Human APOE ɛ3 and APOE ɛ4 Alleles Have Differential Effects on Mouse Olfactory Epithelium

Selection of mice

To assess the effect of human APOE genotype on the olfactory epithelia of mice, two strains of gene-targeted mice on a C57BL/6 background were used. In E3 mice the human APOE ɛ3 allele replaces the mouse Apoe gene and in E4 mice the human APOE ɛ4 allele replaces the mouse Apoe gene. The olfactory epithelium shows little sexual dimorphism under most conditions, including the same sex housing conditions used for this study [60, 61], so both male and female mice were used in this study. Mice were treated according to an approved Institutional Animal Care and Use protocol.

Tissue lysis and RNA extraction

Dissections of olfactory epithelia typically include small amounts of neighboring cells from respiratory epithelia and from the underlying lamina propria, cartilage, and bone. For this reason, these dissected samples are properly called olfactory mucosae. Olfactory mucosae were dissected and placed into 500μl of lysis buffer from the RNeasy Mini Kit (Cat.no. 74004, Qiagen, Hilden, Germany) in a 1.5 mL tube on ice. Tissues were homogenized using the Tissue Ruptor (Cat. no. A662282, Omni International-GLH, Kennesaw, Georgia) for 50 s, and the tube was returned to ice. Total RNA was extracted from these lysed tissue samples using RNeasy Mini Kit according to manufacturer’s instructions (Qiagen). Total RNA was eluted in 50μl RNAse free water and stored at –80°C. The integrity and purity of RNA was measured with the Agilent Bioanalyzer 2100 using an RNA nano Chip kit (Agilent Technologies Inc., Palo Alto, CA). RNA integrity values (RIN) > 7 were considered of sufficient quality for RNA sequencing.

RNA-Seq

RNA-Seq was performed on four E3 samples (1 male, 3 females) and six E4 samples (3 males, 3 females) by Novogene (Novogene Corporation Inc, Sacramento, CA). RNA-Seq reads in the form of compressed Fastq files were uploaded to the Illumina BaseSpace Sequencing Hub (Illumina Inc., San Diego, CA) and were aligned to the mouse genome (build mm10) using the STAR Aligner v2.5.2a. After alignment, differential mRNA abundance was assessed using DESeq2 with Fragments Per Kilobase per Million mapped reads (FPKM) normalization. Loss of a male sample in processing led to fewer male E4 samples, causing us to reject significant differences in four Y-chromosome mRNAs (Eif2s3y, Uty, Ddx3y, and Kdm5d). Log₂(Fold Difference) and -log(Padj) values were used for visualization of differences in transcript abundance in the form of volcano plots using Tableau software (Seattle, WA). For calculating the number of expressed genes, a threshold of FPKM > 5 was used. RNA-Seq data are available at Gene Expression Omnibus, accession number GSE181038.

Functional bioinformatics

Messenger RNAs showing significant differences were analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7 [62]. The functional annotation clustering tool was used to look for enrichment of ontology annotations among Mus musculus genes. A Benjamini-Hochberg corrected threshold of p < 0.05 was applied.

Immunohistochemistry

Female E3 and E4 mice were paired across genotype to compensate for differences in timing and conditions of dissection of snouts. These were used to prepare coronal cryosections on microscope slides (Cat. no. 12-550-15, Fisher Scientific, Pittsburgh, PA) as described previously [63–67]. These frozen sections were thawed rapidly and permeabilized in phosphate buffered saline (PBS) pH 7.4 (Cat.no.14200, Gibco, Waltham, MA) containing 1% Triton X-100 for 30 min. 500μl of blocking buffer (2% BSA and 0.4% Triton X-100 in PBS) was applied to each slide for 1 h at room temperature followed by incubation overnight at 4°C in 1 : 200 dilutions of primary antibody in blocking buffer. The primary antibodies were directed against the active fragment of caspase-3 (Casp3) (Cat.no. 9661L, Cell Signaling Technology Inc., Danvers, MA) and against phosphorylated histone H3 (Cat.no. 06-570, EMD Millipore Biosciences, Billerica, MA). The specificities of these antibodies have been demonstrated previously [68–71]. Sections were incubated in washing buffer (0.05% Tween-20 in PBS) for 30 min followed by 90 min in a Cy3-conjugated donkey anti-rabbit IgG secondary antibody (Cat.no.711-165-152, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) diluted 1 : 1000 in blocking buffer. The sections were then placed in washing buffer for 30 min. Counterstaining for nuclei was done for 5 min using Hoechst 33342 (Cat.no. H3570, Invitrogen, Carlsbad, CA) diluted 1 : 1000 in PBS and then the sections were rinsed with 1x PBS. Slides were mounted with an anti-fade mounting media, Vectashield (Cat. no. ZG1028, Vector Laboratories, Burlingame, CA), covered with a glass cover slip (Cat.no.48393241, VWR International, Radnor, PA) and sealed with nail polish. Fluorescent images were obtained on the Nikon Eclipse Ti-U inverted microscope with a SPOT camera and Spot software version 5.6 (SPOT Imaging, Sterling Heights, MI).

To avoid bias in selecting images used to quantify the frequency of immunoreactive cells, either brightfield or Hoechst 33342 fluorescence was used to locate olfactory epithelium in tissue sections and to select regions for imaging. Only then was antigen immunoreactivity viewed and images acquired using 10X or 20X objectives. At least 10 images per antigen per mouse were taken. Counts of immunoreactive cells in these images were done blind. Counts of cells immunoreactive for active Caspase-3 were made from the OSN cell body layers of the epithelium while counts for phosphorylated histone H3 were made from the basal cell layer. Counts were normalized by dividing by the total length of epithelium in the image and compared across genotype (n = 3).

Metabolic measurements

Mice of both sexes were weighed and administered an oral gavage (approximately 200μl per mouse) of 2-[1,2-³H (N)]-deoxy-D-glucose (Cat. No. NET328A001MC, Perkin Elmer, Waltham, MA) diluted in D-glucose solution (2 mg glucose /g body weight). Each E3 mouse was paired with an E4 mouse for age, sex, and time of gavage. Nine pairs of E3 and E4 mice were tested (four female pairs, five male pairs), with four pairs on one day and five on another. Thirty minutes after gavage, mice were administered a lethal dose of ketamine–xylazine–acepromazine cocktail (0.01 ml/g of 25 mg/ml ketamine (Covetrus, Dublin, OH), 0.625 mg/ml acepromazine (Covetrus), 3.125 mg/ml xylazine (Sigma-Aldrich, St. Louis, MO)) via intraperitoneal injection and olfactory mucosae were dissected. Tissue was homogenized in 4 ml of PBS. These homogenates were transferred to a scintillation vial, 1 ml of Optiphase scintillation fluid (Perkin Elmer) was added, and samples were measured on a liquid scintillation counter (Beckman Coulter, Inc., Brea, California). Counts were normalized to body weight.

Cellular respiration in dissociated olfactory mucosal cells

Seven male mice and five female mice of each genotype were used for this experiment. Olfactory mucosae were dissected from the nasal cavity and placed into a 2 mL collection tube containing 700μL of oxygenated low calcium saline solution (110 mM NaCl, 25 mM NaHCO₃, 5 mM KCl, 5 mM N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 1 mM MgSO₄, 4.8 mM CaCl₂, 5 mM EGTA, 10 mM NaOH, 10 mM glucose). The tissue was homogenized (Omni International GLH) at a speed level 1 for 60 s. The homogenized tissue was then transferred into a new pre-cooled 1.5 mL tube containing 150μL of oxygenated low calcium saline solution containing 5 mM cysteine and supplemented with 21 units of papain (Cat. No. P4762-1G, Sigma-Aldrich) and 0.3 units of DNase I (Cat. No. AM2222, Ambion Inc., Austin, Texas). The mixture in the tube were mixed by inverting 2-3 times and incubated for 37°C for 15 min. Then 450μL of non-oxygenated low calcium saline solution supplemented with 18,000 units of trypsin (Cat. No. T0303-1G, Sigma-Aldrich) and 0.9 units of DNase I, and the tube was mixed by inverting 2-3 times and incubated at 37°C for 5 min. The digested tissue sample was transferred into fresh culture tube (Cat. No. 60825-414, VWR) containing 3 mL of cold HBSS (Cat. No. 14025-092, Life Technologies, Carlsbad, CA) containing 6 units of DNase I. The digested tissue was homogenized at speed level 1 for 20 s and centrifuged for 10 min at 200 x g (Cat. no. 5810R, Eppendorf, Hamburg, Germany). The supernatant was discarded, and the content were transferred to a fresh culture tube containing 3ml of cold HBSS containing 6 units of DNase I. The above procedure was repeated one more time and finally after decanting the supernatant each pellet was resuspended into 800μl of Seahorse XF Assay medium and further filtered through a cell-strainer cap into a 5 mL polystyrene round-bottom tube (Cat. no. 352235, Corning Life Sciences, Tewksbury, MA). The tube was incubated at 37°C for 2 min and cells were counted on a BIO-RAD TC20™ Automated Cell Counter (Hercules, CA).

Cellular respiration measurements were done immediately after cell dissociation using a Seahorse XFe96 (Agilent Technologies Inc., Santa Clara, CA). The final cell pellets of acutely dissociated olfactory mucosal cells were resuspended in 1 ml XF assay media (Cat. no.103680, Agilent Technologies) containing 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate. Live cell counting was done using trypan blue staining. Based on these counts, 120,000 live cells in 175μl media were plated per well on a 96-well Seahorse cell culture plate coated with Cell-Tak (Cat. no. 354240, Corning). Once cells were added to wells, plates were spun at 300 x g for 5 min and then incubated at 37°C for 30 min. Injection ports of a Seahorse cartridge, hydrated on the previous day per manufacturer instructions, were loaded and the assay was performed as per the Mito Stress Test protocol. The substrates/inhibitors/uncouplers were prepared in the reconstituted XF assay media and injected such that the cells were sequentially exposed to 2.5μM Oligomycin, 1μM FCCP (2-[2-4-(trifluoromethoxy) phenyl] hydrazinylidene] - propanedinitrile), 0.1μM Rotenone+10 mM Succinate, and 1μM antimycin-A injected through Seahorse cartridge ports A, B, C, and D respectively. Oxygen consumption rates were measured after each injection and the respiratory parameters were determined using the Mito Stress test output format.

Statistical analyses

For immunohistochemistry, cell counts from paired E3 and E4 mice were generated by a person blind to genotype and then analyzed by one-sided paired t-tests (n = 3). The increased risk of LOAD associated with the APOE ɛ4 allele predicts loss of neurons in E4 mice, justifying one-sided tests. For glucose uptake the normalized counts from paired E3 and E4 mice were analyzed by one-sided paired t-tests (n = 9). The presence of APOE ɛ4 alleles is associated with decreased glucose uptake in other tissues, justifying one-sided tests [54, 72–76]. For each quantitative outcome of the cellular respiration measures, an analysis of covariance (ANCOVA) model was fit to test for differences between the two genotypes studied. Due to limitations on sample processing, the mice from each genotype group were split into four subgroups (3 mice per genotype for each subgroup) whose measurements were made on separate days. To account for any resulting variability in the outcome variables, we adjusted for the day of data collection in each model. Shapiro-Wilk and Levene tests were used in each case to check the assumptions of normality and homogeneity of variances, respectively. Graphical plots of the cellular respiration measures were also generated to assess model assumptions and genotype differences. No evidence of lack of normality or heteroscedasticity was found. Across these analyses, a p-value < 0.05 was considered statistically significant. All analyses were completed in R, version 4.0.3 (R Foundation for Statistical Computing; Vienna, Austria).

Differences in the transcriptomes of olfactory mucosae in adult E3 and E4 mice

The olfactory system is highly susceptible to AD and this susceptibility may include peripheral effects in the olfactory epithelium [45, 77]. A pilot experiment done on young adult mice (age 3 months) using E3 and E4 mice on a mixed genetic background (C57BL/6:FVB) identified hundreds (741) of differentially abundant mRNAs, suggesting that the olfactory epithelia of mice expressing human ɛ3 and ɛ4 alleles may be phenotypically different (Supplementary Figure 1; Supplementary Table 1). To test whether these differences arise from differential effects of the human alleles rather than genetic variation we assessed the transcriptome of olfactory mucosal samples from E3 and E4 mice on pure C57BL/6 genetic backgrounds at a mature adult age (6 months) when early events associated with LOAD are likely to be detectable. Samples of whole olfactory mucosae at this age contained mRNAs from 16,107 genes detected above background (mean FPKM > 5), consistent with previous measures of the transcriptome of this tissue [65, 79].

We detected 121 mRNAs that differed in abundance (Fig. 1; Supplementary Table 2). Of these, 75 are primarily expressed in mature OSNs [65, 81], with 46 being less abundant and 29 more abundant in E4 samples than E3 samples. This distribution is not strongly biased and only includes a few of the hundreds of mRNAs specific to mature OSNs, indicating that a difference in the proportion of mature OSNs in the olfactory epithelium is not the reason these mRNAs differed. Six of the mRNAs are expressed primarily by immature OSNs. Thirty-four of the mRNAs are found primarily in non-neuronal cells of the olfactory epithelium. Of these, 15 were less abundant and 19 more abundant in E4 mice. As with mature OSNs, this distribution indicates that the mRNA abundance differences are not due to differences in the abundance or proportions of non-neuronal cells in the samples.

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Fig. 1

Differentially abundant mRNAs between E4 and E3 olfactory mucosae as detected by DESeq2.

Although some differentially abundant mRNAs come from genes on chromosome 7, such as Ndufa3 and Alkbh6, they are distant from the modified Apoe locus on this chromosome, and we do not detect a bias toward effects on mRNAs from genes located on chromosome 7. For example, more differentially abundant mRNAs arise from genes on chromosomes 2 and 6 than from chromosome 7.

The mRNA abundance differences between E3 and E4 samples include mRNAs encoding proteins whose functional relationships are potentially related to increased risk of damage to neurons. Functional bioinformatics revealed overrepresented functional ontology annotations (Table 1) when all 121 differentially abundant mRNAs are assessed together and when these mRNAs are grouped into subsets according to which olfactory mucosal cell types express them most strongly. The latter is possible because the transcriptomes of three cell populations in the olfactory epithelium have been defined in detail: mature OSNs, immature OSNs, and the pooled population of non-neuronal cells in this tissue [65, 80]. Analyses of the mRNAs primarily expressed in this non-neuronal population and more abundant in E4 samples revealed an overrepresented serine protease inhibitor annotation due to a group of four mRNAs (Serpina3m, Serpina3n, Agt, and Slpi). Functional bioinformatics of every level of analysis, from the entire set of 121 differentially abundant mRNAs down to mature OSN mRNAs less abundant in E4 mice, revealed that olfactory transduction is an overrepresented annotation due to differences in the abundance of 17 odorant receptor mRNAs. Oxidoreductase was also an overrepresented annotation due to a set of 10 mRNAs that are less abundant in E4 samples (Table 1). These mRNAs encode either oxidoreductases or proteins that regulate oxidoreductases. If we exclude odorant receptors and restrict functional bioinformatics analysis to the 77 mRNAs that are not significant in previously published data from cortex, this overrepresentation of oxidoreductases remains.

Cellular respiration is not affected at 6 months of age

The lower abundance of a set of 10 mRNAs related to oxidoreductases in E4 samples suggested decreased capacity in some cellular metabolism pathways in cells from the olfactory mucosae of E4 mice compared to E3 mice. Several of these mRNAs are abundant in OSNs (Adi1, Alkbh6, Gapdhs, Gpx1, Gpx4, Nnt, and Pcyox1) and others are expressed primarily in non-neuronal cells (Aldh1a7, Cbr2, and Cyp2e1). Of the encoded proteins, five are known to reside in mitochondria or have effects on mitochondria (Cbr2, Cyp2e1, Gpx1, Gpx4, and Nnt), suggesting potential for effects on cellular respiration [82–86]. We investigated this hypothesis using Seahorse technology to measure respiration in cells acutely dissociated from olfactory mucosae. Mito Stress test experiments found no significant differences in any parameter of cellular respiration (Fig. 2; Supplementary Table 3).

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Fig. 2

Cellular respiration measures show no differences between E4 and E3 olfactory mucosae. A) To illustrate Mito Stress Test oxygen consumption rate (OCR) measurements, data from the two samples of cells acutely dissociated from olfactory mucosae, one each from E4 and E3 mice, are shown. Oligo, oligomycin; Rot+Succ, mixture of rotenone and succinate; Anti-A, antimycin-A. Measures of basal respiration (B), mitochondrial proton leak (C), maximum respiration (D), spare respiratory capacity (E), non-mitochondrial oxygen consumption (F), ATP production (G), coupling efficiency (H), and spare respiratory capacity (I) for E4 and E3 cell samples. The p-values are from analysis of covariance.

Glucose hypometabolism in E4 olfactory mucosae at 6 months of age

Glucose hypometabolism is perhaps the earliest symptom of a nervous system at risk for LOAD [54, 72–76]. We note that the mRNA with the second largest positive difference between E4 and E3 is Asns. Asns is an asparagine synthase whose expression increases when glucose availability is reduced [87]. Asns mRNA should increase when glucose uptake is reduced, predicting that E4 olfactory mucosae have lower glucose uptake than E3 olfactory mucosae. Another clue potentially linking transcriptome differences with glucose hypometabolism in olfactory mucosae is a decrease in Gapdhs mRNA. Knockdown of Gapdhs mRNA in melanoma cells decreases glucose uptake [88]. When we measured glucose uptake in samples of olfactory mucosae at age 6 months, we detected less uptake in E4 samples relative to E3 samples (Fig. 3; Supplementary Table 4). Since Asns is broadly expressed in mature OSNs, immature OSNs, and non-neuronal cells of the olfactory epithelium [65], these data do not yet point toward differences in glucose uptake in any particular cell type.

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Fig. 3

Glucose (2- [1,2-³H (N)]-deoxy-d-glucose) uptake was lower in olfactory mucosal samples from E4 mice compared to E3 mice (one-sided paired t-test; n = 9). To minimize temporal variation, each E4 mouse was paired with an E3 mouse. Effect size is displayed as the confidence interval around the paired mean difference.

OSN apoptosis increases in E4 mice at 10 months of age

AD leads to loss of neurons in several regions of the human brain, including regions that process olfactory signals [37–39]. Human OSNs from people suffering from AD show morphological differences from non-AD individuals, consistent with olfactory epithelium pathology [41]. Furthermore, OSNs are susceptible to apoptosis at very early ages in mouse models of FAD [45]. Based on these findings we hypothesized that the E4 mouse model of increased risk for LOAD would show evidence of increased OSN apoptosis relative to E3 mice. In a paired design experiment, we performed immunostaining for the active fragment of Casp3, a marker of OSN apoptosis, in olfactory epithelia of E3 and E4 mice. We found a significant difference in the frequency of Casp3 immunoreactivity in OSNs of E4 mice compared to E3 mice at 10 months of age but not at 6 months of age (Fig. 4A-C; Supplementary Table 5). These data indicate that human E4 does not begin to have measurable effects on mouse OSN survival until middle age.

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Fig. 4

The rate of OSN apoptosis, as measured by the frequency of OSNs immunoreactive for the active fragment of Casp3, is greater in E4 mice than in E3 mice at age 10 months (one-sided paired t-tests). A. Example of a Casp3-immunoreactive OSN in an E3 mouse at age 6 months. B, C) The frequency of Casp3 immunoreactive OSNs differ at age 10 months but not at age 6 months. D) Example of phosphorylated histone H3 (pH-3) immunoreactive basal cells in an E3 mouse at age 6 months. E, F) The frequency of basal cell proliferation, as measured by pH-3 immunoreactivity does not differ at either age.

The olfactory epithelium continuously replaces OSNs throughout life from basal stem and progenitor cells. When OSN loss increases substantially the rate of basal cell proliferation increases to maintain the size of the OSN population [89]. Whether small increases in the rate of OSN apoptosis would increase basal cell proliferation is uncertain. In undamaged olfactory epithelia of wild-type mice, a peak of apoptosis occurs at the boundary between the basal cell layer and the immature OSN layer, consistent with overproduction of basal cells under laboratory housing conditions [48]. This suggests that small increases in OSN apoptosis might not lead to increased basal cell proliferation. We used immunoreactivity for phosphorylation of histone H3, a highly specific marker of mitosis, to assess basal cell proliferation. These data revealed no difference in basal cell proliferation in the olfactory epithelia of E4 mice compared to E3 mice at either 6 or 10 months of age (Fig. 4C-F).