8. Alzheimer 3

Question 1

reasons why cholesterol and vascular dysfunction is linked to AD (8)

Answer 1

1. high serum LDL during midlife
2. ApoE4, Trem2, ApoJ, ABCA7
3. atherosclerosis
4. T2 diabetes
5. smoking
6. hypertension
7. hyperlipidemia
8. obesity

Question 2

2 genetic variants that are protective against AD

Answer 2

1. ApoE2 variant is neutral and associated with longevity of AD and protects from cognitive decline
2. APP mutation statistically protects from AD

Question 3

Effect of presenilin mutation

Answer 3

usually gives early onset AD

Question 4

Describe effect of presenilin mutation with 2 ApoE3 ChC mutations

Answer 4

• lots of plaques and less tau but cognitively healthy
• therefore, ChC mutation overrides AD pathology from the presenilin mutation

Question 5

why did we look at centenarians for AD?

Answer 5

there is association of CETP variants with longevity, cognitive function, and memory in centenarians

Question 6

lipoprotein plasma profile associated with longevity and preservation of cognitive function

Answer 6

healthy people with high HDL and almost no LDL had mutation where CETP was not spliced –> modulated age-related cognitive function

Question 7

correlation of CETP genotype with AD/memory

Answer 7

CETP modifies the effect of memory decline with at least 1 allele of ApoE4

Question 8

describe the memory performance of different ApoE4 and CETP variant combinations

Answer 8

ApoE4 carriers with WT CETP or heterozygous CETP mutation declined FASTER

ApoE4 carriers with homozygous CETP mutation had normal memory performance –> therefore, CETP variant overrides negative effects

Question 9

is CETP protective in early or late AD?

Answer 9

CETP is protective in early cognitive decline in ApoE4, not protective in late stages

Question 10

why is CETP studied extensively?

Answer 10

CETP is implicated in CVD and people want to study CVD

Question 11

What is CETP?

Answer 11

Serum glycoprotein secreted by the liver

Question 12

What is the role of CETP?

Answer 12

Carries CE from HDL –> LDL
Carries TAG from LDL –> LDL

Pro-atherosclerotic bc increases LDL

Question 13

difficulty in studying CETP in mice? why does this cause problems in AD studies?

Answer 13

mice don’t express CETP and have very low VLDL and LDL

problem in many AD studies bc they ignored the fact that mice have very low LDL

Question 14

2 hypotheses about CETP and AD and 2 research questions

Answer 14

1. CETP activity contributes to AD
2. CETP inhibition may delay or prevent AD onset in ApoE4 carriers
3. How does CETP affect cholesterol distribution in a mouse?
4. Does CETP inhibition rescue AD pathology?

Question 15

what is the first step in investigating CETP in AD?

Answer 15

confirm a cholesterol diet induces CETP

Question 16

experiment: confirm cholesterol diet induces CETP

Answer 16

compare humanCETP transgenic mice to WT controls

2 months old –> fed high cholesterol diet
5 months old:
- control diet had inactive CETP in liver and baseline activity in plasma
- high cholesterol diet had active CETP in liver and increased activity in plasma

Question 17

once it was confirmed that cholesterol diet induces CETP, what is the next step?

Answer 17

look at lipoprotein profiles in hCETP transgenic mice

Question 18

results of experiment: lipoprotein profiles in hCETP transgenic mice

Answer 18

• HDL levels decrease a little bit with CETP on both diets
• LDL levels increase A LOT with CETP on high cholesterol diet

Therefore, hCETP transgenic mice have humanized lipoprotein profile

Question 19

what is MALDI-IMS? how does it work (4 steps)?

Answer 19

Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spec

1. section brain
2. put sections on a grid with a matrix for MS
3. ionize with a laser beam and get masses of specific molecules
4. look at MS to see how specific mass is distributed across the brain

Question 20

what is the purpose of the matrix in MALDI-IMS?

Answer 20

protects organic tissue from laser beam

Question 21

what are the 2 ways we can confirm that CETP increases cholesterol?

Answer 21

1. MALDI-IMS of brain lipids
2. stain brain with filipin

Question 22

results of MALDI-IMS of brain lipids

Answer 22

CETP mice on both diets have more cholesterol –> therefore CETP increases cholesterol in the brain

Question 23

what is filipin?

Answer 23

binds cholesterol and stains it blue

Question 24

results of filipin stain

Answer 24

More cholesterol staining in CETP transgenic mice, more with high cholesterol diet

Question 25

CETP and inflammation levels in periphery vs brain

Answer 25

high CETP and inflammation in liver high CETP and less inflammation in brain

Question 26

why do we look at integrity of BBB? how does this work?

Answer 26

to ensure that the CETP models on both diets can maintain the integrity of BBB Inject Evans Blue into plasma (v large molecule) it will stain all tissue except for the brain bc of BBB --> if brain is blue, there is breakdown of BBB

Question 27

describe drug repurposing with CETP inhibitors

Answer 27

CETP inhibitors have been developed for CVD so now we can test for AD

Question 28

4 benefits of drug repurposing

Answer 28

1. toxicity testing in humans already done 2. faster progression through clinical trials 3. saves lots of time and money 4. adverse effects/side effects/tolerability in humans is likely known

Question 29

why should we test whether CETP inhibitors can enter the brain?

Answer 29

because AD is a disease of the brain so must be able to control CETP in brain

Question 30

experiment: can CETP inhibitor enter the brain?

Answer 30

1 IV dose of drug - detectable levels in brain!

Question 31

what type of mice is best to use for testing CETP inhibitors in AD?

Answer 31

cross APP transgenic mice with hCETP transgenic mice and put on high cholesterol die

Question 32

experiment: CETP inhibition in plasma

Answer 32

measure CETP inhibition in plasma by measuring the amount of lipids transferred - WT and APP mice have no CETP activity - CETP and A/C mice have CETP and drug inhibits it! Therefore, drug inhibits CETP activity and transfer of lipids

Question 33

experiment: quantification of HDL, LDL in plasma

Answer 33

- WT and APP mice have no change in LDL or HDL - CETP and A/C mice have increased HDL and decreased LDL with drug Therefore, drug and mouse model work!

Question 34

how can we measure memory performance?

Answer 34

train mouse with 2 same objects --> next day, replace with 1 new object --> measure amount of time spent investigating new object mouse should spend more time looking at new object than something they already know

Question 35

experiment: CETP inhibitors and memory performance - how does this relate to centenarian studies?

Answer 35

in CETP and A/C mice with drug --> increased memory performance therefore, in A/C, drug overrides the negative effect from APP matches centenarian studies!

Question 36

correlation of LDL/HDL vs cognitive performance

Answer 36

increased cognition: - increased HDL - decreased LDL

Question 37

experiment: AB quantification in brain

Answer 37

no difference in AB levels in APP vs A/C mice and treated vs untreated therefore, no association with AB and cognition --> DISPROVES amyloid hypothesis

Question 38

experiment: cholesterol in hippocampus

Answer 38

use filipin to measure amount of cholesterol in CETP and A/C mice, increased cholesterol in brain

Question 39

describe cholesterol in the brain normally

Answer 39

brain has 25% of whole body cholesterol --> effluxed from brain by conversion to 24S-hydroxy-cholesterol

Question 40

why do CETP transgenic mice have more cholesterol in the brain?

Answer 40

detected lower levels of 24S-hydroxy-cholesterol --> therefore there is less cholesterol efflux from the brain and therefore high cholesterol in the brain

Question 41

3 conclusions of CETP as modulator of AD pathology

Answer 41

1. mutating CETP can delay conversion from early to late AD, depending on ApoE4 2. brain cholesterol levels increase in CETP transgenic mice 3. CETP inhibitor can maintain memory

Question 42

current hypothesis about CETP as a modulator of AD pathology

Answer 42

CETP redistributes cholesterol in the brain in an UNBENEFICIAL way that interferes with something in the brain to cause impaired cognition --> CETP inhibition rescues this

Question 43

2 future directions regarding CETP as a modulator of AD pathology

Answer 43

1. is it peripheral or central CETP? 2. how are CETP and ApoE4 connected?

Question 44

CETP as a drug target

Answer 44

1. What is the evidence that inhibition will cure the disease? - We know CETP inhibition is associated with longevity and humans with CETP deficiency have no cognitive impairment - SNPs, mutations, and KO in humans can tell us that cognitive performance can be maintained 2. What is the evidence that the target is druggable? - CETP is in blood --> very druggable in blood - But unsure if CETP is druggable in brain bc hard to measure CETP activity/lipid transfer - We already know that many drugs can alter CETP and modify lipid profile 3. What is the evidence of selectivity? - CETP is upregulated in high cholesterol diet and we know high cholesterol diet is risk factor - CETP is in many places so could give off-target effects BUT mostly active in plasma where drug would stay - + unique activity profile with pretty good evidence of selectivity