Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein‐specific antibodies

Abstract

SummaryAlzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age‐related and brain region‐specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP‐transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP‐transgenic mouse and one APP‐transgenic rat model. We observed remarkable differences in expression levels and brain region‐specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP‐transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.

Defined astrocytic expression of human amyloid precursor protein in Tg2576 mouse brain

“…So far, to the best of our knowledge, no glial expression of hAPP in Tg2576 brain was demonstrated. However, using a novel antibody that differentiates between endogenous mouse and hAPP (Höfling et al, ), we here report the presence of nonneuronal cells immunoreactive for hAPP in a brain region‐specific manner. To identify the glial cell types expressing the transgene, double immunofluorescent labelings of hAPP with cell type‐specific marker proteins were performed in brains of 3‐ and 18‐month‐old Tg2576 mice and analyzed by confocal laser scanning microscopy.…”

“…The specificity and applicability of the 1D1 antibody has been extensively characterized recently (Höfling et al, ). It allows differentiation between endogenous mouse and transgenic human APP and binds an extracellular N‐terminal hAPP epitope between amino acids 40 and 64.…”

“…It allows differentiation between endogenous mouse and transgenic human APP and binds an extracellular N‐terminal hAPP epitope between amino acids 40 and 64. Thus, it does not detect Abeta peptides, but rather the hAPP and can be applied for immunocytochemistry, immunohistochemistry, immunoprecipitation and Western blot as well as FACS analyses (Höfling et al, ). Importantly, using Western blot analyses, robust hAPP bands at approximately 100 kDa were present in brain tissue homogenates of APP‐transgenic Tg2576, 3xTg and I5 mice, whereas bands were neither detected at molecular weights of Abeta peptides or C‐terminal APP fragments nor in wild type mouse brain tissue (Supporting Information Figure S1).…”

“…With regard to the amyloid pathology, a large magnitude of human APP (hAPP) constructs with mutations within the Abeta sequence or close to the beta cleavage site of the APP was used to generate transgenic mice that develop AD‐typical histopathology. These constructs differ with regard to the hAPP isoform used (APP695, APP751, APP770), the mutations introduced (Swedish, London, Dutch, Florida, Iberian, Arctic, Indiana, Iowa) and the promoter‐driving transgene expression (PDGF‐β, Thy1, CMV/β‐actin, CaMKII‐α, prion protein, neuron‐specific enolase) (Höfling et al, ). As a result, the onset and degree of amyloid pathology, neuronal and synaptic loss as well as changes in long‐term potentiation, neuronal network activity, and the degree of cognitive impairment differ substantially between the animal models established.…”

APP mouse models for Alzheimer's disease preclinical studies

“…Different Tg mice use different promoters to drive APP transgene expression that may affect Aβ in vivo propagation (Jucker & Walker, 2013). Höfling et al (2016) showed differences in expression level and brain regional patterning of exogenous APP among different APP‐Tg mouse lines.…”