Therapeutic strategies for Alzheimer’s disease

Currently, two types of drugs are authorized for the treatment of AD. The first treatment is based on the inhibition of acetyl cholinesterase (AChE), because many symptoms of dementia are closely related to cholinergic dysfunction. Until now, four cholinesterase inhibitors have been approved for the treatment of mild AD: tacrine, donepezil, rivastigmine and galantamine. The treatment with these drugs produces modest symptomatic improvement in patients, but it does not slow the progression of the disease [88-90]. An alternative treatment is performed with memantine, a N-methyl-D-aspartate (NMDA) receptor antagonist, which appears to prevent the neuronal excitotoxic effect exerted by high levels of glutamate. Memantine has been approved for the treatment of moderate to severe Alzheimer dementia [91-93].

Several therapeutic strategies have been proposed based on the knowledge of Aβ formation and the effects of soluble Aβ oligomers on the synaptic function. Inhibition of β- and γ-secretase should decrease Aβ generation, but might cause unwanted side effects. Another therapeutic strategy would consist in lowering the level of soluble Aβ oligomers [79].

Aβ immunotherapy is a promising strategy for reducing the level of Aβ in the brain.

Immunological approaches have been proposed as a strategy for diagnostics and treatment of neurodegenerative diseases. Two types of antibodies specific to Aβ have been identified and characterized to dissagregate and/or to inhibit amyloid fibril deposition: (i), Aβ-plaque specific antibodies resulting from active immunization with Aβ(1-42), that disaggregate Aβ-plaques; and (ii), physiological Aβ-autoantibodies that inhibit fibril formation. Aβ-plaque specific antibodies generated by active immunization with Aβ(1-42) and Aβ-derived aggregates have been shown to reduce the neurotoxicity and reverse the memory deficits in APP-transgenic mice, and are

epitope peptide contains the residues (4-10) (FRHDSGY), which are accessible in Aβ(1-42) as well as in oligomeric and protofibrillar Aβ. Although an early clinical trial of immunization of AD patients with Aβ(1-42) indicated initial good tolerability, the clinical evaluation was stopped after approximately 6% of the vaccinated patients developed severe meningo-encephalitic inflammations [94-97]. In contrast, physiological Aβ-autoantibodies isolated from serum of healthy individuals and AD patients were found to specifically recognize the C-terminal part of Aβ. The carboxy-terminal epitope comprises residues (21-37) of Aβ and thus the Aβ-aggregation domain. Aβ-autoantibodies lead to a shift of Aβ from the central Aβ pool into the periphery and interfere with the early steps of plaque development (i.e. they abolish oligomerization of Aβ) [98]. A potential therapeutic concept for AD is passive immunization with intravenous immunoglobulin (IVIg) containing naturally occurring Aβ-autoantibodies [99].

The differential recognition of the C-terminal Aβ-autoantibody epitope and the N-terminal plaque-specific epitope provided the basis for the main goals of the present dissertation.

Unpaired variable domains of single heavy chain antibody (VHH) fragments (nanobodies) (Figure 9), have recently been considered to have high potential for various medical applications. Because of their capability to prevent the unfolding of amyloidogenic protein variants or even clear existing aggregates in vitro, nanobodies are expected to be a therapeutic alternative for treating amyloidosis disorders such as AD. VHH, termed nanobody, represents the smallest antigen binding unit with a molecular size of ~15 kDa, in comparison to single-chain antibody fragments consisting of variable domains of heavy and light chains connected by a peptide linker (scFv, 30 kDa), to Fab fragments (60 kDa) and to the whole IgG antibodies (150 kDa) [100]. Nanobodies have favourable properties for biophysical studies, including small size, high solubility and stability [101-103]. The concept of single-domain antibody (dAb) was first introduced by Ward et al. [104]. The discovery of camelid heavy-chain antibodies naturally devoided of light chains opened up a new opportunity to develop dAb with improved properties [103]. Camelid VHHs display similar functional characteristics with respect to specificity and affinity compared to classical antibodies [105, 106]. Dromedary subclasses were originally named IgG1,

IgG2 and IgG3 in order of decreasing molecular weight. The sera from camels (Camelus bactrianus) and llamas contained similar heavy–chain antibodies.

Compared to dromedaries, a slightly lower percentage of heavy-chain antibodies are observed in llamas.

V

C_H3 C_H2

V_HH

blood collection

V

C_H3 C_H2

V_HH

V

C_H3 C_H2

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Figure 9: Heavy-chain antibody from Llama Glama obtained after immunization of the animal. VHH, called nanobody, represents the smallest fragment of a single-chain antibody.

The present dissertation is focused on the characterization of Aβ-nanobodies and the elucidation of the β-amyloid epitope peptide recognized by the Aβ-nanobodies.

Human Cystatin C (HCC) (Figure 10) with a molecular weight of 13 kDa is the main cysteine protease inhibitor in mammalian body fluids [107] and has been found in high concentrations in cerebrospinal fluid (CSF). Immunohistological studies have shown that the protease inhibitor Cystatin C and other proteins such as apolipoprotein E, clusterin, transthyretin and gelsolin co-deposit with Aβ [108-110]. A wide spectrum of biological activities has been associated with HCC, such as modulation of neuropeptide activation and neurite proliferation [111, 112]. While wild type Cystatin C shows no aggregation tendency, the naturally occurring mutant L68Q presents a high tendency to form amyloid fibrils, causing hereditary cerebral hemorrhage of the amyloidosis-Icelandic type. The presence of HCC in Aβ-plaques has been suggested to result from its binding to APP. Alternatively, HCC may bind to Aβ prior to the secretion or following the deposition in brain. Sastre et al. found that the association of HCC with Aβ causes an inhibition of fibril formation and suggested an N-terminal Aβ-sequence to be responsible for the interaction, with formation of a stoichiometric HCC-Aβ complex [111]. Human Cystatin C has a protective role in Alzheimer’s disease, by preventing the formation of the toxic forms of Aβ and by direct protection of neuronal cells from Aβ toxicity [113-115].

β1 β4 β3 β2 β5

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C L2 L1

β1 β4 β3 β2 β5

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C L2 L1

Figure 10: Ribbon representation of human Cystatin C (HCC) (pdb File 1 CEW). Secondary structure of HCC: L1: loop 1; L2: loop 2; α1: α - helix 1; α2 : α-helix 2; β1 – β5: β-sheet 1 – 5.

The identification and characterization of the binding epitope of HCC in the central domain of Aβ was studied in the present dissertation. The protease inhibitor has an important role in the aggregation process and amyloidogenesis. Blocking the hydrophobic core of the HCC, the Aβ oligomerization can be inhibited and the fibril formation can be regulated. Moreover, the identification of the binding site in HCC is important for the oligomerization studies of Cystatin C. New oligomerization inhibitors may be designed based on the HCC-epitope.