I am Tosifa Memon, currently a phD candidate in Biology department at the University of Utah. I worked on this proposal for my qualifying exam and got the opportunity to present it to my thesis committee. Abstract: The root extract of Withania somnifera (WS), traditionally known to enhance memory, has been reported to improve cognitive and behavioral deficits in mice models of Alzheimer’s disease (AD). WS treatment was also found effective in reducing brain depositions and promoting peripheral clearance of toxic amyloid- (A) peptides which can otherwise interfere with normal neuronal function. How WS treatment exerts these beneficial effects remains unexplored and demands further investigation. Interestingly, Sehgal et al. study also found that WS treatment enhanced expression of liver low density lipoprotein-related protein (LRP1) along with plasma soluble form of LRP (sLRP), key players in peripheral Aβ clearance. Conversely, downregulation of liver LRP1 abrogated the therapeutic effects of WS extract and reduced plasma sLRP level. This finding not only highlights LRP1 as a potential target in AD treatment but also hints at WS components as potential leads for drug discovery. Therefore, it is imperative to know how treatment with WS root extract induces liver LRP1 expression. WS extract possesses hypocholesteremic and hypoglycemic activity. This may offer some explanation to therapeutic effects of WS treatment as abnormal cholesterol metabolism and insulin-resistance are known risk-factors in AD onset and progression. Like WS extract, cholesterol and insulin can also regulate LRP1 expression. Depletion in cell cholesterol, most likely by inhibiting bile acid (BA) reabsorption, can upregulate LRP1 mRNA expression and sLRP release while insulin stimulation promotes translocation of LRP1 from cytosol to plasma membrane (PM). Therefore, it is possible that WS induces LRP1 mRNA expression and sLRP release by inhibiting intestinal BA reabsorption and consequent depletion in liver cell cholesterol. In addition, effective peripheral Aclearance by WS will depend on PM expression of liver LRP1 more than its mRNA expression. Hence, it is possible that WS treatment promotes LRP1 translocation to PM by improving insulin signaling and sensitivity. Aim 1: To determine whether treatment with WS inhibits intestinal BA reabsorption leading to reduced cell cholesterol and increased LRP1 expression in liver cells Aim 2: To determine whether treatment with WS improves insulin sensitivity and signalling leading to enhanced liver LRP1 PM expression and A uptake from plasma.

1. Mechanism(s) underlying the therapeutic
effects of Withania somnifera in Alzheimer’s
disease
Tosifa Memon
11/06/2012
1

2. Alzheimer’s Disease (AD)
• Progressive deterioration in memory, cognition,
and behavior
• Pathological hallmarks
– Amyloid Plaques
– Neurofibrillary tangles
• Current treatment
– Symptomatic
2

3. Withania somnifera (WS) treatment
LRP1 (LDLR-related protein): Endocytic scavenging receptor, transporter,
and signaling molecule
sLRP : Soluble form of LRP1
3
Sehgal et al. PNAS (2012)

4. How does enhanced expression of liver LRP1 reduce
plaques in brain?
Liver LRP1 & plasma sLRP promote
A clearance from blood
Shift in equilibrium of A level
between brain & blood
Efflux of A from brain to blood via
LRP1 at blood-brain-barrier (BBB)
4
Sagare et al. Pharmacology & Therapeutics 2012

5. Liver LRP1
WS expression A
treatment + clearance
? Plasma sLRP
level
Question: How does WS treatment enhance
liver LRP1 expression?
5

6. How are cholesterol and insulin related to LRP1
expression?
Visavadiya et al. Moon et al.
Phytomedicine Metabolism Clinical &
(2007) Experimental (2011)
Low liver
cholesterol
Increase in
WS
liver LRP1
treatment
expression
Insulin
Anwer et al. Basic Tamaki et al. The
& Clinical American Society
Pharmacology for Pharmacology
and Toxicology and Experimental
(2008) Therapeutics (2007)
6

7. WS
treatment
Three know strategies to lower liver cholesterol:
1. Inhibit cholesterol absorption
2. Inhibit cholesterol biosynthesis
Low liver 3. Inhibit bile acid reabsorption
cholesterol
Insulin
signaling Increase in liver A
Increase in liver LRP1
LRP1 plasma- clearance
mRNA and protein
Translocation membrane (PM) and sLRP
expression
expression release
7

8. Cholesterol-lowering strategies
Absorbed
from diet
Biosynthesis
Cholesterol
Metabolism
Liver cell 8

9. Which cholesterol-lowering strategy
applies to WS?
BARI: Bile Acid Reabsorption Inhibitor
Ezetimibe: Cholesterol Absorption Inhibitor
Statin: HMG CoA reductase inhibitor 9

10. Withanolides: Main active constituent of WS extract
Lactone
Steroid core
Cholesterol Bile acid (BA)
10

11. Hypothetic Model:
Hypothesis 1 Mechanism(s) underlying
WS WS effects on LRP1
treatment expression and A
clearance
Hypothesis 2
Low liver
cholesterol
Insulin
signaling Increase in liver A
Increase in liver LRP1
LRP1 plasma- clearance
mRNA and protein
Translocation membrane (PM) and sLRP
expression
expression release
11

12. Experimental method
WS ? Liver Plasma A
treatment LRP1 + sLRP clearance
expression level
WS semipurified • mRNA using • Immunoblot • Plasma and
extract RT-PCR brain A
• Daily dose of • Total-cell and levels using
1g/kg body PM protein Enzyme-
weight using linked
• 75% immunoblot immunoso
withanolides rbent assay
and 20% (ELISA)
AD mice model: APP/PS1
withanosides
• Cognitive deficits by 8 months
• Middle (10-13 months) and Old-aged (22-23 months)
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13. Aim 1: Test if WS treatment inhibits BA reabsorption
Increases
Inhibits liver LRP1
WS intestinal BA Lowers liver mRNA and
treatment reabsorption cholesterol protein
expression
1. Test effects of WS treatment on BA and cholesterol levels
(in vivo)
2. Test effects of WS treatment on LRP1 expression by
replenishing lost BA (in vivo)
3. Test effects of WS on BA transport/reabsorption (in vitro)
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14. Objective 1.1: Test effects of WS treatment on BA and
cholesterol levels
Vehicle WS extract BARI (2164U90)
4 - 14 days 4 - 14 days 4 - 14 days
APP/PS1:
Fecal, plasma (portal vein), and liver BA level
Liver cholesterol level
Using enzymatic colorimetric assay
Liver LRP1 mRNA, and total-cell and PM protein expression
Plasma sLRP level
Plasma and brain A level
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15. Objective 1.1: Possible outcomes
If WS treatment inhibits BA reabsorption then:
• Fecal BA content will increase
– Will correlate with increase in liver LRP1 mRNA and total cell-protein expression
• Plasma BA level will decrease
• Liver cholesterol level will decrease
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16. Objective 1.2: Test effects of WS treatment on LRP1
expression by replenishing lost BA
WS extract Vehicle BARI (2164U90)
7 and 14 days 7 and 14 days 7 and 14 days
APP/PS1
Saline infusion BA infusion Saline infusion BA infusion
Intravenous infusion with exogenous BA [taurocholate (Tch)] at the rate of 100
nmol/min/100g
Fecal BA content
Liver cholesterol level
Liver LRP1 mRNA, and total-cell and PM protein expression
Plasma sLRP levels
Plasma and brain A levels
16

17. Objective 1.2: Possible outcomes
If WS inhibits BA reabsorption then exogenous BA (Tch):
• Will abolish effects of WS on liver LRP1 expression, plasma sLRP level, and A
clearance
• Will not lower liver cholesterol
• Will not affect fecal BA content
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18. Objective 1.3: Test effects of WS on BA
transport/reabsorption in vitro
BA transporter: Ileal apical sodium-dependent BA transporter (ASBT, aka
IBAT)
Caco-2 (human colonic adenocarcinoma cell-line): Well-characterized for
intestinal proteins and transporters including ASBT
Passive BA transport: Measure by substituting NaCl with KCl in the media
Incubate with WS (5 to 100 5 M [14C] Aliquots at 30, 60, Measure
g/ml) or BARI (30 M) or Tch 90, and 120 min radioactivitity
vehicle for 30 min
At the end of an
experiment,
collect apical
media, basolateral
media, and cells
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19. Objective 1.3: Possible outcomes
WS treatment may inhibit BA transport (either passive or
through ASBT) in a concentration-dependent manner
BA transport via ASBT = Total transport – passive transport
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20. If WS inhibits BA reabsorption then….
Increases
Inhibits liver LRP1
WS intestinal BA Lowers liver mRNA and
treatment reabsorption cholesterol protein
expression
1. WS treatment, like BARI, will increase fecal BA content
– Will correlate with increase in LRP1 mRNA expression
2. Exogenous BA infusion will abolish the effects of WS
treatment on LRP1 expression
3. WS treatment, like BARI, will inhibit BA transport across
caco-2 cells
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21. Aim 2: Test if WS treatment improves insulin signaling
Increases liver A
WS Improves clearance
insulin LRP1 PM
treatment expression and sLRP
signaling release
• Insulin sensitivity
• Plasma insulin level
1. Test effects of WS treatment on insulin levels and sensitivity
(in vivo)
2. Test effects of WS treatment on LRP1 PM expression and A
clearance under insulin-depleted condition (in vivo)
3. Test effects of WS on insulin-dependent LRP1 PM expression
and A uptake (in vitro)
21

22. Objective 2.1: Test effects of WS treatment on insulin levels and
sensitivity
Vehicle WS extract
7 - 28 days 7 - 28 days
WT APP/PS1 WT APP/PS1
Oral glucose tolerance test (OGTT): A measure of insulin sensitivity in vivo
Fasted mice will be fed glucose solution followed blood collection at 0 (fasting), 15,
30, 60, and 120 min
Plasma insulin level: Using ELISA
Liver LRP1 mRNA, and total-cell and PM protein expression
Plasma sLRP level
Plasma and brain A level 22

23. Objective 2.1: Possible outcomes
Improved insulin signaling during WS treatment:
• Will correlate with increase in A clearance
• Will correlate with increase in liver LRP1 PM protein expression
• Will not correlate with increase in liver LRP1 mRNA and total-cell protein
expression
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24. Objective 2.2: Test effects of WS treatment on LRP1 PM
expression and A clearance under insulin-depleted condition
Vehicle WS extract
7 - 28 days 7 - 28 days
APP/PS1
Control STZ Control STZ
Insulin depletion using streptozotocin (STZ)
Blood glucose level >300 mg/dl will be used for WS treatment in STZ group
Liver LRP1 mRNA, and total-cell and PM protein expression
Plasma sLRP level
Plasma and brain A level 24

25. Objective 2.2: Possible outcomes
Insulin-depletion during WS treatment:
• Will abolish effects of WS on liver LRP1 PM protein expression, A
clearance, and sLRP release
• Will not affect liver LRP1 mRNA and total-cell protein expression
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26. Objective 2.3: Test effects of WS on insulin-dependent LRP1 PM
expression and A uptake in vitro
30 min pre- Vehicle WS (5-100 g/mL)
incubation:
Cultured liver cells
- + - +
from APP/PS1 insulin insulin insulin insulin
or WT
Lysed liver cells and culture media will be collected to meausure:
• LRP1 mRNA, and total-cell and PM protein expression
• [125I] A uptake
30 min incubation Collect media Measure
with [125I] A and lyse cells radioactivity
26

27. Objective 2.3: Possible outcomes
WS in cultured liver cells of APP/PS1:
• Will increase insulin-dependent LRP1 PM expression
• Will increase A uptake
• Will not increase LRP1 mRNA and total-cell protein expression
Vehicle - insulin
A uptake
Vehicle + insulin
WS - insulin
WS + insulin
WT APP/PS1 27

28. If WS improves insulin signaling then…
Increases liver A
WS Improves clearance
insulin LRP1 PM
treatment expression and sLRP
signaling release
1. WS treatment will improve glucose tolerance and insulin
sensitivity
– Will correlate with increase in LRP1 PM expression
2. Insulin-depletion during WS treatment will abolish the
effects of WS on LRP1 PM expression and A clearance
3. WS treatment will improve insulin-dependent LRP1 PM
expression and A uptake
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29. Hypothetic Model:
Mechanism(s) underlying
Aim 1 WS WS effects on LRP1
treatment expression and A
clearance
Aim 2
Low liver
cholesterol
Insulin
signaling Increase in liver A
Increase in liver LRP1
LRP1 plasma- clearance
mRNA and protein
Translocation membrane (PM) and sLRP
expression
expression release
29

30. Thank You!
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