Proteolysis in the formation of amyloid fibrils in Alzheimer's disease

Proteolysis in the formation of amyloid fibrils in Alzheimer's disease

300 NEUROBIOLOGYOF AGING. VOLUME ]i. 19~¢!J ABSTRACTS OF SE(?OND INTERNATIONALCONFERENCEON ALZHEIMER'S DISEASE BRAIN AMYI.OIDOSIS an accumulation of...

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300

NEUROBIOLOGYOF AGING. VOLUME ]i. 19~¢!J ABSTRACTS OF SE(?OND INTERNATIONALCONFERENCEON ALZHEIMER'S DISEASE BRAIN AMYI.OIDOSIS

an accumulation of the abnormal metabolites in the cytoplasm in the form of inclusions such as those found in lipidoses, dissimilar to the extracellular amyloid f i b r i l s . Someadditional factors which result in an immunological response must also be considered.

194 PROTEOL¥SIS

OF AMYLOID FIBRILS P. Mehlhaff. University of California, San Diego, School of Medicine, Department of Pathology, La Jolla, California 92093 USA. The formation of amyloid fibrils can occur by a variety of potential physicochemical mechanisms. The first mechanism described (Glenner et al., Science 174:712, 1971) was by proteolysis of an amyloidogenic precursor e.g. an immunoglobulin light polypeptide chain or Bence Jones protein. The two ma~or sites of amyloid fibril deposition in Alzheimer's disease are the cerebral vaaculature and the plaque cores. The 4.2 kd B protein (~P) (Glenner and Wong, Biochem. Biophys. Res. C o ~ u n . 120:885, 1984) comprises the amyloid fibril protein in both of these sites and derives apparently from the 79 kd B protein gene product (BPP) presumably by proteolysis. From current available amino acid sequence data the Nterminus of the 8P results from proteolysis of the methlonyl-aepartyl bond (residues 596 and 597 of the BPP}. Since BPP is a normally occurring protein (serum or tissue), it is assumed that creation of amyloid fibrils in normal individuals is prevented i) by prior proteolytic cleavage within the 8P region (SPP residues 597-638) or 2) by inhibition of Met-Asp (residues 596 and 597) proteolysis. Conversely amyloid fibril formation may be caused 1) by enzyme induction of Met-Asp proteolysis or 2) by inhibition of proteolytic cleavage of the internal BP sequence of BPP. The possibility that the site of cleavage to form the C-terminus of the 5P (residues 635 to 639 of BPP) is crucial in amyloidogenesis is also apparent. These possibilities lead to the conclusion that, if Met-Asp proteolytic cleavage of BPP in an Alzheimer's patient can be inhibited, formation of amyloid fibrils in cerebral vessels and plaque cores could be thwarted. This also provides a potential therapeutic approach to Alzheimer's disease. IN ALZHEIMERtS

IN THE FORMATION

DISEASE.

*G.G.

Glenner,

195 Co-occurrence of two amylold proteins, geisolln and beta-protein, in a patient with familial emy#oidoele, Rnnish Type, and Alzhelmeds disuse *B. Frangtone, M. Haltis, J. Ghlso, S. Kluru and F. Pr~lt. Department of Pathology, NY Unlverldty Medical Center, U.S.A., Department of Pathology and Neurology, Unlvertd~ of Heislnkl, Heisintd, Finland F!~

arid ~

~

Finni~t type, is clinically characterized by cranial neuropethy

corneal dystrophy. It Is an autosomal dominant form of systlmlatlc

arnyloidosls wlk'h small deposits of congophllic material occurring In rno~ tissues, In ~ with blood vessel wails and basement ~ , Including the petneur~ sheett~ of cranial and peripheral nerves. Am~o~ fllatls

weremaraotedImmtheIddneyofe pattentVUO,and rab~ antiaerumrabedagainst a 12 kOa pur~d amyloidsubun#dlst~ayedstrong immunohtstochemlCeireactivity w~h the ~

depots.

The andrm terminal sequence of the amyloid protein to poeiUon 15 (ATEVPVSWESTFNNGD) showed homology with gdsolin, • 93 kDa plasma ixotsln. GidloUlt, a ~ r a g u l a t s d actin-moduisting protein, is found as both an Intrinsic cytol01asndcprotein and as a secreted i~,~mmaprotein. Tha low molecular we~nt of the ~ ~ protein In relation to geisolin Indtcatee that the emylold peptide is a degladatlon product, starting at position 173 of the mature g~solin molecule, as deo~¢ed by the mRNA Patient VUO had ~ 1 Abdleimer's Disease, and i m m u ~ studies revealed neuritic pklques and neuroflbrhry tangles reacting with to tNli,emyleld I~NMn end paired helical filaments. These ~udlm show film two distln~ types of Inherited amyloidosls can coexist In a patient although the relationship Is stilt unclear.

196 MUTATION IN THE A L Z H E I M E R ' S D I S E A S E AMYIA)ID G E N E IN PATIENTS WITH H ~ I E D I T A R Y CEREBRAL H E M O R R H A G E WITH AMYLOIDOSIS DUTCH TYPE

*E. Levy,] M.D. Carman, 2 I.J. Fernandez-Madrid, 1 I. Lieberburg, 2 M.D. Power,2 S.G. Van Duinen,3 G.Th.A.M. Bots,3 W. Luyendijk,3 and B. Frangione. 1 1New York Univ. Medical Center, New York, NY, 2Athena Neurosciences, So. San Francisco, CA. 3Univ. Medical Center, Leiden, The Netherlands Hereditary cerebral hemorrhage with amyloidosis, of Dutch type (HCHWA-D), is a distinct autosomal dominant form of familial amyloidosis with predominantly cerebrovascular involvement. The amyloid protein that precipitates in the cerebral vessel walls of HCHWA-D patients contains 39 residues and is similar to the amyloid protein described in vessel walls (beta-protein) and senile plaques (A4) in brains of Alzheimer's disease, Down's syndrome and sporadic cerebral amyloid angiopathy patients. The cloning and sequencing of the two exons encoding the amyloid protein from brain tissue of two H C H W A - D patients reveal a mutation (C instead of G), causing a single amino acid substitution (GLN instead of GLU) at position 22 of the amyloid protein. The normal and the mutated genes, present in both patients, were cloned and sequenced from isolated genomic DNA. The base substitution abolishes an Mbo II restriction site, creating a restriction fragment length polymorphism (RFLP) marker for the disease. The mutation may account for the unique deposition of the amyloid protein in the cerebral vessel walls of HCHWA-D patients, leading to cerebral hemorrhages and early death. These findings can be used to establish a diagnostic assay in high risk populations and for prenatal evaluation. 197 SOLUBLE DERIVATIVES OF THE 8 AMYLOID PROTEIN PRECURSOR IN CEREBROSPINAL FLUID ARE ALTERED IN NORMAL AGING AND TO A GREATER EXTENT IN ALZHEIMER'S DISEASE M.R. Palmert, M.L. Cohen, V. Frazzini, M. Usiak, R. Mayeux, M. Raskind, W. Tourtellotte, and *S.G. Younkin. Case Western Reserve University, Cleveland, OH; Columbia University, New York, NY; University of Washington, Seattle, WA; and VAMC Wadsworth, Los Angeles, CA. Using antisera to peptides in the 8 amyloid protein precursor (BAPP), we have identified soluble ~125, ~105, and ~25 kDa 8APP derivatives that lack the carboxyl terminus of the full-length, membrane* associated forms. The ~125 kDa form contains the Kunitz protease inhibitor domain, but the ~105 and ~25 kDa proteins lack this insert. All three forms are readily detected in human CSF. By purifying the three soluble BAPP derivatives from human cerebrospinal fluid (CSF) and sequencing their amino termini, we have established that each has the amino terminus predicted from 8APP cDNAs after removal of a 17 amino acid signal sequence. We are currently adapting our purification procedure to isolate these proteins from human brain tissue in order to obtain the large amounts of soluble 8APP derivatives necessary for further analyses. In addition, we are using an expanded set of antisera to search for 8APP derivatives that could have gone undetected in our initial assessment. We have recently shown that both the -125 and ~105 kDa 8APP derivatives are specifically labeled by several antisera to the 8 amyloid protein (SAP). This finding indicates that both the KPI-containing (~125 kDa) form and the KPI-free (~105 kDa) forms contain all or part of the 6AP sequence. Moreover it suggests that generation of the SAP [which deposits as amyloid in Alzheimer'S disease (AD)] could be driven by relatively subtle changes in the proteolytic events that produce or process soluble 8APP derivatives. To evaluate i3APP processing in AD, we have quantitated the ~125, ~105, and -25 kDa amino-terminal derivatives in CSF samples from 24 AD patients and 12 controls. Our analysis shows (i) that, in AD, there is a significant decrease in the relative amount of the ~105 kDa form and a corresponding significant increase in the relative amount of the ~25 kDa form, (ii) that these changes correlate with the mental status of the AD patients, and (iii) that the same changes occur to a lesser extent in elderly as compared with young control patients. These observations indicate that 8APP processing changes in normal individuals as they age and to a greater extent in those who develop AD. The changes in i3APP derivatives that we have observed in CSF could have far reaching implications because they can be measured in living patients and may reflect fundamental mechanisms responsible for amyloid deposition.

198 CLONING PEPTIDE

AND EXPRESSIOH O F A SECR~'IU~ A 4 A M Y L O I D PRECURSOR. *J.N. OCTAVE, F. d e SAUVAGE, E.