First printed in R&D Systems' 2001 Catalog.
Alzheimer's Disease (AD) is a neurodegenerative disease of the central nervous system associated with progressive memory loss resulting in dementia. Two pathological characteristics are observed in AD patients at autopsy: extracellular plaques and intracellular tangles in the hippocampus, cerebral cortex, and other areas of the brain essential for cognitive function. Plaques are formed mostly from the deposition of amyloid ß (Aß), a peptide derived from amyloid precursor protein (APP). Filamentous tangles are formed from paired helical filaments composed of neurofilament and hyperphosphorylated tau protein, a microtubule-associated protein. It is not clear, however, whether these two pathological changes are the markers or the causes of AD. Late-onset/sporadic AD has virtually identical pathology to early-onset/familial AD (FAD), thus suggesting common pathogenic pathways for both forms of AD. To date, genetic studies have revealed four genes that may be linked to autosomal dominant or familial early-onset AD (FAD).1-6 These four genes include: amyloid precursor protein (APP), presenilin 1 (PS1), presenilin 2 (PS2), and apolipoprotein E (ApoE). All mutations associated with APP and PS proteins can lead to an increase in the production of Aß peptides, specifically the more amyloidogenic form, Aß42. In addition to genetic influences on amyloid plaque and intracellular tangle formation, environmental factors (e.g., cytokines, neurotoxins, etc.) may also play important roles in the development and progression of AD.7
|Fig. 1. Intracellular tangles found within neurons of brain regions essential for cognitive function are one of the pathological characteristics associated with AD. Filamentous tangles are formed from paired helical fragments (PHF) composed of neurofilament and hyperphosphorylated tau protein. Polymerization of hyperphosphorylated tau protein leads to PHF formation. PHF can also be modified by glycosylation and ubiquitination.|
APP is an integral membrane protein, occurring in different isoforms. The common isoforms contain 695 (APP695), 751 (APP751) and 771 (APP771) amino acids (aa), respectively. Among these isoforms, APP695 is the major isoform and is expressed exclusively in neurons.8-10 In contrast, APP751 and APP770 are expressed in both neural and non-neural cells.1 The primary structure of APP has a signal sequence, a large extramembranous N-terminal region, a single transmembrane domain, and a small 47 aa residue cytoplasmic C-terminal tail.11 The APP proteins mature in the endoplasmic reticulum and Golgi apparatus and exhibit post-translational modifications, including phosphorylation, glycosylation and sulfation.12-16
Proteolytic cleavage of APP results in generation of Aß peptides of various lengths. Ab peptides are normally soluble monomers that circulate at low levels in cerebrospinal fluid and blood. In the brains of AD patients, formation of insoluble, fibrillar plaques is facilitated by an increase and accumulation of Aß peptides. The predominant form of Aß peptides found within conditioned cell culture media and cerebrospinal fluid is the shorter Aß40 peptide.17-19 Aß42, however, is the Aß peptide form initially deposited within the extracellular plaques of AD patients. This may be explained by the following. All FAD-linked mutations identified within APP lead to the increased production of Aß42.1 Additionally, Aß42 tends to aggregate at a faster rate and at lower concentrations than the Aß40 form.20-22
Three proteases, a-, ß- and ?-secretases, are involved in APP cleavage (see references 23-25 for reviews). At the cell surface, APP undergoes proteolysis by an α-secretase that cleaves between Lys687 and Leu688 thus releasing a large, soluble ectodomain (α-APP). The C-terminal fragment (83 aa, ~10 kDa) is retained within the cell membrane. This fragment can then be cleaved by ?-secretase at aa residues 711 or 713 within the APP transmembrane domain thereby releasing the p3 peptide. Alternatively, uncleaved cell surface APP can be internalized by endocytosis via coated vesicles in the distal cytoplasmic domain. The full-length APP can then be trafficked to later endosomes and lysosomes for degradation or transferred to early endosomes for generation of Aß peptides. In the early endosomes, APP is cleaved by ß-secretase after Met671, creating a membrane-retained C-terminal fragment (99 aa, ~12 kDa). Cleavage by ß-secretase exhibits relatively rigid primary aa sequence requirements (i.e., between Met671 and Asp672 of APP). At the membrane surface, the 12 kDa C-terminal fragment can then be further cleaved by ?-secretase within the hydrophobic transmembrane domain at either Val711 or Ile713 thus releasing an Aß peptide (i.e., either Aß40 and Aß42).
Identification and characterization of the ß- and ?-secretases have been important areas of focus in AD research. Although several candidates have been suggested for ß-secretase, BACE is the only one identified having complete ß-secretase activity.26 Cloning and expression of the enzyme reveals that the human brain ß-secretase/BACE is a membrane-bound aspartic proteinase.27, 28 The ?-secretase has not been definitively identified yet. Numerous studies, however, have linked ?-secretase and PS1 as either the same enzyme molecule or cofactors within the same complex.29-36
PS1 and PS2 are integral membrane proteins that contain multiple transmembrane domains.37, 38 PS1 and PS2 have a similar predicted structure and share 67% aa identity.4-6 Both proteins are predominantly located within the endoplasmic reticulum (ER) and early Golgi apparatus.39-42 They are primarily expressed in neurons and are ubiquitously expressed within the brain.39,40,43 Endogenous PS1 and PS2 proteins are proteolytically cleaved to generate two polypeptides. The 46 kDa PS1 protein is cleaved to yield a 28 kDa N-terminal fragment (NTF) and an 18 kDa C-terminal fragment (CTF), whereas the 55 kDa PS2 protein is cleaved to yield a 35 kDa NTF and a 20 kDa CTF.44 The predominant species of both PS1 and PS2 observed in both cultured mammalian cells and the brain are the processed fragments. Full-length PS1 has been found only in transfected cell lines and transgenic mice that overexpress PS1.45
|Fig. 2. PS1 (presenilin-1) plays a role in ?-secretase cleavage of APP (amyloid precursor protein). BACE/ß-secretase (blue) cleaves the APP precursor protein after Met671, creating a membrane-retained C-terminal fragment. This fragment can then be further cleaved by γ-secretase (purple) within the hydrophobic transmembrane domain to release Aß. PS1 is an integral membrane protein with multiple transmembrane domains that has been linked with ?-secretase activity. Further research is necessary in order to determine whether PS1 can either directly regulate ?-secretase activity as a cofactor within a protein complex or serve as the actual protease itself.|
The exact functions associated with PS proteins have not been fully characterized yet. 46-49 PS1 is required for proper formation of the axial skeleton and is involved in normal neurogenesis and survival of progenitor cells and neurons in specific brain regions.50 PS proteins have also been proposed to function in the control of apoptosis.50-55 As mentioned previously, PS1 is also involved in ?-secretase activity.29-36 Additionally, the binding of PS proteins to APP may play an important role in inducing intercellular signaling.56
The majority of early onset FAD cases are caused by mutations within the PS genes. More than forty mutations have been described in the gene for PS1 that can subsequently result in FAD.4, 35, 49 Mutations in both PS1 and PS2 are associated with an increased production of the Aß42 peptide.4, 5, 35, 58-61 Aß42, the more amyloidogenic form of Aß., can aggregate to form diffuse and neuritic amyloid plaques, thus suggesting that the influence of PS proteins on the production of Aß42 may be an initiating event for developing AD.21, 48 Mutations in the PS1 gene may also facilitate neuronal apoptosis by destabilizing ß-catenin (i.e., part of the PS protein complex), thus predisposing individuals to early onset FAD.55, 57
The risk factor and mean age of onset for late-onset AD is influenced by the inheritance of specific apoE alleles (for reviews, see references 62-65). ApoE is a 34 kDa protein existing as three major isoforms, E2 (Cys158), E3 (Cys112 and Arg158) and E4 (Arg112). Among the three isoforms, ApoE3 is the most common representing ~78% of the total forms, whereas ApoE4 represents 15% and ApoE2 represents 7%. The proportion of different isoforms varies between racial and ethnic groups. ApoE plays an important role in lipid transport in human blood and other body fluids.65 It participates in plasma lipoprotein metabolism, cholesterol homeostasis and local lipid transport processes (for a review, see reference 66). ApoE is produced by various cell types, including liver, kidney, fat cells and macrophages. In the brain, it is primarily synthesized and secreted by astrocytes67, 68 and plays a major role in lipid transport within the central nervous systems.69 Occurrence of the ApoE4 isoform is significantly associated with late-onset AD.3,70-72 The exact role ApoE4 plays, however, in the pathogenesis of AD is not clear. ApoE may be involved in the formation of amyloid plaques or tangles by interacting with Aß or tau proteins.73 Its expression is considered as a risk factor for AD that is not necessarily sufficient for development of disease.
Cytokines also play critical roles in the development and progression of AD. Cells associated with extracellular plaques within the brains of AD patients can produce a variety of cytokines and other related proteins that can ultimately influence plaque and tangle formation. Additionally, Aß itself can stimulate microglia, astrocytes and oligodendrocytes to secrete proinflammatory cytokines, chemokines, and reactive oxygen species (ROS) which can lead to neuronal damage. Several cytokines have been associated with AD development and progression, such as IL-1, IL-6, TGF-ß and TNF-a (for reviews, see references 72-75). For example, a differential expression profile of various TGF-ß isotypes can be observed within AD plaques, neuronal tangles and the cells associated with senile plaques, thus suggesting a role for these cytokines in promoting lesion development.76 The expression of and additional cytokine, HGF, is increased within senile plaques, potentially as a function of gliosis and microglial proliferation.77
|Fig. 3. Astrocytes and microglial cells associated with Aß plaques can release a variety of cytokines and other factors. Aß itself can also stimulate cells associated with plaques to release cytokines, chemokines and reactive oxygen species (ROS). These cytokines can initiate a complex of interactions, such as upregulation of expression and processing of APP, induction of cytokine overexpression (i.e., autocrine and/or paracrine loops), and recruitment of immune cells, which may all lead to degeneration of specific neuronal populations within the AD-affected brain.|
Cytokines typically associated with amyloid plaques (e.g., IL-1, IL-6 and TNF-a) may influence the expression of additional factors associated with the pathogenesis of AD. IL-1, IL-6 and TNF-a can stimulate in vitro glial and neuronal cell cultures to secrete complement proteins.78 Elevated levels of IL-1 present in AD brain tissue can also influence expression of the neurite extension factor S100ß by activated astrocytes.79, 80 Upregulation of S100ß may then lead to stimulation of neurite growth and eventual neuritic plaque formation. IL-1α also plays a role in regulating heparan sulfate proteoglycan (HSPG) synthesis in AD.81 HSPGs are also tightly associated with Aß and may be important for Aß peptide aggregation within the brains of AD patients.82-84