5. Alzheimer's disease

Alzheimer's disease, AD, is one of the most common neurodegenative maladies in Western societies. Clinical symptoms occur between the ages of 60-70y. This disease, for which no effective treatment is currently available, initially presents with symptoms of memory loss, after which a progressive decline of both cognitive and motor function occurs. Both genetic and environmental factors are implicated in its development.

Females are more susceptible than males, which may be attributable to the higher constitutive activity of the synaptic zinc transporter ZnT3. Studies showed that female mice exhibited age-dependent hyperactivity of the ZnT3 transporter which was associated with increased Aβ deposition.

The amyloidosis which occurs in AD involves abnormal protein processing; a structural transition of a polypeptide chain from a natively folded protein to one with an improperly folded conformation which leads to inefficient protein degradation. Truncated proteins are formed, and the resulting peptides tend to aggregate.  There is considerable evidence that defective homeostasis of redox-active metals, i.e. iron and copper, together with oxidative stress, contribute to the neuropathology of AD. The characteristic histology of AD is the deposition of both the amyloid peptide, Aβ, as neurotic plaques, Figure 8a and of the protein tau, as neurofribrillary tangles, Figure 8b predominantly in the cerebral cortex and hippocampus.

Amyloid precursor protein, APP, a type I membrane protein, resembles a cell surface receptor and is physiologically processed by site specific proteolysis. APP is cleaved by α-secretase, ( within the Aβ domain between Lys⁶⁸⁷ and Leu⁶⁸⁸), to yield APPsαand the C-terminal fragment containing p3, Figure 9. The production of the amyloid peptide, Aβ, is thus precluded. The membrane anchored α-carboxy terminal fragment, α-CTF, is then cleaved by γ-secretase within the membrane, releasing p3 peptide and the APP intracellular domain (AICD), Figure 9, (Wilquet and De Strooper, 2004). The presence of increasing amounts of iron may alter α-secretase activity; one hypothesis suggested that iron might be required as a co-factor or be an allosteric modifier of α-secretase activity. Iron may also decrease α-secretase cleavage rates.

In amyloidogenesis, the APP is cleaved sequentially by the proteolytic enzymes β-secretase (aspartyl protease, BACE or Asp-20) and then by γ-secretase. β-secretase has a C-terminal transmembrane domain and two active site motifs located in the luminal domain. Beta secretase cleaves APP between Met ⁶⁷¹ and Asp⁶⁷² and yields APPβs and C99 fragments.  The enzyme γ- secretase, a multi-subunit complex, (containing presenilins 1 and 2, (PS1 and PS2)), will then cleave APPβs to produce β-amyloid peptide, Aβ, (Aβ₄₂ and Aβ₄₀) and AICD.  Some recent studies indicate that there may be other factors involved in the action of PSs on the intramembranous proteolysis of APP. Mutations in PS1 or PS2 genes will increase the production of the toxic Aβ ₄₂ .

In individuals with AD, the aggregated and soluble fractions of Aβ are markedly increased,  the two β-amyloid peptides, Aβ₄₂ and Aβ_40, migrate from the cell to form aggregates, fibrils and eventually neuritic plaques. The structure of Aβ₄₀ consists of two helices spanning residues 15-23 and 31-35, which are separated by a disordered region. The Aβ₄₂ peptide adopts a regular type I β-turn, yielding a well defined tertiary structure (Crescenzi et al., 2002), Figure 10, which shows similarities with the fusion domain of the haemagglutinin of the influenza virus.

Aβ accumulation and aggregation is considered to be the initiating factor in AD pathogenesis although it is known that such deposition occurs over many years, if not decades, prior to the clinical cognitive impairment. Aβ may be oxidised within the membrane, perhaps as a result of the increased Cu and Fe levels in the brain, (Bush, 2003), from where it is ultimately liberated in a soluble form, to precipitate in the amyloid plaques. Aβ peptides will increase calcium influx through voltage gated calcium channels (N and L types) by reducing magnesium blockade of NMDA receptors, as well as forming cation-selective ion channels after Aβ peptide incorporation into the cellular membrane, thereby increasing excitotoxicity. Aβpeptides may interfere with long-term hippocampal potentiation and cause synaptic dysfunction in Alzheimer's disease.

It is unknown whether Aβ, which is continuously secreted under normal physiological conditions, may have a physiological role, possibly functioning as an antioxidant. There is an inverse relationship between Aβ content and in vivo oxidative damage, suggesting that Aβ might be a modulator of ROS generation. The precipitation of Aβ into plaques, associated with increased levels of metal ions, may be an efficient means of presentation to phagocytic cells for its removal from the cell,  Other physiological functions assigned to A<β are as a superoxide scavenger (SOD activity), a cholesterol binding molecule, and an acute phase reactant (reviewed in Obrenovich et al., 2002). Many forms of stress, including head injury and trauma, may be regulated via β-amyloid. .

Tau proteins are highly hydrophilic microtubule-associated proteins.. There are six isoforms of tau, ranging between 352 and 441 amino acids in length, which are produced as a result of alternative mRNA slicing from a single gene on chromosome 17, and are differentially expressed in neurons. In the normal brain, the functions of tau include stabilisation of axonal microtubules, modulation of signal transduction and regulation of vesicle transport. Tau protein monomers can polymerise to form fibrils known as paired helical filaments (aggregated hyperphosphorylated tau protein), which become the building blocks for the neurofibrillary tangles, neuropil threads and dystrophic neuritis that accumulate in AD. Tau is a natively unfolded protein, containing neither α-helix nor β-sheet, which regulates microtubule function in the axon. It shows an even distribution of hydrophobic fragments along its sequence with up to four repeats of approx 30 residues in its central region. Peptides from tau are more soluble than Aβ. Short synthetic peptides spanning tau repeat sequences are able to aggregate as β-structures. A peptide from the C-terminus (residues 423-441) forms a regular helix in an organic solvent/aqueous mixture, with a helical stabilising C-capping motif (Esposito et al., 2000).

NFTs contain redox active iron. Accumulation of tau in neurofibrillary tangles is associated with the induction of haem oxygenase 1, HO-1 a potent antioxidant which plays an important role in metabolising haem released from damaged mitochondria. HO-1 will reduce oxidative damage but Fe²⁺ will be released which may participate in Fenton chemistry to produce hydroxyl radicals. Tau within the neurofibrillary tangles is oxidatively damaged. High levels of Zn, Cu and Fe are constitutively found in the neocortical regions which are most prone to AD pathology. Enriched amounts of copper, iron and zinc are also present in the insoluble amyloid plaques in post mortem AD brain. Alternatively is has been suggested that the formation of the β-sheet configuration of Aβ may actually be a protective mechanism. It maybe that the increased synthesis of APP and Aβ is an attempt by the brain cells to detoxify the elevated levels of redox active metals, copper and iron; other studies suggest that zinc and copper are inhibitory and prevent β-sheet formation. Membrane-bound Aβ may be damaged by metal induced reactive oxygen species  prior to their liberation from the membrane, and consequently precipitated by zinc which is released from synaptic vesicles. Figure 11. In vitro, zinc rapidly accelerates Aβ aggregation, the zinc being associated with the N-terminal region of Aβ which has an autonomous zinc binding domain. Zinc induces conformational change of the 1-16 N-terminal region of AP3. .  In adult rat brain, pools of zinc are detectable within glutamatergic synaptic vesicles in the neocortex which represents 30% of total brain zinc. Zinc is released during synaptic transmission, possibly in conjunction with glutamate, and induces cerebral Aβ deposition in a transgenic mouse model for AD. The transporter ZnT3 carries zinc into vesicles such that its genetic ablation will inhibit Aβ deposition in Tg2576 mice.

Iron may modulate APP processing, by virtue of the .presence of a putative iron response element in APP mRNA (based on sequence homology). The IRE was mapped within the 5'-untranslated regions (5'-UTR) of the APP transcript (+51 to +94) from the 5'cap site. Figure 12. The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain (+101 to +146). In response to intracellular iron chelation, translation of APP was selectively down regulated, thereby causing a striking decrease in the production of APP _sol. Iron influx reversed this inhibition, by a pathway similar to iron control of the translation of the ferritin-L mRNAs by iron responsive elements in its 5'UTRs. . In addition, increase in cytokine production, namely IL-1 increased IRP binding to the APP 5'-UTR, thereby decreasing APP production. When the APP cRNA probe is mutated in the core IRE domain, IRP binding is abolished. In addition binding of the IRP to the IRE might interfere with APP translation and translocation across the endoplasmic reticulum membrane. This interference could be significant since α-secretase activity has been shown to require membrane bound APP. The role played by IRP-2 in Alzheimer's disease remains undefined but may be more important than was previously thought.

Aβ has a very effective binding domain for copper in its N-terminal domain and can bind copper in nmol amounts, Figure 13. It is unclear whether APP or Aβ, when associated with copper, are in fact neuronal metallochaperones. Knock out and knock in mice for APP show that in the former, cerebral cortex copper levels are increased, whereas in the latter reduced copper levels were assayed. Copper was also influential in APP processing in the cell, copper will reduce levels of Aβ and cause an increase in the secretion of the APP ectodomain.