Pathophysiological characteristics and therapeutic perspectives of

“She sits on the bed with a helpless expression. What is your name? Auguste. Last name? Auguste. What is your husband’s name? Auguste, I think.” [50]

It has been already a century ago since the German psychiatrist Alois Alzheimer presented the case of Auguste D., a 51-year-old lady who had shown progressive loss of cognitive functions and psychosocial competence. A. Alzheimer described for the first time the clinical picture of presenile dementia as well as the histological findings of amyloid plaques, neurofibrillary tangles and arteriosclerotic changes.

Alzheimer´s disease is clinically characterized by a progressive decline of cognitive functions from mild forgetfulness and cognitive impairment, to widespread loss of memory, language and logical thinking having impact on the ability to perform everyday activities and changing the patient’s behavior. Death occurs, on average, 10 years after the diagnosis. In addition to its direct effects on patients, advanced AD loads a tremendous burden on family caregivers and causes substantial nursing costs for the society [51]. Due to the increase of life expectancy of the population, the absolute number of people afflicted by AD is expected to grow substantially. It is estimated that there are currently 26 million people worldwide suffering of Alzheimer´s disease, and the global prevalence is expected to increase to more than 100 million by 2050.

Current medications approved for the treatment of Alzheimer´s disease are based on the modulation of neurotransmission. Acethylcholinesterase (AchE) inhibitors attempt to address the cholinergic deficits seen in AD and are used for mild to moderate cases. Memantine an (N-methyl-D-aspartate)-receptor antagonist that has been used for the treatment of moderate to severe Alzheimer dementia aims to prevent the neuronal excitotoxic effect exerted by high levels of glutamate. Although producing moderate symptomatic improvements of the cognitive function, none of these drugs appears to be able to cure Alzheimer´s disease [52].

Hence, an enormous need exists for the development of new medications for AD with strong disease-modifying properties, and research is focused on the development of new therapeutic strategies that target the underlying pathogenic mechanisms of Alzheimer´s disease.

A comparative examination of the brains from AD patients and normal elderly individuals reveals a dramatic loss of brain tissue [53]. Shrinkage of the brain is extremely severe in the hippocampus, temporal and parietal lobes and is mainly observed in the widened cortical sulci and ventricular dilatation as depicted in Figure 8b. The histopathological hallmarks of Alzheimer´s disease are loss of cholinergic and glutamatergic neurons, intracellular and extracellular deposits of proteins and microvascular angiopathy. Many neurons in the brain regions typically affected in AD contain abnormal protein deposits called neurofibrillary tangles that occupy much of the perinuclear cytoplasm (see Figure 8a). The neurofibrillary tangles consist of microtubule-associated protein Tau in abnormally phosphorylated form [54]. The in vitro phosphorylation of tau has been reported to inhibit the polymerization of tubulin [55] into the microtubules. Microtubules are crucially important structures which run through the cell and are involved in axonal transport, synaptic transmission, cell support and shape.

a) b)

Normal Alzheimer´s Disease Normal Alzheimer´s Disease

Normal Alzheimer´s Disease Normal Alzheimer´s Disease

Figure 8: Pathophysiological characteristics of Alzheimer´s Disease compared with a healthy individual: a) neurofibrillary tangles and amyloid plaques; b) brain cross section showing atrophy of the brain tissue affecting predominantly the language and memory lobes. Copyright © 2000-2009 American Health Foundation. All rights reserved.

The extracellular deposits are referred to as neuritic or senile plaques and consist of aggregated amyloid-ß protein [56] surrounded by astrocytes and neurites emanating from local neurons. Microvascular angiopathy caused by the deposition of amyloid-ß protein on the walls of the arterioles and venules was found outside the brain as well as within the cerebral cortex of the brains from patients with Alzheimer´s disease [57, 58]. Cerebral amyloid angiopathy can lead to hemorrhages which may contribute to the cognitive decline [59]. The hypothesis that states the fundamental role of the overproduction and accumulation of Aß in senile plaques in the pathology of AD has been extesively studied in the last two decades. An overview concerning the origin of amyloid-ß protein and the accumulation in senile plaques as well as the main therapeutic strategies that are currently pursued will be discussed in the following sections of the introduction. In Alzheimer´s disease, excessive activation of NMDA receptors by L-Glutamate (L-Glu) is believed to cause elevated cytosolic Ca²⁺ which then initiates pathological events that ultimately lead to neurodegeneration [52, 60].

Amyloid-ß (Aß) was first sequenced from the meningeal blood vessels of AD patients and individuals with Down syndrome by George G. Glenner [58, 61] and then identified in the senile plaques [62]. Aß is proteolytically cleaved from the amyloid precursor protein (APP) that contains a single transmembrane domain, with a longer N-terminal amino acid sequence emanating out of the cell and a shorter C-terminal domain jutting into the cytosol. APP is encoded by a gene located on the chromosome 21 [63-65] and although is produced by many cells and tissues its precise biological role has remained unknown. Several forms of APP that differ mainly at the amino-terminal end of the sequence have been described to arise by alternative splicing: APP-695, APP-751 and APP-770 [66-68]. The enzymes that play a central role in the proteolytic processing of APP are α-, β- and γ-secretases (see Figure 9). The proteolytic cleavage by α-secretase occurs 12 amino acids NH2 -terminal to the transmembrane domain and releases a large soluble fragment (α-APPs) into the extracellular space. The 83-amino acid residue COOH-terminal fragment is retained in the membrane and is further cleaved by γ-secretase, generating the p3 peptide fragment and a 57/59 amino acid residue carboxy-terminal fragment (CT57/59). Alternatively, APP is cleaved 16 amino acids N-terminal to the α-secretase cleavage site by ß-secretase releasing ß-APPs into the extracellular space and retaining a 99-amino-acid residue in the membrane. The cleavage by

γ-secretase produces a 40/42 peptide fragment referred to as Aß and the CT57/59.

The α-secretase activity was described to be exerted by three related metalloproteases of the ADAM (a disintegrin and metalloprotease) family, ADAM-9 [69], ADAM-10 [70] and ADAM-17 [71]. Two aspartyl enzymes responsible for the ß-secretase cleavage have been identified in 1999 referred to as BACE (ßAPP cleaving enzyme) [72-75] and BACE-2 [76, 77]. BACE activity can also generate fragments of APP cleaved at secondary sites such as Glu11 within the Aß sequence [78]. The second enzymatic activity required for Aß generation is exerted heterogeneously by γ-secretase. Most of the full-length Aß species produced is a 40-residue peptide (Aß40), whereas a smaller proportion is a 42-40-residue carboxy-terminal form.

Figure 9: Proteolytic processing of APP. The conjoint cleavage of APP by α- and γ-secretase produces the harmless fragment p3, the carboxy-terminal C57/59 and the longer soluble α-APP; Alternatively the cleavage by ß- and γ-secretase releases the 40/42 residues long fragment called Aß that is prone to aggregation.

Under normal circumstances, Aß generated in the CNS is cleared with a half-life of 1-2 h [79]. Initially, Aß41-2 which is more prone to aggregation than Aß40 is deposited in diffuse (nonfibrillar) plaques with little or no detectable neuritic dystrophy. The mechanism through which the aggregated Aß exerts its toxic effects is still

cause damage to cultured neuronal cells [80, 81]. However, more recent findings suggest that soluble oligomeric prefibrillar forms of Aß may represent the neurotoxic species that causes neurotoxicity and synaptic dysfunction [82, 83].

The dominating hypothesis concerning the mechanisms leading to Alzheimer’s disease assigns a central role of the accumulation of Aß in brain to the initiation of a cascade of pathological events that ultimately lead to neurodegeneration and dementia [52, 84]. A first argument supporting a causal role of Aß in Alzheimer´s disease came from the identification of the APP gene locus on chromosome 21 and the earlier finding that individuals affected by Down syndrome posses 3 copies of the chromosome 21 and develop invariably AD pathology. Additionally, several mutations that are responsible for early-onset forms of familial Alzheimer’s disease (FAD) have been identified in the APP gene. These mutations are located directly adjacent close to the ß- and γ-secretase cleavage sites favoring the proteolytic processing of APP and leading to increased Aß production. Mutations in the genes encoding PS1 and PS2 are also responsible for elevated production of Aß. Increased Aß plaque deposition in brain has been associated with the presence of apolipoprotein E (apoE)-protein although the mechanism remains unknown [85]. Recent data showed a reduction of Aß deposition in the offspring by crossing APP overexpressing transgenic mice with apoE-deficient mice [86].