Mechanism(s) underlying the therapeutic effects of Withania somnifera in Alzheimer’s

disease

Tosifa Memon

11/06/2012

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Alzheimer’s Disease (AD)

• Progressive deterioration in memory, cognition, and behavior

• Pathological hallmarks– Amyloid Plaques

– Neurofibrillary tangles

• Current treatment– Symptomatic

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Withania somnifera (WS) treatment

3Sehgal et al. PNAS (2012)

LRP1 (LDLR-related protein): Endocytic scavenging receptor, transporter, and signaling moleculesLRP : Soluble form of LRP1

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How does enhanced expression of liver LRP1 reduce plaques in brain?

Sagare et al. Pharmacology & Therapeutics 2012

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)

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Question: How does WS treatment enhance liver LRP1 expression?

WS treatment

Liver LRP1 expression

+Plasma sLRP

level

Aclearance

?

How are cholesterol and insulin related to LRP1 expression?

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WS treatment

Increase in liver LRP1 expression

Low liver cholesterol

Insulin Anwer et al. Basic & Clinical Pharmacology and Toxicology (2008)

Visavadiya et al. Phytomedicine(2007)

Moon et al.Metabolism Clinical & Experimental (2011)

Tamaki et al. The American Society for Pharmacology and Experimental Therapeutics (2007)

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WS treatment

Increase in liver LRP1 mRNA and protein

expression

Increase in liver LRP1 plasma-

membrane (PM) expression

Aclearance and sLRPrelease

Low liver cholesterol

Insulin signaling

Translocation

Three know strategies to lower liver cholesterol:1. Inhibit cholesterol absorption2. Inhibit cholesterol biosynthesis3. Inhibit bile acid reabsorption

Cholesterol-lowering strategies

8Liver cell

Cholesterol

Absorbed from diet

Biosynthesis

Metabolism

Which cholesterol-lowering strategy applies to WS?

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BARI: Bile Acid Reabsorption InhibitorEzetimibe: Cholesterol Absorption InhibitorStatin: HMG CoA reductase inhibitor

Withanolides: Main active constituent of WS extract

Cholesterol Bile acid (BA)

Steroid core

Lactone

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WS treatment

Increase in liver LRP1 mRNA and protein

expression

Increase in liver LRP1 plasma-

membrane (PM) expression

Aclearance and sLRPrelease

Low liver cholesterol

Hypothetic Model: Mechanism(s) underlying

WS effects on LRP1 expression and A

clearance

Hypothesis 2

Insulin signaling

Translocation

Hypothesis 1

Experimental method

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WS treatment

LiverLRP1

expression

Aclearance

? Plasma sLRPlevel

+

WS semipurified extract

• Daily dose of 1g/kg body weight

• 75% withanolidesand 20% withanosides

• mRNA using RT-PCR

• Total-cell and PM protein using immunoblot

• Immunoblot • Plasma and brain Alevels using Enzyme-linked immunosorbent assay (ELISA)

AD mice model: APP/PS1

• Cognitive deficits by 8 months

• Middle (10-13 months) and Old-aged (22-23 months)

Aim 1: Test if WS treatment inhibits BA reabsorption

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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WS treatment

Increases liver LRP1 mRNA and

protein expression

Inhibits intestinal BA reabsorption

Lowers liver cholesterol

Objective 1.1: Test effects of WS treatment on BA and cholesterol levels

Fecal, plasma (portal vein), and liver BA levelLiver cholesterol level

Using enzymatic colorimetric assay

Liver LRP1 mRNA, and total-cell and PM protein expressionPlasma sLRP level

Plasma and brain A level14

WS extract4 - 14 days

BARI (2164U90) 4 - 14 days

APP/PS1:

Vehicle 4 - 14 days

Objective 1.1: Possible outcomes

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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

Fecal BA content

Liver cholesterol level

Liver LRP1 mRNA, and total-cell and PM protein expression

Plasma sLRP levels

Plasma and brain A levels

Objective 1.2: Test effects of WS treatment on LRP1 expression by replenishing lost BA

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WS extract7 and 14 days

BARI (2164U90) 7 and 14 days

BA infusion BA infusion

Intravenous infusion with exogenous BA [taurocholate (Tch)] at the rate of 100 nmol/min/100g

Saline infusion

Vehicle7 and 14 days

APP/PS1

Saline infusion

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

Objective 1.2: Possible outcomes

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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

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5 M [14C] Tch

Incubate with WS (5 to 100 g/ml) or BARI (30 M) or

vehicle for 30 min

Aliquots at 30, 60, 90, and 120 min

Measure radioactivitity

At the end of an experiment, collect apical

media, basolateralmedia, and cells

Passive BA transport: Measure by substituting NaCl with KCl in the media

Objective 1.3: Possible outcomes

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WS treatment may inhibit BA transport (either passive or through ASBT) in a concentration-dependent manner

BA transport via ASBT = Total transport – passive transport

If WS inhibits BA reabsorption then….

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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WS treatment

Increases liver LRP1 mRNA and

protein expression

Inhibits intestinal BA reabsorption

Lowers liver cholesterol

Aim 2: Test if WS treatment improves insulin signaling

1. Test effects of WS treatment on insulin levels and sensitivity (in vivo)

2. Test effects of WS treatment on LRP1 PM expression and Aclearance under insulin-depleted condition (in vivo)

3. Test effects of WS on insulin-dependent LRP1 PM expression and A uptake (in vitro)

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WS treatment

Increases liver LRP1 PM

expression

Improves insulin

signaling

Aclearance and sLRPrelease

• Insulin sensitivity• Plasma insulin level

Objective 2.1: Test effects of WS treatment on insulin levels and sensitivity

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

WS extract7 - 28 days

Vehicle7 - 28 days

APP/PS1 APP/PS1WT WT

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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Objective 2.2: Test effects of WS treatment on LRP1 PM expression and A clearance under insulin-depleted condition

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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

WS extract7 - 28 days

Vehicle7 - 28 days

STZ STZControl Control

APP/PS1

Objective 2.2: Possible outcomes

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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

Objective 2.3: Test effects of WS on insulin-dependent LRP1 PM expression and A uptake in vitro

Lysed liver cells and culture media will be collected to meausure:

• LRP1 mRNA, and total-cell and PM protein expression

• [125I] A uptake

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-insulin

Cultured liver cells from APP/PS1

or WT

WS (5-100 g/mL) Vehicle30 min pre-incubation:

+ insulin

-insulin

+ insulin

30 min incubation with [125I] A

Collect media and lyse cells

Measure radioactivity

Objective 2.3: Possible outcomes

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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

WT APP/PS1

Vehicle - insulin

Vehicle + insulin

WS - insulin

WS + insulinAu

pta

ke

If WS improves insulin signaling then…

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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WS treatment

Increases liver LRP1 PM

expression

Improves insulin

signaling

Aclearance and sLRPrelease

29

WS treatment

Increase in liver LRP1 mRNA and protein

expression

Increase in liver LRP1 plasma-

membrane (PM) expression

Aclearance and sLRPrelease

Low liver cholesterol

Hypothetic Model: Mechanism(s) underlying

WS effects on LRP1 expression and A

clearance

Aim 1

Aim 2

Insulin signaling

Translocation

Thank You!

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