Regular articleEntorhinal cortical defects in Tg2576 mice are present as early as 2–4 months of age
Introduction
Alzheimer's disease (AD), the primary cause of dementia, is characterized by 2 types of neuropathology: the accumulation of amyloid-β (Aβ) plaque caused by overproduction of Aβ (Aβ) and neurofibrillary tangles caused by hyperphosphorylation of tau. Progressive memory impairment and neurodegeneration are also hallmarks of AD.
The hippocampal-entorhinal circuitry appears to be particularly vulnerable in AD (Braak and Braak, 1996, Morrison and Hof, 2002), and several studies suggest that the entorhinal cortex (EC) is one of the structures that is affected early in AD (Bobinski et al., 1999, Braak and Braak, 1991, Braak and Braak, 1996, DeToledo-Morrell et al., 2007, Hof, 1997, Scharfman and Chao, 2013). The first neurons in the EC to deteriorate appear to be the stellate cells in layer II (Braak and Braak, 1985, Braak and Braak, 1991, Kordower et al., 2001, Stranahan and Mattson, 2010), which form the perforant path projection to the dentate gyrus (DG). Based on experiments in mice, it has been suggested that Aβ and tau pathology originates in the EC and subsequently spreads to the hippocampus by a trans-synaptic mechanism (De Calignon et al., 2012, Harris et al., 2010, Lazarov et al., 2002, Liu et al., 2012).
These findings are important because they suggest that prevention of EC neuropathology could prevent or delay AD. However, it is unclear why the EC is vulnerable and why it is susceptible early in AD. To gain insight into these questions, we used the Tg2576 mouse model of AD neuropathology. The Tg2576 mouse uses the hamster prion promoter to overexpress human amyloid precursor protein (hAPP) 695 with the mutation of a Swedish family with AD (K670N/M671L) and progressive Aβ pathology occurs after 6 months of age (Hsiao et al., 1996, Irizarry et al., 1997, Kawarabayashi et al., 2001, Lee and Han, 2013, Westerman et al., 2002).
Notably, one study showed with immunohistochemistry that Aβ is detectable in the olfactory bulb by 3–4 months of age (Wesson et al., 2010) suggesting that Aβ neuropathology develops earlier than 6 months of age. In fact, there is some evidence that structural and plasticity defects in the hippocampus occur in Tg2576 mice before 6 months of age; there was a decrease in spine density of granule cells (GCs), the principal cell of the DG, in 4-month-old mice (Jacobsen et al., 2006). There also was a deficit in long-term potentiation of the perforant path projection to the GCs (Jacobsen et al., 2006). Also, in 3-month-old Tg2576 mice, there was a decrease in synaptic contacts and increase in synaptic length in stratum lacunosum-moleculare of CA1 (Balietti et al., 2013). These changes could originate in the EC because the perforant path projection to the DG was affected, and there were defects in the area where the perforant path projects to CA1.
To address early changes in the EC in Tg2576 mice, we used mice that were younger than 4 months old, timing most of the analyses for an age after puberty (2 months old) to prevent puberty-associated changes from complicating the analysis. We also chose assays that are uncommon—but ones that we thought would be ideal to probe the EC—to determine if atypical approaches would better detect defects in the EC of young mice. For example, rather than using whole brain homogenate, we microdissected the EC and other regions and performed a sandwich enzyme-linked immunosorbent assay (ELISA) to detect soluble human Aβ40 and Aβ42. In addition, instead of probing behavior with the Morris water maze, the object placement (OP) task was used to examine behavior because the EC is known to be involved in this task (Parron and Save, 2004, Steffenach et al., 2005, Witter and Moser, 2006). Immunohistochemistry using an antibody to a neuronal nuclear antigen (NeuN) was also used because NeuN expression decreases with neurotrophin deficits in the EC (Duffy et al., 2011). Other methods were also used, such as examining the staining of myelin using histochemistry.
The behavioral and anatomical data suggested that there were impairments in the EC of Tg2576 mice at <4 months of age, so electrophysiological recordings from the EC were made in slices to gain more insight. We found evidence of increased excitability, supporting previous suggestions that epileptiform activity is a characteristic of mouse models of AD where familial mutations of hAPP are overexpressed (Noebels, 2011, Palop and Mucke, 2009). Furthermore, the data support the idea that defects that are relevant to AD occur in the EC (Chin et al., 2007, Francis et al., 2012). They suggest that impairments occur very early in life but they may require assays other than those that are commonly used to be detected.
Section snippets
Animals
The experimental procedures were carried out in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at The Nathan Kline Institute. Tg2576 mice (Hsiao et al., 1996) and wild-type (WT) littermates were bred on a mixed C57BL/6 and SJLF1/J background (Wesson and Wilson, 2011, Wesson et al., 2011). Mice were housed 3–4/cage, in standard mouse cages with corncob bedding and a 12-hour light-dark cycle (lights on, 7 AM). Food
Results
The average age of all mice used in this study was 2.9 ± 0.04 months (range 1.4–3.8 months; n = 151). There was no significant age difference between WT (3.0 ± 0.05 months, n = 69) and Tg2576 mice (2.8 ± 0.06 months, n = 82; Student t test, p = 0.089).
Discussion
The results demonstrate that abnormalities develop in the EC of Tg2576 mice at extremely early ages using assays that are sensitive to EC impairments such as OP, NeuN expression, myelin uptake, and recordings from the EC in slices. Thus, young Tg2576 mice had impaired OP, weak EC NeuN-ir, EC cells showed myelin uptake, and evoked responses in the EC exhibited abnormal excitability. The alterations in the EC developed when human Aβ40 and Aβ42 were detected but before Aβ plaque pathology.
These
Disclosure statement
The authors have no actual or potential conflicts of interest to disclose.
Acknowledgements
The authors thank Thomas Radman and Charlie Schroeder for implementation of current source density analysis. This study was supported by the Alzheimer's Association (NESAD-12-239551), New York Office of Mental Health, the NYU Langone Medical Center of Excellence Seed Grant program, National Institutes of Health MH-084215 and P01 AG017617.
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