5 SUMMARY
Besides Aβ staring with an aspartate at position 1, a variety of modified and truncated Aβ forms have been detected in AD brains. Together with full length Aβ1-42 and Aβ1-40, N-truncated AβpE3-42 and Aβ4-42 are major variants in AD (Bayer and Wirths, 2014). Aβ4-42
has been identified as a particular abundant Aβ species in the hippocampus and cortex of AD patients and was discovered as early as 1985 (Masters et al., 1985; Portelius et al., 2010). Sedimentation studies indicate that Aβ4-42 exhibits fast aggregation kinetics (Pike et al., 1995b). Interestingly, short-term treatment of primary cortical neurons revealed that Aβ4-42 is as toxic as pyroglutamate Aβ3-42 and Aβ1-42. Moreover, treatment of wild-type mice using intraventricular Aβ injection with Aβ4-42 induced significant working memory deficits.
These deficits were similar to the ones induced through pyroglutamate Aβ3-42 and Aβ1-42 (Bouter et al., 2013). However, relatively little is known about the contribution of Aβ4-42 to the development and progression of AD. In this study, it could be shown that Aβ4-x precedes AβpE3-x accumulation in the brain of young 5XFAD mice by using the novel antibody NT4X-167. Moreover, Aβ4-x could be detected together with Aβ1-x intraneuronal in cortical neurons of six weeks old homozygous 5XFAD mice. The results emphasize the significance of Aβ4-42 in AD pathology.
In the present work, the effects of chronic exposure of Aβ4-42 in vivo were studied.
Nearly all transgenic mouse models for AD rely on artificial combinations of mutations.
However, the vast majority of AD patients do not possess mutations and suffer from the sporadic form of the disease (Elder et al., 2010; Zetterberg and Mattsson, 2014).
Therefore, direct expression of a specific Aβ species in vivo may demonstrate a more physiologically relevant AD model. Given these considerations the Tg4-42 mouse model, that exclusively expresses human Aβ4-42, was recently generated in our lab. (Bouter et al., 2013). Here, the new homozygous Tg4-42 line was generated (Tg4-42hom) in addition to heterozygous mice. Using immunohistochemistry, it could be shown that Tg4-42 and Tg4-42hom mice develop region-specific intraneuronal accumulation of Aβ accompanied by gliosis. Tg4-42 and Tg4-42hom mice showed strong intraneuronal Aβ immunoreactivity predominantly in the CA1 pyramidal cell layer of the hippocampus beginning at two
5 Summary
months of age. Intraneuronal Aβ was also detected in Tg4-42 mice in the occipital cortex, piriform cortex, striatum and superior colliculus starting a two months of age. Aβ4-42 expression in the CA1 declined during aging in Tg4-42 and Tg4-42hom mice. The decrease of Aβ expression can be attributed to the extensive neuron loss in this brain region. Long-term exposure to N-truncated Aβ4-42 induced an age- and dose-dependent neuron loss in hemizygous and homozygous Tg4-42 mice in the CA1 layer of the hippocampus. At eight months unbiased stereology revealed a 38 % neuron loss in hemizygous Tg4-42 mice and a 65 % neuron loss in Tg4-42hom. Furthermore, 12-month-old Tg4-42 mice showed a 51 % decrease in neuron number in the CA1 compared to same-aged WT.
The behavior analysis of Tg4-42 and Tg4-42hom mice revealed that mice develop no deficits in either motor function or anxiety-behavior that could prevent or potentially confound memory testing of these mice. Strikingly, mice showed hippocampus-dependant memory deficits similar to AD patients. Tg4-42 and Tg4-42hom were profoundly impaired in their spatial reference memory. Moreover, aged Tg4-42 showed a decline in contextual fear memory. The over-expression of Aβ4-42 in this mouse model induces severe age-dependant memory deficits that can be attributed to the massive neuron loss in the hippocampus.
In summary, the pathology of Tg4-42 mice is unique in its massive neuron loss and
severe memory behavior deficits induced through intraneuronal accumulation of N-terminally truncated Aβ4-42 albeit without plaque formation. It can be stated that Tg4-42
mice emphasize the toxicity of Aβ4-42 in vivo. Moreover, the resulting neurodegeneration associated with neuron loss, gliosis and memory deficits appears to be a direct consequence of intracellular neurotoxicity of Aβ. In summary, Tg4-42 is a valid AD mouse model showing key features of sporadic AD.
One of the central research questions on the etiology of AD is the elucidation of the molecular signatures triggered by the amyloid cascade of pathological events. Next generation sequencing allows the identification of genes involved in disease processes in an unbiased manner. A comparative gene expression analysis of brain tissue of Tg4-42 and 5XFAD mice was performed using next-generation sequencing. As stated before, Tg4.42 mice express N-truncated Aβ4-42 and develop intraneuronal Aβ aggregation, neuron loss and behavioral deficits albeit without plaque formation. The widely used 5XFAD model, which is based on the expression of mutant amyloid precursor protein and presenilin-1 genes, is a typical model for early plaque formation, intraneuronal Aβ aggregation, neuron loss and behavioral deficits (Oakley et al., 2006; Jawhar et al., 2010).
Interestingly, the results showed that learning and memory deficits in the Morris water maze and fear conditioning tasks in Tg4-42 mice at twelve months of age are similar to
5 Summary
the deficits in 5XFAD animals. This suggested that comparative gene expression analysis between the models would allow the dissection of plaque-related and -unrelated disease relevant factors. Therefore brain transcriptomes of young and old Tg4-42, 5XFAD, and wild-type control mice were analyzed by deep-sequencing of non-ribosomal RNA (RNA-Seq). Differentially expressed genes represented by more than 200 deep sequencing reads (DEGs) were verified by qRT-PCR. Nineteen DEGs were identified in presymptomatic young 5XFAD mice, while no DEGs could be isolated in young Tg4-42 mice. In the aged cohort, 131 DEGs were found in 5XFAD and 56 DEGs in Tg4-42 mice.
Intriguingly, 36 DEGs were identified in both mouse models indicating common disease pathways associated with behavioral deficits and neuron loss. The pool of genes that showed differential expression exclusively in Tg4-42 is likely associated to soluble Aβ as no extracellular plaques are found in this model. In addition, the robust CA1 neuron loss could also contribute to the differential expression profile as 5XFAD mice display no neuron loss in this brain region. Many of the DEGs specific to the 5XFAD model belong to neuroinflammatory processes typically associated with plaques. As Tg4-42 mice do not develop any plaques, but still show massive neuron loss, it can be concluded that only the DEGs common to both models together with those specific to Tg4-42 are defining the molecular signature underlying memory decline in AD.
Taken together, the results of this thesis demonstrate Aβ4-42 as a toxic Aβ variant that likely plays a dominant role in triggering AD pathology.