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Background: Hyposmia in Alzheimer’s disease (AD) is a typical early symptom according to numerous previous clinical studies. Although amyloid-β (Aβ), which is one of the toxic factors upregulated early in AD, has been identified in many studies, even in the peripheral areas. The pathology involving olfactory sensory neurons (OSNs) remains poorly understood.
Methods: Here, we focused on peripheral olfactory sensory neurons (OSNs) and delved deeper into the direct relationship between pathophysiological and behavioral results using odorants. We also confirmed histologically the pathological changes in three-month-old 5xFAD mouse models, which recapitulates AD pathology. We introduced a numeric scale histologically to compare physiological phenomenon and local tissue lesions regardless of the anatomical plane.
Results: We observed the odorant group that the 5xFAD mice showed reduced responses to odorants. These also did not physiologically activate OSNs that propagate their axons to the ventral olfactory bulb. Interestingly, the amount of accumulated amyloid-β (Aβ) was high in the OSNs located in the olfactory epithelial ectoturbinate and the ventral olfactory bulb glomeruli. We also observed irreversible damage to the ectoturbinate of the olfactory epithelium by measuring the impaired neuronal turnover ratio from the basal cells to the matured OSNs.
Conclusions: Our results showed that partial and asymmetrical accumulation of Aβ coincided with physiologically and structurally damaged areas in the peripheral olfactory system, which evoked hyporeactivity to some odorants. Taken together, partial olfactory dysfunction closely-associated with peripheral OSN’s loss could be a leading cause of AD-related hyposmia, a characteristic of early AD.
Keywords
Alzheimer’s disease, Olfactory dysfunction, β-amyloid, 5xFAD, Olfactory sensory neuron, Zonal organization, Odor detection test, Ca2+ imaging, Topographic analysis, Neuronal turnover
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- Movie1WTmineralwater.avi
Movie S1. Representative clip of trained moving mouse for analysis of odor detection test using DeepLabCut. Four points of interest (POIs) that we tracked in each frame were the nose (blue), ears (light blue and yellow), and tail (red). Randomly selected 180 frames and manually labeled POIs in those frames, and used them to train and test a neural network model implemented in DeepLabCut. Evaluation of labeling accuracy was achieved by comparing the labels acquired from the convolutional neural network on the test set with manual labels. The model was then used to evaluate all frames in each group of the 20 videos used for training. The resulting x and y coordinates corresponding to the middle position of four POIs within each frame were used to determine location.
- SonetalARTSupplementarymaterialsminorrev.pdf
Figure S1. Verification of early-stage AD phenotype in 5xFAD transgenic mice. (a) Illustration of the time course for Y-maze and Morris water maze test. (b) Basic mobility tests using Y-maze showed the total number of arm entries (WT, n = 23; 5xFAD, n = 16). (c–d) Spontaneous alternation test using the Y-maze and Morris water maze test was performed to evaluate working memory (two-month: WT, n = 23; 5xFAD, n = 16, four-month: WT, n = 6; 5xFAD, n = 6, six-month: WT, n = 10; 5xFAD, n = 10). (c) A spontaneous alternation test using Y-maze was performed and the number of entries to another arm was measured (top). Scheme illustration (bottom). (d) The Morris water maze task was performed and the ratio of escape latency (WT/5xFAD) was measured (top). Scheme illustration (bottom). (e) Illustration of experimental timepoints identified in this research based on the result interpretation. One-way ANOVA was performed for statistical analysis. All data presented as mean ± SEMs. Statistical significances are noted [ns, non-significant; ***P < 0.001]. Alzheimer’s disease (AD), wild-type (WT), five familial AD mutations (5xFAD). Figure S2. Clustering based on spatial information of the activity maps and signal size in olfactory synapses. (a) A schematic diagram of the olfactometer. Compressed air was used as the carrier gas. olfactometer delivered mixed air and saturated with odorant vapor in the odor applicator. The flow rates of the air and the odorant vapor were controlled by a flow meter and a syringe pump, respectively. Turning-off of the suction to the outer barrel releases odorant from the end of the applicator. (b) In order to confirm the correlation with the IHC results to be followed, we analyzed the (ΔF/F0) change across the whole OB. The entire analysis area of OB is divided equally among 10 sections from the anterior to posterior and 180 section from dorsal to ventral within a lateral olfactory bulb view, respectively. (c–e) The spatial information of a widely used inbred strain of mouse (C57BL/6). (c) Illustration of OSNs-derived input signal map based on calcium activity of individual odorants. (d) Clustering based on the spatial correlation between each calcium activity pattern with angle glomeruli located in Fig. 2. (e) Each odorant was divided into two groups; lyral [L], acetophenone [A], and eugenol [E] (group A); geraniol [G], allyl phenylacetate [AP], heptanoic acid [HA], and heptanal [H] (group B). (f) Representative spatial information from WT and 5xFAD mice. Olfactory sensory neurons (OSNs), wild-type (WT), five familial AD mutations (5xFAD). Figure S3. The immunoreactivity of Aβ isoforms in the coronal section of the olfactory bulb. (a) Illustration of criteria to indicate the relative angular position of olfactory synapses. (b) The immunoreactivity of Aβ isoforms in the coronal section of the olfactory bulb (green); (Top) anti-A11 (oligomeric), (Middle) anti-6E10 (Aβ1-16), (Bottom) anti-D54D2 (Aβ1-40, Aβ1-42), and DAPI (blue). Asterisk (*): glomerular layer, Hash (#): granule cell layer. Scale bar: 500 μm. Wild-type (WT), five familial AD mutations (5xFAD). Figure S4. The angle-matched spatial correlation between A11 level in 5xFAD and calcium activity in WT. Scatter diagrams displaying correlations between each variable and linear regression analysis with 95% confidence intervals were used (gray dashed line). (a) Correlation with lyral, acetophenone, and eugenol. (b) Correlation with geraniol, allyl phenylacetate, heptanoic acid, and heptanal. Wild-type (WT), five familial AD mutations (5xFAD), Alzheimer’s disease (AD). Figure S5. Cell death of the OB. The TUNEL-positive signal in the coronal section of olfactory bulb (green). (Top) Domain section image, (bottom) magnified cropped image from upper domain section image, and DAPI (blue). Asterisk (*): glomerular layer, Hash (#): granule cell layer, circle (O): TUNEL positive cell. Scale bar: 100 μm. Wild-type (WT), five familial AD mutations (5xFAD). Figure S6. Intrinsic turnover characteristics of OSNs. All experimental subjects in this figure were two-month C57BL/6 mice, a widely used inbred strain. (a–c) Analysis of OSNs proliferation and differentiation (“turn”). (a) Representative IHC data quantifying BrdU (+) cells by time after BrdU intraperitoneal (IP) injection between turbinate ecto and endo. (b) Comparative quantification one day after injection between ecto and endoturbinate. (c) Differentiation ratio through quantification by migration degree by time after injection between the ecto and endoturbinate. (d–e) Analysis of the death of OSNs (“over”). (d) TUNEL (+) cells indicated as IF data in each turbinate and (e) comparative quantification. Data are represented as mean ± SEM from three independent experiments. For statistical analysis, a two-tailed unpaired t-test was performed using Prism software (GraphPad software, USA). Statistical significances are noted (*P < 0.05, ***P < 0.001). Olfactory sensory neurons (OSNs), wild-type (WT), five familial AD mutations (5xFAD), Alzheimer’s disease (AD).
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View the published article at Alzheimer's Research & Therapy.
Version 3
Posted 02 Dec, 2020
No community comments so far
Editorial decision: Accept
On 19 Nov, 2020
Editor assigned
On 18 Nov, 2020
Submission checks complete
On 18 Nov, 2020
Editor invited
On 18 Nov, 2020
No comments provided
Review #3 received
Received 14 Nov, 2020
Review #2 received
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Reviewer #3 agreed
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Reviewer #2 agreed
On 06 Nov, 2020
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On 03 Nov, 2020
Reviewers invited
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Editorial decision: Major Revision
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On 28 Aug, 2020
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Received 21 Aug, 2020
Reviewer #1 agreed
On 11 Aug, 2020
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License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Alzheimer's Research & Therapy.
Background: Hyposmia in Alzheimer’s disease (AD) is a typical early symptom according to numerous previous clinical studies. Although amyloid-β (Aβ), which is one of the toxic factors upregulated early in AD, has been identified in many studies, even in the peripheral areas. The pathology involving olfactory sensory neurons (OSNs) remains poorly understood.
Methods: Here, we focused on peripheral olfactory sensory neurons (OSNs) and delved deeper into the direct relationship between pathophysiological and behavioral results using odorants. We also confirmed histologically the pathological changes in three-month-old 5xFAD mouse models, which recapitulates AD pathology. We introduced a numeric scale histologically to compare physiological phenomenon and local tissue lesions regardless of the anatomical plane.
Results: We observed the odorant group that the 5xFAD mice showed reduced responses to odorants. These also did not physiologically activate OSNs that propagate their axons to the ventral olfactory bulb. Interestingly, the amount of accumulated amyloid-β (Aβ) was high in the OSNs located in the olfactory epithelial ectoturbinate and the ventral olfactory bulb glomeruli. We also observed irreversible damage to the ectoturbinate of the olfactory epithelium by measuring the impaired neuronal turnover ratio from the basal cells to the matured OSNs.
Conclusions: Our results showed that partial and asymmetrical accumulation of Aβ coincided with physiologically and structurally damaged areas in the peripheral olfactory system, which evoked hyporeactivity to some odorants. Taken together, partial olfactory dysfunction closely-associated with peripheral OSN’s loss could be a leading cause of AD-related hyposmia, a characteristic of early AD.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- Movie1WTmineralwater.avi
Movie S1. Representative clip of trained moving mouse for analysis of odor detection test using DeepLabCut. Four points of interest (POIs) that we tracked in each frame were the nose (blue), ears (light blue and yellow), and tail (red). Randomly selected 180 frames and manually labeled POIs in those frames, and used them to train and test a neural network model implemented in DeepLabCut. Evaluation of labeling accuracy was achieved by comparing the labels acquired from the convolutional neural network on the test set with manual labels. The model was then used to evaluate all frames in each group of the 20 videos used for training. The resulting x and y coordinates corresponding to the middle position of four POIs within each frame were used to determine location.
- SonetalARTSupplementarymaterialsminorrev.pdf
Figure S1. Verification of early-stage AD phenotype in 5xFAD transgenic mice. (a) Illustration of the time course for Y-maze and Morris water maze test. (b) Basic mobility tests using Y-maze showed the total number of arm entries (WT, n = 23; 5xFAD, n = 16). (c–d) Spontaneous alternation test using the Y-maze and Morris water maze test was performed to evaluate working memory (two-month: WT, n = 23; 5xFAD, n = 16, four-month: WT, n = 6; 5xFAD, n = 6, six-month: WT, n = 10; 5xFAD, n = 10). (c) A spontaneous alternation test using Y-maze was performed and the number of entries to another arm was measured (top). Scheme illustration (bottom). (d) The Morris water maze task was performed and the ratio of escape latency (WT/5xFAD) was measured (top). Scheme illustration (bottom). (e) Illustration of experimental timepoints identified in this research based on the result interpretation. One-way ANOVA was performed for statistical analysis. All data presented as mean ± SEMs. Statistical significances are noted [ns, non-significant; ***P < 0.001]. Alzheimer’s disease (AD), wild-type (WT), five familial AD mutations (5xFAD). Figure S2. Clustering based on spatial information of the activity maps and signal size in olfactory synapses. (a) A schematic diagram of the olfactometer. Compressed air was used as the carrier gas. olfactometer delivered mixed air and saturated with odorant vapor in the odor applicator. The flow rates of the air and the odorant vapor were controlled by a flow meter and a syringe pump, respectively. Turning-off of the suction to the outer barrel releases odorant from the end of the applicator. (b) In order to confirm the correlation with the IHC results to be followed, we analyzed the (ΔF/F0) change across the whole OB. The entire analysis area of OB is divided equally among 10 sections from the anterior to posterior and 180 section from dorsal to ventral within a lateral olfactory bulb view, respectively. (c–e) The spatial information of a widely used inbred strain of mouse (C57BL/6). (c) Illustration of OSNs-derived input signal map based on calcium activity of individual odorants. (d) Clustering based on the spatial correlation between each calcium activity pattern with angle glomeruli located in Fig. 2. (e) Each odorant was divided into two groups; lyral [L], acetophenone [A], and eugenol [E] (group A); geraniol [G], allyl phenylacetate [AP], heptanoic acid [HA], and heptanal [H] (group B). (f) Representative spatial information from WT and 5xFAD mice. Olfactory sensory neurons (OSNs), wild-type (WT), five familial AD mutations (5xFAD). Figure S3. The immunoreactivity of Aβ isoforms in the coronal section of the olfactory bulb. (a) Illustration of criteria to indicate the relative angular position of olfactory synapses. (b) The immunoreactivity of Aβ isoforms in the coronal section of the olfactory bulb (green); (Top) anti-A11 (oligomeric), (Middle) anti-6E10 (Aβ1-16), (Bottom) anti-D54D2 (Aβ1-40, Aβ1-42), and DAPI (blue). Asterisk (*): glomerular layer, Hash (#): granule cell layer. Scale bar: 500 μm. Wild-type (WT), five familial AD mutations (5xFAD). Figure S4. The angle-matched spatial correlation between A11 level in 5xFAD and calcium activity in WT. Scatter diagrams displaying correlations between each variable and linear regression analysis with 95% confidence intervals were used (gray dashed line). (a) Correlation with lyral, acetophenone, and eugenol. (b) Correlation with geraniol, allyl phenylacetate, heptanoic acid, and heptanal. Wild-type (WT), five familial AD mutations (5xFAD), Alzheimer’s disease (AD). Figure S5. Cell death of the OB. The TUNEL-positive signal in the coronal section of olfactory bulb (green). (Top) Domain section image, (bottom) magnified cropped image from upper domain section image, and DAPI (blue). Asterisk (*): glomerular layer, Hash (#): granule cell layer, circle (O): TUNEL positive cell. Scale bar: 100 μm. Wild-type (WT), five familial AD mutations (5xFAD). Figure S6. Intrinsic turnover characteristics of OSNs. All experimental subjects in this figure were two-month C57BL/6 mice, a widely used inbred strain. (a–c) Analysis of OSNs proliferation and differentiation (“turn”). (a) Representative IHC data quantifying BrdU (+) cells by time after BrdU intraperitoneal (IP) injection between turbinate ecto and endo. (b) Comparative quantification one day after injection between ecto and endoturbinate. (c) Differentiation ratio through quantification by migration degree by time after injection between the ecto and endoturbinate. (d–e) Analysis of the death of OSNs (“over”). (d) TUNEL (+) cells indicated as IF data in each turbinate and (e) comparative quantification. Data are represented as mean ± SEM from three independent experiments. For statistical analysis, a two-tailed unpaired t-test was performed using Prism software (GraphPad software, USA). Statistical significances are noted (*P < 0.05, ***P < 0.001). Olfactory sensory neurons (OSNs), wild-type (WT), five familial AD mutations (5xFAD), Alzheimer’s disease (AD).
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