1000263

research-article2021
JDRXXX10.1177/00220345211000263Journal of Dental ResearchA<span class="symbol" cstyle="symbol">β</span> Accumulation in Vmo

Research Reports: Biological

Journal of Dental Research
1­–8
Aβ Accumulation in Vmo Contributes to © International & American Associations
for Dental Research 2021

Masticatory Dysfunction in 5XFAD Mice Article reuse guidelines:

sagepub.com/journals-permissions
DOI: 10.1177/00220345211000263
https://doi.org/10.1177/00220345211000263
journals.sagepub.com/home/jdr

H.B. Kim1*, D. Kim2*, H. Kim3, W. Kim1, S. Chung1, S.H. Lee2, H.R. Kim4,
and S.B. Oh1,2,3

Abstract
Alzheimer’s disease (AD) shows various symptoms that reflect cognitive impairment and loss of neural circuit integrity. Sensory
dysfunctions such as olfactory and ocular pathology are also observed and used as indicators for early detection of AD. Although
mastication is suggested to correlate with AD progression, changes in the masticatory system have yet to be established in transgenic
animal models of AD. In the present study, we have assessed pathologic hallmarks of AD with the masticatory behavior of 5XFAD
mice. We found that masticatory efficiency and maximum biting force were decreased in 5XFAD mice, with no significant change in
general motor function. Immunohistochemical analysis revealed significant accumulation of Aβ (amyloid β), increased microglia number,
and cell death in Vmo (trigeminal motor nucleus) as compared with other cranial motor nuclei that innervate the orofacial region.
Masseter muscle weight and muscle fiber size were also decreased in 5XFAD mice. Taken together, our results demonstrate that
Aβ accumulation in Vmo contributes to masticatory dysfunction in 5XFAD mice, suggesting a close association between masticatory
dysfunction and dementia.

Keywords: amyloid β, dementia, trigeminal motor nucleus, masseter muscle, Alzheimer’s disease, mastication

Introduction neurogenesis, and spatial memory test performance have been

Alzheimer’s disease (AD) is one of the most common causes
of dementia that results from the progressive degeneration of
the central nervous system (Rosen et al. 1984). AD is generally
thought to result from neuronal death caused by the accumula-
tion of amyloid β (Aβ) protein. Aβ, the neurotoxic peptide that
is cleaved from amyloid precursor protein, can aggregate to
form Aβ plaques that are commonly found in the brain of
patients with AD (He et al. 2018). Brain imaging of patients
with AD has revealed degeneration of symptom-related brain
regions (e.g., hippocampus), and this may cause memory dete-
rioration (Deweer et al. 1995). Aβ accumulations are also seen
in the temporal lobe, which may correlate to the sensory and
motor dysfunctions seen in patients with AD, such as olfactory,
ocular, and gait problems (O’Keeffe et al. 1996; Mesholam
et al. 1998; Lewis et al. 2006; Frost et al. 2010; He et al. 2018).
However, Aβ accumulation has yet to be addressed in the oro-
facial sensorimotor system despite the correlation observed in
oral dysfunction and cognitive impairment (Onozuka et al.
2000; Campos et al. 2017).
Recent clinical studies have found significant cognitive
decline in patients with AD, with oral impairments and masti-
catory malfunctions including oral dyskinesia, periodontitis,
and tooth loss (Singhrao et al. 2014; Campos et al. 2017).
Furthermore, a meta-analysis of cohort studies found that indi-
viduals with <20 teeth were more likely to develop cognitive
impairment and dementia, supporting the close correlation
between tooth loss and cognitive decline (Cerutti-Kopplin
et al. 2016). In various animal models of altered mastication,
decreases in hippocampal neuron number, synaptic activity,
consistently reported (Piancino et al. 2020). Thus, disturbed
mastication can negatively affect not only the normal function
of the hippocampus but also hippocampus-dependent memory
and cognition. However, Aβ accumulation or neuronal loss in
mastication-related brainstem regions has yet to be observed in
patients with AD or in a 5XFAD animal model of AD (Parvizi
et al. 2001; Uematsu et al. 2018). Amyloid precursor protein
and calpain-I are highly expressed and extensively colocalized
in the trigeminal motor nucleus (Vmo) as compared with the
other cranial motor nuclei, and they are observed in brain
regions with Aβ deposition in patients with AD (Siman et al.
1
Department of Brain and Cognitive Sciences, College of Natural
Sciences, Seoul National University, Seoul, Republic of Korea
2
Dental Research Institute and Department of Neurobiology and
Physiology, School of Dentistry, Seoul National University, Seoul,
Republic of Korea
3
Interdisciplinary Program in Neuroscience, Seoul National University,
Seoul, Republic of Korea
4
College of Dentistry, Dankook University, Cheonan, Republic of Korea
*Authors contributing equally to this article.
A supplemental appendix to this article is available online.
Corresponding Authors:
S.B. Oh, Dental Research Institute and Department of Neurobiology and
Physiology, School of Dentistry, Seoul National University, 101 Daehak-
ro, Jongno-gu, Seoul, 03080, Republic of Korea.
Email: odolbae@snu.ac.kr
H.R. Kim, College of Dentistry, Dankook University, Cheonan 31116,
Republic of Korea.
Email: hrkimdp@dankook.ac.kr
2 Journal of Dental Research 00(0)
1990). This suggests a correlation between Aβ accumulation in
Vmo and masticatory dysfunction.
Transgenic AD mouse models were created to replicate the
degenerative brain progression of human AD through the
genetic mutation of amyloid precursor protein and/or preseni-
lin to increase Aβ40/42 production (Oakley et al. 2006). Of the
many transgenic AD models, 5XFAD (Tg6799) mouse is well
known to cause Aβ pathology as early as 1 to 2 mo of age and
impairment of spatial working memory by 6 mo (Lewis et al.
2006; Devi and Ohno 2010; Eimer and Vassar 2013). In this
study, we investigated whether Vmo manifests Aβ pathology
in 5XFAD mice and, if so, whether Aβ pathology of Vmo is
associated with masticatory dysfunction in an early stage of
dementia (5 mo old).
Materials and Methods
Mice
All experiments were performed with 5-mo-old male 5XFAD
mice (Tg6799; stock 006554, Jackson Laboratory) and littermate
mice, maintained in a temperature-controlled room (23 ± 1 °C,
12/12 h light/dark cycle) with standard laboratory chow (pellet
diet) and water ad libitum unless otherwise noted. All experimen-
tal procedures were approved by the Institutional Animal Care
and Use Committee at Seoul National University. This study con-
formed to the ARRIVE guidelines (Animal Research: Reporting
In Vivo Experiments) for preclinical animal studies.
Animal Behavior Tests
For the masticatory behavior test, all mice were fasted 24 h and
then habituated for 1 h in a clear acrylic cage. Animals were
exposed to chow that was wrapped with aluminum foil except
1 cm of the end. The masticatory behavior was recorded by
video camera for 1 h. The chow-approaching number was
defined as the sum of the individual events that the mouse bit
and masticated the chow in a sequence. The chow-approaching
time was defined as the total time that the mouse spent on the
biting-and-masticating sequence. Chow approach without a
biting-and-mastication sequence, such as sniffing and touch-
ing, was all excluded from data analysis. Mice body weight
and chow weight were measured before and after the mastica-
tory efficiency test to obtain chow consumption per body
weight. All analysis was done blind to the experimental groups
to avoid experimental bias.
The maximal biting force of the mice was recorded with a
Piezo sensor (FlexiForce, A201; Tekscan) by mildly restraining
the mouse with a hand for free head movement and by placing the
tip of the Piezo sensor in front of the snout, leaving it to freely
bite with incisors. The biting force was measured for 200 s per
mouse, and the data were digitalized by FlexiForce software. The
maximal biting force was determined from the highest score.
A rotarod test was performed as previously described
(Singh et al. 2018) with modifications. The rotarod retention
time was automatically measured from start to animal fall from
the horizontal bar by the rotarod apparatus.
Immunohistochemistry
The samples were prepared following the method previously
described (Lee et al. 2020). Brain sections were blocked for 2 h
at room temperature with 4% normal horse serum (NHS) in
0.3% Triton-X in phosphate-buffered saline (PBST) and then
incubated with primary antibodies: mouse anti-Aβ monoclonal
antibody (4G8, SIG-39220; BioLegend), goat anti-choline acet-
yltransferase (ChAT, AB144P; Merck Millipore), rabbit anti-
cleaved caspase-3 (cas-3, 9664; Cell Signaling Technology),
and rabbit anti-Iba-1 (Iba-1, 019-19741; FujiFilm) in 4% NHS
in 0.3% PBST for 2 d at room temperature. The sections were
washed with phosphate-buffered saline (PBS) and then incu-
bated with secondary antibodies: donkey anti-goat cy3 (705-
165-147; Jackson Laboratory), donkey anti-rabbit cy3
(711-165-152; Jackson Laboratory), and donkey anti-mouse
Alexa Fluor 488 (715-545-150; Jackson Laboratory) in 0.3%
PBST for 2 h at room temperature. For the Aβ plaque staining,
1% thioflavin S (T-1892; Sigma) was applied for 12 min and
then washed with PBS.
Muscle sections were blocked with 2% NHS in 0.3% PBST
for 2 h and incubated with rabbit anti-laminin polyclonal anti-
body (ab11575; Abcam) in 2% NHS in 0.3% PBST for 2 d at
room temperature. The sections were washed with PBS and
incubated with donkey anti-rabbit Alexa Fluor 488 (711-545-
152; Jackson Laboratory) in 0.3% PBST for 2 h at room
temperature.
Hematoxylin and Eosin Staining
The samples of mandible and masseter muscle were fixed with
formalin and sequentially dehydrated with 80% to100% etha-
nol in increasing concentration. The tissues were then infil-
trated with resin (Technovit 7200; Heraeus Kulzer GmbH).
The tissue blocks were sectioned in 30-μm thickness.
Micro–computed tomography
The head specimens of 5XFAD and littermates were sagittally
split into 2 symmetrical parts, which were then postfixed in 4%
paraformaldehyde at 4 °C for 2 d. Scanning of the first man-
dibular molar of 5XFAD and littermate mice was performed by
Skyscan 1172 (Bruker). The scanning parameters were 6.92-
μm pixel size, 70-kV x-ray voltage, 141-μA electric current,
and 0.5-mm aluminum filter. All data sets were reconstructed
with NRecon software (SkyScan). By using the protocol modi-
fied from the quantitative analysis of rat jawbone microstruc-
tures (Chatterjee et al. 2017), the volume of the whole first
molar, crown, and canal from each genotype was analyzed by
CT Analyzer (version 1.18.8.0; Bruker).
Data Analysis and Statistics
All histologic analyses were processed by ZEN 3.2 Blue
(Zeiss) and ImageJ (National Institutes of Health) software.
All statistics were performed by Prism software (version
6.01; GraphPad). Comparison among multiple groups was
Aβ Accumulation in Vmo 3

A
Chow consumption/body weight B C

** **

Chow approaching time (sec)

0.025 250 80

Chow approaching number

(8) (8)
0.020 (7) 200
60 (8)
0.015 150
(8) 40
0.010 100 (8)

20
0.005 50

0.000 0 0
Littermate 5XFAD Littermate 5XFAD Littermate 5XFAD

D E

Rotarod retention time (sec)

150
* 60
Maxium biting force (unit)

(12) (12)
(12)
100 40
(12)

50 20

0 0
Littermate 5XFAD Littermate 5XFAD

Figure 1.  Measurement of masticatory efficiency in 5XFAD mice. (A) Weight of chow fed to mice was divided by the body weight of the individual
mice before the experiment (P = .4153, n = 7 or 8/group). (B) Total time spent chow feeding (P = .0012, n = 8/group) and (C) the chow-approaching
number (P = .0029, n = 8/group). (D) Maximal biting force (P = .0469, n = 12/group) and (E) rotarod body performance test (P = .8443, n = 12/group).
(A–E) The 5XFAD mice versus littermate controls. All n values are presented in parentheses. Student’s t test. Mean ± SEM. *P < .05. **P < .01.

made by 1-way analysis of variance (ANOVA) with Bonferroni mice (Fig. 1B, C). Next, we compared the maximal biting
post hoc test, and comparison between 2 groups was made with force, and 5XFAD mice showed significantly decreased maxi-
a Student’s t test. Differences with P values <.05 were regarded mal biting force than the littermate mice (Student’s t test,
as statistically significant. P = .0469, n = 12/group; Fig. 1D). However, when we measured
the general motor activity of the mice by the rotarod perfor-
mance test, there was no difference in the retention time
Supplemental Information between the groups (Student’s t test, P = 0.8443, n = 12/group;
Detailed materials and methods are included in the Appendix. Fig. 1E).

Results Aβ Accumulated Specifically in Vmo of 5XFAD Mice

5XFAD Mice Showed Decreased Masticatory We then investigated the Aβ accumulation in various cranial
motor nuclei, such as Vmo, which directly innervates to the
Function masticatory muscle; the facial motor nucleus (7th cranial motor
We used 5-mo-old 5XFAD mice when the animals were known nucleus) and the hypoglossal motor nucleus (12th cranial
to have cognitive impairment (Lewis et al. 2006). We first motor nucleus), which are involved in the movement of the
examined the masticatory function of 5XFAD mice by measur- orofacial region; and the dorsal motor nucleus of the vagus
ing the chow consumption behavior after 24-h fasting. There nerve (10th cranial motor nucleus) as the control cranial motor
was no difference in chow consumption between 5XFAD and nucleus (Nakamura et al. 2004). We identified motor neurons
littermate mice (Student’s t test, P = .4153, n = 7 or 8/group; with cholinergic neuronal marker (ChAT), and Aβ accumula-
Fig. 1A), but the total time spent in chow-feeding behavior tion was determined by immunostaining with anti-Aβ 17-24
(Student’s t test, P = .0012, n = 8/group) and the total number of antibody (clone 4G8; Tousseyn et al. 2015) and thioflavin S
approaches (Student’s t test, P = .0029, n = 8/group) were sig- fluorescent dye for targeting Aβ plaque (Yuan and Grutzendler
nificantly increased in the 5XFAD mice versus the littermate 2016; Fig. 2A). The staining showed a distinct accumulation of
4 Journal of Dental Research 00(0)

no accumulation of Aβ was found in the littermate

A mice (Fig. 2A). We examined Aβ plaque accumu-
lation in the subregions of Vmo in 5XFAD mice—
the ventromedial Vmo (VM Vmo; jaw-opening
muscle) and the dorsolateral Vmo (DL Vmo; jaw-
closing muscle)—and observed significantly
increased Aβ plaque accumulation in the DL Vmo
as compared with VM Vmo (1-way ANOVA: F[2,
6] = 9.231, P = .0148 with Bonferroni post test,
n = 3; Fig. 2D, E).

Preferential Microglial Activation

and Cell Death of Vmo in 5XFAD Mice
B C
Since toxic Aβ oligomers have been proposed to
0.8
(6)
150
(9)
cause brain inflammation and neuronal degenera-
A plagues / area (mm2)
ChAT+ A + / ChAT+

0.6
tion (Butterfield and Lashuel 2010; Grimaldi et al.
100 2018), we next examined the microglial activation
0.4 and cell death of Vmo neurons in 5XFAD mice.
0.2 (6)
50
(6)
When we immunostained microglia with Iba-1 as
(6)
(6) (6)
a surrogate of microglial activation, we observed
(6)
0.0
5N 7N 10N 12N
0
5N 7N 10N 12N
an increased number of microglia in Vmo as com-
pared with the other nuclei in the 5XFAD mice
D E (Student’s t test, P = .0035, n = 3 or 4/group; Fig.
Trigeminal motor nucleus
Rostral Caudal 3B). The percentage of activated microglia with
deramification and swelling of cell body (Fig. 3C)
200
(3)
was also significantly increased in Vmo versus the
(3)
150 other nuclei (1-way ANOVA: F[3, 12] = 46.33,
(3)
P < .0001 with Bonferroni posttest, n = 4/group;
100
Fig. 3D). The number of activated microglia in
50 Vmo was also significantly increased in the
5XFAD mice as compared with the littermate mice
0
Whole
Vmo
DL
Vmo
VM
Vmo
(Student’s t test, P = .0002, n = 3 or 4/group; Fig.
3E). To confirm the neuronal degeneration of
Jaw opening (VM Vmo) Jaw closing (DL Vmo)
Vmo, we analyzed immunoreactive cells with
active caspase-3, a cell death marker, which were
Figure 2. Amyloid β (Aβ) accumulation in the cranial motor nuclei of 5XFAD mice.
found only in Vmo of the 5XFAD mice (Fig. 3F).
(A) Staining of Aβ (purple), thioflavin S (green), and ChAT (choline acetyltransferase;
red). Arrowhead (white) indicates colocalization of ChAT, thioflavin S, and Aβ The number of ChAT-positive cells was signifi-
staining. Squarebox shows zoomed-in image of arrowhead region. Scale bar (white): cantly decreased in the 5XFAD mice as compared
50 μm. (B) Quantitative data show ratio of Aβ+:ChAT+ neurons. One-way analysis of with the littermate mice (Student’s t test, P = .0173,
variance (ANOVA): F(3, 20) = 83.35, P < .0001, with Bonferroni post test. Mean ± SEM.
****P < .0001. n = 6 mice/group. (C) Number of Aβ plaque in ChAT+ area from cranial n = 5 or 6/group; Fig. 3G), whereas the total size of
nuclei (5N, 7N, 10N, and 12N). Trigeminal motor nucleus (Vmo), 5N; facial motor Vmo did not differ between the groups (Student’s
nucleus, 7N; vagus nucleus, 10N; hypoglossal nucleus, 12N. One-way ANOVA: F(3, t test, P = .3203, n = 5 or 6/group; Fig. 3H).
20) = 67.24, P < .0001, with Bonferroni post test. Mean ± SEM. ****P < .0001. n = 6 to
9 mice/group. (D) Staining of thioflavin S (green) and ChAT (red) in rostral to caudal
Vmo. The dotted line (white) represents the ventromedial (VM) Vmo region innervating
the jaw-opening muscle, and the solid line (white) represents the dorsolateral (DL)
Masseter Muscle Weight and Fiber
Vmo region innervating the jaw-closing muscle. Scale bar (white): 200 μm. (E) Number Thickness Were Decreased
of Aβ plaque in ChAT+ area from whole Vmo, VM Vmo, and DL Vmo. One-way
ANOVA: F(2, 6) = 9.231, P = .0148, with Bonferroni post test. Mean ± SEM. *P < .05. in 5XFAD Mice
n = 3. All n values are presented in parentheses.
Finally, we investigated the changes of the masti-
catory muscle innervated by Vmo neurons and
Aβ plaque in a ChAT-positive area of Vmo as compared with observed a significant decrease in weight (Student’s t test,
the other cranial nuclei in 5XFAD mice (Fig. 2B; 1-way P = .0208, n = 5/group; Fig. 4A) and area (Student’s t test,
ANOVA: F[3, 20] = 83.35, P < .0001 with Bonferroni post test, P = .0428, n = 5/group; Fig. 4B, C) of masseter muscle in the
n = 6/group; Fig. 2C; 1-way ANOVA: F[3, 20] =  67.24, 5XFAD mice as compared with the littermate mice. The
P < .0001 with Bonferroni post test, n = 6 to 9/group). However, thickness of masseter muscle fiber was also significantly
Aβ Accumulation in Vmo 5

A 5N 7N 10N & 12N B

Littermate
300 (4)

Total microglia number /

5XFAD
Littermate

(4)
(4)
(3)

area (mm2)
200 (3)
(3)
(3) (4)
100
5XFAD

0
5N 7N 10N 12N
Iba-1 ChAT

C D E
50

Activated microglia number/

(4)

/ total microglia number (%)

50
(4) 40
Activated microglia 40

area (mm2)
30
30
20
(4)
20
10 (3)
10 (4) (4)
0
0
Iba-1 ChAT 5N 7N 10N 12N
Littermate 5XFAD

F 5N 7N 10N & 12N G H

ChAT positive cell / area (mm2)

Trigeminal nucleus size (mm2)

350 0.3
Littermate

(6) (6)
280 (5)
(5)
0.2
210

140
5XFAD

0.1
70

0 0.0
Active caspase-3 ChAT Littermate 5XFAD Littermate 5XFAD

Figure 3.  Brain inflammation and cell death in the cranial motor nuclei of 5XFAD mice. (A) Immunostaining of ChAT (choline acetyltransferase;
green) and IBa1 (red). Scale bar (white): 50 μm. (B) Total number of microglia/area (mm2) (P = .0035, n = 3 or 4/group). (C) Immunostaining of microglia
in 5N: activated (asterisk) and deactivated (arrowhead) form. Scale bar (white): 50 μm. (D) Percentage of activated microglia to the total number of
microglia in 5N, 7N, 10N, and 12N. One-way analysis of variance: F(3, 12) = 46.33, P < .0001, with Bonferroni post test. Mean ± SEM. n = 4/group. (E)
Activated microglia per area unit (mm2). P = .0002. n = 3 or 4/group. (F) Staining of ChAT (green) and caspase-3 (red). Chat+ and caspase-3+ cells are
presented in arrows. Scale bar (white): 50 μm. (G) Number of ChAT+ cells per area unit (mm2) in 5N (P = .0173, n = 5 or 6/group) and (H) size of the
5N (mm2; P = .3203, n = 5 or 6/group). Trigeminal motor nucleus, 5N; facial motor nucleus, 7N; vagus nucleus (dorsal-motor), 10N; hypoglossal nucleus,
12N. All n values are presented in parentheses. Mean ± SEM. Student’s t test.

decreased in 5XFAD mice as compared with the littermate innervated by other cranial nuclei, the posterior belly of the
mice, thus showing higher proportion of smaller sized-mus- digastric muscle innervated by the facial motor nucleus
cle fibers (Student’s t test, P = .0338, n = 5/group; Fig. 4D–F). (Student’s t test, P = .1754, n = 3; Appendix Fig. 1A, B) and
However, when we compared the fiber size of the muscles the hyoglossus muscle innervated by the hypoglossal nucleus
6 Journal of Dental Research 00(0)

A B Littermate 5XFAD C
0.25
Masseter muscle weight (g)

10

Masseter muscle area (mm2)

0.20
(5)
(5) 8 (5)
0.15 (5) 6

0.10 4

0.05 2

0.00 0
1mm 1mm
Littermate 5XFAD Littermate 5XFAD

D E F
Masseter muscle
Average muscle fiber size ( m2) 1500 20
Littermate 5XFAD
(5) Littermate (5)
15

Proportion (% )
1000 5XFAD (5)
(5)
10

500
5

100 m 100 m 0 0
Littermate 5XFAD 0 1500 3000 4500
Muscle cell size (um 2)

G Crown Root Whole H I J

0.20 0.15
Littermate

1.0
(5)

Whole tooth volume (mm3)

(4)
Crown volume (mm3)

(4)
Root volume (mm3)

(5) (5) (4)

0.8
0.15
0.10
0.6
0.10
0.4
0.05
5XFAD

0.05
0.2

0.00 0.00 0.0

Littermate 5XFAD Littermate 5XFAD Littermate 5XFAD

Figure 4.  Histologic characterization of masseter muscle in 5XFAD mice. (A) Masseter muscle weight (P = .0208, n = 5/group). (B) Hematoxylin and
eosin staining of whole maxillary and mandibular coronal section and (C) masseter muscle area (P = .0428, n = 5/group). (D) Representative image
of cross section of masseter muscle stained with laminin (green). (E) Analysis of masseter muscle fiber diameter (P = .0338, n = 5/group) and (F)
size distribution of masseter muscle fiber diameter. (G) Micro–computed tomography and 3-dimensional volumetric measurements of (H) crown
(P = .7056, n = 4 or 5/group), (I) tooth root (P = .4985, n = 4 or 5/group), and (J) whole first mandibular (P = .8302, n = 4 or 5/group) in 5XFAD mice and
littermate. All n values are presented in parentheses. Mean ± SEM. Student’s t test. *P < .05. **P < .01.

(Student’s t test, P =.6278, n = 3; Appendix Fig. 1C, D) Discussion

showed no difference in average muscle fiber size between
5XFAD and littermate mice. To investigate the changes in In this study, we found decreased masticatory efficiency and
masticatory system components other than the muscle, we maximum biting force in 5XFAD mice, without a significant
measured volumetric changes in various components of the change in general motor function. Excessive accumulation of
mandibular first molar (Fig. 4G). The crown (Student’s t test, Aβ, increased number of microglia, and regional cell death
P = .7056, n = 4 or 5/group), root (Student’s t test, P = .4985, were also observed in Vmo, which innervates jaw-closing and
n 
=  4 or 5/group), and whole volume (Student’s t test, jaw-opening muscles (including the masseter muscle), in com-
P = .8302, n = 4 or 5/group) of the first molar were not parison with the other cranial motor nuclei. Furthermore, the
­different between the groups (Fig. 4H–J). weight and muscle fiber size of the masseter muscle, but not
Aβ Accumulation in Vmo 7
the posterior belly of the digastric muscle nor the hyoglossus
muscle, were significantly decreased in 5XFAD mice as com-
pared with littermates. These results suggest a correlation
between specific Aβ accumulation in Vmo and masticatory
dysfunction in AD.
In our study, we first examined the outcome of significant
Aβ accumulation in Vmo to the masticatory function via sets
of behavioral tests. Mastication efficiency was measured when
it naturally occurred. Although the chow intake did not differ
between the groups, 5XFAD mice showed an increase in total
time spent chow feeding and in number of feeding behaviors
(Fig. 1B–D). These results indicate that 5XFAD mice may
have decreased masticatory function, which is consistent with
clinical research showing a functional decline of mastication in
patients with AD, such as increased masticatory cycle time,
slow masticatory velocity, and decreased biting force (Karlsson
and Carlsson 1990). The decreased biting force also indicates
the disrupted masticatory function in 5XFAD mice (Fig. 1D).
However, aging might also contribute to the masticatory dys-
function in 5XFAD mice. It is well established that masseter
muscle fiber types change with aging (Grünheid et al. 2009)
and that sarcopenia is observed in masseter muscles in the
elderly (Sakuma and Yamaguchi 2018). Further works on the
detailed analysis of muscle fiber types may help to rule out
the aging factor. Interestingly, there was no difference in
rotarod retention time between 5XFAD mice and littermates
(Fig. 1E), which is consistent with a recent report showing no
cell deaths observed in the motor neuron of the cervical spinal
cord of 6-mo-old 5XFAD mice as compared with nontrans-
genic littermates (Chu et al. 2017). These results suggest that
the masticatory dysfunction seen in 5-mo-old 5XFAD mice
occurs prior to cerebellar or spinal cord degeneration.
Aβ, the pathologic hallmark of AD, is highly correlated
with the pathologic process of neural degeneration. The
regional profile of Aβ accumulation in brain structures has
been studied with the purpose of interpreting functional altera-
tion and progression of the disease stage. For example, Aβ
accumulation in cortical regions and the hippocampus has been
implicated in cognitive deficiency and decreased function of
spatial memory (Lewis et al. 2006). Furthermore, several stud-
ies have shown that olfactory malfunction in AD is caused by
Aβ accumulation in the somatostatin-positive neuronal popu-
lation of the olfactory bulb (Mesholam et al. 1998; Yoo et al.
2017). In agreement with the study that reported Aβ accumula-
tion in Vmo of 3xTGAD mice (Overk et al. 2009), our result
demonstrated specific accumulation of Aβ and plaque forma-
tion in Vmo. When we compared DL Vmo (which innervates
jaw-closing muscles) and VM Vmo (which innervates jaw-
opening muscles; Travers 2015), Aβ plaque accumulation was
significantly higher in DL Vmo (Fig. 2). A degenerative event,
such as brain inflammation with microglial activation and
caspase-3 expression, was also consistently present with Aβ
accumulation (Fig. 3).
The process of mastication results from pattern generation in
the brainstem, and jaw-closing and jaw-opening muscles par-
ticipate and cooperate with each other during the mastication
process. Although we have focused our works on the masseter
muscle based on Aβ accumulation in DL Vmo rather than VM
Vmo, it is likely that jaw-opening muscles are also affected by
Aβ- or mastication-induced pathogenesis in AD. It was interest-
ing to note that Aβ accumulation in other cranial motor nuclei,
such as facial nucleus and hypoglossal nucleus which innervate
the orofacial region, was relatively limited, and their target
muscles, posterior belly of the digastric muscle and hyoglossus
muscle, also showed comparable fiber size between 5XFAD
and littermate mice (Appendix Fig. 1). Thus, masticatory func-
tion seems to be more disturbed than any other motor functions
in the orofacial area in 5XFAD mice.
Although there is a lack of direct clinical evidence showing
the accumulation of Aβ in Vmo, the possibility of occasional
Aβ accumulation in Vmo due to increased expression and
extensive colocalization of amyloid precursor protein and
calpain-I remains high (Siman et al. 1990). Although we found
masticatory dysfunctions in 5XFAD mice, we should be care-
ful in translating our results directly to clinical observation of
masticatory efficiency in patients with AD. However, clinical
research indeed showed the functional decline of mastication
in patients with AD (Karlsson and Carlsson 1990). Further
clinical studies are thus required to show correlation between
pathogenesis of AD and masticatory dysfunction.
Given the degeneration of Vmo neurons, our final question
was whether Vmo degeneration is accompanied by any impair-
ments of the peripheral masticatory system. Our results showed
decreased masseter muscle weight and atrophy based on the
histologic analysis (Fig. 4A–F). A recent study in DstGt homo-
zygous mice (model of sensory and autonomic neuropathy
type VI) showed cell death in Vmo motoneurons with masseter
muscle atrophy and weak activity of electromyography
(Hossain et al. 2018). This phenomenon could be interpreted
from a study showing muscle weakness caused by denervation
of motoneuron and presynaptic dysfunction of the neuromus-
cular junction (Kong et al. 2009). In addition, Aβ is known to
produce cytotoxic effects and attenuate excitatory synaptic
transmission (Karlsson and Carlsson 1990; Terry et al. 1991).
Taken together, Aβ accumulation in Vmo neurons may lead to
neuronal cell death that decreases the innervation of motoneu-
rons to masseter muscle or disrupts presynaptic modulations
that alter muscle morphology.
In conclusion, specific accumulation of Aβ in Vmo is
strongly associated with decreased masticatory efficiency in
5XFAD mice. This may explain the masticatory dysfunction
seen in patients with AD, suggesting a close relationship
between mastication and AD.
Author Contributions
H.B. Kim, contributed to conception, design, and data acquisition,
drafted and critically revised the manuscript; D. Kim, contributed
to conception and design, drafted and critically revised the manu-
script; H. Kim, W. Kim, contributed to data acquisition and analy-
sis, drafted and critically revised the manuscript; S. Chung, S.H.
Lee, contributed to data analysis and interpretation, drafted and
critically revised the manuscript; H.R. Kim, contributed to
8 Journal of Dental Research 00(0)
conception and data interpretation, drafted and critically revised
the manuscript; S.B. Oh, contributed to conception, design, and
data interpretation, drafted and critically revised the manuscript.
All authors gave final approval and agree to be accountable for all
aspects of the work.
Acknowledgments
The animals used in the present study were kindly provided by
Dr. Inhee Mook-Jung (Seoul National University, Republic of
Korea).
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
This research was supported by the National Research Foundation
of Korea grant (2016M3A9B6021209, 2017M3C7A1025602,
2018R1A5A2024418 to S.B.O. and 2017M3A9E4047243 to
H.R.K.) funded by the Korean government (Ministry of Science
and ICT).
References
Butterfield SM, Lashuel HA. 2010. Amyloidogenic protein–membrane inter-
actions: mechanistic insight from model systems. Angew Chem Int Ed.
49(33):5628–5654.
Campos CH, Ribeiro GR, Costa JL, Rodrigues Garcia RC. 2017. Correlation
of cognitive and masticatory function in Alzheimer’s disease. Clin Oral
Investig. 21(2):573–578.
Cerutti-Kopplin D, Feine J, Padilha D, De Souza R, Ahmadi M, Rompré P,
Booij L, Emami E. 2016. Tooth loss increases the risk of diminished cogni-
tive function: a systematic review and meta-analysis. JDR Clin Trans Res.
1(1):10–19.
Chatterjee M, Faot F, Correa C, Duyck J, Naert I, Vandamme K. 2017. A robust
methodology for the quantitative assessment of the rat jawbone microstruc-
ture. Int J Oral Sci. 9(2):87–94.
Chu T-H, Cummins K, Sparling JS, Tsutsui S, Brideau C, Nilsson KPR, Joseph
JT, Stys PK. 2017. Axonal and myelinic pathology in 5XFAD Alzheimer’s
mouse spinal cord. PLoS One. 12(11):e0188218.
Devi L, Ohno M. 2010. Phospho-eiF2α level is important for determining
abilities of BACE1 reduction to rescue cholinergic neurodegeneration and
memory defects in 5XFAD mice. PLoS One. 5(9):e12974.
Deweer B, Lehericy S, Pillon B, Baulac M, Chiras J, Marsault C, Agid Y,
Dubois B. 1995. Memory disorders in probable Alzheimer’s disease: the
role of hippocampal atrophy as shown with MRI. J Neurol Neurosurg
Psychiatry. 58(5):590–597.
Eimer WA, Vassar R. 2013. Neuron loss in the 5XFAD mouse model of
Alzheimer’s disease correlates with intraneuronal Aβ42 accumulation and
caspase-3 activation. Mol Neurodegener. 8:2.
Frost S, Martins RN, Kanagasingam Y. 2010. Ocular biomarkers for early
detection of Alzheimer’s disease. J Alzheimers Dis. 22(1):1–16.
Grimaldi A, Brighi C, Peruzzi G, Ragozzino D, Bonanni V, Limatola C, Ruocco
G, Di Angelantonio S. 2018. Inflammation, neurodegeneration and protein
aggregation in the retina as ocular biomarkers for Alzheimer’s disease in
the 3xTg-AD mouse model. Cell Death Dis. 9(6):685.
Grünheid T, Langenbach GE, Korfage JA, Zentner A, Van Eijden TM. 2009.
The adaptive response of jaw muscles to varying functional demands. Eur
J Orthod. 31(6):596–612.
He Z, Guo JL, McBride JD, Narasimhan S, Kim H, Changolkar L, Zhang
B, Gathagan RJ, Yue C, Dengler C. 2018. Amyloid-β plaques enhance
Alzheimer’s brain tau-seeded pathologies by facilitating neuritic plaque tau
aggregation. Nat Med. 24(1):29–38.
Hossain MI, Horie M, Yoshioka N, Kurose M, Yamamura K, Takebayashi H.
2018. Motoneuron degeneration in the trigeminal motor nucleus innervat-
ing the masseter muscle in dystonia musculorum mice. Neurochem Int.
119:159–170.
Karlsson S, Carlsson GE. 1990. Characteristics of mandibular masticatory
movement in young and elderly dentate subjects. J Dent Res. 69(2):473–
476.
Kong L, Wang X, Choe DW, Polley M, Burnett BG, Bosch-Marce M, Griffin
JW, Rich MM, Sumner CJ. 2009. Impaired synaptic vesicle release and
immaturity of neuromuscular junctions in spinal muscular atrophy mice.
J Neurosci. 29(3):842–851.
Lee P, Lee J, Kim H, Lee J, Oh S. 2020. TRPM8 mediates hyperosmotic stim-
uli-induced nociception in dental afferents. J Dent Res. 99(1):107–114.
Lewis H, Beher D, Cookson N, Oakley A, Piggott M, Morris CM, Jaros E,
Perry R, Ince P, Kenny RA, et al. 2006. Quantification of Alzheimer pathol-
ogy in ageing and dementia: age-related accumulation of amyloid-beta(42)
peptide in vascular dementia. Neuropathol Appl Neurobiol. 32(2):103–118.
Mesholam RI, Moberg PJ, Mahr RN, Doty RL. 1998. Olfaction in neurodegen-
erative disease: a meta-analysis of olfactory functioning in Alzheimer’s and
Parkinson’s diseases. Arch Neurol. 55(1):84–90.
Nakamura Y, Katakura N, Nakajima M, Liu J. 2004. Rhythm generation for
food-ingestive movements. Prog Brain Res. 143:97–103.
Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, Guillozet-Bongaarts A,
Ohno M, Disterhoft J, Van Eldik L, et al. 2006. Intraneuronal beta-amyloid
aggregates, neurodegeneration, and neuron loss in transgenic mice with five
familial Alzheimer’s disease mutations: potential factors in amyloid plaque
formation. J Neurosci. 26(40):10129–10140.
O’Keeffe ST, Kazeem H, Philpott RM, Playfer JR, Gosney M, Lye M. 1996.
Gait disturbance in Alzheimer’s disease: a clinical study. Age Ageing.
25(4):313–316.
Onozuka M, Watanabe K, Nagasaki S, Jiang Y, Ozono S, Nishiyama K,
Kawase T, Karasawa N, Nagatsu I. 2000. Impairment of spatial memory
and changes in astroglial responsiveness following loss of molar teeth in
aged SAMP8 mice. Behav Brain Res. 108(2):145–155.
Overk CR, Kelley CM, Mufson EJ. 2009. Brainstem Alzheimer’s-like pathol-
ogy in the triple transgenic mouse model of Alzheimer’s disease. Neurobiol
Dis. 35(3):415–425.
Parvizi J, Van Hoesen GW, Damasio A. 2001. The selective vulnerability of
brainstem nuclei to Alzheimer’s disease. Ann Neurol. 49(1):53–66.
Piancino MG, Tortarolo A, Polimeni A, Bramanti E, Bramanti P. 2020. Altered
mastication adversely impacts morpho-functional features of the hippocam-
pus: a systematic review on animal studies in three different experimental
conditions involving the masticatory function. PLoS One. 15(8):e0237872.
Rosen WG, Mohs RC, Davis KL. 1984. A new rating scale for Alzheimer’s
disease. Am J Psychiatry. 141(11):1356–1364.
Sakuma K, Yamaguchi A. 2018. Recent advances in pharmacological,
hormonal, and nutritional intervention for sarcopenia. Pflügers Arch.
470(3):449–460.
Siman R, Card J, Davis L. 1990. Proteolytic processing of beta-amyloid precur-
sor by calpain I. J Neurosci. 10(7):2400–2411.
Singh S, Mishra A, Mohanbhai SJ, Tiwari V, Chaturvedi RK, Khurana S,
Shukla S. 2018. Axin-2 knockdown promote mitochondrial biogenesis and
dopaminergic neurogenesis by regulating Wnt/β-catenin signaling in rat
model of Parkinson’s disease. Free Radic Biol Med. 129:73–87.
Singhrao SK, Harding A, Simmons T, Robinson S, Kesavalu L, Crean S. 2014.
Oral inflammation, tooth loss, risk factors, and association with progression
of Alzheimer’s disease. J Alzheimers Dis. 42(3):723–737.
Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA,
Katzman R. 1991. Physical basis of cognitive alterations in Alzheimer’s
disease: synapse loss is the major correlate of cognitive impairment. Ann
Neurol. 30(4):572–580.
Tousseyn T, Bajsarowicz K, Sánchez H, Gheyara A, Oehler A, Geschwind
M, DeArmond B, DeArmond SJ. 2015. Prion disease induces Alzheimer
disease—like neuropathologic changes. J Neuropathol Exp Neurol.
74(9):873–888.
Travers JB. 2015. Oromotor nuclei. In: Paxinos G, editor. The rat nervous sys-
tem. Elsevier. Chapter 11, p. 223–245.
Uematsu M, Nakamura A, Ebashi M, Hirokawa K, Takahashi R, Uchihara
T. 2018. Brainstem tau pathology in Alzheimer’s disease is character-
ized by increase of three repeat tau and independent of amyloid β. Acta
Neuropathol Commun. 6(1):1.
Yoo S-J, Lee J-H, Kim SY, Son G, Kim JY, Cho B, Yu S-W, Chang K-A,
Suh Y-H, Moon C. 2017. Differential spatial expression of peripheral olfac-
tory neuron-derived bace1 induces olfactory impairment by region-specific
accumulation of β-amyloid oligomer. Cell Death Dis. 8(8):e2977.
Yuan P, Grutzendler J. 2016. Attenuation of β-amyloid deposition and neu-
rotoxicity by chemogenetic modulation of neural activity. J Neurosci.
36(2):632–641.