To learn more and watch the webinar on demand, go to: https://insidescientific.com/webinar/eeg-monitoring-approaches-to-predict-learning-and-memory-changes-in-early-alzheimers-disease/ A major goal of Alzheimer’s disease research is to find factors that may be predictive of later cognitive decline. Environmental factors such as dietary deficiency or exposure to toxins may have little detectable impact in young and middle-aged brains yet still accelerate disease pathways to contribute to earlier onset of neurodegenerative disease over time. Understanding the mechanisms involved could lead to new preventative strategies to slow the onset of cognitive decline or may suggest new pathways for pharmacological interventions in later life. During this webinar, Dr. Harrison discusses how even seemingly mild changes in neural signaling can be detected through altered EEG activity and correlated with poorer performance on tasks of learning and memory. In addition, she demonstrates how vitamin C deficiency and exposure to toxins can impact glutamate uptake and clearance. Using mouse models of beta-amyloid aggregation and wild-type controls at different ages, the Harrison lab uses molecular biology, behavioral and electrophysical techniques to understand triggers for onset of cognitive decline even in the absence of high levels of classical Alzheimer’s disease neuropathology. Key Topics Include: - Demonstrate that small changes in neural activity are correlated with significant changes in behavior - Demonstrate the utility of using long-term measurement of EEG in the home-cage rather than smaller snapshots of neural activity - Demonstrate how different factors of the exposome – diet and environmental toxins – can impact onset of Alzheimer’s disease and cognitive decline

1. EEG Monitoring Approaches to
Predict Learning and Memory
Changes in Early Alzheimer’s Disease
Fiona Harrison, PhD
Associate Professor
Department of Medicine
Vanderbilt University Medical Center

2. During this webinar, Dr. Harrison discusses how even seemingly mild
changes in neural signaling can be detected through altered EEG
activity and correlated with poorer performance on tasks of learning
and memory. In addition, she demonstrates how vitamin C deficiency
and exposure to toxins can impact glutamate uptake and clearance.
EEG Monitoring Approaches to
Predict Learning and Memory
Changes in Early Alzheimer’s Disease

3. EEG monitoring approaches
to predict learning and
memory changes in early
Alzheimer’s disease
Fiona E Harrison, Ph.D.
Associate Professor, Medicine
Vanderbilt University Medical Center

4. Alzheimer’s
disease
Defining Pathology of Alzheimer’s Disease
How important are beta-amyloid plaques in cognitive decline?

5. Rodrigue 2012 PMID: 22302550
Lack of consistent correlation between 𝝱-amyloid neuropathology and cognition in humans

6. Aβ 1-16 (33.1.1) antibody
12-month-old
9-month-old
CRND8 mice
3-month-old
Hanna 2012 PMID: 22697412 Cacucci 2008 PMID: 18505838
Tg2576 mice
16-month-old
Congo Red
Stronger correlation between 𝝱-amyloid neuropathology and cognition in mice

7. Multiple contributing factors to cognitive decline
Exercise
Education and Career
Lifetime nutrition
?
?
?
Cognitive decline
Basic pathology
Environmental toxins
Health history
Sleep

8. Multiple contributing factors to cognitive decline
Neuroinflammation
Microglia
Astrocytes
Oxidative damage
Mitochondrial dysfunction
DNA damage and repair
Vascular changes
Metabolic dysregulation
?
?
?
Cognitive decline
Basic pathology
Neurotransmitter changes
Cholinergic system
Dopaminergic system
Glutamate & GABAergic systems

9. Increased hyperexcitability, epilepsy and seizures in Alzheimer’s disease
• Patients with MCI or AD with epilepsy or subclinical epileptiform activity presented earlier onset
of cognitive decline (5-8 years compared to non-epileptic controls).
Epileptiform EEG activity observed in about 41% of non AD MCI patients
More than 50% of seizures with MCI are non convulsive.
Vossel 2013 PMID: 23835471
• Clinically silent hippocampal seizures and epileptiform spikes during sleep in AD patients with no
history of seizures
Lam 2017 PMID: 28459436
Early AD pathology
Neuronal hyperactivation
Increased epileptic activity
Faster accumulation of pathology
More rapid cognitive decline

10. Increased hyperexcitability, epilepsy
and seizures in Alzheimer’s disease
mouse models
Mice overexpressing FAD-mutant genes have
spontaneous epileptiform discharges (“spikes”)
and/or seizures detectable by EEG recordings
Palop & Mucke PMID: 27829687

11. APP/PS1 4-9 months had higher incidence of epileptiform-like discharges
Seizure events - interictal spikes, sharp waves, or polyspikes
Reyes-Marin and Nunez 2017 PMID: 28963050
Interictal spikes during rapid-eye movement (REM) sleep in 5-week-old Tg2576
Kam 2016 PMID: 26818394
5 weeks old 7 months old
Increased hyperexcitability, epilepsy
and seizures in Alzheimer’s disease
mouse models

12. Hypothesis – Hyperexcitability is sufficient to induce
cognitive decline in the presence of early AD pathology
Experimental design:
Kainic acid – analog of excitatory amino acid neurotransmitter glutamate
Binds kainate receptors leading to excitatory postsynaptic potentials (EPSPs)
High concentrations (20-40mg/kg) induce seizures overstimulate neurons to death
Wild-type
APP/PSEN1
Kainic acid
Sub-acute or chronic
Low dose (5-10 mg/kg)
Long term potentiation (LTP)
Learning and Memory
EEG
3-6 months
Kainic acid

13. Sub-acute kainic acid dosing disrupts long term potentiation in 3-month-old APP/PSEN1 mice
Wilcox et al Under Review NBD
Wild-type
APP/PSEN1
Saline Kainic acid
Data generated by Dr. William Nobis Lab

14. 0 15 30 45 60 75
0
50
100
150
200
250
LTP SC-CA1
Time (Mins)
%Baseline
slope
(mV/ms)
TBS
Wild-type
APP/PSEN1
Saline Kainic acid
Sub-acute kainic acid dosing disrupts long term potentiation in 3-month-old APP/PSEN1 mice
Wilcox et al Under Review NBD Data generated by Dr. William Nobis Lab

15. 0 5 10 15
0
50
100
# Kainic acid (10 mg/kg) injections
Percent
survival
Wild-type Saline
Wild-type KA
APP/PSEN1 Saline
APP/PSEN1 KA
Chronic kainic acid dosing increases mortality 5-month-old APP/PSEN1 mice
Day 1 Day 2 Day 3 Day 4 Day 5
0
10
20
30
Escape
latency
(s)
0
10
20
30
40
Time
in
quadrant
(s)
*** ***
***
*
***
***
***
***
* ***
**
***
a a a a
Saline KA
Wild-type
Saline KA
APP/PSEN1
Wild-type Saline APP/PSEN1 KA
Morris water maze
Hidden platform acquisition
Wilcox et al Under Review NBD

16. Day 1 Day 2 Day 3
0
10
20
30
40
Escape
latency
(s)
0
10
20
30
Time
in
quadrant
(s)
*** **
*
*
b b b
Saline KA
Wild-type
Saline KA
APP/PSEN1
Previous Target quadrant
Target quadrant
Non-Target quadrant
Wild-type APP/PSEN1
Saline
Kainic
acid
Morris water maze - Reversal Learning
Wilcox et al Under Review NBD
Chronic kainic acid dosing impairs memory retention in complex tasks

17. 0 5 10 15 20
0
2
4
6
8
10
Bouts
of
Low
Mobility
* *
0
50
100
150
Total
bouts
of
low
mobility
***
**
Saline KA
Wild-type
Saline KA
APP/PSEN1
*
APP/PSEN1 mice are more sensitive to kainic acid induced immobility
Force plate actimetry
Wild-type Saline
Wild-type KA
APP/PSEN1 Saline
APP/PSEN1 KA
Wilcox et al Under Review NBD

18. 3x3mm
1x1mm
Tail extension
Rigid posture
Wilcox et al Under Review NBD
APP/PSEN1 mice are more sensitive to kainic acid induced EEG abnormalities

19. 0
400
800
1200
Spikes
Saline Kainic acid
Wk 1 Wk 4 Wk 1 Wk 4
0
20
40
60
Spike
trains
Saline Kainic acid
Wk 1 Wk 4 Wk 1 Wk 4
Wild-type Saline
Wild-type KA
APP/PSEN1 Saline
APP/PSEN1 KA
Wilcox et al Under Review NBD
APP/PSEN1 mice are more sensitive to kainic acid induced EEG abnormalities

20. Normal EEG
EEG Frequency
Wave Dominance
Association
Delta (<4 Hz) Deep Sleep
Theta (4-8 Hz) Sleep, relaxation
Alpha (8-12 Hz) Resting wakefulness
Beta (16-25 Hz) Active wake & sedation
Gamma (25-50 Hz) Complex tasks

21. APP/PSEN1 mice have altered wave forms
0
25
50
75
100
Time
(%
of
2hr)
Wake
NREM
REM
Saline KA Saline KA
Wild-type APP/PSEN1
50
75
100
Time
(%
of
2hr)
Wake
NREM
REM
https://support.datasci.com/
Wilcox et al Under Review NBD

22. REM Sleep
Non-REM Sleep
Wake
APP/PSEN1 mice have altered wave forms
Wilcox et al Under Review NBD

23. B
a
s
e
l
i
n
e
C
u
e
B
a
s
e
l
i
n
e
C
u
e
B
a
s
e
l
i
n
e
C
u
e
B
a
s
e
l
i
n
e
C
u
e
0
30
60
90
120
Time
freezing
(s)
a a b
B
aseline
C
ontext
B
aseline
C
ontext
B
aseline
C
ontext
B
aseline
C
ontext
0
50
100
150
200
250
Time
freezing
(s)
a a a b
Chronic kainic acid dosing impairs memory retention in complex tasks
Training, day 1 Context test, day 2 Cue test, day 2
n.s.
Wilcox et al Under Review NBD

24. Abdul 2009 PMID: 19828810 Garcia-Esparcia 2018 PMID: 29755340
Alzheimer’s disease is associated with altered glutamate transport

25. 0
1
2
3
Protein
normalized
to
WT
Sal
Saline KA
Wild-type
Saline KA
APP/PSEN1
Coomassie – total protein
GLT-1
62 kDa
GLAST
60 kDA
GFAP
51 kDA
Decreased GLT-1 in APP/PSEN1 cortex exacerbated by chronic kainic acid
Wilcox et al Under Review NBD
0.0
0.5
1.0
1.5
Protein
normalized
to
WT
Sal
Saline KA
Wild-type
Saline KA
APP/PSEN1
**
+++
+++
0.0
0.5
1.0
1.5
Protein
normalized
to
WT
Sal
Saline KA
Wild-type
Saline KA
APP/PSEN1

26. Dietary factors
Traumatic brain injury
Flying foxes
BMAA
Blue Mussels
Domoic acid
Air Pollution
Manganese
Acute illness
Hypothesis – Environmental triggers of glutamate
dysfunction will accelerate cognitive decline

27. Hypothesis – Environmental triggers of glutamate
dysfunction will accelerate cognitive decline
Ascorbic acid / Vitamin C
C6H8O6
RDI 45-120 mg/d
repletion 300-500 mg/d
Levine 1996 PMID:8623000
Depletion <28 uM
Deficient <10 uM

28. Wilson 2000 PMID: 10700597
Astrocyte Ascorbate-Glutamate Exchange

29. Faster latency to ‘head bob’ Brain Vitamin C decreased by 50%
Mi 2018; PMID: 30172223
Vitamin C depletion increases sensitivity to 10 mg/kg kainic acid in gulo-/- mice

30. Warner et al PMID: 25616451
Vitamin C depletion increases epileptiform abnormalities in SVCT2+/- APP/PSEN1 mice
Kainic acid, 10mg/kg
1 hour post injection
Tethered EEG system
Wild-type
APP/PSEN1
Neuron Vitamin C
decreased by ~ 20%

31. Hypothesis: Chronic vitamin C depletion will increase epileptiform
discharges in APP/PSEN1 mice
High Ascorbic acid
1.0 g/L
4.5 months
19 months
Low Ascorbic acid
0.03 g/L
Weeks
3x3mm
1x1mm
Surgery EEG
0 1 2 3 4 5 6
Gulo-/-
Wild-type
Gulo-/-
APP/PSEN1
High Vitamin C Low Vitamin C
Within-subjects design

32. Conclusions
• Sub-clinical and non-convulsive seizure activity are an early
part of AD pathophysiology
• EEG markers may be a useful early predictive of later
cognitive decline
• Diet and environmental exposures may contribute to
cumulative damage throughout life
• Focus on early neuropathological changes may provide better
options for intervention

33. 33
FUNDING
R01 ES031401 & AG038739 to FEH
I01 CX001610 to James May, M.D.
HARRISON LAB
Brittany Spitznagel, Ph.D.
Jordyn Wilcox, Ph.D.
David Consoli
Rebecca Buchanan
Amanda Marino
Adriana Tienda
Shilpy Dixit Ph.D.
Deborah Mi M.D.
VANDERBILT MOUSE
NEUROBEHAVIOR CORE
John Allison
Krista Paffenroth @VitaminSeer
@WoManganese
@DavidCConsoli
@Becca_Buchanan
@LabNobis
NOBIS LAB
William P Nobis, M.D., Ph.D.
Benjamin Owen, Ph.D.

34. Thanks for participating!
Before you go…
• To learn more and watch the webinar, go to:
insidescientific.com
• Interested in learning more about DSI’s solutions for
Alzheimer’s research? Visit: www.datasci.com