Research ArticleHuman Apolipoprotein E Genotype Differentially Affects Olfactory Behavior and Sensory Physiology in Mice
Introduction
Apolipoprotein E (ApoE) is the primary carrier of cholesterol within the brain, and ApoE genotype is an important determinant of an individual’s risk for developing Alzheimer’s disease (AD) (Corder et al., 1993, Farrer et al., 1997, Bu, 2009, Liu et al., 2013). Three alleles of ApoE occur in humans: ε2 (cys112, cys158; ∼6% of the ApoE alleles in the population), ε3 (cys112, arg158; the most abundant allele at ∼80%), and ε4 (arg112, arg158; ∼14%) (Mahley, 1988, Mahley and Rall, 2000). ApoE4, in a dose-dependent manner, is the single most important genetic risk-factor for AD (Corder et al., 1993, Farrer et al., 1997, Bu, 2009, Liu et al., 2013). ApoE3 is viewed as the neutral allele in terms of neurodegenerative risk (Corder et al., 1993, Mahley and Rall, 2000, Liu et al., 2013) (and most closely resembles murine ApoE (Raffai et al., 2001)). ApoE2 is associated with a lower risk of AD-related neurodegeneration (Corder et al., 1994, Liu et al., 2013), delayed age of onset of AD, and a greater likelihood of survival to advanced age compared to the ApoE3 and ApoE4 alleles [reviewed in (Suri et al., 2013)]. In addition to AD, there is increasing evidence that ApoE is involved in several other disorders, with ApoE4 often exerting a deleterious and ApoE2 a protective effect; while not all studies have found a difference between ApoE2 and ApoE3 carriers [reviewed in (Suri et al., 2013)], both ApoE4 and ApoE2 appear to be associated with hemorrhagic and ischemic cerebrovascular disease [reviewed in (Liu et al., 2013, Suri et al., 2013, Lopez et al., 2014)], with a high risk of argyrophilic grain disease and frontotemporal dementia [reviewed in (Suri et al., 2013)]. ApoE2 also appears to confer an increased incidence and severity of posttraumatic stress disorder (PTSD) (Freeman et al., 2005, Kim et al., 2013, Johnson et al., 2015). While the effects of ApoE4 have been extensively studied, the inconsistencies among a small number of studies regarding the effect of ApoE2 may in part be due to challenges related to its low frequency allele.
While ApoE genotype may affect a myriad of neural circuits and functions, the olfactory system and olfactory perception appear to be a unique and early biomarker of ApoE genotype. In humans, ApoE4 carriers show early emergence of olfactory dysfunction (Price et al., 1991, Bacon et al., 1998, Mesholam et al., 1998, Murphy et al., 1998, Graves et al., 1999, Gilbert and Murphy, 2004, Josefsson et al., 2017, Peng et al., 2017), show impaired odor identification (Murphy et al., 1998, Olofsson et al., 2010, Olofsson et al., 2016), and modified olfactory related evoked potentials (Kowalewski and Murphy, 2012, Morgan and Murphy, 2012) prior to other forms of cognitive impairment, including AD impairment associated with amyloid β and tau pathology. In mice, ApoE4 impairs short-term odor memory and induces olfactory system hyperexcitability including in the olfactory bulb (OB) and piriform cortex (PCX) (Peng et al., 2017) as well as the lateral entorhinal cortex (Nuriel et al., 2017) The olfactory system is suitable for assessing links between cell biology, circuit function and behavior given its relatively simple circuitry and the availability of reliable, robust behavioral assays in both humans and non-human animals. ApoE is important for neuroregeneration of the rodent olfactory system (Nathan et al., 2005), and ApoE knock-out mice show impaired olfactory detection (Nathan et al., 2004). This olfactory dysfunction is associated with neurophysiological (Mesholam et al., 1998, Corby et al., 2012, Kowalewski and Murphy, 2012, Peng et al., 2017), structural (Tanaka et al., 1998, Hashimoto et al., 2001) and cellular changes (Tsuboi et al., 2003, Nathan et al., 2005, Hussain et al., 2013) in olfactory regions of the brain. Understanding how ApoE isoforms may influence olfactory system function and perception would provide important insights into ApoE4 as a major risk factor for olfactory and cognitive decline.
Here, we directly compared olfactory behavior and olfactory system physiology across all three ApoE genotypes in mice that are homozygous for human ApoE2, ApoE3, or ApoE4. We hypothesized a genotype-dependent gradient in odor habituation and olfactory system excitability, such that, for example, ApoE4 mice would display hyper-excitability and impaired behavioral habituation, and ApoE2 mice hypo-excitability and increased behavioral habituation, relative to ApoE3 mice. The results were in line with our hypotheses and they extend previous work (Peng et al., 2017).
Section snippets
Study approval
All animal procedures were performed in accordance with the Nathan S. Kline Institute (NKI) for Psychiatric Research Institutional Animal Care Committee’s approval.
Mice
Mice used in this experiment were homozygous for human ApoE2, ApoE3, and ApoE4 genes on a C57BL/6 background which are from long-standing colonies at NKI. These targeted-replacement mice express human ApoE under the control of the endogenous murine promoter (Sullivan et al., 1997), which allows for the expression of human ApoE at
Olfactory behavior
Independent of age or genotype, mice readily investigated novel odors presented to their homecage, suggesting no difference in behavioral odor responsiveness. As shown in Fig. 1, there was no age (F(1,34) = 0.001, p = 0.97) or genotype (F(2,34) = 1.3, p = 0.3) difference in the amount of time spent investigating the initial presentation of a novel odor.
In contrast, odor habituation was significantly affected by genotype (Fig. 2). For habituation, the analysis strategy was informed by prior
Discussion
Despite olfaction emerging as a system of importance to study the behavioral and physiological effects of ApoE, research remains limited. The present results demonstrate that while the initial tendency of mice to investigate novel odors is unaffected by ApoE genotype, odor habituation is impaired in E4 relative to E2 mice, with E3 mice presenting an intermediate functional level. Memory impairment in this simple task emerges in E4 mice by 6 months, as previously reported (Peng et al., 2017).
Acknowledgments
We thank Dr. Monika Pawlik for her expert assistance with our mouse colonies. This work was supported by the NIH (P01 AG017617 to EL and PMM, and R01 AG057517 to EL, PMM, and DAW; KP was additionally supported by an NIH postdoctoral research training grant T32-AG052909). JKO was supported by a Pro Futura Scientia VII fellowship and research grants from the Marianne and Marcus Wallenberg Foundation (MMW 2014:0178) and the Swedish Foundation for the Humanities and Social Sciences (M14–0375:1).
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