Lectures 5-6 (Sarah Ryan)

Question 1

What does Motor neuron disease mean

Week 3 - Motor Neuron Disease 1

Answer 1

It is an umbrella term for a group of neurodegenerative diseases

Question 2

What is another name for ALS

Week 3 - Motor Neuron Disease 1

Answer 2

Lou Gehrigs disease

Question 3

What does ALS stand for

Week 3 - Motor Neuron Disease 1

Answer 3

Amyotrophic lateral sclerosis

Question 4

How does ALS progress?

(Not in terms of individual symptoms but in pathway)

Week 3 - Motor Neuron Disease 1

Answer 4

Motor neuron degeneration →reduced signals to muscles → NMJ disrupted → muscle wasting and weakness → progressive paralysis

(→ = leads to ) (NMJ = Neuromuscular junction)

Question 5

What happens after 2-5 years of the symptomatic onset of ALS

Week 3 - Motor Neuron Disease 1

Answer 5

Death, its fatal after 2-5 years

Question 6

What effective treatments (if any) exist for ALS)

Week 3 - Motor Neuron Disease 1

Answer 6

None, more research is needed

Question 7

What are the two distinctions of onset for clincal symptoms of ALS

(two types of onsets elicit different symptoms, what are the types)

Week 3 - Motor Neuron Disease 1

Answer 7

Spinal/limb onset and bulbar onset symptoms
* spinal/limb onset involves onset of muscle weakness from the limbs/spine and spreading to the midsection of the body

• bulbar onset involves onset of muscle weakness from the bulbar region (near brainstem) and spreading downwards to the rest of the body

Question 8

What are the clinical symptons of Limb/spinal onset ALS

(4)

Week 3 - Motor Neuron Disease 1

Answer 8

• Muscle stiffness (spasticity) and cramps
• muscle twitching (fasiculations)
• weakness in legs, arms, hand and feet
• poor grip strength

Question 9

What are the clinical symptoms of bulbar onset ALS

(7)

Week 3 - Motor Neuron Disease 1

Answer 9

• Degeneration of corticobulbar tract
• Wasting/weakness/fasciculations
• Difficulty swallowing (dysphagia)
• Pooling of saliva (sialorrhoea)
• Spasm of vocal cords (laryngyospasm)
• Slow or slurred speech
• Worse prognosis due to respiratory infections

Question 10

What motor neurons are effected in ALS

Week 3 - Motor Neuron Disease 1

Answer 10

Both upper and lower motor neurons, effects corticospinal tract in upper motor neuron transmission

Question 11

What are some cognitve symptons of ALS

(5)

Week 3 - Motor Neuron Disease 1

Answer 11

• Episodic memory impairments
• 15% develop frontotemporal dementia (characterised by degeneration of neurons in frontal region of brain)
• Atrophy of frontal and temporal lobes
• Personality changes (potential to be aggressive/ irrational/impulsive)
• language disorder (may have issues expressing themselves or understanding language)

Question 12

What percentage of ALS cases are hereditary

Week 3 - Motor Neuron Disease 1

Answer 12

5-10% (the rest are sporadic)

Question 13

Define SALS and FALS

Week 3 - Motor Neuron Disease 1

Answer 13

sporadic ALS and Familial ALS

sporadic ALS refers to sporadic onset cases

familial ALS refers to hereditary/ familial cases

Question 14

Is ALS a homogenous or heterogenous disease

Week 3 - Motor Neuron Disease 1

Answer 14

A very heterogenous disease

Question 15

What can some mutations that cause ALS also cause

Week 3 - Motor Neuron Disease 1

Answer 15

The development of FTD (frontotemporal dementia)

Question 16

What are three Mutations that can cause ALS onset?

Week 3 - Motor Neuron Disease 1

Answer 16

• SOD1 mutations
• C9orf72 mutations
• TARDBP mutations

Question 17

What percent of cases do SOD1 Mutations account for

(familial and sporadic)

Week 3 - Motor Neuron Disease 1

Answer 17

• Accounts for 10-20% of familial cases & 1-2% of sporadic cases

Question 18

What is the regular function of SOD1 and what does the mutation do to it

Week 3 - Motor Neuron Disease 1

Answer 18

• Antioxidant enzyme - breaks down superoxide radicals
• the mutation is a loss of its antioxidant function, making it incapable of breaking down superoxide radicals (which need to be broken down otherwise they can lead to oxidative damage)

Question 19

How is the SOD1 mutation expressed across cells

Week 3 - Motor Neuron Disease 1

Answer 19

Ubiquitously expressed, expressed in all cells at once

Question 20

Where is the SOD1 mutation found?

Week 3 - Motor Neuron Disease 1

Answer 20

Mainly in cytosol (component of cytoplasm) but can also be found in mitochondria, nucleus and ER

Question 21

IS a singular or many SOD1 mutations linked to ALS and what type of mutations are they

Week 3 - Motor Neuron Disease 1

Answer 21

Many, mostly being single amino acid subsitutions (missense mutations)

Question 22

What fraction of total ALS cases does the C9orf72 mutation account for

Week 3 - Motor Neuron Disease 1

Answer 22

Most common cause of ALS - 1/12 of ALL cases

Question 23

Besides ALS, what do C9orf72 mutations cause

Week 3 - Motor Neuron Disease 1

Answer 23

Frontotemporal dementia (FTD)

Question 24

What actually happens in C9orf72 mutations to cause ALS

(hint:bases)

Week 3 - Motor Neuron Disease 1

Answer 24

The same 6 bases (GGGGCC) get repeated over and over again thousands of times when theyre not supposed to

This is called a large hexanucleotide repeat expansion

Question 25

In relation to the C9orf72 mutations

Which regions does the hexanucleotide repeat expansion appear in, and what impact (if any) does this have on the wild type copy

Week 3 - Motor Neuron Disease 1

Answer 25

• The repeat is in non-coding regions (Intron 1/promoter regions)
• Isnt interrupting the normal sequence of the wild type copy, but you do get LESS of the wild type - Patients express 50% less C9orf72

Question 26

In relation to the C9orf72 mutations

What happens when the hexanucleotide expression gets transcribed

Week 3 - Motor Neuron Disease 1

Answer 26

• You get translation of the repeat that wouldnt happen in any form in another person, the G bases interact and form a G quadruplex structure which tricks the body into thinking theres a start codon where there is not
• This is translated to produce 5 repetitive dipeptides - These are novel proteins that dont exist in any form in a healthy brain

Question 27

What does TARDBP encode for when healthy

Week 3 - Motor Neuron Disease 1

Answer 27

TDP-43 (transactive response DNA binding protein 43)

Question 28

Where is TARDPBP mainly found

Week 3 - Motor Neuron Disease 1

Answer 28

In the nucleus

Question 29

What is TARDBP (and TDP-43) responsible for

Week 3 - Motor Neuron Disease 1

Answer 29

DNA repair and RNA processing : regulation of transcription, translation, splicing

Question 30

How many mutations of TARDBP are linked to ALS and where do they primarily occur

Week 3 - Motor Neuron Disease 1

Answer 30

• > 40 mutations (mostly missense/substitution) linked to ALS
• vast majority occur in glycine rich domain

Question 31

What is the rarity of TARDBP mutations in causing ALS

Week 3 - Motor Neuron Disease 1

Answer 31

rare - 1-3% of ALL ALS cases

Question 32

Which mutations causing ALS are also linked to frontotemporal dementia

Week 3 - Motor Neuron Disease 1

Answer 32

C9orf72 and TARDBP mutations

Question 33

What happens to motor neurons in ALS and how can this be identified

(2)

Week 3 - Motor Neuron Disease 1

Answer 33

• Motor neurons die - there is neurodegeneration in the motor cortex of an ALS patient
• There is an indent near the precentral gyrus because motor neurons in that region have died

Question 34

What are the most common protein inclusions in ALS

Week 3 - Motor Neuron Disease 1

Answer 34

SOD1 and TDP43

(They are NOT found at the same time)

Question 35

What is protein aggregation

Week 3 - Motor Neuron Disease 1

Answer 35

The formation of insoluble intraneuronal protein aggregates/inclusions due to proteins being ubiquitinated & phosphorylated

Question 36

What is the rarity of protein aggregation in Neurodegenerative diseases

Week 3 - Motor Neuron Disease 1

Answer 36

Common to all neurodegenerative diseases

Question 37

What types of inclusions do SOD1 cases of ALS feature

Week 3 - Motor Neuron Disease 1

Answer 37

All SOD1 cases have SOD1 inclusions

(rare in sporadic cases as SOD1 doesnt typically cause sporadic ALS)

Question 38

When does the TDP protein inclusion appear in ALS cases

(two main points)

Week 3 - Motor Neuron Disease 1

Answer 38

• In the majority of sporadic cases
• appears in all mutations of TARDBP and C9orf72 as well as several others

Question 39

When does the C9orf72 dipeptide protein inclusion appear in ALS cases

Week 3 - Motor Neuron Disease 1

Answer 39

Aggregate only in C9orf72 mutations

Question 40

What are the three main ways we can study ALS from humans?

Answer 40

Post-mortem patient tissue (brains and spinal cord)

Clinical imaging (e.g., PET)

Patient blood/CSF samples.

Question 41

What are the limitations to using post-mortem tissue to study ALS?

Answer 41

Cannot manipulate the system with drugs/genetics/tesing function.

You’re looking at the end stage of the disease - cannot see the process or early stages of ALS onset.

Question 42

What are the three main In vitro disease models?

Answer 42

Immortalised cell lines

Human iPSC-derived neurons (or other cells)

Primary cell cultures from animals.

Question 43

Outline key info about immortalised cell lines as a method of study in ALS.

Answer 43

Immortalised cells are modified on purpose, or taken from a cancer - leading them to always being replicating.

However, these cells don’t always work in the same way that they do in the brain.

You can get neuron like cells that have synapses and perform like neurons.

+ cheap and easy to use.

• not exactly like real cells, can act differently meaning that you need to use converging methods of study for best results.

Question 44

Outline the basic process of creating Human iPSC-derived neurons, why are they good for ALS study?

Answer 44

They are induced Pluripotent Skin Cells (iPSC)

METHOD:
Take a skin sample, isolate fibroblasts then treat them with signalling factors that turn them back into pluripotent stem cells.

Then use the right combination of growth factors and differentiate them into any cell (such as cortical motor neurons, astrocytes etc)

WHY THEY ARE GOOD FOR STUDY:
We can take skin cells from ALS and Control patients and grow motor neurons to do experiments on.

Question 45

Outline the basic process of collecting primary cell cultures from animals (mouse as example).

Name a pro and con of using them.

Answer 45

Take the thigh bone of a dead mouse and flush out the marrow.

From this you can use the macrophages to study on.

(+) High yield from this.

(-) Not a lot of time to use these cells as they will die.

Question 46

What are the 4 main In vivo disease models used to study ALS?

(as in what animal models are used)

Answer 46

Rodents

Drosophila

Zebrafish

C. elegans

Question 47

Why can we use In vivo models to study ALS?

How can we ensure validity?

Answer 47

There is a lot of conservation of the genome in these organisms and the basic cell biology can be similar making them good models (neurons act very similarily across most organisms)

Testing the same hypothesis on many organisms and finding the same things in all of them shows that it is likely to be the same in humans (convergent evidence)

Question 48

What are the two main types of transgenic models of ALS?

Answer 48

Knockout animals/cell lines

Knock-in/overexpression models.

Question 49

What are the two main use cases for using knockout animals/cell lines in studying ALS?

Answer 49

Investigating normal function

Investigating whether loss of function causes diseases.

Question 50

What are the 4 main examples of knock-in/overexpression models used in ALS?

Answer 50

SOD 1 G93A mice.

TARDBP mutations OR overexpression of human WT TDP-43

C9orf72 expansions GGGGCC expansions

Artificail expression of a single C9orf dipeptide (e.g., poly-GR)

Question 51

What can knock-out models of ALS tell us?

Answer 51

Firstly, it tells us about the normal function of the gene.

If the knock out shows phenotypic expression similar/the same as the ALS symptoms then it shows that a LoF in that gene is likely to be causing the disease/symptoms.
- Such as SOD1 loss causing toxicity.

If not, then we can begin to research a toxic GoF in this gene instead.

Question 52

What does G93A mean in relation to SOD1 G93A?

Answer 52

Glycine at amino acid 93 has turned into a Alanine.

Question 53

What is TARDBP and what role does it play?

Why is it related to ALS?

Answer 53

TDP-43 is encoded by the TARDBP gene, which is a highly conserved and ubiquitously expressed protein localized primarily within the nucleus, and functions in transcription and RNA splicing regulation.

Research has found it to be associated with ALS.

Question 54

What are C9orf72 expansions?

Answer 54

Repeat expansions in the promoter region of C9orf72.

Question 55

Outline the results of the study in transgenic mice with the C9orf72 dipeptide expansion (poly-GR).

Answer 55

Pictures of the cortex and hippocampus that are stained green (or red…) show lower density of cell bodies in GFP-(GR)100 mice - in comparison to controls.

This shows that the dipeptide repeat of Glycine-Argenine is sufficient to cause neurodegeneration in mice.

Question 56

What did the rotor-rod task with SOD1 mutant mice show?

Answer 56

Mutant mice (induced ALS) showed significant motor deficits in this task when compared with WT control mice.

Suggests that SOD1 is implicated in motor control/neurodegeneration.

Question 57

What is the role of SOD1?

What does it’s LoF lead to?

Answer 57

Superoxide dismutase 1 (SOD1) is as an antioxidant enzyme protecting the cell from reactive oxygen species toxicity.

(SOD1 binds to promoters and regulates expression of genes involved in oxidative stress responses).

Therefore, the LoF subjects motor neurons to toxicity that can lead to neurodegeneration, and therefore MN degeneration and loss of motor control.

Question 58

What are the three main causes of ALS that Sarah mentioned?

(There are more - these were the ones we went in detail in)

Answer 58

Glutamate excitotoxicity (EAAT2)

Dysfunction and Neurodegeneration of Microglia.

Altered axonal transport.

Question 59

Outline the key information of how glutamate excitotoxicity occurs.

Answer 59

Glutamate is an excitatory neurotransmitter that binds to AMPA, NMDA and kainate receptors - all leading to Ca2+ influx.

The process of Glutamate reuptake is regulated heavily via EAATs (excitatory amino acid transporters).

This is because too much glutamate = too much calcium in cytosol = absolute carnage (upon checking I don’t think this is technical term).

This is because Calcium is involved in mediating many processes.

Question 60

What are EAAT’s?

Answer 60

Excitatory amino acid transporters.

They uptake glutamate and are found on the pre/post synpatic neurons and astrocytes.

Question 61

What does glutamate excitotoxicity cause in cells? (5 in detail)

Answer 61

• Activation of signalling processes/enzymes (such as proteases, endonucleases, phosphlipases) - all of which can damage the cell.
• Damage to the cytoskeleton, membrane, DNA etc.
• Mitchondria releasing toxic free radicals.
• Caspace cleavage which leds to apoptosis.
  (Caspases (proteolytic enzymes) are activated during apoptosis and cleave specific proteins, resulting in the irreversible commitment to cell death)
• (Apoptosis means the death of cells which occurs as a normal and controlled part of an organism’s growth or development.)

Question 62

What are the overarching models that provide evidence for glutamate excitotoxicity in ALS? (3)

Answer 62

In ALS patients/patient post-mortem tissue.

In cell culture

In mice

Question 63

What is the evidence for glutamate excitotoxicity in ALS from ALS patients/patient post-mortem tissue?

Answer 63

There is increased glutamate in patient plasma and CSF.

There is reduced expression of glutamate transporters (e.g., EAAT2) on astrocytes in ALS patient motor cortex and spinal cord.

• Leading to too much glutamate in extracellular space, and therefore, too much calcium (causing the carnage)

Question 64

What is the evidence for glutamate excitotoxicity in ALS from cell culture research?

Answer 64

Cultured motor neurons particularly vulnerable to excitotoxicity compared to other neuronal subtypes.

Patient CSF is toxic to cultured motor neurons.

• So more susceptible to glutamate excitotoxicity = could explain MN selectivity.
• Shows that there is something in patient CSF that directly damages MN’s, which is aleviated with glutamate receptor antagonists. (shows at least a mediating role).

Question 65

What is the evidence for glutamate excitotoxicity in ALS from mice research?

Answer 65

EAAT2 knockout exacerbates motor impairments and reduces survival in mSOD1 mice.

AND

Increasing EAAT2 expression alleviates motor impairments and increases survival in mice (compared to the knockout mice).

Question 66

What is the significance of Riluzole? What can it tell us?

Answer 66

It is the only ALS approved drug and has only modest therapeutic benefit (very diriy drug).

It reduces glutamate transmission but isn’t a whole solution/cure…

Therefore, there must be other mechanisms at play.

Question 67

Outline the key information about the process of axonal transport. (Healthy)

Answer 67

Most proteins synthesised in cell body, lipids, mitochondria and vesicles all need transporting along neural cells.

As MN’s are very long, this process is more important/needs to be regulated).

Microtubulin (tubulin polymers) act as “train tracks” to which motor proteins, such as kinesins and dyneins, bind cargoes and “walk” along them - powered by the hydrolysis of ATP.

(Good video on slide if you want to see the process)

Question 68

What are the two types of axonal transport?

Answer 68

Anterograde = away from the cell body

Retrograde = towards the cell body

(Good video on slide if you want to see the process)

Question 69

What can fluorescently tagging vesciles show us?

Answer 69

We can watch how they move down the cell/axon and quantify the speed and volume of the vesicles moving.

This information can then be used to compare between ALS patients and healthy controls - is there anything wrong in ALS patients?

(Good video on slide if you want to see this in action)

Question 70

What are the two main types of evidence that axonal transport is impaired in ALS?

Answer 70

Evidence in patients.

Evidence in mice and cells.

Question 71

What is the patient evidence for axonal transport impairments could cause ALS?

Answer 71

Mutations in kinesin (axonal transport protein) family linked to ALS (KIR1A, KIR5A)

EM (electron microscopy) and histopathology of ALS patient tissue found abnormal acculumulation of vesciles, lysosomes, mitochondria, neurofilaments, microtubules etc. in motor neurons.

Good evidence because they both show that damage to transport/stopping transport can be a cause for ALS.

Question 72

What is the evidence in mice and cells that axonal transport impairments can cause ALS?

Answer 72

SOD1 and TARDBP mutant mice and cells have axonal transport defects.

Cargoes are fluorescently labelled and tracked with live-imaging, and are shown to move more slowly or not at all in ALS models.

(Recent research shows similar phenotype caused by C9orf72 expansion).

Question 73

Outline the methods and results of PART 1 Fumagali et al (2021) into C9orf72 expansion impairing axonal transport.

finding IF, not the cause…

Answer 73

METHODS:
- Used patient iPSC-derived motor neurons
- Fluorescently labelled mitchondria and live-imaged them.
- Measured how many mitochondria were motile.

RESULTS:
- In C9orf72 patients there were significantly less motile mitochondria.
- In addition, of those moving mitochondria, the C9orf92 expansion group showed significantly higher % of mitochondria that were pausing.

Shows that there are big axonal transport defecits due to C9orf92 expansions.

Question 74

Outline the methods and results of PART 2 Fumagali et al (2021) into C9orf72 expansion impairing axonal transport.

Answer 74

METHODS:
- Tested 3 dipeptide repeats (GP, GR, PR).
- Took only control MN’s and then treated them with synthetic dipeptide repeat proteins.
- Did this to see if it was the dipeptide repeats causing the reduced motility.
- Used low, medium and high concentrations for each protein.

RESULTS:
- In GR and PR there was a reduction in motility of mitochondria, showing that these two dipeptide repeats caused the phenotype.
- GP did not.

Question 75

Outline the methods and results of PART 3 Fumagali et al (2021) into C9orf72 expansion impairing axonal transport.

Western Block

Answer 75

METHODS:
- They took the DPR’s that showed significant decrease in motility (GR and PR) - as well as GP (no impact on motility in previous experiment).
- Tested how they aggregated in the cytoplasm of MN’s and what impact this might have with various different protein components of axonal transport system.

• Mushed up all the cells (with the DPRs) into a homogenised liquid.
• Then mix with antibody coated “beads” - specific to GR, GP or PR
• Any of the cell homogenate will stick to antibodies along with anything bound to it in a complex.
• Then washed everything else off, performed a western block and saw the results…

RESULTS:
- (See slide for picture)
- Two C9orf92 dipeptides = GR and PR, are physically binding to protein components of the axonal transport system.
- Making them no longer free to do their job, inhibiting axonal transport.

Question 76

Why is neuroinflammation interesting when studying ALS?

Answer 76

AS neuroinflammation is common in all neurodegenerative diseases.

Question 77

How is microgliosis observed in the brain of ALS patients, what is it correlated with and what does lead to increases of?

Answer 77

Observed by immunohistochemistry and PET imaging.

It is correlated to severity of disease progression.

Increased pro-inflammatory cytokines in CSF

Question 78

What is Microgliosis?

Answer 78

Microgliosis is an intense reaction of CNS microglia to pathogens.

One of the characteristic features of microgliosis is an increase in the number of activated microglia at the site of lesion.

Question 79

What are pro-inflammatory cytokines?

Answer 79

Proinflammatory cytokines are small proteins released by cells of the immune system, mainly white blood cells, in response to foreign invaders like bacteria, viruses, or damaged tissues.

They act as chemical messengers, triggering a cascade of events that initiate and orchestrate inflammation, a crucial defense mechanism of the body.

Question 80

What is the main inflammatory cytokine implicated in ALS research?

Mentioned in lecture the most

Answer 80

IL-1Beta

Question 81

What did a PET scan between controls and ALS patients show about microglia?

Answer 81

There are more microglia in patients and more activation of them.

Question 82

What did research into morphological changes in ALS microglia show? What methods did they use to determine this?

Answer 82

METHODS:
- Compared WT and SOD1 mutation patients.
- They stained microglia processes green and then did a SHOL analysis.

RESULTS:
- Found that in SOD1 patients the microglia showed a more amoeboid shape, which shows more activation.
- Suggests increased microglial activation in SOD1 ALS.

Question 83

Is there any evidence that inflammation causes damage in ALS? If so, what is it?

Answer 83

Yes, genetic evidence supports key role of microglia/neuroinflammation is causing/contributing to the damage during ALS.

Genes such as:
- TBK1
- OPTN1
- VCP mutations (coded by VPS35)

Question 84

Outline the ways that neuroinflammation can occur in ALS?

Think toxicity

Answer 84

Inflammatory cytokines can be toxic to neurons.

ALS microglia kill cells when they are not functioning properly and can be very toxic

Question 85

What is mSOD1?

Answer 85

mutant Superoxide Dimutase 1 - found in ALS patients.

Question 86

What is the relevance of IL-2 - what does it stand for?

Answer 86

Interleukin 2 is a treatment that has been shown in recent trials to target microglia inflammation and show survival benefits from its application.

Question 87

Detail the findings about mSOD1’s effect on motor neurons under different conditions.

Answer 87

mSOD1 motor neurons were protected against degeneration by co-culturing with WT microglia.

However,

mSOD1 microglia are toxic to cultured neurons.

Provides further evidence for toxic microglia being a harmful cause of toxicity in ALS.

Question 88

What is the role of the NLRP3 inflammasome?

Generally WT

Answer 88

It is a large, intracellular multiprotein complex(s) playing a key role in the innate immune system and inflammatory response.

It acts as a sensor and activator, initiating the release of potent inflammatory molecules called pro-inflammatory cytokines in response to various threats.

Question 89

What is the basic process of how the NLRP3 inflammasome works?

Answer 89

Activation of inflammasome leads to caspase-1 activation.

Caspase-1 cleaves pro-interleukin 1beta (pro-IL-1Beta) to IL-1Beta

IL-1Beta is a inflammatory cytokine

Therefore, NLRP3 leads to the release of inflammatory cytokines - resulting in inflammation.

Question 90

What has NLRP3 inhibition been shown to do in mice (in what condition)?

Answer 90

NLRP3 inhibition rescues cognition in Alzheimer’s Rodents.

Question 91

What was the AIM of Sarah’s ALS study? (Lecturer)

Answer 91

To find whether the DPR’s (transcribed by the C9orf72 expansion) also activate the NLRP3 inflammasome, causing the release of IL-1Beta and thus an inflammatory reponse.

Question 92

Describe the methods and results of PART 1 of Sarah (lecturers) ALS study. (First three stages)

NLRP3

Answer 92

METHODS:
- Cultured mice macrophages in a dish and treated them with synthetic dipeptides.

RESULTS:
- Found that GR causes big increases in IL-1Beta being released in the culture media.

Have to doubt data so did another experiment

METHODS:
- Repeated with different types of microphages to make sure it wasn’t some cell specific artefact.
- They also used a drug that is a selective NLRP3 inhibitor

RESULTS:
- Found that GR causes big increases in IL-1Beta being released in the culture media - they found the same result.
- The selective NLRP3 inhibitor blocked the response - showing that the increased response was definitely from the NLRP3 inflammasome.

METHODS:
- Tested the dose dependent response of GR, in intervals of 1, 3, 10 and 30uM.

RESULTS:
- Found a dose dependent response as the more GR introduced, the more IL-1beta more produced.

The more IL-1Beta, the more inflammasome activation

(Looks like a longer flashcard then it is, go look at slide 23 of MND 2 lecture to see graphs)

Question 93

Describe the methods and results of PART 2 of Sarah (lecturers) ALS study.

Answer 93

Following from the results of the previous stage that GR increased the IL-1Beta release and that there was a dose-dependent relationship…

METHODS:
- They used ELISA (Enzyme-Linked Immunosorbent Assay) to detect whether it was a mature form of/or precursor protein for IL-1Beta.

RESULTS:
- A 17kDa line in GR shows that it was the mature form of IL-1Beta being measured (as this was expected size of protein).

Therefore, western block confirms that GR causes cleavage of pro-IL-1Beta to IL-1Beta (inflammatory cytokine).

Question 94

Describe the methods and results of PART 3 of Sarah (lecturers) ALS study.

Answer 94

Following from the results that western block confirms that GR causes cleavage of pro-IL-1Beta to IL-1Beta (inflammatory cytokine)…

METHODS:
- Used immunofluoresence to stain green ASC in mouse hippocampul slice cultures.
- Used GR and AP to see if the response was DPR specific.
- Also used MCC950 (NLRP3 inhibitor) in a condition to see effect on results.
- Used imaging and counting of ASC specks to conclude results.

RESULTS:
Imaging:
- Can see that when GR is added, you get intense spots of green where all the ASC proteins from the cytoplasm come together to form the inflammasome complex.
ASC specks:
- Found that there was significantly more with GR.
- Found NO specs were formed with AP (therefore it is DPR specific - GR)
- Found when treating with MCC950, less ASC specs form, so it is via NLRP3 that this mechanism occurs

Question 95

What is the process of forming the NLRP3 inflammasome?

Answer 95

1. Upon NLRP3 activation, it undergoes changes that allow it to bind to ASC through their respective protein domains (PYD).
   - This interaction is essential for recruiting other inflammasome components, like pro-caspase-1.
2. Once NLRP3 and ASC are linked, they form a seed that attracts pro-caspase-1.
   These three proteins form a multi-protein complex known as the NLRP3 inflammasome.
3. Within the inflammasome, pro-caspase-1 is activated by NLRP3, cleaving pro-inflammatory cytokine precursors like pro-IL-1β and pro-IL-18 into their active forms.
4. These active cytokines are then released from the cell, triggering various immune responses like inflammation and fever.

(NLRP3: Acts as the sensor, detecting a variety of danger signals within the cell (e.g., bacterial toxins, damaged cells).
ASC: Serves as the platform, providing a structure for inflammasome assembly.)

Question 96

What did a NLPR3 knockout study show in mice? Why is this exciting?

Answer 96

Found that this knockout rescues motility in the ALS mouse phenotype as no inflammation could be formed.

This suggests that NLRP3 inflammasome is ACTIVELY a part of neurodegeneration in ALS.

Many drugs inhibit NLRP3 so this is an exciting potential therapeutic target.