AMYLOID IN ALZHEIMER'S DISEASE

Amyloidosis

Amyloid deposits found in many organs having similar histological staining properties ie red-green birefringence with Congo red

This pictorial property because of a high content of B-structure

Amyloid proteins all fragments derived by proteolysis from a larger precursor protein

e.g. serum amyloid component immunoglobulin

The constituent proteins can be of different origins

Primary amyloidosis

When the amyloid deposition causes disease all or often due to a mutation in the precursor protein causing abnormal proteolytic processing

e.g. some cases of familial Alzheimer’s disease

hereditary cerebral haemorrhage with amyloidosis of Dutch type (HCHWA-D)

Secondary amyloidosis

When amyloid deposition is the result of other disease

e.g. other cases of FAD and sporadic AD

NFT are amyloid

Neurofibrillary tangles are also Congophilic and sometimes described as amyloid

This has led to much confusion about the relationship of NFT to extracellular amyloid but they are composed of different proteins

Preferable to reserve the term amyloid to describe the extracellular deposits of B-amyloid in plaque cores and blood vessels and use NFT and PHF to describe the intraneuronal and host tangle protein deposits which are, nevertheless, an amyloid according to their histochemical staining properties

Amyloid deposits also found in

· Normal old age

· Down's syndrome

· ALS-Parkinsonism-Dementia complex of Guam (diffuse plaques)

· Dementia pugilistica (diffuse plaques)

· .Hereditary cerebral haemonhage with amyloidosis of Dutch type (HCHWA-D) (vascular)

· .Sporadic cerebral angiopathy (vascular)

In aged animals of several other species, including non-human primates, polar bears, dogs; no evidence of tau pathology (Selkoe et al. 1987)

Scrapie -type plaques

Different from AD senile plaques

Found in the sporigiform encephalopathies or prion diseases:

· Creuttfeldt-Jakob disease (CJD)

· Kuru

· Gerstmarin~Straussler-Scheinken syndrome (GSS) .

· Scrapie

· Bovine spongiform encephalopathy (BSE)

Aluminium in plaque cores

Edwardson and colleagues isolated plaque cores stripped of protein (Candy et at., 1986) Remainder of the core is inorganic - Al, Si

Earlier studies claimed that tangle-bearing neurones in AD and ALS-Parkinsonism-Dementia complex of Guam also contain elevated levels of Al

Al is neurotoxic (e.g. dialysis encephalopathy)

But most Al is excreted and very little crosses the blood brain barrier

In plasma Al bound to transferrin and accumulates in rat brain in areas with highest levels of transferrin receptors (Pulien et at., 1990)

Epidemiological studies - small increased risk of. dementia in geographical areas with high Al in drinking water - studies are greatly criticised (Martyn et at., 1989)

Amyoid deposits

Gleririer and Wong (c. 1984) isolated amyloid from menirigeal blood vessels from AD brain obtained partial sequence of a protein that they called B-protein

Masters and Beyreuther and colleagues (c. 1985) isolated a 4,200 M protein from plaque cores - same partial sequence as the B-protein - called A₄-protein

Protein now often called B/A₄-amyloid protein, AB (Abeta), (or variants on this name)

AB is approximatety 3943 amino acids long but has ragged ends

Amyloid deposits are filamentous and insoluble in nondenaturing aqueous buffers

Dissolve in formic acid - used in the extraction and purification of AB

Synthetic peptides corresponding to residues 1-28 of AB form amyoid fibrils in vitro, thus AB determines structure of amyloid fibrils

Antibodies to synthetic peptides corresponding to different segments of the AB stain plaque cores and vascular deposits of amyloid

Treatment of sections with formic acid before staining with these abs reveals many "diffuse plaques" or "diffuse deposits"

diffuse plaques occur in the cerebellum as well as widely distributed throughout other areas of brain

Many intellectually intact brains have abundant diffuse plaques

Diffuse plaques are probably not damaging

Diffuse plaques are often presumed to be precursors of the classical plaque - but riot proven

Down's syndrome brains at around age 20 yr have diffuse deposits but no neuritic involvement nor NFT

Thus, AB deposition taken to be first manifestation of AD pathology (qooyama et at., 1990)

The amyloidal precursor protein (APP) - (Fig 1)

AB cDNA now cloned:

Part of a much larger precursor protein (105 130000 apparent M,)

Membrane glycoprotein (Kang et at 1987 Tanzi el at , 1987; Goidgaber et at., 1987; Ponte et at., 1988; Kitaguchi et al., 1988; Tarizi et at., 1988)

Alternative splicing of mRNA generates at least 8 alternative isoforms

Some have extra domains inserted on the estracellular side of the membrane

One extra domain homologous with Kunitz senne protease inhibitors (KPI)

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- a serine protease inhibitor

Another small 19 amino acid domain is homologous to MRC OX~2

Principal membrane-anchored forms are APP₆96 APP₇₁₄, APP₇₅1, APP₇₇0

These forms also exist lacking exon 15 between KPI and AB (Mo nnin.g. et al., 1992; Ohgami et al., 1993)

A secreted APP isoform with the KPI insert lacking the C-terminal 208 amino acids, including the AB sequence and transmembrane domain, this region is replaced by 20 different C-terminal residues (de-Sauvage and Octave, 1989)

APP has N- and 0-linked sugars and has 0-sulphated tyrosine

APP gene is located on chromosome 21

The APP gene partial sequence was published in 1990 (Yoshikai et al., 1990)

The gene has 18 exons

The KPI and MRC OX~2 domains are individual exons

AB is encoded across the boundary of exons 16 & 17

An AP-1 binding site, heat shock and cytokine regulatory element consensus sequences are in the 5'-prime noncoding region

TPA induces 3.7-fold increase in expression but heat shock only a 1.2-fold increase

Secreted APP is probably produced by alternative splicing of an exon located between exons 13 and 14 but not yet found

APP is a member of a family of homologous proteins

Other family members lack AB sequence

They are:

APL1 - human Wasco et a , 1992)

APL2 - human Wasco el al , 1993)

APPH - human (Sprecher et al., 1993)

APPL - drosophila - human APP rescues mutants (Luo et al., 1992)

Expression of App forms in brain and other tissues

Expression of APP₇14 is only very low (Golde et al 1990)

Brain Spleen Thyrnus Kidney Muscle Heart Liver

APP695 +++ (+) (+) (+) (+) (+) (+)

APP₇₅₁ +++ ++ + +++ + ++ +

APP₇₇₀ ++ ++ + +++ + ++ +

In brain, APP is made mainly in neurones

Because APP forms include forms with KPI, it has been suggested that the formation of the AB and hence amyloid deposits may result from an imbalance in the proportion of APP forms expressed in the brain, assuming that the KPI has a role in self regulating APP proteolysis

Down's cases develop amyloid deposits in their twenties

Down's have an extra copy of APP gene on the third copy of chromosome 21; this leads to elevated expression of APP and urn e et a , 1 8 )

Numerous studies of Northern blotting and in situ hybrid isation have attempted to measure levels of expression of different APP isoforms in different tissues and in different brain areas in control and AD cases

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Some reports claim the ratio of APP695/(APP₇₅₁ + APP₇₁₀) is:

- higher in AD brain regions affected by pathology

- other studies claim the ratio is lower when compared to control brain

There is agreement on some facts and apparent discrepancies on others

An apparently thorough study using PCR to measure the different mRNA species is that of Golde et al (1990); this paper cites most of the preceding studies

Allsop claims APP695 exclusively gives rise to the AB in brain and CSF because APP8 (see below) with N-terminal AB sequences in CSF & brain always contains KPI domain (Kametani et al., 1993)

Several trarisgenic mice with various constructs of APP have been reported but only one model expressing elevated APP₇₇₀ under a neuronal promoter is reliably accepted as exhibiting amyloid deposits (Quon et al., 1991). However, these deppslts are diffuse and not Congophilic and most closely resemble the diffuse deposits in human brain, i.e. not pathogenic

Transgenic mice with a truncated human APP construct (C-terminal 100 amino acids) starting at the N-terminus of the AB sequence and running through to the C-terminus of APP, were reported to have senile plaques and NFTs very similar tn those in AD (Kawabata et al., 1991). However, this report has now been retracted (Kawabata et al., 1992)

Peripheral deposition of amyloid in skin, subcutaneous tissue and intestine in AD has been claimed (Joachim et a)., 1989). This implies amyloid may be blood- rather than brainderived. But, so far, no confirmatory reports

The APP gene

On chromosome 21

Down's syndrome is trisomy chromosome 21

Down's after age 35yr have a pathology indistinguishable from AD

Thus, is the AD pathology in Down's due to a gene dosage effect?

Therefore, might familial AD be due to a mutation in a gene on chromosome 21 such that the effects are the same as an extra copy of this same gene in Down's?

Molecular genetic analysis of familial AD initially showed:

An AD gene on chromosome 21 but apparently not APP gene

Genetic heterogeneity of FAD ie there may be more than one AD gene on chromosome 21 and there may be more than one disease causing allele of a given gene (St George Hyslop et al 1990)

Sequence studies of APP gene has shown 3 different point mutations in the membrane spanning domain of APP, 2 amino

acids beyond the C-terminal cleavage srte for AB ri a few European and Japanese families (Chartier Harlin et al. , 1991;

Goate et al., 1991; Murrell. et al., 1991)

These mutations are at position 717 of the APP molecule (numbering of APP₇₇₀) and are changes of Val to lie, Gly or Phe

A double mutation of the two residues immediately preceding the N-terminus of AB within APP (Lys-Met to Arg Leu) has been found to segregate with AD in 2 Swedish pedigrees (Mullan et al., 1992)

The mutation in hereditary cerebral haemonhage with amyloidosis of Dutch type - HCHWAD

2 families in Holland

Affected individuals suffer fatal cerebral. haemorrhage in middle life and have massive deposits of AB in cerebral vessels but no classical plaques although some diffuse-like plaques

No NFT

No or few neu urological or psychiatric symptoms

Gene is now known to be the APP gene Van-Broeckhoven et al., 1990)

Mutation is within the AB sequence with Glu₂₂ (position 693 of APP₇₇₀) mutated to GIn

Another Dutch family with a point mutation in the preceding amino acid of Ala 21 to Val₂₁ (position 692) has been described

and this apparently produces a mixed AD and HCHWA~D pathology and dementia (Hendricks et al., 1992)

Metabolism of APP (Selkoe, 1993) (Fig 2)

In cultured cells, APP has a half-life of approx 20-30 min (Weidemarin et al., 1989)

Some APP is processed such that the major part of the extracellular domain is secreted -

al. 1990; Wang et al. 1991)

In cultured neurones, some APP present in clathrincoated vesicles and late endosomes (Ferreira et al., 1993)

Sisodia et al (1990; 1991; 1992) and Esch et al (1990) have shown that in cultured cells, APP is cleaved wlthin the AB sequence