Troy Gurney1,2, Marielle Weintraub1,3, Jordon White1,3, Laurel

Characterizing Age-Dependent Aβ Plaque Deposition and Cognitive Deficiency in
Alzheimer’s Disease 5xFAD Transgenic Mice
1,3
White ,
1,2
Gardner ,
1,2
Parnell ,
Marielle
Jordon
Laurel
Thomas
Jessica
1,2
1,2
1,2
1,2
Kelsey Paulhaus , Stephanie Turner , Amy Hardy , and Michael Chumley
of Aging Collaborative,
2Department
3
of Biology and Department of Psychology, Texas Christian University, Fort Worth, TX 76129
Abstract
Methods
Alzheimer’s disease (AD,) a neurodegenerative disorder with no known
cure or treatment, is the most prevalent form of age-related dementia. Using the
5xFAD transgenic mouse model, our lab was able to characterize age-dependent
brain amyloidosis in hippocampus tissue as well as associated cognitive
deficits. The 5xFAD mouse expresses familial Alzheimer’s disease through 5
mutations, three mutations in beta-amyloid precursor protein (APP) and two in
presenilin-1 (PS1). This results in accelerated Alzheimer’s-like pathogenesis
including amyloid-beta (Aβ) plaque accumulation around 2 months of age and
hippocampus synaptic dysfunction around 4 months of age. An established
contextual fear conditioning protocol was used to demonstrate hippocampus
impairment in the 5xFAD mice at ages of 3, 6, and 9 months. Our protocol
revealed no significant difference in contextual memory in the 3- and 6-month
old groups, but showed a marked difference between transgenic positive
(5xFAD) and negative (control) mice at 9 months of age. Additionally, using a
radial arm water maze protocol, we were able to show age-dependent working
memory impairment. Brains were removed by perfusion, sliced by vibratome,
and stained for Aβ using thioflavin-S following these paradigms. Through this
study, it was shown that as the 5xFAD mice age, increased extracellular Aβ
plaque deposition occurs, which is thought to result in behavioral changes and
memory loss.
Contextual Fear Conditioning: Animals were placed in a the CFC paradigm
which included a context chamber containing polka dot walls and a
peppermint scent. On training day, animals were subjected to a 180s
acclimation period followed by a 2s stimulus, followed by a 1 minute interval
period, and another 2s stimulus. On testing day, animals entered the same
context chamber and percent freezing time was monitored and recorded for 3
min.
Radial Arm Water Maze: Animals were placed in a circular pool containing an
8-armed metal apparatus. Animals were given 60s to locate their specific goal
arm containing an escape platform. On training day (blocks 1-5), the
platform alternated between visible and hidden, for each trial. On testing day
(blocks 6-10), the platform was hidden throughout all 15 trials. Errors, which
included entering the wrong arm or pausing >15s, and time to reach the goal
arm were recorded. Triangulation to the goal arm was guided by multiple
extra-maze cues.
Thioflavin-S Staining: Post-vibratome hippocampal brain sections (40μm) were
stained for the presence of Aβ extracellular plaques.
3 Month Old
Results
Introduction
% Time Freezing
•  In the 5xFAD model, these mutations result in rapid accumulation and
deposition of extracellular amyloid beta plaques beginning around 2 months
of age (1).
5xFAD+
*
30
20
•  As hypothesized, FAD+ animals, 3 months of age, did not appear to have
learning deficits as compared to age matched control animals.
3
6
9
3
6
Testing
60
40
*
4
Errors
50
•  However, although expected to have congitive defcitis, 6 month FAD+
animals did not display learning deficit during CFC testing.
9
Figure 1. 9mo 5xFAD+ mice displayed cognitive deficits in CFC. There were no significant differences
between any groups during training day of CFC. Interestingly, during testing, CFC freeszing behavior
was significantly impaired in 9mo 5xFAD+ mice, indicative of cognitive deficits. *= p<0.05; bars
represent ±SEM,
6
5xFAD6
NEG
2
*
4
5xFAD+
POS
*
*
*
2
NEG
POS
Blocks
0
9
10
10
8
6 9
10
7
5 8
4 7
3 6
2 5
0
•  Through this study we were able to characterize the 5xFAD transgenic model
of AD. Using two hippocampus dependent tasks and Aβ plaque staining, we
were able to show age-dependent cognitive deficits and pathology.
References
1 4
0
3
20
•  Aβ plaque formation was detected, starting at 3 months, in the cortex of
5xFAD+ mice. Aβ plaque formations increased with age, and as
demonstrated in the 6, 9, and 12 month images, were detected in both the
cortex and hippocampus.
*
30
2
•  Age-dependent extracellular Aß plaque accumulation in 5xFAD hippocampal
tissue was visualized by Thioflavin-S staining.
70
Figure 3. In the 3 month old 5xFAD+ mice, Aβ plaque formation (inset - green arrow) can be detected
in the cortex. In 6-12 month old 5xFAD+ mice, Aβ plaque formation can be detected in both the cortex
and the hippocampus. Thioflavin S (green) detects Aβ aggregates. TO-PRO 3 (blue) labels nuclei.
•  Animal 9 mos of age displayed sugnfict learning deficits in the hippocampusdependent tasks, contextual fear conditiong. (Fig. 1) and radial arm maze
(Fig. 2).
40
Training
1
% Time Freezing
•  In this study, we attempted to replicate existing data and characterize the agedependent cognitive decline seen in the 5xFAD model utilizing both the
contextual fear conditioning and the radial arm water maze paradigms.
50
0
Errors
•  Memory impairment and behavioral changes have been described by
numerous studies (1-3), and occur as early as 4 months of age in 5xFAD
mice (1).
12 Month Old
•  We were interested in exploring the age dependent AD pathology and
associated hippocampus-dependent cognitive deficits.
10
•  Through synaptic dysfunctions, plaque deposition is believed to result in
cognitive deficits associated with contextual and working memory (1).
9 Month Old
•  5xFAD exhibit accelerated accumulation of Aβ around 2 months of age
5xFAD-
60
•  The transgenic 5xFAD mouse models familial Alzheimer’s through
expression of 5 mutations, 3 in the beta-amyloid precursor protein (APP) and
2 in presenilin-1 (PS1).
6 Month Old
Conclusion
70
•  Alzheimer’s disease is the most prevalent neurodegenerative disorder causing
age-related dementia.
1,2
Mussato ,
WildType
1 Neurobiology
1,3
Weintraub ,
5xFAD
Troy
1,2
Gurney ,
Blocks
Figure 2.Training
9mo 5xFAD+ mice displayed cognitiveTesting
deficits in Radial Arm Water Maze. Each Block consists
of the number of errors averaged over 3 trials, therefore, 30 trials total per mouse. 5xFAD+ represents
transgenic positive 5xFAD mice, while 5xFAD- represents non-transgenic control mice. Number of
errors, which are representative of cognitive deficits, remained significantly higher in 9mo 5xFAD+ mice.
*= p<0.05; bars represent ±SEM,
1.  Jawhar, S., (2011). The 5XFAD mouse model (Ph.D.), Georg August University.
2.  Ohno, M. (2009). Neurobiol Learn Mem, 92: p. 455-459
3.  Kimura, R. & Ohno, M. (2009). Neobiol Dis., 33: p. 229-235