**5.2 Amyloid-β**

222 Basic and Clinical Endocrinology Up-to-Date

the Chinese famine, fetal and infant exposure to undernutrition also resulted in increased risk for metabolic syndrome (Li et al. 2010; Li et al. 2011; Yang et al. 2008; Luo et al. 2006). In summary, IGF-1 signalling during early development is defined by the number of brain IGF-1Rs or their sensitivity and determines endocrine as well as metabolic function in later

Alzheimer's disease (AD) is a chronic and progressiv neurodegenerative disease and the most common form of dementia leading to the loss of cognitive abilities and finally to death

AD was first described by Alois Alzheimer, a German physician, in 1906 (Alzheimer et al. 1995). The disease is characterised by β-amyloid accumulation, formation of extracellular amyloid plaques as well as neurofibrillary tangles. The β-amyloid plaques mainly contain aggregated amyloid-β (Aβ) peptides (Masters et al. 1985). In contrast, the main components of neurofibrillary tangles are hyperphosphorylated and aggregated tau proteins (Ross et al., 2005). The aggregation of Aβ is thought to be the molecular basis of neurodegenration in AD

The tau proteins consist of a N-terminal projection domain, a short tail sequence and a Cterminal domain with microtubule-binding (MTB) repeats. Six isoforms of tau are known in the human brain. These isoforms emerge from alternative splicing of exons 2, 3 and 10. Exon 2 and 3 encode N-terminal parts of tau and exon 10 codes for an additional MTB repeat. Thus, tau can present three or four MTB repeats (Ballatore, Lee, and Trojanowski 2007; Goedert and Spillantini 2006). Tau is predominantly located in the axons of neurons (Hirokawa et al. 1996) and is to less extend found in dendrites (Ittner et al. 2010). The function of tau is yet not completely understood, but it might influence the stabilisation of microtubules and regulation of axonal transport (Gotz, Ittner, and Kins 2006). Tau is phosporylated at several sites via kinases like glycogen synthase kinase 3 (GSK-3β), cyclin-dependent kinase 5 (Cdk5), c-Jun N-

**brain IGF-1 function in the adult brain**

• preservation of neuronal

olfactory bulb • brain vessel growth • spatial learning and other cognitive functions

lifespan

plasticity, for example in the

• neuroprotecetive in ischemic or traumatic injuries and several neurological disorders • reduced signalling extends

life possibly playing a key-role in pathogenesis of age-associated diseases.

**IGF-1 function in the developing** 

• growth and differentiation

• enhancement of myelination • neuroendocrine regulation of

• neuronal polarity and

 synapse formation • growth of oligodendrocytes

metabolism

Table 1. Function of IGF-1 in the brain

**5. IGF-1 in Alzheimer's disease** 

(Citron 2002; Cole et al. 2007).

(Masters et al. 1985).

**5.1 Tau** 

of neurons

Aβ is generated by proteolytic cleavage of the amyloid precursor protein (APP), a type-1 integral membrane protein. APP was first described and cloned in 1987 (Kang et al. 1987; Tanzi et al. 1987; Goldgaber et al. 1987; Robakis et al. 1987). The APP gene is located on chromosome 21. Hence, patients with trisomy 21 show a higher risk to develop Alzheimer´s disease, because of the additional APP allele. Accordingly, the duplication of the isolated APP gene causes cerebral amyloid angiopathy and amyloidosis suggesting that increased APP expression itself is sufficient to cause Alzheimer-like pathology (Rovelet-Lecrux et al. 2006; Sleegers et al. 2006). Another risk factor for AD are mutations of the APP gene (Vassar 2004; Bertram and Tanzi 2005). APP contains a N-terminal extracellular domain and a shorter C-terminal cytoplasmic domain. Alternative splicing of the APP gene results in different isoforms of APP which are distinguishable by length. APP with 751 and 770 (APP751 and APP770) amino acids mainly occur in non-neuronal tissue. APP695 is mainly localised in neurons (Kang and Muller-Hill 1990). The function of APP and the APP-like proteins (APLP) is not clear yet. These proteins are possibly involved in cell adhesion, apoptosis and axonal transport.

The β-secretase BACE1 (β-site APP-cleaving enzyme) plays an essential role in the production of Aβ. It cleaves APP at Asp+1 at the N-terminus. The resulting fragments are called APPsβ and the C-terminal fragment C99. Upon cleavage of C99 by the γ-secretase, a complex formed by presenilin, nicastrin, Aph-1 and Pen-2, Aβ peptides (4 kDa) and the APP intracellular domain (AICD) with a size of 6 kDa are generated. Aβ-peptides mainly occur in two variants: Aβ40 which ends at residue 40 and Aβ42 ending at residue 42 after cleavage. Predominantly, the Aβ42 is prone to aggregate and forms toxic oligomers. Furthermore, APP is cleaved by the α-secretases ADAM10 (a disintegrin and metalloproteinase-like 10) or TACE (tumour necrosis factor-alpha convertase). This results in the C-terminal fragment C83 and APPsα. The cleavage of APP by α- or β-secretase is dependent on the competition between both enzymes. In case the β-secretase cleavage of APP increases, α-secretase processing decreases and vice versa (Vassar et al. 1999; Skovronsky et al. 2000) (Figure 3).

In a healthy brain, there is more production of Aβ40 (~90 %) than there is of Aβ42 (~5-10 %) (Walsh and Selkoe 2007). The accumulation of Aβ42 is an important step in the formation of amyloid plaques (Iwatsubo et al. 1994). The Aβ42:Aβ40 ratio is a diagnostic tool for APP processing and development of AD (Haass and Selkoe 2007).

In addition to age-associated Aβ42 accumulation, mutations in presenilin 1, presenilin 2 and the APP gene lead to familiar early-onset AD (Tabaton and Tamagno 2007; Sherrington et al.

Role of Central Insulin-Like Growth Factor-1

**5.3 IR, IGF-1R signalling and Alzheimer's disease** 

Aβ42 peptides are released.

models (Freude et al. 2009).

neurodegeneration (Sotthibundhu et al. 2008).

al. 2009).

Receptor Signalling in Ageing and Endocrine Regulation 225

APP might be cleaved via the α-, β- or γ-secretase. α-secretase cleavage generates a membrane bound C83 fragment and sAPPα. In case C83 is proteolytically cleaved by the γsecretase the P3 fragment occurs. β-secreatse processing produces the fragments sAPPβ and C99. In case of a simultaneous or subsequent cleavage via β- and γ- secretase the Aβ40 or

The IR and IGF-1R signalling pathway is disturbed in the central nervous system (CNS) of AD patients (Frolich et al. 1998; Frolich et al. 1999; Moloney et al. 2010). Analysis of the mRNA level of insulin and the IR showed a decrease of about 80% in AD patients. Additionally, the expression of the IGF-1R was reduced in AD brains compared to controls (Moloney et al. 2010; Rivera et al. 2005). In contrast, the IGF-1 serum levels of AD patients are increased indicating IGF-1 resistance in AD (Rivera et al. 2005; Vardy et al. 2007). Furthermore, IRS-1 and -2 expression is reduced in AD brains and phosphorylation of IRS-1 at Ser312 and Ser616 is increased, which decreases IRS-1 action characterising AD as "brain type" diabetes (Pilcher 2006)**.** Thus, brains of AD patients are insulin and IGF-1 resistant. Whether these changes are cause or consequence of neurodegeneration is a matter of debate. IGF-1 knockout mice display increase of tau phosphorylation at Ser396 and Ser202 while the tau protein level was not influenced (Cheng et al. 2005). In NIRKO mice, the brain-specific IR knockout mice, tau was hyperphosphorylated at Thr231 (Schubert et al. 2004), whereas IRS-2 knockout mice showed hyperphosphorylation at Ser202 (Schubert et al. 2003). The different phosphorylation patterns of tau in different insulin and IGF-1 resistant mouse models indicate that additional factors may play a role for tau phosphorylation in these

Tg2576 mice express the Swedish mutation of APP (APPsw) and are an established mouse model for analysing amyloid pathology (Vassar et al. 1999; De Strooper 2003; Harada et al. 2006). IRS-2 (IRS-2-/-) or neuron specific IGF-1R knockout (nIGF-1R-/-) in Tg2576 mice protects these mice from premature death and decreases Aβ-accumulation (Freude et

BACE-1 and Presenilin-1/-2, which cleave APP and generate neurotoxic Aβ42, are possible targets for AD treatment since β-secretase cleavage is the rate limiting step of Aβ generation. During ageing, the expression of the neurotrophin receptor tyrosine kinase receptor A (TrkA) and the p75 neurotrophin receptor (p75NTR) changes considerably. Whereas TrkA receptor expression decreases, the p75 neurotrophin receptor increases with age. Human neuroblastoma cells SHSY5Y and primary cultured neurons showed a switch from TrkA to p75NTR expression after treatment with IGF-1 (Costantini, Scrable, and Puglielli 2006). This increases BACE-1 activity via hydrolysis of sphingomyelin and release of ceramide stabilising BACE-1 (Puglielli 2008; Puglielli et al. 2003). It has been shown that embryonic hippocampal neurons treated with Aβ42 as ligand of p75NTR cause cell death. Neurons, which are deficient in p75NTR and also treated with Aβ42, show less cell death. This may represent the molecular mechanism linking IR and IGF-1R signalling pathway to ageing and

In *Caenorhabditis elegans* the knockdown of DAF-2, the orthologue of mammalian IR and IGF-1R, reduces Aβ42 toxicity (Cohen et al. 2006). This reduced Aβ42 toxicity results from the activity of the downstream transcription factors DAF-16, the orthologue of mammalian FoxO1 and 3a as well as heat shock transcription factor-1 (HSF-1) (Hsu, Murphy, and Kenyon 2003; Birkenkamp and Coffer 2003; Cohen et al. 2006). The

1996; Tanzi et al. 1992; Schellenberg et al. 1992; Van Broeckhoven et al. 1992; St George-Hyslop et al. 1992; Rogaev et al. 1995).

The toxic effect of Aβ is not fully understood yet, but might be induced via generation of ion channels, membrane disruption, oxidative stress, induction of apoptosis and inflammation (Hardy and Selkoe 2002; Nakagawa et al. 2000; Soto 2003; Roberson and Mucke 2006). The Aβ aggregation process produces different intermediates. Aβ monomers are soluble and amphipathic with an α-helical conformation and kink regions in water-alcohol mixture (Coles et al. 1998; Crescenzi et al. 2002). Aβ40 displays a random coil structure in aqueous solution (Zhang et al. 2000) and Aβ42 shows β-sheet structure at physiological conditions (Barrow and Zagorski 1991). Aβ dimers are located intracellular *in vivo* and show a hydrophobic core (Roher et al. 1996). Small Aβ oligomers are highly cytotoxic compared to mature Aβ fibrils (Dahlgren et al. 2002; McLean et al. 1999; Cleary et al. 2005; Lesne et al. 2006). The so called Aβ-derived diffusible ligands (ADDLs) show no fibrillar structure and are neurotoxic in a size of about 17 to 42 kDa (Chromy et al. 2003; Klein, Stine, and Teplow 2004; Lambert et al. 1998). The levels of ADDLs are linked to cognitive impairments in AD (Georganopoulou et al. 2005). Aβ protofibrils are the precursors of Aβ fibrils. These protofibrils are present as rod-like and flexible structures. The dyes Congo red and thioflavin T bind to the core of the protofibrils, which indicates a high level of β-sheets (Harper et al. 1999; Arimon et al. 2005; Harper et al. 1997; Kheterpal et al. 2003; Walsh et al. 1997; Williams et al. 2005). Aβ fibrils are insoluble, thermodynamically stable aggregates containing repeats of β-sheets (Ross and Poirier 2005). They also bind Congo red and thioflavin T (Klunk, Jacob, and Mason 1999; LeVine 1999). The amyloid plaques are extracellular aggregates of insoluble Aβ fibrils (Muller-Hill and Beyreuther 1989). These plaques are surrounded by activated microglia, astrocytes and dystrophic dendrites (Selkoe 2004).

Different clinical studies revealed an association of AD and type 2 diabetes (Janson et al. 2004; Ott et al. 1999; Stewart and Liolitsa 1999). A connection of glucose intolerance, impairment of insulin secretion and the risk to develop AD was recently discovered (Ott et al. 1996; Luchsinger et al. 2004; Ronnemaa et al. 2008). Furthermore, AD patients develop more frequently impaired glucose tolerance and type 2 diabetes (Janson et al. 2004) indicating that IR/IGF-1R signalling might influence AD pathogenesis.

Fig. 3. Processing of amyloid precursor protein (APP).

1996; Tanzi et al. 1992; Schellenberg et al. 1992; Van Broeckhoven et al. 1992; St George-

The toxic effect of Aβ is not fully understood yet, but might be induced via generation of ion channels, membrane disruption, oxidative stress, induction of apoptosis and inflammation (Hardy and Selkoe 2002; Nakagawa et al. 2000; Soto 2003; Roberson and Mucke 2006). The Aβ aggregation process produces different intermediates. Aβ monomers are soluble and amphipathic with an α-helical conformation and kink regions in water-alcohol mixture (Coles et al. 1998; Crescenzi et al. 2002). Aβ40 displays a random coil structure in aqueous solution (Zhang et al. 2000) and Aβ42 shows β-sheet structure at physiological conditions (Barrow and Zagorski 1991). Aβ dimers are located intracellular *in vivo* and show a hydrophobic core (Roher et al. 1996). Small Aβ oligomers are highly cytotoxic compared to mature Aβ fibrils (Dahlgren et al. 2002; McLean et al. 1999; Cleary et al. 2005; Lesne et al. 2006). The so called Aβ-derived diffusible ligands (ADDLs) show no fibrillar structure and are neurotoxic in a size of about 17 to 42 kDa (Chromy et al. 2003; Klein, Stine, and Teplow 2004; Lambert et al. 1998). The levels of ADDLs are linked to cognitive impairments in AD (Georganopoulou et al. 2005). Aβ protofibrils are the precursors of Aβ fibrils. These protofibrils are present as rod-like and flexible structures. The dyes Congo red and thioflavin T bind to the core of the protofibrils, which indicates a high level of β-sheets (Harper et al. 1999; Arimon et al. 2005; Harper et al. 1997; Kheterpal et al. 2003; Walsh et al. 1997; Williams et al. 2005). Aβ fibrils are insoluble, thermodynamically stable aggregates containing repeats of β-sheets (Ross and Poirier 2005). They also bind Congo red and thioflavin T (Klunk, Jacob, and Mason 1999; LeVine 1999). The amyloid plaques are extracellular aggregates of insoluble Aβ fibrils (Muller-Hill and Beyreuther 1989). These plaques are surrounded by

Different clinical studies revealed an association of AD and type 2 diabetes (Janson et al. 2004; Ott et al. 1999; Stewart and Liolitsa 1999). A connection of glucose intolerance, impairment of insulin secretion and the risk to develop AD was recently discovered (Ott et al. 1996; Luchsinger et al. 2004; Ronnemaa et al. 2008). Furthermore, AD patients develop more frequently impaired glucose tolerance and type 2 diabetes (Janson et al. 2004)

C83

γ-secretase

P3

α-secretase

sAPPα

activated microglia, astrocytes and dystrophic dendrites (Selkoe 2004).

indicating that IR/IGF-1R signalling might influence AD pathogenesis.

β-secretase

Fig. 3. Processing of amyloid precursor protein (APP).

C99

γ-secretase

Aβ42

Aβ40

sAPPβ

Hyslop et al. 1992; Rogaev et al. 1995).

APP might be cleaved via the α-, β- or γ-secretase. α-secretase cleavage generates a membrane bound C83 fragment and sAPPα. In case C83 is proteolytically cleaved by the γsecretase the P3 fragment occurs. β-secreatse processing produces the fragments sAPPβ and C99. In case of a simultaneous or subsequent cleavage via β- and γ- secretase the Aβ40 or Aβ42 peptides are released.
