Amyloid: Int. J. Exp. Clin. Invest. 4, 233-239 (1997)

Hypothesis: p amyloid precursor protein is a key sorting and targeting receptor for neuropeptidases Richard E. Finel and Carmela R. Abraham2

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Department of Biochemistry, Boston University School of Medicine, Boston, MA 021 18, ENR VA Hospital, Bedford, MA 01730, USA Departments of Biochemistry and Medicine, Boston University School of Medicine, Boston, MA 021 18, USA

KEY WORDS: Alzheimer 5 diseuse; umyloid P; secretase; synaptic transmission, neuropeptides, protease

ABBREVIATIONS: AP = amyloid beta; APPP = p amyloid precursor protein; AD = Alzheimer’s disease; APPLP2 = P-amyloidprecursor- like protein 2; ApoE = upolipoprotein E; ER = endoplasmic reticulum; KPI = Kunitz protease inhibitor; CNS = central nervous system; LRP = lipoprotein receptor related protein

Abstract

A testable hypothesis, that the P-amyloid precursor protein (APPP) is a key sorting and targeting receptor for neuropeptidases and other proteases in the secretory sys- tem, is developed. After delivery of these neuropeptidases to the synapse, ApPP is cleaved by an extracellular pro- tease called an a-secretase which generates a non- amyloidogenic C-terminal fragment. This cleavage which occurs after depolarization of the presynaptic membrane and concomitant neurotransmitter release, frees the A pPP bound peptidases to hydrolyse neuropeptides, analogous to acetylcholine hydrolysis by acetylcholinesterase. Some of these bound neuropeptidases with p-secretase activity can also cleave A PPP to generate a C-terminal amyloidogenic fragment which can then be processed further to generate amyloid beta, a key pathogenic molecule in Alzheimer S disease. We discuss possible explanations for why mutations in APPP itselj presenilins 1 and 2 or apolipoprotein E can either cause or produce high risk fo r Alzheimer S disease; in light of our hypothesis.

hile it is now certain that amyloid beta (A@), a metabolite of the P amyloid precursor protein

W ( A P P P 1 and the major protein component of amy- loid plaques is a key to the pathogenesis of Alzheimer’s disease (AD), it is not clear what the role of ApPP is in the normal myelinated projection neuron, the class of cell that is predominantly affected in this disease. In this article, we propose a novel function for this protein which also may provide insight into the pathogenesis of AD. We also dis- cuss how other proteins implicated in the pathogenesis of AD might interact with APPP.

A variety of studies have indicated that APPP is expressed to a significant extent in projection neurons in the cortex and other regions of the central nervous system (CNS) including the retinal ganglion cells, the only output cells of the retina whose axons comprise the optic nerve1. Neurons predominantly express the smallest isoforrn of APPP, APPP,,5 which lacks the so-called Kunitz protease inhibitor (KPI) domain, a domain which can specifically bind to and inhibit a variety of serine protease?. Glial cells of the CNS also produce and release APPP, but they produce mostly the KPI containing isoforms, APPP,,, , and APPP,,,, as well as a small quantity of APPP,,51.

Correspondence: Dr. Richard E. Fine, Department of Biochemistry, K- 124C, Boston University School of Medicine, 80 East Concord Street, Boston, MA02118, USA Tel: 617-638-4190 Fax: 617-638-5339 E-mail: fine@med-biochm.bu.edu

Submitted: November 26, 1996 Revision Accepted: April 24, 1997

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As part of our efforts to understand the sorting and transport of membrane proteins in projection neurons in vivo, we have carried out studies concerning the synthesis, axonal transport and processing of ApPP in the living adult rabbit retinal ganglion cells/optic n e r ~ e ’ ~ ~ . We have found that the APPP,,5 form of A p P P is synthesized in significant quantities in retinal ganglion cells and is transported down the axon in small transport vesicles via a kinesin mediated mechanism, APPP then fuses with the plasma membrane of the axon and the nerve terminal. It appears that ApPP is a relatively stable protein in these cells with a half life of - 12 h, when compared to tissue cultured cells in which the half life of ApPP is less than 1 h 4.

As shown by studies in which 35S-methionine, cysteine is injected into the rabbit vitreous, APPP,,, is synthesized in retinal ganglion cells and rapidly transported into the axons which make up the optic nerve’, Within 2.5 h after synthesis, both amyloidogenic (containing the AP sequence) and non amyloidogenic C-terminal fragments are generated’. At least a portion of these metabolites are generated in the transport vesicles while the other portion may be produced after arrival at the plasma membrane. The fact that we detect both amyloidogenic and non-amyloidogenic A p P P fragments, suggests that these neurons are a potential source of AP. We have in fact, recently detected AP,., in the optic nerve employing a sandwich ELISA assay (Fine, R. and Barten, D., unpublished result).

While we see that a significant quantity of ApPP is found in transport vesicles which enter the axon rapidly after synthesis in small intermediate density vesicles, a signifi- cant fraction of newly synthesized APPP is associated with a heavier density vesicular fraction that is only detectable in the axon beginning about 5 h after %-met, cys injection’ . This labeled peak persists in the axon for up to 24 h while the intermediate fraction disappears rapidly. Since we have previously shown that substance P containing vesicles are transported into the axon in vesicles with similar biochemi- cal and kinetic properties’, we postulated that a portion of ApPP is packaged in regulated substance P containing secretory vesicles and transported down the axon to the nerve terminals with “delayed export” kinetics.

Recently, however, evidence has been presented that ApPP is “transcytosed” in cultured hippocampal neurons6.’, and these groups have demonstrated that ApPP is initially axonally transported. Then, however, a portion of the ApPP molecules which have reached the axonal plasma membrane are internalized and retrogradely transported back to the cell body and dendrites. Presumably this process could be repeated through several cycles, although this was not shown in these studies.

In view of this evidence, we now feel that the most reasonable interpretation of our data in the in vivo optic nerve is that the high density peak of ApPP and its metabolites

represents a recycling retrograde population of endosomes which carries ApPP from the presynaptic endings and axonal plasma membrane back to the somatodendritic compartment. The fact that APPPpersists in the retinal membranes, which contain both the retinal ganglion cell bodies and dendrites, for more than 24 h after %-met, cys injection is in agreement with this interpretation’.

If ApPP is a recycling plasma membrane protein, what is its function? We would postulate that APPP, as well as other homologues including APPLPl and APPLP2, serve as sorting and transport receptors for peptidases and proteases in order to keep these proteins separate from their substrates, neuropeptides. By binding to these proteins in the Golgi [although it is possible that this interaction occurs even earlier in the secretory pathway i.e. in the endoplasmic reticulum (ER)], this interaction would accomplish two purposes; reversibly inactivating the peptidases until they are released from ApPP and separating them from their substrates which are packaged in “regulated secretory vesicles” [Figure 11. As we and others have demonstrated, ApPP does indeed appear to be segregated from synaptic vesicle markers’.*. Once the APPPand its bound peptidase(s) reach the synaptic plasma membrane, they stay bound until the nerve depolarizes and cytoplasmic Ca++ rises in the terminal, thereby triggering exocytosis [Figure 21. First, classical transmitter containing vesicles are released. Some examples of classical neurotransmitters are glutamate,

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FIGURE 1. Sorting of neuropeptides away from neuropeptidases via the latters’ interaction with ApPP. Sorting of the neuropeptides ( ) and neuropeptidases (I) via their interaction with ApPP ( I ) is shown here to occur in the trans Golgi network (TGN) although it may occur in an earlier Golgi compartment. The neuropeptides are postulated to be sorted into clathrin coated vesicles which become mature secretory granules. ApPP and the bound neuropeptidases are postulated to be sorted into non-clathrin coated vesicles which lose their coats to become constitutive secretory vesicles, fusing with the synaptic plasma membrane.

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acetylcholine and dopamine. The released neurotransmitter can act both on autoreceptors at the presynaptic ending and on postsynaptic receptors. Both receptor classes can activate an a-secretase activity. Many studies using tissue cultured cells have indicated that this activity is stimulated by

Pre

post

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FIGURE 2. APPP-neuropeptidase interactions at the synapse.

a) Upon depolarization of the presynaptic membrane resulting in Ca++ influx into the presynaptic cytoplasm, classical neurotransmitters (0) are released and interact with pre and postsynaptic receptors (Y). Increased cytosolic free Ca++ concomitantly activates a- secretase activities in both pre and post synaptic compartments.

b) The activated a-secretase cleaves APPP (I) thereby releasing the neuropeptidase (I).

c) If presynaptic Ca++ levels rise even higher resulting from a higher frequency of depolarization, neuropeptides are released. The unbound neuropeptidase molecules in the synaptic cleft can cleave neuropeptide molecules ( ) thereby inactivating them. Uncleaved neuropeptides can bind to their receptors (Y). Some uncleaved ApPP molecules still bound to their neuropeptidase are internalized via clathrin coated vesicles that form after synaptic vesicle exocytosis.

essentially all types of neurotransmitters, as well as cytokines, growth factors, etc9.I0. Recently it was shown using brain tissue slices that electrical stimulation or addi- tion of muscarinic acetylcholine agonists, stimulates ApPP cleavage via a-secretase activation".

The or-secretase(s) has not been isolated but it is known that its activation results in the cleavage of ApPP between residues 16 and 17 of the AP sequence and the release of a large N-terminal ApPP fragment (APPPs)". Besides pre- venting the accumulation of an amyloidogenic C- terminal fragment of APPP, we postulate that the cleavage of ApPP results in the release and/or activation of peptidases and pro- teases bound to APPP. These can now inactivate neuroac- tive peptides which are released if sufficient [Ca+*II is reached during neuronal depolarization. Thus, this system could be thought of as analogous to the mechanism by which synap- tic acetylcholinesterase hydrolyses acetylcholine. The hy- drolysed ApPP is presumably further metabolized to smaller peptides in the cleft, where they can serve as attachment and or growth f a c t o r ~ ~ g ~ ' ~ . ~ ~ , or APPPs is reinternalized via the low density lipoprotein receptor related protein".

If ApPP molecules and their bound peptidases are not hydrolysed quickly (probably within seconds after insertion in the plasma membrane), they are reinternalized via clathrin coated vesicles and recycled back to the cell Once they arrive at the cell body, they can be recycled back to the axon, targeted to lysosomes for degradation or transferred to a somatodendritic endosome to be transported to the somato-dendritic somatic plasma membrane6,'. Presumably, each ApPP molecule in a long projection neuron such as a rabbit retinal ganglion cell, can undergo several such cycles since it appears from our experiments that the lifetime of an ApPP molecule can be greater than 12 h'.3.

We have recently shown that several relatively stable metabolic products of ApPP are rapidly generated following synthesis in the retinal ganglion cell bodies and transport into the optic nerve3. At least a portion of these products are generated in secretory vesicles, while the remainder are pro- duced at the cell surface or following reuptake. One of these fragments appears amyloidogenic i.e. containing the whole AP sequence. These fragments disappear with similar ki- netics to those of full length ApPP itself.

What might be the mechanism by which the amyloidogenic C-terminal fragment is generated? One in- teresting possibility stems from the hypothesized function of ApPP as a sorting and transport receptor for neuropeptidases. This function requires the formation of a relatively tight but reversible interaction between ApPP and the molecules it carries [Figure 31. It is likely that this inter- action involves the active site of the enzyme. We envision that since the interaction between the two molecules is pro- longed, up to aday or more, occasionally the enzyme attacks ApPP and cleaves it. We would postulate that one or more

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of the neuropeptidases carried by APPP binds to the unique AP sequence and cleaves at or near the N-terminal amino acids, acting as a P-secretase. In fact one of us (CRA) has isolated several peptidases capable of generating the 99 aminoacid C- terminal AP containing fragment from APPP itself. One of these proteins is the human homologue of a well characterized rat metalloendopeptidase”. A second is a cysteine protease which in the presence of millimolar Ca++ forms a SDS stable complex with APPPI9. Whether or not this protein is a neuropeptidase has not been tested yet.

A third class of protease which could also be neuropeptidase and which can cleave APPP are serine pro- teases. Two members of this family which can cleave APPP and generate the C- terminal AP containing fragment have been characterized20.2’. It is possible that one or both of these molecules can bind the KPI domain on the 751 and 770

amino acid containing isoforms of APPP. It has been previ- ously shown that APPP,,, and AP,,, can reversibly bind serine proteases of both the trypsin and chymotrypsin classesz2.

While the amyloidogenic C-terminal fragment of ApPP appears to be generated fairly frequently by accidental cleav- age of ApPP by neuropeptidases with p secretase activity3, most of these C-terminal fragments are completely metabo- lized, presumably in lysosomes. However, based on cell cul- ture models, it is likely that AP is produced and secreted following the cleavage of a larger C-terminal fragment by the so-called gamma secretase. The compartment where this cleavage occurs is unknown, although it is likely to be either in the secretory compartment or in an endosome; not how- ever in a lysosomerev1n23.

The majority of the AP molecules produced are AP,, and are found in significant amounts in the cerebrospinz fluid and in much smaller quantities in the blood. They are presumably metabolized either in the aforementioned com- partments after uptake into cells, or extracellularly. 8.

C., 4

Pathogenesis of APPP metabolism leading to amyloid deposition and Alzheimer’s Disease

While under normal physiologic conditions, ApPP is completely metabolized; in AD, Down’s Syndrome and to a lesser extent in “normal” aging, AP is deposited in the extracellular spaces of the brain and in blood vessels. Based on what is known about the cell biology of ApPP metabolism together with recent identification of genes which cause or increase the risk of AD, we can make some educated guesses about the processes occurring in cells which lead to the development of AD.

1 ) Mutations in APPP itself. Mutations at either end of the AP sequence, as well as at residue 21 can produce early onset AD which segregates as an autosornal dominant dis- order. In the Swedish mutation, it has been shown that cells produce a much greater quantity of AP than do normal fi-

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7 - FIGURE 3. A possible mechanism for the generation of amyloidogenic C-terminal fragments of ApPP by a bound neuropeptidase with p-secretase activity. a) ApPP binds to a neuropeptidase in a tight but reversible manner. b) Avariety of agents which raise CaF including neurotransmitters, trophic peptides, cytokines, etc. stimulate the a-secretase, resulting in the cleavage of ApPP after amino 16 of the Ap peptide sequence. This cleavage liberates the neuropeptidase and prevents the formation of Ap. c) If ApPP is not cleaved while residing in the plasma membrane, it and its bound neuropeptidase are internalized into small vesicles and recycled to the cell body. d) Occasionally the neuropeptidase will cleave ApPP at the N-terminus of the Ap sequence, generating an amyloidogenic C-terminal fragment, ApPPc’. A subsequent enzymatic cleavage by a y secretase will generate Ap.

broblastsz4. In the 717 mutations, a similar amount of AP is produced, but a much higher percentage is the AP,,,,,, form which is much more amyloidogenic than the AP,, and makes up the major deposited species of AP”.

We could imagine that Down’s syndrome victims who invariably develop the neuropathology of AD if they live past 40, express higher quantities of APPP (a chromosome 21 gene). This leads to the production of more total AP which ultimately leads to AP deposition and nerve cell damage.

2) Mutations in multispanning transmembrane proteins called presenilins 1 and 2 found on chromosomes 14 and 1 respecti~ely~~*~’. These mutations also produce an early onset dominant form of AD. The proteins encoded by these two similar genes have unknown functions, but are similar

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to a receptor found in the nematode C. elegans which plays a key role in differentiation of the vulvazs. Fibroblasts from patients with mutant presenilin 1 or 2 produce a higher quan- tity of Ap,.,? than do normalsz9. We can postulate that these “receptors” in some way alter the intracellular trafficking of APPP leading to a higher conversion of ApPP to the amyloidogenic AP,-42.

Very recently, evidence that presenilin 1 appears to be mainly located in the ER and Golgi apparatus has been obtained’O. Since ApPP transits through the ER and Golgi during its passage to the axonal plasma membrane, and potentially during its recycling, may return to the Golgi as do other plasma membrane proteins in neuroendocrine cells3’, the two proteins could directly interact in these com- partmen ts.

3) We would predict that mutations in a, p and y secretases could also lead to AD via an increase in either total AP or in the more aggregating AP,-42. In this regard, the human metalloprotease analogue of the rat metalloendopeptidase E.C.3.4.24.15 which has p-secretase activity17, has been shown to be linked to the chromosome 19 locus associated with late onset AD3’. It is possible that mutations in this protein could lead to a higher P-secretase activity and the generation of more A@. Experiments to detect such mutations are now underway. Likewise, one could imagine that mutations leading to reduced a-secretase activity could also lead to a higher production of AP. Muta- tions in the y-secretase could lead to a greater percentage of cleavage after the 42”d residue of AP rather than after resi- due 40.

4) The apolipoprotein E4 allele. Linkage studies with families having late onset AD implicated a locus on chro- mosome 19. Further mapping demonstrated an association with the apolipoprotein E (ApoE) gene. This locus has been identified as a gene conferring a high risk of developing late onset AD if the E4 allele of the gene is present33. Two copies of E4 increase the risk for AD by more than 10 fold, one copy by 2-3 fold34. E4 is also associated with a greater risk for atherosclerosis and multiinfarct dementia as well as for less common diseases including Pick’s and Lewy body d i ~ e a s e ~ . g . ~ ~ . The E2 allele, on the other hand, appears slightly protective although, because of its rarity, the data so far are not totally convincing.

What is the molecular basis for the higher risk of people with the E4 allele for developing late onset AD? There appear to be several hypotheses, which are not mutually exclusive. The only relevant one for discussion here, is that there is a direct interaction between ApoE and AP leading to a greater aggregation, amyloid formation and neurotoxicity in the case of ApoE4 than E336.

There are several findings that suggest a physiological link between ApoE and APPP metabolism. A recent study has demonstrated that the low density lipoprotein receptor

related protein (LRP) which is found on neurons in the CNS, can internalize both ApoE containing lipoproteins and APPPs through binding to independent domains of LRP. This strongly suggests that after internalization by neurons, ApoE and ApPP reside in the same endosomal compartment. We have recently found that APPP and ApoE are present as well in kinetically and biochemically identical cellular compart- ments in the retinal ganglion axons in v ~ v o ~ ~ . It also appears that APPPs, the N-terminal ApPP fragment generated by a-secretase cleavage of ApPP, is present on ApoE contain- ing lipoprotein particles isolated from the rabbit vitreous (J. Shanmugaratnam and R.E. Fine, unpublished observation).

From these data, we can suggest that differential inter- actions between ApoE4 and ApoE3 and 2 and ApPP can produce different physiological and pathological effects on neurons. Several cell culture studies using peripheral neu- rons and neuronal cell lines have indicated that ApoE3 con- taining lipoproteins provide neurons with the ability to growth long neurites, while ApoE4 containing lipoproteins inhibit process growth3*.”. Microtubules appear to be much more stable when cultured with ApoE3 containing lipopro- teins than with ApoE4 containing lipoprotein^^^.

Because of differential interactions between ApoE3 or E4 and amyloidogenic ApPP C-terminal fragments in endosomal compartments where they both reside for pro- longed times, AP may be produced in greater amounts in neurons from 4 or 4/ 3 individuals. One “molecular” ex- planation for this interaction is that ApoE binding to the N- terminus of amyloidogenic C-terminal ApPP fragments solubilizes them. The solubilized fragments are now a sub- strate for gamma secretases in late endosomes and or lyso- somes. We would envision ApoE4 being a more effective solubilizer than ApoE3 or presenting amyloidogenic C-ter- minal fragments to gamma secretases such that they are cleaved between the 42-43 residue rather than between the 39-40 residues.

Conclusion

We have proposed a testable hypothesis, that ApPP functions as a sorting and targeting receptor for neuropeptidases and other “proteases” in the secretory path- way probably the Golgi andor ER. It binds these enzymes, both to reversibly inactivate them, separate them from their substrates, the neuropeptides, and to deliver them to the synapse. They are then released in order to hydrolyse the neuropeptides analogous to the manner by which acetyl- cholinesterase hydrolyses acetylcholine. This mechanism can explain the reason why ApPP is cleaved in response to depolarization and neurotransmitter release, as well as help- ing to understand why ApPP is cleaved specifically at the

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site which generates the amyloidogenic C- terminal peptide, the rate limiting step in the generation of the “pathogenic” A P molecule.

Acknowledgments

We acknowledge the expert secretarial assistance of Simona Zacarian. This work was supported by grants from the National Institute of Aging, R37AG-05894, P30AG- 13846 and R01AG-09905, the “Alzheimer’s Association/ F.M.Kirby Foundation (1996- 1997) Pilot Research Grant” and the Department of Veterans Affairs.

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