Generation and Characterization of an Abcc1 Humanized Mouse Model (hABCC1flx/flx ) with Knockout Capability.

ATP-binding cassette (ABC) transporters such as ABCB1 (P-glycoprotein), ABCC1 (MRP1), and ABCG2 (BCRP) are well known for their role in rendering cancer cells resistant to chemotherapy. Additionally, recent research provided evidence that, along with other ABC transporters (ABCA1 and ABCA7), they might be cornerstones to tackle neurodegenerative diseases. Overcoming chemoresistance in cancer, understanding drug-drug interactions, and developing efficient and specific drugs that alter ABC transporter function are hindered by a lack of in vivo research models, which are fully predictive for humans. Hence, the humanization of ABC transporters in mice has become a major focus in pharmaceutical and neurodegenerative research. Here, we present a characterization of the first Abcc1 humanized mouse line. To preserve endogenous expression profiles, we chose to generate a knockin mouse model that leads to the expression of a chimeric protein that is fully human except for one amino acid. We found robust mRNA and protein expression within all major organs analyzed (brain, lung, spleen, and kidney). Furthermore, we demonstrate the functionality of the expressed human ABCC1 protein in brain and lungs using functional positron emission tomography imaging in vivo. Through the introduction of loxP sites, we additionally enabled this humanized mouse model for highly sophisticated studies involving cell type-specific transporter ablation. Based on our data, the presented mouse model appears to be a promising tool for the investigation of cell-specific ABCC1 function. It can provide a new basis for better translation of preclinical research.

Expression of endogenous mouse APP modulates ß-amyloid deposition in hAPP-transgenic mice.

Amyloid-ß (Aß) deposition is one of the hallmarks of the amyloid hypothesis in Alzheimer's disease (AD). Mouse models using APP-transgene overexpression to generate amyloid plaques have shown to model only certain parts of the disease. The extent to which the data from mice can be transferred to man remains controversial. Several studies have shown convincing treatment results in reducing Aß and enhancing cognition in mice but failed totally in human. One model-dependent factor has so far been almost completely neglected: the endogenous expression of mouse APP and its effects on the transgenic models and the readout for therapeutic approaches.Here, we report that hAPP-transgenic models of amyloidosis devoid of endogenous mouse APP expression (mAPP-knockout / mAPPko) show increased amounts and higher speed of Aß deposition than controls with mAPP. The number of senile plaques and the level of aggregated hAß were elevated in mAPPko mice, while the deposition in cortical blood vessels was delayed, indicating an alteration in the general aggregation propensity of hAß together with endogenous mAß. Furthermore, the cellular response to Aß deposition was modulated: mAPPko mice developed a pronounced and age-dependent astrogliosis, while microglial association to amyloid plaques was diminished. The expression of human and murine aggregation-prone proteins with differing amino acid sequences within the same mouse model might not only alter the extent of deposition but also modulate the route of pathogenesis, and thus, decisively influence the study outcome, especially in translational research.

Revisiting rodent models: Octodon degus as Alzheimer's disease model?

Alzheimer's disease primarily occurs as sporadic disease and is accompanied with vast socio-economic problems. The mandatory basic research relies on robust and reliable disease models to overcome increasing incidence and emerging social challenges. Rodent models are most efficient, versatile, and predominantly used in research. However, only highly artificial and mostly genetically modified models are available. As these 'engineered' models reproduce only isolated features, researchers demand more suitable models of sporadic neurodegenerative diseases. One very promising animal model was the South American rodent Octodon degus, which was repeatedly described as natural 'sporadic Alzheimer's disease model' with 'Alzheimer's disease-like neuropathology'. To unveil advantages over the 'artificial' mouse models, we re-evaluated the age-dependent, neurohistological changes in young and aged Octodon degus (1 to 5-years-old) bred in a wild-type colony in Germany. In our hands, extensive neuropathological analyses of young and aged animals revealed normal age-related cortical changes without obvious signs for extensive degeneration as seen in patients with dementia. Neither significant neuronal loss nor enhanced microglial activation were observed in aged animals. Silver impregnation methods, conventional, and immunohistological stains as well as biochemical fractionations revealed neither amyloid accumulation nor tangle formation. Phosphoepitope-specific antibodies against tau species displayed similar intraneuronal reactivity in both, young and aged Octodon degus.In contrast to previous results, our study suggests that Octodon degus born and bred in captivity do not inevitably develop cortical amyloidosis, tangle formation or neuronal loss as seen in Alzheimer's disease patients or transgenic disease models.

Chronic Toxoplasma gondii infection enhances ß-amyloid phagocytosis and clearance by recruited monocytes.

INTRODUCTION: Alzheimer's disease (AD) is associated with the accumulation of ß-amyloid (Aß) as senile plaques in the brain, thus leading to neurodegeneration and cognitive impairment. Plaque formation depends not merely on the amount of generated Aß peptides, but more importantly on their effective removal. Chronic infections with neurotropic pathogens, most prominently the parasite Toxoplasma (T.) gondii, are frequent in the elderly, and it has been suggested that the resulting neuroinflammation may influence the course of AD. In the present study, we investigated how chronic T. gondii infection and resulting neuroinflammation affect plaque deposition and removal in a mouse model of AD. RESULTS: Chronic infection with T. gondii was associated with reduced Aß and plaque load in 5xFAD mice. Upon infection, myeloid-derived CCR2(hi) Ly6C(hi) monocytes, CCR2(+) Ly6C(int), and CCR2(+) Ly6C(low) mononuclear cells were recruited to the brain of mice. Compared to microglia, these recruited mononuclear cells showed highly increased phagocytic capacity of Aß ex vivo. The F4/80(+) Ly6C(low) macrophages expressed high levels of Triggering Receptor Expressed on Myeloid cells 2 (TREM2), CD36, and Scavenger Receptor A1 (SCARA1), indicating phagocytic activity. Importantly, selective ablation of CCR2(+) Ly6C(hi) monocytes resulted in an increased amount of Aß in infected mice. Elevated insulin-degrading enzyme (IDE), matrix metalloproteinase 9 (MMP9), as well as immunoproteasome subunits ß1i/LMP2, ß2i/MECL-1, and ß5i/LMP7 mRNA levels in the infected brains indicated increased proteolytic Aß degradation. Particularly, LMP7 was highly expressed by the recruited mononuclear cells in the brain, suggesting a novel mechanism of Aß clearance. CONCLUSIONS: Our results indicate that chronic Toxoplasma infection ameliorates ß-amyloidosis in a murine model of AD by activation of the immune system, specifically by recruitment of Ly6C(hi) monocytes and by enhancement of phagocytosis and degradation of soluble Aß. Our findings provide evidence for a modulatory role of inflammation-induced Aß phagocytosis and degradation by newly recruited peripheral immune cells in the pathophysiology of AD.

Accumulation of murine amyloid-ß mimics early Alzheimer's disease.

Amyloidosis mouse models of Alzheimer's disease are generally established by transgenic approaches leading to an overexpression of mutated human genes that are known to be involved in the generation of amyloid-ß in Alzheimer's families. Although these models made substantial contributions to the current knowledge about the 'amyloid hypothesis' of Alzheimer's disease, the overproduction of amyloid-ß peptides mimics only inherited (familiar) Alzheimer's disease, which accounts for <1% of all patients with Alzheimer's disease. The inherited form is even regarded a 'rare' disease according to the regulations for funding of the European Union (www.erare.eu). Here, we show that mice that are double-deficient for neprilysin (encoded by Mme), one major amyloid-ß-degrading enzyme, and the ABC transporter ABCC1, a major contributor to amyloid-ß clearance from the brain, develop various aspects of sporadic Alzheimer's disease mimicking the clinical stage of mild cognitive impairment. Using behavioural tests, electrophysiology and morphological analyses, we compared different ABC transporter-deficient animals and found that alterations are most prominent in neprilysin × ABCC1 double-deficient mice. We show that these mice have a reduced probability to survive, show increased anxiety in new environments, and have a reduced working memory performance. Furthermore, we detected morphological changes in the hippocampus and amygdala, e.g. astrogliosis and reduced numbers of synapses, leading to defective long-term potentiation in functional measurements. Compared to human, murine amyloid-ß is poorly aggregating, due to changes in three amino acids at N-terminal positions 5, 10, and 13. Interestingly, our findings account for the action of early occurring amyloid-ß species/aggregates, i.e. monomers and small amyloid-ß oligomers. Thus, neprilysin × ABCC1 double-deficient mice present a new model for early effects of amyloid-ß-related mild cognitive impairment that allows investigations without artificial overexpression of inherited Alzheimer's disease genes.

Genomic background-related activation of microglia and reduced ß-amyloidosis in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is by far the most common neurodegenerative disease. AD is histologically characterized not only by extracellular senile plaques and vascular deposits consisting of ß-amyloid (Aß) but also by accompanying neuroinflammatory processes involving the brain's microglia. The importance of the microglia is still in controversial discussion, which currently favors a protective function in disease progression. Recent findings by different research groups highlighted the importance of strain-specific and mitochondria-specific genomic variations in mouse models of cerebral ß-amyloidosis. Here, we want to summarize our previously presented data and add new results that draw attention towards the consideration of strain-specific genomic alterations in the setting of APP transgenes. We present data from APP-transgenic mice in commonly used C57Bl/6J and FVB/N genomic backgrounds and show a direct influence on the kinetics of Aß deposition and the activity of resident microglia. Plaque size, plaque deposition rate and the total amount of Aß are highest in C57Bl/6J mice as compared to the FVB/N genomic background, which can be explained at least partially by a reduced microglia activity towards amyloid deposits in the C57BL/6J strain.