Laboratory Techniques for ALS Research

Question 1

How can modelling of disease help us to bridge the gap between genetics and pathology?

Answer 1

▪️ 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

Question 2

What percentage of ALS cases are familial?

Answer 2

~10%

Question 3

What is Amyotrophic Lateral Sclerosis?

Answer 3

▪️ Progressive, incurable neurodegenerative condition
▪️ Loss of both upper and lower motor neurons leading to loss of innervation and muscle atrophy

Question 4

What are the main symptoms of ALS?

Answer 4

▪️ 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)

Question 5

What are the main subtypes of FTD?

Answer 5

▪️ Behavioural variant (bvFTD)
▪️ Semantic variant (svFTD)
▪️ Progressive non-fluent aphasia (nfPPA)

Question 6

What are the main symptoms of behavioural variant FTD?

Answer 6

▪️ Personality change
▪️ Socially inappropriate behaviour
▪️ Emotional lability

Question 7

When does death typically occur in FTD?

Answer 7

~4 years from symptom onset due to physical deline

Question 8

What percentage of ALS patients present with bvFTD?

Answer 8

~5%

(but many more show subtle cognitive signs)

Question 9

What percentage of FTD cases appear to be familial?

Answer 9

~40%

Question 10

What is linkage analysis?

Answer 10

A technique to map disease-causing gene mutations across generations of high-risk families by identifying genetic markers that are coinherited with the trait

Question 11

Where are aggregations of TDP-43 found in ALS?

Answer 11

In cytoplasm of neurons and glia, particularly in the spinal cord

Question 12

How might TDP-43 mutations cause disease?

Answer 12

▪️ Gain of function AND loss of function
▪️ Aggregations and mislocalisations are toxic
▪️ RNA processing toxicity

Question 13

What is mislocalisation of TDP-43 in ALS?

Answer 13

Depleted from the nucleus and aggregated in the cytoplasm

Question 14

How do you model disease progression?

Answer 14

▪️ Clinical samples
▪️ Animal models (e.g., fruit flies, mice)
▪️ Stem cells and in vitro neurons

Question 15

What are the benefits of fruit flies in neuroscience?

Answer 15

▪️ In vivo
▪️ Cost effective
▪️ No ethics approval - relatively simple to carry out

Question 16

How can fruit flies be used in ALS research?

Answer 16

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

Question 17

What three genes were found using fruit flies to suppress TDP-43 toxicity thus improving the MND phenotype?

Answer 17

▪️ GSK3
▪️ hat-trick
▪️ xmas-2

Question 18

How can we introduce a human-equivalent mutation into a mouse?

Answer 18

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

Question 19

What is Automated Continuous Behavioural Monitoring and what has it found in TDP-43 ALS mice?

Answer 19

▪️ Mice monitored continuously be computer which can determine what its doing
▪️ ALS mutant mice walk less, hang less, but rear up and eat more1

Question 20

What can a rotor rod be used for and what has been found in TPD-43 ALS mice?

Answer 20

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)?

Question 21

How can you measure motor function in mice?

Answer 21

▪️ Automated Continuous Behavioural Monitoring
▪️ Rotor rod
▪️ Electrophysiology
▪️ Laser capture microdissection and RNA sequencing

Question 22

How can electrophysiology be used to assess motor nerve function?

Answer 22

▪️ 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?)

Question 23

What is laser capture microdissection and RNA sequencing and how can it be used in ALS?

Answer 23

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

Question 24

What can be used to investigate behavioural phenotype of TDP-43 mutant mice and what does it assess?

Answer 24

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

Question 25

What deficits were shown in TDP-43 mutant mice on the 5-choice serial reaction time task?

Answer 25

▪️ 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

Question 26

How can the marble burying assay be used to assess TDP-43 mutant mice?

Answer 26

Mutant mice much less interested in burying marbles, worse over time ▪️ Test of perseveration? ▪️ Test of apathy?

Question 27

What is the overall conclusion of the motor and behavioural mice studies on TDP-43 mutation?

Answer 27

Mice get cognitive phenotype as oppose to motor phenotype

Question 28

What was found in histological studies of TDP-43 mutant mice?

Answer 28

▪️ 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

Question 29

What is a Western blot/immunoblotting?

Answer 29

▪️ Separate weights of protein, transfer to membrane ▪️ Add antibody to detect ▪️ Lighter protein = quicker it goes down ▪️ Darker bands = more protein

Question 30

What genetic changes have been found when looking at the RNA sequencing of TDP-43 mutant mice?

Answer 30

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?)

Question 31

What brain changes have been seen in TDP-43 mutant mice?

Answer 31

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

Question 32

What molecular analysis can you conduct in mice models of disease?

Answer 32

▪️ MRS ▪️ Autoradiography Focus on GABA receptors in ALS?

Question 33

How can you investigate brain function in transgenic mice?

Answer 33

▪️ In vivo fMRI looking at glucose metabolism ▪️ Autoradiography of glucose metabolism after animal dies

Question 34

How can stem cells be used for human-derived models of disease?

Answer 34

▪️ Reprogramme skins cells to becomes stem cells (iPSCs ▪️ Can then induce them to become neurons and glia

Question 35

What is the main problem with iPSCs?

Answer 35

Lose epigenetic factors (e.g., smoking, diet, mental health) These are likely key in ALS

Question 36

How might you overcome the problem of epigenetic loss seen with iPSCs?

Answer 36

'Direct' differentiation - programme skin cell to go straight to being a neuron/glia (no stem cell in between)

Question 37

What is the main pathogenic mechanism of SOD1 mutation in ALS?

Answer 37

Toxic gain of function

Question 38

How can gene therapies with ASO be used for ALS?

Answer 38

▪️ 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