbation of Af3 secretion may have w-desirable effect\.

Poster Presentation: Dementias II A DE

NOVO

LOID

FIBRILS

Alzheimer’s

DESIGNED WHICH

Disease

a-HELICAL ARE

NOT

and Related

PEPTIDE

FORMS

Although this treatment had no effect on HSA’s activity, it enhanced the inhibitory potency of BSA and ovalbumin (l&fold and 30.fold decreases in the IC5O values. respectively). Thus, only 50 nM of modified ovalbumin is required to inhibit fibril extension by 50%. As expected, reductton and alkylation of these proteins affects their intrinsic fluorescence. Surprisingly. these modified proteins exhibit a response in the thioflavin ashy which is similar to, but not identical with, the response of amyloid fib&. This suggests that formation of amyloid-like, aggregrated structure may play a role in the inhibitory activities of these den&orated proteins. Tryptic digestion of these denatured proteins led to a complete loss of their inhibitory activities, suggesting that the enhanced inhibitory activity that follows reduction and alkylation is not due to the exposure of linear sequence neo-epitopea. Taken together, these results suggest that the inhibition of fibril extension exhibited by some proteins may be due to generic element? of cecondary or tertiary structure that can he found in both native and non-native proteins.

AMY-

NEUROTOXIC

134pl CHOLINESTERASE TENSIVE EPISODES INSTABILITY

Amyloldwes arc protem depwtwn dlwaws in which otherww soluble proteins or peptide? deposit in tissues in the form of insoluble amyloid fib&. Recent studies have shown that ostensibly nonwmyloldogenic proteins, under certain non-physiologic conditions, form tibrils. We report here that ata, a monomeric u-h&al de now designed peptide, forms rigid, non-branching fibrils 6.10 nm in diameter and of indefinite length, when incubated at 37°C at neutral pH. The assembly process occurs in concert with an a-helix to p-\heet conformattonal change. Surprisingly, unlike Abfibrils, utafibrils are not toxic to cultured rat neurons or PC12 cells. Our results suggest that the potential to form fibrila is not limited to those proteins associated with amyloldoses and that fibril formation alone is not predictive of neurotoxic activity.

IMPROVED METHOD OF PREPARING 1347) AN B-PROTEIN FOR FIBRlLLOGENESIS AND

THE

AMYLOID

NEUROTOXICITY

EXPERIMENTS

Synthetic amyloid P-protein (AB) IS used wdely to study fibril formation and the phyriologic effects of low molecular weight and fibrillar forms of the peptide on cells in culture or in experimental anm~al~. Not infrequently, conflicting results have arisen in these studies, in part due to variation in the starting conformation and assembly state of AP. To awid thew problems. we sought a simple. reliable means of preparing APfor experimental use. WC found that salvation of synthetic peptide with sodium hydroxide (AbXNnOH), followed by lyophdization, produced stocks with superior solubility and fibrillogenesis characteristics. Solubilization of the pretreated material with neutral buffers resulted in a pH transition from - IO.5 to neutral, avoiding the isoelectric point of AP(pl--5.5). at which Abprecipitation and aggregation propensity are maximal. Relatwe to tnfluoroacctate (AOXTFA) or hydrochloric acid (ABXHCI) salts of AP, yields of “low molecular weight Ap’ (monomers and/or dimera) were improved aigniiicantly by NaOH pretreatment. Time-dependent changes in circular dichroism spectra and Congo red dye-binding showed that APXNaOH formed fib& more readily than did the other Appreparatiow and that these fibrils were equally neurotoxic. NaOH pretreatment thus offer\ advantages for the preparation of Aafor biophyaical and physiologic studies.

Brian

DENATURATION

IMPROVES

ULAR

TO INHIBIT

O’Nualloin,

PROTEINS Ronuld

We/x/,

UT Med

THE ABILITIES

OF SOME

GLOB-

AB FIBRILLOGENESIS Cm, Knoxvillr,

TN

We have characterized the bass of the inhibitory activities of native and denatured serum albumms (BSA and HSA) and xrpina (anti-trypsm and ovalbumin) on the rxtenswn phax of AP (l-40) tibtil growth. The native proteins inhibit tibril extension with \mlilar potencies, exhibiting ICSO values for inhibition in the 0.35-1.66 pM range. HSA and antitryphin are about 3.fold more potent inhibitors than BSA and ovalbumin. Although the inhibltors decrease the rate of fibril extension, they do not alter the kinetic order (first-order) of the reaction. The inhibitors are not able to disagregrate Ap fib&. Thmflavin T fluorescence and HPLC solublhty analyses were used to momtor inhibition. To understand the molecular propenn that govern the inhibitory ectivitie\ of BSA. HSA. and ovalbumin, we disrupted their native three-dimensional \tructwes by rcducmg and alkylatlng their disulfide bond\.

INHIBITORS RELATED

IN PEOPLE

WITH

MAY

EXACERBATE

HYPO-

TO NEUROCARDIOVASCULAR DEMENTIA

OBJECTIVE: To determine the frequency of orthostatic hypotension (OH) and carotid sinuh hypersensitivity (CSH) in people with dementia. and to evaluate whether they are exacerbated by cholinesterase inhibitor therapy. BACKGROUND: OH and CSH are referred to generically as Neurocardiovascular Instability (NCVI. Preliminary data indicate that NCVI have a high frequency m both Alzheimer’$ disease (AD) and Dementia with Lewy bodice (DLB). This is important as NCVI is a major cause of syncope and falls in the elderly, with theoretical reasons to anticipate exacerbation with cholinesteraae therapy in dementia. DESIGN/METHODS: Consecutive patients from a dementia case regtster received a standardized evaluation, with operationalized climcal diagnoses of DLB and probable AD (50 cases have come to post-mortem with positive predictive value\ of 92% for DLB and 80% for AD). Patients had a 12 lead ECG, phaaic blood pressure and heart rate measurements during 2 minutes of active standing and during carotid sinus maarage. OH and CSH were diagnowzd according to established criteria. A sub-group of 20 patients were enrolled in a 6 week pharmacological challenge wth doneperil (5mg od for I week, IOmg od for 5 weeks). RESULTS: Forty two DLB patients (age 75.7, 48% male, MMSE 16.3) and 54AD p[atienta (age 80, 33% male, MMSE 17.2) were assessed. Overall 35 (83%) DLB patients and 35 (65%) AD patients (c;hi sq 4. I p=O.O4) had at least one form of NCVI (AD: OH 37% CSH 51% DLB: OH 48%. CSH 58%). NCVI worsened significantly wth donepeztl treatment (wilcoxon r=2.4 p=O.02, with 70% of people developing nw NCVI. Four falls were experienced by 3 patients. all during doneperil treatment and all xwciated with clinically significant changes in NCVI. CONCLUSIONS: NCVI i\ common in dementia patients. Cholmesterase inhibitors worsen NCVI, with potentially important implicatmns in the clinic. Further work needs to evaluate the mechanism\ and determine the clinical signiticance of the observed changes