ND - L4 Flashcards
Topics discussed?
pathways leading to neuronal dysfunction & memory loss
approaches to understanding the molecular & cellular biology in AD
pathways leading to neuronal dysfunction & memory loss?
Abeta
tau
inflammatory
Neurotoxic mechanisms associated with Abeta? (10)
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
Direct reactive oxygen species generation?
Cu(OO) -O2–> Cu(I)+H2O2 –> Cu(II) + OH*
indirect oxidative stress?
NMDA type glutamate receptor modulation
accumulation of intraneuronal Abeta?
inhibits cell metabolism
synaptic toxicity?
NMDA receptor mediated
impairment of vesicle release
inhibition of vesicle trafficking to synapse o inhibition of endocytosis
modulation of extracellular environment
aberrant cell signalling?
Abeta can react with lots of receptors (eg. NMDA acetylcholine fyn kinase)
changes many aspects of cell function
inhibition of glutamate uptake?
Astrocytes regulate extracellular glutamate levels & protect neurons from too much
decreased cellular energy production?
maybe impaired glucose delivery (abeta affects mitochondria)
lack of trophic support from glia?
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
What else does Abeta toxicity depend on? explain experiment
tau interaction with fyn kinase in neuronal dendrites
inflammation neurotoxicity?
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
approaches to understanding the molecular & cellular biology in AD topics?
development of model systems assays for neurotoxicity identification of potential targets cell models animal models
What are some problems with synthetic Abeta peptides?
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?
What is the name of a cell viability assay?
MTT/MTS
Advantages of MTT/MTS?
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
Disadvantages of MTT/MTS?
can’t tell if healthy or dead
replicating –> increases measured viability
toxic to cells
not real time
Approaches to identify pathways in cell models?
knockdown of proteins (Fyn kinase)
inhibition of enzymes (calcineurin inhibitors)
analysis of protein changes induced by amyloid
measuring cell growth, function induced by amyloid
What have MTT/MTS assays of amyloid pepetide toxicity shown?
oligomeric forms toxic
mutation of His residues affects Cu binding (reduced toxicity)
mutation of Tyr10 also reduces toxicity
Optimum drugs for neurodegeneration are?
small cross BBB non-toxic cleared quickly highly specific easy to make in large quantities
Topics of animal models?
AD ‘like’ mice
Tau mice
APP/Tau models
Types of AD ‘like’ mice?
Tg2576: human APP (mutations K595N/M596L) - aggressive onset AD
APP/PS1: APP double mutation and human mutant PS1 - more aggressive
Tau mice?
P301L: human Tau gene mutation
NFTs (dementia) but no amyloid deposits
What do APP/Tau models show?
changes to amyloid in AD are upstream on changes to tau (but tau critical for disease effects)
Other useful models?
knockout mice - compare deletions (most proteins redundant)
ex vivo tissue - brain slices
other animals
