T RANSGENIC MOUSE MODELS OF A LZHEIMER ' S DISEASE

The detection of mutations in the APP and PSEN genes in patients with familial AD led to the generation of a variety of transgenic animal models. A wide range of transgenic mouse models have been developed expressing mutant human APP. The first AD transgenic mouse model based on mutant APP was the PDAPP model. These mice express human APP with the Indiana mutation (V717F) under the platelet derived growth factor promoter. PDAPP mice develop an age-dependant extracellular plaque pathology in addition to dystrophic neurites, astrocytosis and microgliosis. (Games et al., 1995; Reilly et al., 2003). Moreover, they show progressive synaptic loss and develop age-dependant memory deficits without neuron loss (Irizarry et al., 1997; Chen et al., 2000; Dodart et al., 2000). The Tg2576 mouse also possesses a single APP mutation and is one of the most widely studied AD models. Human APP containing the Swedish FAD mutation (K670N/M671L) is overexpressed in these mice under the control of the hamster prion promoter. Tg2576 mice develop an age-dependant plaque pathology along with dystrophic neuritis and gliosis similar to PDAPP mice. A significant plaque pathology in Tg2576 mice can be detected between 11 and 13 months. Strikingly, these mice develop working and spatial reference memory deficits prior to a significant plaque pathology (Hsiao et al., 1996; Holcomb et al., 1998; Elder et al., 2010). Subsequently, several other AD mouse models were generated with human APP containing one or more FAD mutations. Generally speaking, these mice show elevated Aβ levels and extracellular Aβ.

Furthermore, many of these mice also show signs of inflammation and develop behavior and cognitive deficits (Sturchler-Pierrat et al., 1997; Chishti et al., 2001; Games et al., 2006; Elder et al., 2010).

Next to APP transgenic mouse models, many PSEN mice have been developed.

Transgenic mice expressing mutated human PSEN consistently show elevated Aβ₄₂ levels without developing a plaque pathology (Cavanaugh et al., 2014). The lack of plaques may be due to lower levels of Aβ₄₂ in the PSEN lines in comparison to APP models. It could also be explained through the different solubility of human and murine Aβ (Jankowsky et al., 2007). However, a number of PSEN models develop altered axonal transport, impaired calcium homeostasis in the ER and mild cognitive deficits (Duyckaerts et al., 2008).

In order to create a more aggressive AD pathology mouse models with a combination of multiple APP mutations or APP and PSEN mutations were created. Double transgenic mice expressing mutated PSEN and APP develop plaques earlier than single transgenic APP mice and show increased inflammation and behavior deficits (Duff et al., 1996; Holcomb et al., 1998; Casas et al., 2004; Cavanaugh et al., 2014). Although these

1 Introduction

mouse models show a wide spectrum of AD pathology, they lack some key feature of AD including NFT formation. To overcome the lack of NFTs in AD mouse models, FAD and tau mutations from frontotemperal dementia have been combined. The 3xTg model is an example for a triple transgenic mouse model. These mice, generated by injecting two transgenes containing APP-Swedish and P301L mutations into a PS1M146V knock-in mice, show next to plaque depositions, a NFT pathology (Oddo et al., 2003).

Furthermore, most AD mouse models do not exhibit the significant neuron loss seen in AD patients. Neuron loss could be mainly detected in mice with a combination of APP and PSEN mutations. APP751SL/PS1M146L, APP/PS1K1 and 5XFAD mice are some examples for AD models with neuron loss (Casas et al., 2004; Schmitz et al., 2004;

Oakley et al., 2006; Jawhar et al., 2010).

The use of transgenic mouse models has led to new insights regarding the role of intraneuronal Aβ accumulation in AD. Intraneuronal Aβ has been reported in variety of mouse models including Tg2576, 3xTg, APP/PS1K1 and 5XFAD (Casas et al., 2004;

Billings et al., 2005; Oakley et al., 2006; Takahashi et al., 2013). Furthermore, several mouse models showed a link between significant neuron loss and intraneuronal Aβ accumulation (Wirths and Bayer, 2012).

Familial AD accounts for a minor fraction of all AD cases, however all of the previously described AD mouse models rely on one or more FAD mutations. Rodents do not develop a spontaneously AD pathology as they age (Sarasa and Pesini, 2009). To date, there has been nearly no success in the generation of sporadic AD mouse models.

A rodent model that shows at least some aspects of SAD has been generated by intracerebroventricular (icv) injection of streptozotocin (STZ). These mice are generated based on the detection of an insulin-resistant brain state in SAD. STZ is injected to induce such a brain insulin resistant state. These icv-STZ mice show memory and learning deficits, decreased energy metabolism, neuroinflammation, altered synaptic proteins and increased hyperphosphorylated tau in the brain (Salkovic-Petrisic et al., 2006; de la Monte, Suzanne M. and Wands, 2008; Chen et al., 2012; Chen et al., 2013).

Next to mice, a variety of other animals have been proposed to study AD including dogs, rats, rabbits, primates and even dolphins. However, mice remain the most commonly used vertebrates to study AD especially due to their relatively short reproductive cycle, their small size, large litters and easy handling (Sarasa and Pesini, 2009). Several non-vertebrates, like the Caenorhabditis elegans and Drosophila melanogaster have also be used and genetically manipulated to study certain aspects of AD (Wu and Luo, 2005; Prüßing et al., 2013). AD mouse models, as well as other animal models, have highly contributed to the understanding of the pathology in AD. However,

1 Introduction

none of the animal model mimics all human AD features, they rather reflect various parts of the disease and therefore provide insights in different aspects of AD.

1.8.1 The 5XFAD mouse model

The 5XFAD mouse model, first described by Oakley et al. (2006), is a double transgenic APP/PS1 mouse model that coexpresses five familial Alzheimer's disease (FAD) mutations. 5XFAD mice overexpress the 695 amino acids isoform of human APP carrying the Florida (I716V), London (V717I) and Swedish (K670N/M671L) mutations, together with mutant PSEN-1 (M146L; L286V) under the control of the murine Thy-1 promoter (FIGURE 1.8). Mice showed stable germline transmission and coinheritance of the APP and PSEN-1 transgenes over multiple generations. Therefore, mice breed as if they are a single transgenic mouse model (Oakley et al., 2006; Eimer and Vassar, 2013). In order to facilitate a better comparison with other AD mouse models 5XFAD mice, that were originally generated as a C57/B6xSJL strain, were backcrossed onto a C57BL/B6 background (Jawhar et al., 2010).

The Florida, London and the two PSEN mutations result in an increased production of Aβ_x-42, while the Swedish mutation increases the levels of total Aβ. Plaque pathology in these mice starts between two and three months of age and is accompanied by astrocytosis and microgliosis. These features increase massively with age (Oakley et al., 2006). Extracellular plaques have also been detected in the spinal cords of three months old 5XFAD mice (Jawhar et al., 2010). In addition to extracellular plaques, 5XFAD exhibit intracellular Aβ in the cortical Layer 5 and the subiculum starting at 1.5 months of age (Eimer and Vassar, 2013). Intraneuronal Aβ precedes plaque formation and correlates well with neuron loss in these brain regions. The 5XFAD mouse is one of a few mouse models exhibiting significant neuron loss. Unbiased stereology revealed a significant neuron loss in the pyramidal neurons of the Layer 5 cortex and the subiculum starting at 9 months of age (Eimer and Vassar, 2013). Strikingly, the CA1 region of the hippocampus lacking intraneuronal Aβ shows amyloid plaques but no neuron loss (Jawhar et al., 2010).

Furthermore, 5XFAD develop age-dependant synaptic degeneration indicated by the reduction of synaptic markers including synaptophysin, syntaxin, and postsynaptic density protein 95 (Oakley et al., 2006). Swollen presynaptic terminals and axonal processes develop intraneuronal BACE1 and Aβ prior to plaque formation. Swelling and dystrophic changes in these structures are associated with deposition of extracellular Aβ plaques (Zhang et al., 2009).

1 Introduction

5XFAD mice develop age-dependant progressive behavior deficits. Working memory, spatial memory and episodic memory deficits have been described in these mice as well as decreased anxiety. They show among others impairments in the Y-maze, Morris mater maze, the conditioned taste aversion task and contextual fear conditioning (Ohno et al., 2006; Devi and Ohno, 2010; Jawhar et al., 2010; Devi and Ohno, 2013).

Next to cognitive impairments, 5XFAD mice also show a decline in motor function. The motor phenotype correlates well with extensive spinal cord pathology, including age-dependent axonal degeneration and intraneuronal Aβ accumulation (Jawhar et al., 2010).

FIGURE 1.8 The 5XFAD transgenes. Schematic diagram of (A) Thy-1 APP and (B) Thy-1 PSEN-1 transgenes in the 5XFAD mouse model. FAD mutations are indicated by arrows. Abbreviations: Sw

= Swedish mutation; Lon = London mutation; Fl = FL mutation. Figure created after Oakley et al., 2006.