1 INTRODUCTION
1.4 Age-related neurodegeneration of autoimmune nature: Alzheimer’s disease
“Every 70 seconds, someone in America develops Alzheimer’s disease. By mid-century, someone will develop Alzheimer’s every 33 seconds.” [39]
As the life expectancy of individuals has continued to increase, there has been a concomitant increase in diseases primarily associated with appearance late in life. Age-related dementia is a major category of such diseases and Alzheimer’s disease (AD) is one of the most widely known and most widely feared neurodegenerative diseases. The dramatically increased life span has promoted AD to the 6th leading cause of death across all ages in the United States in 2006, whereas in Europe, AD is rated as the 3rd cause of death after heart diseases and cancers in 2009. While the total numbers attributed to other major causes of death such as heart diseases, breast and prostate cancer, and stroke, have declined over the past several years, those due to AD have continued to increase, with recent statistics indicating the rate of death has increased by 47 % from 2000 to 2006 [39].
Alzheimer’s disease is characterized by the gradual loss of memory and other cognitive abilities. Memory difficulties, apathy and depression are often early clinical symptoms, while later symptoms include impaired judgement, disorientation, confusion, and behaviour changes. In advanced Alzheimer’s people need help with bathing, dressing, eating and other daily activities [40].
Although the causes of AD are still poorly understood, most experts agree that Alzheimer’s develops as a result of multiple factors rather than a single cause. The greatest risk factor for AD is advancing age. Most individuals with AD are aged 65 or older, and the risk of developing AD has been reported to increase 50 % by the age of 85 [39] (see following paragraphs for a discussion of genetic mutations of the amyloid precursor protein). A
genetic factor related to increased risk of AD late in life is apolipoprotein-E4 (APOE-e4), one of three common forms of APOE gene, which provides the blueprint for a protein that carries cholesterol in blood. While everyone inherits one of the APOE genes from each parent, individuals that inherit one or both APOE-e4 genes are at higher risk [41, 42].
There is currently no treatment available to stop or slow the cognitive decline in AD. The U.S. Food and Drug Administration has approved five drugs that temporarily alleviate the symptoms of neurodegeneration for 6 to 12 months for about half of the patients. These are acetylcholinesterase inhibitors (Aricept®, Razadyne® and Exelon®) and regulators of glutamate activity (Namenda®), two small molecules involved in learning and memory [43, 44]. Hence, the present lack of effective therapeutic agents requires the exploration of alternative approaches for treatment and prevention.
Post-mortem examination of the brains of AD patients shows dramatic losses of brain mass, most severe in hippocampus, temporal and parietal lobes. Neuronal loss, intra- and extracellular protein accumulation, as well as microvascular angiopathy are histopathological characteristics of AD. Intraneuronal neurofibrillary tangles consisting of filaments of hyperphosphorylated forms of tau proteins twisted around microtubules, cause disintegration and hinder propagation of electric signals [45, 46].
A major pathological feature of AD is the accumulation of extracellular plaques that contain aggregates of the neurotoxic ß-amyloid (Aß) polypeptide as major components, surrounded by astrocytes and activated microglia [47]. Beta-amyloid comprises a group of polypeptides of 38-43 residues with partially N- and C-terminally truncated sequences, which are formed by proteolytic cleavage of the amyloid precursor protein (APP), a transmembrane protein involved in synaptogenesis and neuronal plasticity. As schematically shown in Figure 1.7, overproduction and/or accumulation of Aß results in the formation of extracellular deposits.
Figure 1.7: Proteolytic processing of the amyloid precursor protein (APP695) and formation of the ß-aggregates in AD: (A) transmembrane amyloid precursor protein, (B) proteolytic formation of ß-amyloid peptide, (C) extracellular accumulation and aggregation of Aß-peptide, and (D) schematic representation of proteolytic processing pathways of APP695: “non-amyloidogenic” pathway producing sAPPα and CTFα fragments (left), and “amyloidogenic” pathway, releasing the neurotoxic, plaque forming Aß-peptides (right).
APP was not initially discovered for its physiological role in many organs and cells, but rather because of the characteristic deposition of Aß-containing plaques in AD [48]. The function of APP is only partially understood at present. There are three major isoforms of APP, containing 695, 751 and 770 amino acid residues (designated as APP695, APP751 and APP770, respectively) [49], which are derived from alternative splicing of the mRNA of a single gene located on chromosome 21 [50]. In nerve cells, the predominantly expressed isoform is APP695, while isoforms APP751 and APP770 predominate in other cell types [51]. Three proteolytic enzymes, (denoted α-, ß- and γ-secretases), are involved in the proteolytic degradation of APP and the secretion of soluble APP (sAPP) forms [52, 53]
(see Figure 1.7 D). In the “non-amyloidogenic” pathway, cleavage by α-secretase occurs at the Lys-16 residue downstream of the N-terminal of Aß, releasing the sAPPα fragment and the C-terminal transmembrane fragment of APP, CTFα [54, 55]. The soluble extracellular domain sAPPα derived from the non-amyloidogenic processing pathway, acts as a growth factor in many cell types and promotes neuritogenesis in post-mitotic neurons [49]. In vivo, infusion of sAPPα into the brain increases synaptic density, protects hippocampal neurons against ischemic injury and enhances memory performance [56-58].
Alternatively, cleavage of APP by ß-secretase(s) at the C-terminal end of sAPPß (Lys-Met
in human physiological APP) leads to the formation of the neurotoxic Aß peptide(s), and has been denoted as the “amyloidogenic” pathway. Several pathogenic mutations in the APP gene leading to increased Aß production and early onset of non-age related AD (around the age of 50) have been identified. The "Swedish" mutations comprise the simultaneous substitutions Lys670Asn and Met671Leu in full-length APP770, which were found to enhance the ß-secretase cleavage rate yielding elevated levels of sAPPß. These mutations represented the basis for the development of corresponding transgenic mouse models of AD [59-61].
During transit through the intracellular protein secretory pathway, APP has been shown to undergo multiple post-translational modification, such as by N- and O-glycosylation, phosphorylation and tyrosine sulfation [62-64]. It has been suggested that N- and O- glycosylation of the extracellular domain of APP are prerequisites for phosphorylation of Thr668 of the cytoplasmic domain during neuronal differentiation [65], and for the proteolytic cleavage by secretases [66]. Moreover, mutants defective in O-glycosylation have been reported to exert an altered cellular metabolism compared to wild type, physiological APP695 [66]. The structure-function relation of APP and Aß as its key AD peptide, have not been elucidated in molecular detail. The pathophysiological importance of this relation to Aß and amyloid deposits in neurodegeneration is underlined by the above-mentioned early-onset cases of AD, with mutations in the APP gene chromosome 21, and in presenilin genes that lead to increased production of Aß [67-69]. Furthermore, patients with Trisomy 21 (Down syndrome), having an extra copy of the APP gene, develop large number of plaques at early age [70-72]. These results and the present lack of knowledge of the molecular structure of APP have been a major motivation for the structural studies of APP in this thesis.
1.5 Immunotherapeutic and diagnostic approaches using Aß specific antibodies