1. Thioflavin-S staining in AD mice or NonTg controls 2 day and 7 days post-TBI
Traumatic brain injuries (TBIs) can generate profound
neurodegeneration leading to motor and cognitive
impairments. Tauopathies and amyloid deposits following a
TBI are reminiscent of Alzheimer's Disease (AD); indeed, TBI
is a leading risk factor for sporadic AD. A common pathogenic
feature between TBI and AD is calcium dysregulation through
the ryanodine receptor (RyR), which contributes to TBI
pathology within hours post-injury while sustained
dysregulation leads to structural, synaptic, and plasticity
deficits. Our project investigates the use of novel RyR
stabilizers that have proven highly encouraging in AD mouse
models. We administered a TBI to control and AD mice,
following which animals were treated with novel RyR-
compounds for varying time periods post-injury. Our data
indicate that stabilizing RyR2-calcium signaling after TBI
impedes injury progression and reduces Tau-histopathology.
We hypothesize that by stabilizing aberrant calcium signaling
post-TBI, we may produce immediate and long-term protective
benefits and reduce the conversion to AD.
• A CCI-TBI protocol rapidly induces tau and plaque
pathology in brains of control and AD mice
• AD mice not only show more pathology, but more
aggressive propagation of plaques across hemispheres
• Inhibiting intracellular RyR-calcium release with dantrolene
(ryanodex) or our novel derivatives may be a new
approach to reduce long-term deficits associated with TBI,
and prevent conversion to AD.
• In-vivo imaging in live animal models reveals plaque
formation occurs as early as three days post-TBI.
Treatment with Ryanodex during this period effectively
reduces overall plaque load.
1. Hefendehl J K et al. J. Neurosci. 2011;31:624-629
2. Chakroborty S, et al. (2012) Stabilizing ER Ca2+ Channel Function as an Early
Preventative Strategy for Alzheimer’s Disease. PLoS ONE 7(12): e52056.
References
Targeting intracellular calcium channels as a therapeutic approach
for traumatic brain injury and preventing conversion to AD
Nicolas Kapecki1, Bhargov Desai1, Rosalind Helfrich1, Steffanie Fisher1, Dorothy Kozlowski2, Grace Stutzmann1.1Dept. of Neuroscience, Chicago Medical School, Rosalind Franklin University,
North Chicago, IL; 2Dept. of Biological Sciences, DePaul University, Chicago, IL
Abstract
Materials & Methods
Results cont.Results Results cont.
Conclusion
Figure 2. Dense core plaques change aggregation patterns
over time: AD mice show greater propagation
TAS TBI 2D
NonTg TBI 2-day AD 2-day Sham AD TBI 2-day
NonTg TBI 7-day AD 7-day sham AD TBI 7-day
TAS Control 7D
As expected, the AD
strains of mice have
increased plaque
pathology compared to
the control non-
transgenic strain.
Pathology is further
significantly increased
when AD mice are
subjected to a TBI.
A notable finding
shown on the graph is
increased propagation
of pathology to the
contralateral site of
injury in AD mice.
0
5
10
15
20
25
30
35
%threshold
Phospho-Tau staining
NTg NTg TgCRND8 TgCRND8
Sal CK013 Sal CK013
*
Figure 4. Novel drug development for TBI: Negative allosteric
modulators targeting RyR2 reduce phospho-tau in AD mice
CK013
Increased TBI Histopathology in AD mice
Figure 1. Phospho-Tau staining is increased 3 days post -
TBI in AD brains relative to nontransgenic (NonTg) controls
Traumatic Brain Injury Model (Controlled Cortical Impact (CCI)):
Mice are deeply anesthetized with isoflurane and positioned in a
stereotaxic frame. A 3 mm craniotomy is made overlying the
hippocampus without disturbing the dura. The brain surface is
impacted using a 2.0 mm tip at 1.0 mm depth, at 3.0 m/s terminal
velocity, with 250 msec duration (Benchmark Impactor). Control sham
animals are anesthetized, placed in the stereotaxic frame, and have
the scalp opened and sutured .
Mouse Models:
TgCRND8, APP/PS1, and 3xTg-AD mouse models of Alzheimer's
disease are used, and their age-matched non-transgenic (NonTg)
controls (ages 2-4 months of age).
2-photon In Vivo Imaging of Tau Pathology
Under isoflurane anesthesia, mice are fitted with a chronic cranial
window after the CCI procedure. For imaging, mice are injected i.p.
with methoxy-XO4 (10mg/kg; dense core plaque stain) and placed in
a head stabilizing device mounted on the stage of the 2-photon
microscope. Under 2-photon laser excitation (780 nm), we image the
distribution of cortical amyloid and tau aggregates, and track
proliferation over time.
Calcium Regulation and Immunostaining:
Mice are injected ip (10mg/kg) with dantrolene 5x/week for 4 weeks.
Mice are then transcardially perfused with 4% paraformaldehyde, post
fixed for 24 hours, and 30 micron thick sections are cut, rinsed, and
prepared for standard immunohistochemistry. Primary antibodies
against phospho-tau (CP-13, Covance) and are diluted 1:500.
Conventional Immunohistochemistry protocols were used (see
Chakroborty et al., 2012)
Thioflavin-S staining against dense core plaques: Free-floating
hippocampal sections were washed with TBS (4 x 3 minutes). Then
soaked in 0.5% thioflavin S (in 50/50 ethyl alcohol/distilled water,
Sigma-Aldrich) for 10 minutes followed by 2 x 3 minute washes with
50% ethyl alcohol and again with TBS (2 x 3 minutes), mounted with
minimal drying, and coverslipped with anti-fade mounting medium
PVA-DABCO for microscopy.
Association cortex
Red: CP13 phospho-tau staining; Blue: Dapi/cell nuclei
NonTg TBI AD TBI
Dentate gyrus
Cortex
TgCRND8-Sham TBI-Saline TBI-Ryanodex
Figure 3. 30 day-treatment with Ryanodex (RyR inhibitor)
reduces tau pathology in control and TgCRND8 AD mice
subjected to TBI
The amount of ThioS plaques significantly increases (2 v. 7 day
comparison) on the side of the brain contralateral to the impact. This
phenomenon is not seen in the control transgenic strains.
Decreased Tau Pathology with RyR-Drug Therapy In Vivo 2-Photon Imaging Tracking Plaque
Pathology and Clearance with RyR-drugs
Treatment with
a calcium
channel
stabilizer
(dantrolene/
ryanodex) for
30 days after a
mild CCI-TBI
markedly
reduces the
amount of
pathological
plaques in the
dentate gyrus
(DG) of the
hippocampus.
Figure 5. In vivo 2-photon imaging of TAS-AD Mouse
Cortex
Above, we show reduced phospho-tau staining in AD mice treated with our
dantrolene-derivative, CK013 (4 weeks, 10 mg/kg, ip). These novel
derivatives were created to better cross the blood brain barrier and target
brain regions affected by AD. *=p<0.05, one way ANOVA, n=3-5 mice/group.
Sham TBI Sham TBI
NonTg TgCRND8 AD
Sal RyR Sal RyR Sal RyR Sal RyR
0
0.0209 0.6880
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
In Vivo Plaques 3-day post TBI Ryanodex
Treatment
NonTg Ryano 3-day
TAS Ryano 3-day
TAS Saline 3-day
NonTg Ryano TAS Ryano TAS Saline
TAS Saline 3-day TAS Saline 3-day TAS Saline 3-day
TAS Ryanodex 3-day NonTg Ryanodex 3-day
Above, In Vivo plaque staining is shown using 2-Photon
microscopic imaging. Quantitative analysis of random image
sampling reveals reduced plaque loads in animals treated with
Ryanodex 3 days post-TBI.
Thioflavin-S staining in AD mice or NonTg controls 2-day
and 7-day post-TBI