ApoE4 is a risk factor for AD

Numerous genetic studies have revealed that inheritance of ApoE4 allele increases the risk and rate of progression of late-onset Alzheimer disease, LOAD [1-3]. The human APOE gene exists as three polymorphic alleles—ε2, ε3 and ε4—which have population frequencies of 8.4%, 77.9% and 13.7%, respectively [4]. However, the frequency of the ε4 allele is dramatically increased to ~40% in patients with AD (4). Additionally, 65-80% of AD patients have at least one ApoE4 allele. Inheritance of a single ApoE ε4 variant increases a person’s risk of developing AD by a factor of three in men and four in women, and having two copies of the ε4 allele increases risk by up to 15-fold compared to persons without the ε4 variant [4,5]. Furthermore, ApoE4 inheritance decreases the age of onset of AD [1,6-8].

Apolipoprotein E (ApoE) mediates lipid transport from one tissue or cell type to another [9,10], thus participates in the regulation of lipid homeostasis. In peripheral tissues, ApoE is mainly produced by the liver and macrophages, and mediates cholesterol metabolism in an isoformdependent manner. ApoE4 is associated with hypercholesterolemia, which is a risk factor for atherosclerosis, coronary heart disease and stroke [9,10]. In the CNS, ApoE is mainly produced by astrocytes, but also can be expressed by oligodendrocytes, and ependymal layer cells [11,12]. Cholesterol is transported to neurons by ApoE via ApoE receptors, which are members of the low-density lipoprotein receptor (LDLR) family [13]. Increasing evidence suggests that under diverse pathophysiological conditions, CNS neurons also express ApoE, although at lower levels than astrocytes [14-17]. The cellular origin of ApoE appears to influence its effects on AD pathology [18,19].

Neurofibrillary tangles and amyloid plaques, two neuropathological hallmarks of AD, are increased in brain samples from ApoE4 carriers as compared to non-ApoE4 carriers [3,7]. Both plaques and tangles appear earlier in ApoE4 carriers as compared to non-carriers of ApoE4. In addition, AD patients with ApoE4 genotype showed widespread degeneration of neurons in areas of the brain related to learning and memory, compared to non-ApoE4 patients [20]. Although various hypotheses have been proposed to explain the relationship between ApoE4 and AD, the mechanism by which ApoE4 leads to AD in humans, if at all, is still unclear. A comprehensive study of the pathophysiological effect of ApoE4 on AD progression is hampered by ethical limitations to sampling of brain tissues in living persons as they progress through preclinical to clinical stages of the disease. The early involvement of the more accessible olfactory pathways by AD biology, therefore offers some hope that the olfactory paradigm can be applied to investigative efforts aimed at elucidating preclinical pathophysiology of disease progression in people with genetic risk of AD. This review examines the scientific premise in support of the validity of the olfactory paradigm for investigation of early and progressive AD in ApoE4 carriers. To provide a template for interpreting data on ApoE-related changes in olfaction in AD, we first provide an overview of basic neurobiology of olfaction and a brief description of methods used for assessment of olfaction. This is followed by the review of extant data that examined whether the olfactory system reliably reflects the effect of apoE variations on AD vulnerability.

Neurobiology of olfaction

The olfactory system enables us to perceive smell from the environment, and flavors from food. Loss of olfaction is linked to many neurodegenerative diseases, including Parkinson’s disease (PD), fronto-temporal dementia (FTD) and AD [21,22]. The olfactory system begins with the olfactory mucosa, a pseudostratified columnar epithelium in the posterior region of the nasal cavity, from which odor information is projected into the olfactory bulb (OB) at the base of the brain (Figure 1). In all mammals, nasal airflow carries odorants into contact with olfactory receptors (ORs) located on the cilia of olfactory receptor neurons (ORNs) in the nasal olfactory mucosa. ORNs express only one OR type [23], and ORNs expressing the same OR innervate up to two glomeruli per OB [24]. Odorant binding with an OR triggers a G-coupled protein–mediated intracellular signaling cascade, ultimately producing an action potential [25,26]. The ORs possess unique chemosensory tuning properties [27,28], that provide a first step at which the olfactory system can sort the limitless number of odorants it may encounter. Action potentials in the ORNs transmit odorant information into discrete zones in the OB whose activation is dictated by nasal airflow [29-31]. These zones in the OB form a “spatial map” of odorant information [32-35] and are modulated by local glomerular layer neurons. This type of organization is believed to be important for the most basic aspects of olfactory perception, including odor perception and discrimination [36-38]. The OB is a six-layer structure in which the sequential stages of odor information processing take place (Figure 1). The axons of ORNs form the olfactory nerve layer of the OB [36-38]. Secondary olfactory neurons, called mitral cells (MCs) located in the mitral cell layer of OB, and tufted cells (TCs) located in the external plexiform layer, all innervate OB glomeruli. Another major cell population in the OB are the granule cells, which are axon-less interneurons organized in patchy aggregated rows in the most central cell layer of the OB (interneuron). The apical dendrites of granule cells form synapsis with MCs and TCs. Granule cells also receive centrifugal input from some secondary olfactory structures and display broader odor-tuning characteristics than the upstream MCs and TCs [39]. Granule cells are mostly GABAergic and glutamatergic. In many mammalian species granule cells are constantly renewed by neurogenesis during adulthood [40-43]. The activity of MCs, TCs, and interneurons in the OB is subject to neuromodulation [44]. The OB receives dense noradrenergic projections from the locus ceruleus, cholinergic input from the horizontal limb of diagonal band of Broca, and serotoninergic afferents from the medial and dorsal raphe nuclei. Axons from MC and TC converge to form the lateral olfactory tract, whose distal projections innervate a variety of secondary olfactory structures, including the anterior olfactory nucleus (AON), piriform cortex, olfactory tubercle, the lateral entorhinal cortex, and paraamygdaloid complex (Figure 2). These secondary olfactory structures are regarded as the primary olfactory cortex. Cells in the AON cells are highly responsive to odor stimulation [45] and they are believed to aid in intra-nostril odor localization [46]. The piriform cortex is well-known for its role in modifying the processing of odors based upon experience and learning [47-51]. Olfactory tubercle neurons are reliably activated by tasks that assess odor valence, motivated behaviors, and acquisition of rewards, suggesting important roles of the olfactory tubercle in guiding hedonic and valence-dependent responses to odors [52,53]. Neurons within these secondary olfactory structures project into tertiary olfactory structures, which include the orbitofrontal cortex, the insular cortex, and the dorsal hippocampus [54]. Of particular relevance to AD, the entorhinal cortex innervates the hippocampus via the perforant pathway [55]. Additionally, thalamic regions receive olfactory information from several of the secondary olfactory structures, including the AON, piriform cortex, and olfactory tubercle. Olfactory information is also transmitted to the hypothalamus via the amygdaloid complex [56]. These foregoing discussions suggest that odor experience is intertwined with motivated behavior, emotion and cognition through overlap neural circuitry.

Figure 1: Schematic diagram of the olfactory neuroepithelium (OE) and the olfactory bulb (OB). The OE consists of cells at different stages of differentiation, including the proliferating progenitor cells (yellow color), the postmitotic immature olfactory neurons (pink color) and the olfactory sensory, OSN (also known as olfactory receptor neurons, ORN). Axons from the OSN pierce through the cribriform plate at the base of the skull to enter the OB, where they form the olfactory nerve layer. The OB, above the OE shows the laminar organization, the major cell types and the basic neuronal circuits. Interneurons shown are the granule cells (across different layers) and the periglomerular cells in the glomerular layer (GL). Efferent neurons of the olfactory bulb are tuffed and mitral cells.

Figure 2: Simplified diagram of brain regions involved in the processing of olfactory information. The lateral olfactory tract project odor information into the primary olfactory cortex, which include the anterior olfactory nucleus (not shown), piriform cortex, olfactory tubercle, amygdaloid complex, and entorhinal cortex. From these primary olfactory cortical regions, odor information is projected to the thalamus, orbitofrontal cortex, insula cortex and hippocampus.

Generally, psychophysical tasks of olfaction are based upon the presentation of odors to a test subject, followed by examination of the subject’s responses to questions about some characteristics of the odorants presented. Olfactory psychophysical tasks most commonly measured in studies of olfaction in aging include odor identification, odor memory, odor discrimination and odor threshold sensitivity tasks [57-59]. Other methods which have been employed for clinical assessment of olfaction include surveys of subjective rating of olfactory ability and quality of life [60] and assessments of odor familiarity [61] and hedonics [62].

Odor identification tasks

During odor identification tests, odorants are presented sequentially to test subjects at supra-threshold concentrations, and subjects are required to identify each odor from a list of descriptors. This forced choice procedure is aimed at minimizing subjects' response bias. Odor identification tests are considered the most advanced type of test depiction of higher-order cortical functions among other psychophysical tests [63-65]. However, a major problem of odor identification test is that it correlates with the verbal abilities of the subject and has a strong cultural precondition, as not all odors are known equally well in various cultural groups. The most widely used identification tests include the 40-odorant University of Pennsylvania Smell Identification Test (UPSIT) [59] and the Sniffin’ Sticks Identification Test [58]. However, several other tools are available, such as the 3-item Quick Smell Identification Test, Q-SIT [66]; the 12-item Brief Smell Identification Test, B-SIT [67]; the Smell Diskettes Olfaction Test [68]; the San Diego Odor Identification Test, (SDOIT), which consists of 8 common household odorants presented in opaque jars and includes a picture board to assist odor identification [20]; the Brief (Cross-Cultural) Smell Identification Test [67]; and the Scandinavian Odor Identification Test [69]. Additionally, odor identification can be assessed using flow dilution olfactometers, such as the T&T Olfactometer [70].

Odor discrimination tasks

In odor discrimination procedures, different odorants are presented to subjects at supra-threshold concentrations, and subjects have to determine which of the odorants smell different [71]. This task examines the ability to distinguish between odors, not to recognize or identify them. In clinical applications discrimination tests are used in combination with identification and threshold tests. The Sniffin’ Sticks Test (SST) discrimination task involves a triple-forced-choice procedure. Per triplet, two distracter pens encompass identical smells, while the respective third pen (the clue) contains a different odor. The number of correctly identified clues represents the discrimination score [71].

Odor threshold tasks

The principle behind the threshold tests is that a subject is repeatedly exposed to ascending and descending concentrations of the same odorant and is required to identify the least detectable concentration for the index odor (usually n-butanol or phenyl-ethyl-alcohol) [72]. The widespread use of the latter odorant in odor threshold applications is based on the premise that it more selectively activates olfactory receptors than the trigeminal receptors in the nose [72]. This task is normally associated with the peripheral part of the olfactory pathway [73], and can be measured by means of the T&T olfactometer [70], the Threshold Test of the Sniffin’ Sticks Extended Test [71], and the Smell Threshold Test (STT) [74].

Odor memory tasks

Perhaps due to their relative complexity, odor memory tasks were introduced more recently, compared to threshold and identification tasks. Most clinical applications of odor memory use a recognition task carried out in two stages: the acquisition and the recognition stages. During the acquisition stage, the subject is asked to smell a small set of common household odors at intervals of 30 seconds to control for odor adaptation. This is followed by the recognition stage whereby the subject would be required to recognize the previously presented odors among distractor odors [75]. The total number of correctly recognized, percent of odors in the acquisition stage that was correctly identified, and recognition errors are outcome variables of odor memory tests commonly used in clinical studies [75,76].

Neuroimaging procedures, including structural and functional magnetic resonance imaging, Positron Emission Tests (PET), Single-Photon Emission Computed Tomography (SPECT), and Electroencephalography (EEG), have been used in neuroscience research to characterize the neurobiology of olfactory processing. The imaging modalities provide good spatial localizations of regions relevant to olfaction, while the EEG applications reveal the sequence of neuronal activations with high temporal resolution [77]. However, due to its relative simplicity, neurobiological investigations of the relationship between ApoE and olfaction have almost exclusively employed EEG methods.

To characterize EEG patterns of olfactory perception, researchers either place electrodes intranasally to acquire odor-induced electrophysiological activity locally in the olfactory epithelium (i.e., electro-olfactography, EOG), or acquire cortical activations during odor exposure, through placement of electrodes on the scalp (i.e., odor event-related potentials, OERPs). These methods provide more objective measures of olfactory function, independent of patients’ response bias. Compared to EOG, OERPs are more commonly assessed in clinical populations, and their absence is often a strong indicator of olfactory loss [78]. Odor event-related potentials (OERPs) result from the sequential activation of different brain areas, beginning from olfactory bulbs and tracts and involving the orbitofrontal and insular cortices, along with rostrum-medial regions of the temporal lobe [79]. In most OERP designs, three scalp electrodes placed along the midline – frontal (Fz), central (Cz), and parietal (Pz) – regions allow for examination of relative activations of the cortical fronto-centroparietal regions by olfactory stimuli [80]. Odor event-related potentials consist of a series of positive and negative voltage deflections, which are related to a set of underlying components. Most components are referred to by a letter (N/P) indicating polarity (negative/positive), followed by a number indicating either the latency in milliseconds or the component's ordinal position in the waveform. For instance, a negative-going peak that often occurs about 100 ms after a stimulus is presented is often called the N100 or N1. Typically, N1 is followed by a positive peak, known as the P200 or P2. The specified latencies for OERP components are often quite variable. For example, the P300 (P3) component may exhibit a peak anywhere between 250 ms – 700 ms [81]. These OERP parameters are important variables in studies investigating the influence of ApoE polymorphisms in olfactory functions and in AD.

ApoE4 association with olfactory dysfunction in AD

An overview of results from studies investigating the association between apoE polymorphisms and olfactory functions is depicted in Table 1. As shown, deficits in odor fluency, odor identification, odor recognition memory, and odor threshold sensitivity have all been associated with inheritance of the ApoE4 genotype in several studies [20,82-86]. These impairments in olfactory psychophysics are observed early in the course of AD, even before the onset of clinical dementia [20,86]. Non-demented carriers of ApoE4 polymorphism showed significant decline in olfactory processing as compared to individuals without ApoE4 allele [87,88]. Importantly, patients with increased brain atrophy have greater olfactory impairment [89], indicating that olfactory function is further diminished as AD progresses. In family studies, siblings of AD probands had lower olfactory identification scores compared to siblings of control probands [90]. Moreover, within families, siblings with ApoE4 allele showed greater deficits in odor identification tests than siblings without ApoE4 allele. This finding of cosegregation between ApoE4 and olfactory identification deficits suggests that odor identification deficits may reflect early disease expression in individuals at increased risk for developing the disease [90]. Data from longitudinal studies provide additional supporting evidence of the association between ApoE4 inheritance and poor scores in olfactory tests, but more importantly highlights the predictive effect of baseline ApoE4 status on progression of olfactory loss over a 4-year period [83]. A recent study suggests that domains of high-order olfactory functioning, like odor identification and remote memory measured by odor familiarity ratings, may be more impaired in AD E4/4 homozygotes compared to E3/4 heterozygotes and E3/3 homozygotes [91]. These deficits give insight into how the presence of two E4 alleles may differentially affect the progression of AD. Down Syndrome involves trisomy of chromosome 21, where the gene for Amyloid Precursor Protein (APP) is located. Down Syndrome (DS) represents premature aging disorder and autopsy studies show that by age 40; the brains of almost all individuals with DS have significant levels of plaques and tangles, which are definitive neuropathological features of AD. These consistent brain changes notwithstanding, development of AD is not uniform in DS [92]. Interestingly, individuals with Down Syndrome (DS) who are carriers of ApoE4 allele, exhibit significantly greater deficits in odor identification than those who are negative for the allele [93]. It implies therefore, that olfactory tests and ApoE4 genotyping may contribute to improved prediction of AD risks in DS populations.

Table 1: ApoE olfaction studies.

Olfactory event-related potentials (OERPs) have also demonstrated high sensitivity to subtle changes in olfactory functioning, and to AD and ApoE status [94-96]. A growing number of clinical studies have shown that brain changes associated with ApoE4 allele are captured much earlier in age through OERP recordings than through psychophysical tests of olfaction, thereby suggesting the potential utility of OERP for identification of preclinical stages of AD [82,97-99].

Mechanistic studies linking ApoE4 to olfactory function

Despite consistent evidence of the robust relationship between olfactory dysfunction, ApoE4 inheritance and AD, the mechanism underlying these relationships are not fully understood. However, murine models of ApoE are beginning to illuminate our understanding of the role of ApoE in olfactory structure and function. The results of mechanistic studies in mice and from in vitro biochemical assays are highlighted in Table 2. ApoE deficiency in apoE KO mice leads to deficits in several tasks of olfactory function, suggesting an important role of ApoE in the mice olfactory system [100,101]. Previous studies showed that ApoE is expressed at high levels by a variety of cell types in the olfactory epithelium (OE). In particular, high expression of ApoE in the basal cells and adjoining lamina propria of the OE suggests that ApoE may play a role in the differentiation, maturation and axonal growth of ORNs, perhaps by recycling lipids from degenerating ORN for use by growing axons [102]. Indeed, studies of ApoE KO mice provide support that ApoE plays an important role in olfactory nerve regeneration [103]. Another group of investigators examined the effects of ApoE isoforms on neuronal differentiation and neurite outgrowth in OE explant cultures [104]. They discovered that ApoE2 and apoE3 stimulate neurite outgrowth in OE cultures by interacting with the lipoprotein receptor, LRP. ApoE4, the isoform associated with AD, failed to promote neurite outgrowth, signifying a potential mechanism whereby ApoE4 may lead to olfactory dysfunction in AD patients [104]. Moreover, Nathan et al. [101] investigated the involvement of ApoE in propagating regeneration of OE cells by inducing OE lesions in ApoE and WT mice [101]. The results revealed that ApoE expression in the OE is highly regulated during the entire course of OE reconstitution post injury, and that ApoE deficiency in ApoE KO mice leads to delayed recovery of mature OMP+ cells in the reconstituting OE [105]. Glomerular Synaptophysin (Syn) density, measured by immunohistochemistry, was lower in KO mice at all time points following the lesion [105]. This lower concentration of whole bulb Syn paralleled the slower recovery of glomerular area in KO mice. In the absence of ApoE, synaptic recovery in whole bulb samples was significantly delayed compared to WT mice [106]. This study highlights the important role of ApoE in neuronal differentiation. It is noteworthy that ApoE has also been shown to modulate other molecular factors that are important for neurogenesis, including WNT2 and granulin [107-109]. Some studies have identified a close relationship between estrogen and apolipoprotein E (ApoE) in the central nervous system, and neuroprotective the role of hormone therapy (HT) in several neurological disorders. Estrogen and ApoE function synergistically to minimize the loss of mature sensory neurons and synapses in OB, and OE following ovariectomy [110-113].

Table 2: ApoE mice studies.

Gaps in current studies and future directions

Current diagnosis of Alzheimer’s disease (AD) is based on clinical examination, neuropsychological testing and brain imaging; however, a definite diagnosis can only be made by postmortem examination. Although brain imaging and cerebrospinal fluid biomarkers are applied in patients with mild or questionable symptoms to increase the level of diagnostic certainty, and peripheral bio-fluids are largely investigated, no definitive diagnostic tests are available yet. Biomarkers that reliably predict development of AD would greatly assist preventative and management treatments. This review demonstrates the potential relevance of olfactory system for both biomarker and pathophysiology studies of AD progression. However, many questions must still be answered. It is unclear how to identify and differentiate age-related olfactory changes and olfactory dysfunction caused by disease. Smell loss is also associated with other neurodegenerative disorders, like Parkinson’s disease. Olfactory testing would need to be used with other biomarkers, specific to each disease, or olfactory changes in each disease have to be better specified. While the role of ApoE in olfactory neurogenesis has been reasonably demonstrated in basic studies, it remains uncertain how ApoE4 promotes AD amyloid and tau pathology in the olfactory system, especially in OB tissue. There are numerous studies examining interactions between ApoE4 protein and Amyloid or Tau proteins in cortical and hippocampal tissues [114]. ApoE is thought to be involved in plaque formation, but how exactly ApoE is involved in pathogenesis of AD is not well understood. The hypothesis gaining widespread support is that ApoE is involved in deposition or clearance of Abeta by direct protein-to-protein interaction. When associated with lipid, ApoE4 bound preferentially to an intermediate aggregated form of Abeta and had higher avidity than did ApoE2 or ApoE3 [115]. The tenability of this hypothesis in olfactory tissues has not been studied. Moreover, mechanistic correlations between ApoE and olfaction in AD to date are performed in animal models, not in humans. There are substantial differences between olfactory systems in rodents and humans. Further research is necessary to clarify these uncertainties. Patient-derived olfactory neurons offer an excellent alternative tool to study correlations between ApoE4 and olfactory impairment. Moreover, these olfactory cells can be collected from people at high risk (e.g. ApoE4 carriers), particularly those who display progressive impairments in psychophysical and physiological tests of olfaction. With future accomplishment of these goals in mind, it is reasonable to anticipate that combination of psychophysical, neuroimaging, electrophysiological and molecular studies of olfactory tissues may hold promise for characterization preclinical stages of the disease in people at risk, such as the case of ApoE4 inheritance.

This review summarizes the research showing that ApoE4 is a significant player in olfactory impairment, an early AD symptom. Furthermore, we discussed the mechanistic studies that have been evaluated in ApoE KO mice. Our review stresses the importance of olfactory function as a biomarker of AD, and a potential useful test for prediction of AD development in those with genetic (i.e., ApoE4) risk. Olfactory tests should be incorporated in the assessment of populations at high risk for dementia, like ApoE4 alleles for early recognition of AD and successful intervention.

Sources of Funding/Disclaimers This project has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. Funding support for olfactory testing, and for the enrollment and neuropsychological testing of the control subjects is through USPHS grant MH-091460 (PI, Nwulia). This publication does not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Disclaimer: Dr. Kapetanovic contributed to this article as part of his official duties while working in the NIMH Intramural Office.

We are grateful to Ms. McLean and Dr. Rai at The Translational Neuroscience Laboratory of Howard University, for their assistance with the editorial changes made to this review article.

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