Pharmacological studies reveal APP and Aβ have interactions with glutamate and calcium, cytokines, copper/zinc chelators, secretases and presenilins, nicotinic receptors, acetycholinesterase, neurotrophins, non-steroidal anti-inflame-matory drugs, monoclonal antibodies to Aβ, protease inhibitors, oestrogen, homocysteine, immediate early genes such as c-fos or c-jun and cholesterol. These functional and pharmacological observations highlight the need for greater understanding of APP and Aβ in brain function and have implications for clinical trials.
Glutamate activates ion channel family receptors and G protein coupled receptors which modulate excitatory synaptic transmission through transduction pathways [
The glutamatergic system has been implicated in the pathogenesis of AD through an interaction which enhances the neurotoxicity of the amyloidogenic fragment Aβ of the APP gene [
The amyloidogenic Aβ 1-42, the amyloidogenic peptide product of the amyloid precursor protein damages and kills neurons possibly through an effect on the membrane lipid peroxidation, impaired ion-motive ATPases, glucose and glutamate transporters making nerve cells vulnerable to the excitotoxic effects of glutamate [
Soluble forms of the amyloid precursor protein may inhibit the damaging effects of presenilin 1 mutations by influencing the effects of NF-kappa-B and calcium homeostasis induced by these mutations [
Apolipoprotein E, a risk factor for AD, might increase intracellular calcium and through the NMDA mechanism might damage neurons and encourage calcium influx [
Two studies have reported that NMDA receptors are absent or decreased from cortical and hippocampal regions in AD [16,17]. Other investigations suggest that the degree of cell loss is the reason for this receptor reduction [18,19]. The binding of L-3H-glutamate to the NMDA receptor on synaptic membranes from the hippocampus, fronto-temporal cortex and parietal cortex was unchanged in comparison to the reduction achieved in D-3H-aspartate binding [
Studies have investigated the expression of glutamate receptor genes using in situ hybridisation in human disorders and experimental animal models. In a transgenic mouse model carrying the mutated APP gene (the Swedish double mutation) there was no significant change in NR1 mRNA or autoradiographic receptor binding in the hippocampus and other brain regions [
There is intriguing molecular diversity within the NMDA receptor system which implies that therapy for neurological disorders might be directed towards specific receptors in localized brain regions in cells involving specific heteromers of the NMDA receptor [
Amyloid precursor-like protein 2 (APLP2) belongs to the same family of proteins as APP. APLP2 expression was observed in rat cortical neurones after treatment with glutamate. Lactate dehydrogenase was present in the medium which is indicative of neuronal damage but APLP2 expression was diminished. These observations were not seen with N-methyl-D-aspartate receptor antagonist pre-treatment (MK-801) [
In vitro, glutamate receptors promote the nonamyloidogenic APP processing pathway (sAPP). In vivo, intrahippocampal injection of guinea pigs with mGluR agonist 1S, 3R-ACPD resulted in CA1 neurodegeneration. Intraneuronal granules were found in degenerating neurons of the hippocampus after immunocytochemistry with Aβ antibodies. Guinea pigs injected with NMDA displayed neurodegeneration with immunoreactivity to Aβ [
The possibility that modification of the glutametegic system might be useful in AD has been supported by the beneficial effects of memantine, a non-competitive NMDA antagonist which leads to functional improvement, reduces dependence and clinical deterioration in patients with moderate to severe AD [27,28].
A number of in vitro studies have suggested an interaction between interleukin 1 (IL-1) and APP. IL-1 has been shown to stimulate the APP promoter [
The transition metal ions Cu2+, Zn2+ and Fe3+, are found at elevated levels in the neocortex of AD brains and in higher concentrations within the amyloid plaques [
Treatment with metal chelators induced Aβ aggregates in vitro resulting in the solubilization of Aβ [39,40]. Aβ was also solubilized by metal chelators in post-mortem brain tissue [
The specific reduction of APP-bound copper (II) to copper (I) by APP [
Presenilin has homology to Notch genes. Notch genes are involved in intracellular signalling and development, and may have important roles in the physiological regulation of differentiation within the haemopoietic system— functional properties which may limit the development of compounds which antagonize the actions of presenilin proteins. Mutagenesis experiments of two transmembrane aspartates in PS1 and PS2 abrogate γ-secretase activity and the production of Aβ, suggesting that aspartate sites are critical in the proteolytic cleavage of APP [
There may be a stoichometric interaction between APP and presenilin as both of these proteins form complexes with each other in living cells [53,54]. There may also be an interaction between these proteins at the cell surface which may be important in cell-cell adhesion and signalling which might activate tyrosine kinase [
β-secretase activity could be targeted therapeutically in AD, such that its inhibition would lead to decreased levels of Aβ. A study of 61 AD patients and 33 controls measured the cleavage of APP β-amyloid fragment by β-secretase (BACE) in frontal temporal and cerebellar regions [
Cholinergic activity is known to be hypoactive in most regions of an AD brain and substances which inhibit acetylcholinesterase (AChE) are now used as a treatment for mild to moderate AD [57-63]. AchE expression is higher in and around neuritic plaques of the AD brain. Beta-actin promoter was used in transgenic mouse brain to increase the level of the APP C-terminal 100 amino fragment (APP CT100). APP CT100 and Aβ levels were increased in the brain along with AchE isoforms. AchE was shown to increase with increasing Aβ [
Cleavage of APP can result in either soluble APP or insoluble APP (Aβ-component of neuritic plaques). It is thought that the pathogenesis of AD results from low levels of soluble APP and protein kinase C (PKC). One study with primary cultures rat basal forebrain found that AChE inhibitors (ambenonium and metrifonate) increased PKC levels and cell-associated APP levels in cells and in the medium. The increase in PKC levels and cell-associated APP levels results in an increase in α-secretase activity resulting in an elevation of N-terminal APP, reducing Aβ [57,58,66-68].
Nerve growth factor (NGF) has been shown to be influential in APP processing in vitro. NGF drug treatments could limit cholinergic hypoactivity in the cortex of AD affected brains. TrkA (tyrosine kinase A NGF receptor) has been shown to increase APP processing and p75NTR (neurotrophin receptor) affects APP transcription [
Ibuprofen is a non-steroidal anti-inflammatory drug (NSAID) that delays amyloid deposition in transgenic mice [
Nonsteroidal anti-inflammatory drugs influence the development of AD [
APP processing and formation of amyloidogenic APP holoprotein is enhanced by neurotransmitters such as prostaglandins and norepinephrine by elevating cellular cAMP levels. The conversion of APP to its soluble form is enhanced by neurotransmitters that stimulate phosphatidylinositol hydrolysis by activating muscarinic, serotoninergic or metabotropic glutamate receptors. A study has found that some neuroimmunophilin ligands (cyclosporin A and FK506) inhibited the over expression of APP by prostaglandin E2. This in turn reduced the synthesis of amyloidogenic APP holoprotein. Nonsteroidal inflammatory agents and cyclooxygenase-2 (COX- 2) inhibitors might inhibit the accumulation of amyloid plaques in AD by reducing the levels of amyloidogenic APP holoprotein as observed in cultured neurons or astrocytes and promote neuronal regeneration [
The immune system may contribute an important role in AD. Inflammatory proteins (such as eicosanoids, cytokines and complement components) are released by microglia and astrocytes of the immune system and are known to be associated with neuritic plaques. Inflammatory proteins are thought to be stimulated by Aβ production and deposition. Aβ is thought to stimulate microglia and astrocytes to release the inflammatory proteins and stimulate a neurotoxic response causing cognitive impairment. Under normal conditions microglia stimulate Aβ-specific T cell production to degrade Aβ.
In cortical astrocyte cultures it was shown that PGE2 receptor activation promotes cAMP and induces APP mRNA and amyloidogenic APP holoprotein production [
Complement proteins are found in amyloid plaques in AD brains. Aβ plays a role in complement activation and perhaps chronic inflammation in AD. Pharmacotherapies that inhibit Aβ complement activation might be useful in AD treatment.
Studies of PDAPP mice (which over express mutant human APP) suggest that Aβ immunisation might treat and prevent the neuro pathological changes of AD [
A monoclonal antibody 22C11 that binds to the extracellular domain of APP was used to determine a possible role of APP in neurons. DNA cleavage and condensation within the nucleus were observed along with neuron degeneration when cortical neurons were exposed to 22C11. The effects of 22C11 were blocked by pretreating the neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluromethyl ketone. GSH ethyl ester (GEE) penetration of the cortical neurons also resulted in the prevention of the effects of 22CC11. 22C11 toxicity was enhanced by incubating the neurons with buthionine sulfoximine (gamma-glutamylcysteinyl synthetase). Neuritic degeneration was observed followed by caspase dependent apoptosis when the monoclonal antibody binds to APP. This is suggestive of the involvement of APP in neuronal cell death in AD [
Trials immunizing patients to moieties of Aβ had to be aborted because of deaths from a T-lymphocyte encephalitis, however, neuritic plaques were reduced at autopsy [76,77].
Aβ is the main constituent of plaques in AD and this peptide is formed by the enzymatic cleavage of the transmembrane protein APP by β- and γ-secretases respectively.
A Swedish pedigree of familial AD identifies a double mutation at the β-secretase cleavage site at the amino terminal of APP (APPSw) [
Cysteine protease inhibitors increased the amount of the APP extracellular domain by twofold. Protease inhibitors increased the appearance of incompletely glycosylated APP and increasing the amount of APP entering the secretory pathway. Cysteine proteases quickly degrade APP molecules [
Metalloendopeptidase EC 3.4.24.15 (MP24.15) promotes the degradation of Aβ [
The proteasome is a multicatalytic complex involved in the degradation of polyubiquitinated proteins. The proteasome modulates the intracellular concentration of presenilins 1 and 2. These two proteins, when mutated, appear responsible for most of early onset forms of AD which is thought to be an effect favouring the deposition of long forms of Aβ leading to amyloidogenesis. Controlling presenilins concentrations could have drastic repercussions on cell physiology as suggested by the observation that proteasome inhibitors drastically potentiate the “pathogenic” presenilin function. The possibility of considering the proteasome as a potential target for therapeutic intervention in AD is important [
To understand the mechanisms of APP degradation, it has been established that in the presence of proteasome inhibitors, the cystolic molecular chaperon Hsc73, interacts with the cytoplasmic domain of APP (carboxy-terminal) which signals lysosomal proteolysis. Hsc73 binds to the various mutated isoforms of APP (as found in the Swedish or Dutch mutations) in equal amounts and this is suggestive of an Hsc73 attachment mechanism dependent on the conformation of the APP secretory cleavage site [
N-acetyl-leucyl-leucyl-norleucinal (ALLN or LLnL) is a calpain inhibitor [84-88] inhibits proteasome activities at high concentrations [
In studies using canine kidney cells (MDCK) and human embryonic kidney cells (HEK) the calpain 1 inhibitor LLnL (ALLN) and lysosomotropic agent ammonium chloride (NH4Cl) were used to inhibit the degradation of PS1 and APP-c100 (containing the Aβ fragment) [94-98]. It was observed that APP-C100 formed a higher molecular mass complex with PS1 fragments. When PS1 was immunoprecipitated, a large amount of APP-C100 followed suit. This is suggestive that PS1 may directly interact with APP-C100 to control Aβ deposition [
In APP processing α-secretase seems to be a Ca2+ dependent protease, as does calpain. In AD protease abnormalities seem to occur in the processing of APP when it is cleaved excessively by β or γ-secretases and α-secretase activity is inactivated. Intracellular Ca2+ imbalance seems to be prevalent in AD [100,101].
One study suggests that cysteine aspartate-specific proteases (caspase) directly contribute to AD pathogenesis. Caspases cleave APP encouraging the amyloidogenic processing pathway of the protein. Caspases also cleave presenilins thus promoting apoptosis. Presenilin C-terminal fragments are known to have antiapoptotic functions [
The presenilin 2 mutation (N141T-PS2) inhibits secretion of the α-secretase cleaved product of APP in human HEK293 cells [
A study using ovarectomized guinea-pigs suggests that the absence of ovarian oestrogen in postmenopausal women might increase Aβ 40 and Aβ 42 concentrations in the brain and 17b-estradiol (E2) treatment decreased Aβ deposition [
Homocysteine is an amino acid which is neurotoxic [110-114] and is known to accumulate in many neurodegenerative disorders including Alzheimer’s disease [115- 117].
Homocysteine is known to augment Aβ neurotoxicity [
The protooncogenes c-fos and c-jun are members of a set of genes known as cellular immediate early genes, and are believed to play an important role in stimulus-transcripttion coupling [
Autosomal dominant mutations in the presenilin 1 gene
(PS1) increased concentrations of Aβ (1-42) in earlyonset AD patients. In transfected cell medium and transgenic mouse brain with PS1 mutations the level of Aβ 11-42 was increased. A human cell line expressing inducible antisense PS1 RNA found that Aβ (42) increased five-fold after 14 days of treatment whilst PS1 holoprotein decreased by 90% [
Manin-Darby canine kidney (MDCK) cells were transfected with cDNA APP with a 42 amino acid truncation at the C-terminus (DeltaC). A unique Aβ sequence was found and immunoprecipitated with an Aβ 17-24-specific monoclonal antibody (4G8) but not with Aβ 1-16-specific monoclonal antibody (BAN50). Treatment of DeltaC MDCK cells with the cholesterol synthesis inhibitor compactin, or the cholesterol binding drug filipin, resulted in the immunoprecipitation of Aβ by BAN50 but not 4 GB. These results suggest that Aβ production is cholesterol-dependent [
Clinical studies have revealed that cholesterol-lowering statin drugs might reduce the risk of AD [
APP and Aβ have many proposed actions in the central nervous system and together probably function as a coupled peptide system with a fundamental role in neuromodulation (