Laboratory Techniques for ALS Research Flashcards
How can modelling of disease help us to bridge the gap between genetics and pathology?
▪️ Patients don’t usually present clinically until late stages of disease progression
▪️ Models can help use understand the middle stages - what goes on between genetics and pathology
What percentage of ALS cases are familial?
~10%
What is Amyotrophic Lateral Sclerosis?
▪️ Progressive, incurable neurodegenerative condition
▪️ Loss of both upper and lower motor neurons leading to loss of innervation and muscle atrophy
What are the main symptoms of ALS?
▪️ Focal onset of weakness/spasticity in limb or bulbar muscles
▪️ Muscle wasting
▪️ Dysarthria
▪️ Dysphagia
▪️ Death due to respiratory failure or infection (~3 years from symptom onset)
What are the main subtypes of FTD?
▪️ Behavioural variant (bvFTD)
▪️ Semantic variant (svFTD)
▪️ Progressive non-fluent aphasia (nfPPA)
What are the main symptoms of behavioural variant FTD?
▪️ Personality change
▪️ Socially inappropriate behaviour
▪️ Emotional lability
When does death typically occur in FTD?
~4 years from symptom onset due to physical deline
What percentage of ALS patients present with bvFTD?
~5%
(but many more show subtle cognitive signs)
What percentage of FTD cases appear to be familial?
~40%
What is linkage analysis?
A technique to map disease-causing gene mutations across generations of high-risk families by identifying genetic markers that are coinherited with the trait
Where are aggregations of TDP-43 found in ALS?
In cytoplasm of neurons and glia, particularly in the spinal cord
How might TDP-43 mutations cause disease?
▪️ Gain of function AND loss of function
▪️ Aggregations and mislocalisations are toxic
▪️ RNA processing toxicity
What is mislocalisation of TDP-43 in ALS?
Depleted from the nucleus and aggregated in the cytoplasm
How do you model disease progression?
▪️ Clinical samples
▪️ Animal models (e.g., fruit flies, mice)
▪️ Stem cells and in vitro neurons
What are the benefits of fruit flies in neuroscience?
▪️ In vivo
▪️ Cost effective
▪️ No ethics approval - relatively simple to carry out
How can fruit flies be used in ALS research?
Flies genetically modified with chemical, crossbred with MND/ALS flies to see if you can change the ALS phenotype
Study neurodegeneration in the drosophila leg
What three genes were found using fruit flies to suppress TDP-43 toxicity thus improving the MND phenotype?
▪️ GSK3
▪️ hat-trick
▪️ xmas-2
How can we introduce a human-equivalent mutation into a mouse?
CRISPR/Cas9
▪️ CAS9 protein cuts DNA, guided to the TDP-43 sequence by guide RNS
▪️ Simultaneously include section of DNA with mutation so that it heals with this included
What is Automated Continuous Behavioural Monitoring and what has it found in TDP-43 ALS mice?
▪️ Mice monitored continuously be computer which can determine what its doing
▪️ ALS mutant mice walk less, hang less, but rear up and eat more1
What can a rotor rod be used for and what has been found in TPD-43 ALS mice?
Assessing motor function in mice - spin them around faster and faster and see how long they can hold on
▪️ Mutants falling off quickly BUT is it because they are fatter (found to be eating more)?
How can you measure motor function in mice?
▪️ Automated Continuous Behavioural Monitoring
▪️ Rotor rod
▪️ Electrophysiology
▪️ Laser capture microdissection and RNA sequencing
How can electrophysiology be used to assess motor nerve function?
▪️ Place needles into their muscles
▪️ Pick up signals indicating loss of innervation or fasciculations (unstable activity)
(No difference found in mutant TDP-43 mice - don’t have a motor problem?)
What is laser capture microdissection and RNA sequencing and how can it be used in ALS?
To investigate problems with motor nerves at the molecular level
▪️ Use laser to chop out motor neurons from tissue from anterior of spinal cord
▪️ Extract RNA
▪️ Look at molecular profile of cells
What can be used to investigate behavioural phenotype of TDP-43 mutant mice and what does it assess?
Mice in operant cage with 5-choice serial reaction time task
▪️ Attention
▪️ Impulsivity
▪️ Perseveration
▪️ Psychomotor speed
All measures of frontal lobe function! - FTD features of TDP-43 disease
What deficits were shown in TDP-43 mutant mice on the 5-choice serial reaction time task?
▪️ Took longer to learn
▪️ Higher level of omissions - not paying attention as well
▪️ Also deficits in memory on novel object recognition test - less good discrimination of novel item after short delay, next to none after a while
How can the marble burying assay be used to assess TDP-43 mutant mice?
Mutant mice much less interested in burying marbles, worse over time
▪️ Test of perseveration?
▪️ Test of apathy?
What is the overall conclusion of the motor and behavioural mice studies on TDP-43 mutation?
Mice get cognitive phenotype as oppose to motor phenotype
What was found in histological studies of TDP-43 mutant mice?
▪️ No difference in number of cells
▪️ No evidence of aggregations or nuclear depletion (distribution of TDP-43)
▪️ BUT there was more of it
▪️ TARDBP gene upregulated
What is a Western blot/immunoblotting?
▪️ Separate weights of protein, transfer to membrane
▪️ Add antibody to detect
▪️ Lighter protein = quicker it goes down
▪️ Darker bands = more protein
What genetic changes have been found when looking at the RNA sequencing of TDP-43 mutant mice?
400 different genes differentially expressed incl:
▪️ Upregulation of TARDBP gene (increase in TDP-43 protein and RNA)
▪️ Downregulation of Pvalb (parvalbumin related to inhibitory interneurons = reduced protection from excitation?)
What brain changes have been seen in TDP-43 mutant mice?
Reduction in volume:
▪️ Front of brain
▪️ Part of cerebellum
▪️ Entorhinal cortex (equivalent of human temporal)
Increase in volume:
▪️ Ventricles (reflect atrophy? ability to drain CSF?)
Parietal relatively preserved
What molecular analysis can you conduct in mice models of disease?
▪️ MRS
▪️ Autoradiography
Focus on GABA receptors in ALS?
How can you investigate brain function in transgenic mice?
▪️ In vivo fMRI looking at glucose metabolism
▪️ Autoradiography of glucose metabolism after animal dies
How can stem cells be used for human-derived models of disease?
▪️ Reprogramme skins cells to becomes stem cells (iPSCs
▪️ Can then induce them to become neurons and glia
What is the main problem with iPSCs?
Lose epigenetic factors (e.g., smoking, diet, mental health)
These are likely key in ALS
How might you overcome the problem of epigenetic loss seen with iPSCs?
‘Direct’ differentiation - programme skin cell to go straight to being a neuron/glia (no stem cell in between)
What is the main pathogenic mechanism of SOD1 mutation in ALS?
Toxic gain of function
How can gene therapies with ASO be used for ALS?
▪️ ASO sequence mimics SOD1 RNA sequence
▪️ Drug binds to RNA
▪️ RNase H from within cell digests RNA - lose both good and bad RNA
▪️ Thus reduce level of toxic protein
▪️ ASO given through monthly lumbar puncture
