ND - L4

Question 1

Topics discussed?

Answer 1

pathways leading to neuronal dysfunction & memory loss

approaches to understanding the molecular & cellular biology in AD

Question 2

pathways leading to neuronal dysfunction & memory loss?

Answer 2

Abeta
tau
inflammatory

Question 3

Neurotoxic mechanisms associated with Abeta? (10)

Answer 3

direct reactive oxygen species generation
indirect oxidative stress
accumulation of intraneuronal Abeta
synaptic toxicity
aberrant cell signalling
inhibition of axonal transport
ER stress
inhibition of glutamate uptake
decreased cellular energy production
lack of trophic support

Question 4

Direct reactive oxygen species generation?

Answer 4

Cu(OO) -O2–> Cu(I)+H2O2 –> Cu(II) + OH*

Question 5

indirect oxidative stress?

Answer 5

NMDA type glutamate receptor modulation

Question 6

accumulation of intraneuronal Abeta?

Answer 6

inhibits cell metabolism

Question 7

synaptic toxicity?

Answer 7

NMDA receptor mediated
impairment of vesicle release
inhibition of vesicle trafficking to synapse o inhibition of endocytosis
modulation of extracellular environment

Question 8

aberrant cell signalling?

Answer 8

Abeta can react with lots of receptors (eg. NMDA acetylcholine fyn kinase)
changes many aspects of cell function

Question 9

inhibition of glutamate uptake?

Answer 9

Astrocytes regulate extracellular glutamate levels & protect neurons from too much

Question 10

decreased cellular energy production?

Answer 10

maybe impaired glucose delivery (abeta affects mitochondria)

Question 11

lack of trophic support from glia?

Answer 11

astrocytes protect neurons from oxidative stress
astrocytes provide nutrients and precursors for
important molecules such as precursors of antioxidant
molecules (eg Glutathione precursor cysteine)
astrocytes regulate levels of metals, co-factors etc
astrocytes secrete growth factors that maintain neurons
changes to astrocytes (eg activation) can result in loss of this support and neuronal dysfunction

Question 12

What else does Abeta toxicity depend on? explain experiment

Answer 12

tau interaction with fyn kinase in neuronal dendrites

Question 13

inflammation neurotoxicity?

Answer 13

resident microglia
invading monocytes
activated astrocytes
production of cytokines and reactive oxygen species that impair neurons
loss of trophic support for neurons
possibly a major role in secondary neurotoxic effects in AD

Question 14

approaches to understanding the molecular & cellular biology in AD topics?

Answer 14

development of model systems
assays for neurotoxicity
identification of potential targets
 cell models
 animal models

Question 15

What are some problems with synthetic Abeta peptides?

Answer 15

contamination with other toxic molecules
normal brain also contains Aβ
separation of different species
Aβ is very ‘sticky’ and aggregates easily
how do you keep it in the form it was purified in?

Question 16

What is the name of a cell viability assay?

Answer 16

MTT/MTS

Question 17

Advantages of MTT/MTS?

Answer 17

subtle measure of cell toxicity (small changes in cell energy levels can be assessed – more relevant to amyloid
toxicity than overt cell death)
rapid, simple assay (few steps) – easy to perform on large numbers of experiments
well known and reproducible

Question 18

Disadvantages of MTT/MTS?

Answer 18

can’t tell if healthy or dead
replicating –> increases measured viability
toxic to cells
not real time

Question 19

Approaches to identify pathways in cell models?

Answer 19

knockdown of proteins (Fyn kinase)
inhibition of enzymes (calcineurin inhibitors)
analysis of protein changes induced by amyloid
measuring cell growth, function induced by amyloid

Question 20

What have MTT/MTS assays of amyloid pepetide toxicity shown?

Answer 20

oligomeric forms toxic
mutation of His residues affects Cu binding (reduced toxicity)
mutation of Tyr10 also reduces toxicity

Question 21

Optimum drugs for neurodegeneration are?

Answer 21

small
cross BBB
non-toxic
cleared quickly
highly specific
easy to make in large quantities

Question 22

Topics of animal models?

Answer 22

AD ‘like’ mice
Tau mice
APP/Tau models

Question 23

Types of AD ‘like’ mice?

Answer 23

Tg2576: human APP (mutations K595N/M596L) - aggressive onset AD

APP/PS1: APP double mutation and human mutant PS1 - more aggressive

Question 24

Tau mice?

Answer 24

P301L: human Tau gene mutation

NFTs (dementia) but no amyloid deposits

Question 25

What do APP/Tau models show?

Answer 25

changes to amyloid in AD are upstream on changes to tau (but tau critical for disease effects)

Question 26

Other useful models?

Answer 26

knockout mice - compare deletions (most proteins redundant)
ex vivo tissue - brain slices
other animals