Chronic CNS Disorders Flashcards
1
Q
When and who by was AD first described?
A
- 1906
- Aloiz Alzheimer
- On a 51 year old patient who had severe behavioural symptoms
- Performed a biopsy and stained the brain to identify amyloid plaques and neurofibrillary tangles
2
Q
What are the two types of AD dependent on age of presentation?
A
- Early onset (<65) familial
- Late-onset (>65) sporadic
3
Q
Outline AD’s characteristics
A
- Progressive
- Memory loss is predominant symptom
- Associated with amyloid plaques and tangles
4
Q
What are the treatment options for AD?
A
- Current treatments (AChE inhibitors, NMDA antagonists) but only offer small symptomatic benefit
- Lecanemab and Donanemab antibodies which are monoclonal antibodies (controversial)
- No treatment to prevent disease
5
Q
What is the cost to society of AD?
A
- > 900,000 UK sufferers (dementia) but 435-640K of these are AD
- Overall cost to society in care costs and lost productivity is 42 bill
- The health and social care costs are higher than cancer and chronic heart disease combined (but less money is allowed for this)
6
Q
What is the earliest symptom of AD?
A
Short-term memory loss
7
Q
What are the advanced symptoms of AD (dependent on the prevalence and dementia type)?
A
- Long-term memory loss
- Confusion
- Language problems
- Mood changes
- Disorganisation
- Withdrawal
- Repetition
- Impulsive behaviour
- Paranoia/delusions
- Eating problems/weight loss
8
Q
What does the marcoscopic pathology look like in AD?
A
- Sulci (gaps) increase in size in the outer regions (atrophy)
- Cortical shrinkage
- Ventricular enlargement (CSF filled chambers)
- Mainly in areas affecting memory and language such as the hippocampus
9
Q
Why is it important that early diagnosis and treatment occurs in AD?
A
The brain is poor at regeneration and making up for lost tissue
So when severe AD, cannot restore the brain to healthy
10
Q
What are the main neuropathological hallmarks of AD?
A
- Amyloid plaques
- Neurofibrillary tangles
- Selective neuronal degeneration
- Synaptic loss
- Inflammation
(this is found in the hippocampus, temporal cortex (also frontal and parietal)
11
Q
How is AD diagnosed?
A
- A set of cognitive tests and sometimes MRI scan
- PET is used mainly in research with a radioactive tracer binding to amyloid (PIB) or fluorodeoxyglucose [18F] tracer
- There are no biomarkers for early diagnosis
- The only definitive diagnosis is post-mortem (as other dementia’s see similar symptoms to AD)
12
Q
What are amyloid plaques?
A
- Insoluble aggregates of beta-amyliod
- Extracellular in the brain parenchyma
13
Q
How are amyloid plaques formed?
A
- Through abnormal cleavage of APP by beta secretase and gamma secretase (PSEN1/2 mutations lead to increase gamma secretase activity)- often familial
- Abnormal clearance of amyloid (monomers to oligomers to proto fibrils, to fibrils to plaques). Oligomers could cause problems in the early stages so by the time there are plaques, the damage has already been done
14
Q
What are neurofibrillary tangles?
A
- Composed of hyperphosphorylated tau
- Intracellular
- Normal function is to bind and stabalise microtubules- when destabalise, things cannot pass from the cell body to the synapse anymore
- NFT develop by an unknown trigger
- No known tau gene mutation in AD (unlike FTD)
15
Q
What is the original amyloid cascade hypothesis?
A
- Aberrant processing of APP
- Increase AB plaque deposition
- Neurofibrillary tangles
- Synaptic and neuronal dysfunction
- Neuronal loss
16
Q
Outline familial AD
A
- Early onset
- Rare- 1-5% of all AD cases
- Genetically inherited
17
Q
What are the mutations involved in familial AD?
A
- APP (Swedish mutation), increased beta secretase activity
- Presenilin 1 or 2 gene, increase gamma activity
- If someone has these mutations, AD will develop, even only one copy
- Clinical trials aimed at AB have so far failed or have limited benefit such as lecanemab and donanemab
18
Q
Outline sporadic AD
A
- Late onset
- Less understood and no known single genetic mutation
19
Q
Outline the genes involved in sporadic AD
A
- Possession of the apolipoprotein E epsilon 4 (APOE) allele significantly increases risk but is not definite
- Often potential risk factors include (atherosclerosis, diabetes, hypertension, obestiy, head injury, infection- all of which have an inflammatory component)
20
Q
What is the suggestion of the amyloid cascade hypothesis of immune system activation?
A
- Activation of microglia and astrocytes, following AB deposition
- The microglia are the first responders to something going wrong and therefore a response to AD pathology
21
Q
What inflammation occurs in the brain of AD patients?
A
- Abnormal expression of proteins associated with the immune response in brain and CSF
- Such as cytokines, chemokines, complement pathway and others (COX2)
- Activation of resident immune cells
22
Q
How was it shown that amyloid beta and inflammation were linked?
A
- PET imaging, a tracer for amyloid and a tracer binding to activated microglia are both injected
- Can see that there is overlap (co-localising) of activated microglia and beta amyloid plaques
- Surrounding the plaques are clusters of microglia
23
Q
How do microglia have a beneficial role in their activation within AD?
A
- Are the main immune cells in the brain (phagocytic)
- The microglia detect the aggregated AB and soluble AB oligomers which may act as DAMPs
- These react with the receptors such as TLRs and CD14 to instruct the microglia to phagocytose AB
- AB degredation via neprilysin and insulin degrading enzyme then occurs
24
Q
How can activated microglia become compromised in AD?
A
- Can deal with the amyloid early on but then become overwhelmed (frsutrated phagocytosis)- they cannot keep up with the problem
-There is AB-dependent impairment of microglial function
25
What do microglia, cultured at certain stages of the disease in AD mice have?
- Decreased phagocytosis
- Reduced capacity to extend processes towards a lesion
- Reduction in levels of AB binding scavenger receptors which help internalise AB
- Reduction in levels of AB degrading enzyme
26
What are some genes involved with immune and microglia function that mutation could increase the risk of AD?
- Clusterin
- CR1 (complement receptor 1)
- CD33 (myeloid cell surface antigen)
- TREM2 (triggering receptor expressed in myeloid cells 2)
- These are expressed on microglia and other myeloid cells
27
What is TREM2's normal function?
- Regulating microglial phagocytosis
- Promotes AB uptake by microglia
- Promotes survival of activated microglia
- If there is mutation, the microglia will not be able to phagocytos as well and increases the risk as the resident immune cells cannot cope as well
28
Outline the detrimental role of microglia activation in AD
- Aggregated AB and solube oligomers act as DAMPs on the receptors (CD14, TLRs for example)
- These acivate the microglia
- The microglia cause the release of pro-inflammatory and neurotoxic factors such as IL-1 and TNFa
- The neurons are sensitive to these factors and will become damages/die
- Neuronal injury releases DAMP signals causing further microglia activation
- There is also excessive complement mediated phagocytosis of synapses by the microglia through CR3 acting on C1q receptor on the neurons.
29
What are astrocytes normal and pathological roles on AD?
- Atrocytes normally support neurons
- Respond to stimuli through reactive gliosis (upregulate GFAP)
- Activated astrocytes may take ip and degrade AB
- Conversion to a reactive state may compromise their supportive role (can't do too many things at once)
- Activated astrocytes release pro-inflammatory cytokines and reactive oxygen species that can be detrimental to neurons
30
What occurs in the periphery during AD (in terms of inflammation)?
- Increases in pro-inflammatory mediators in the circulation (do not have a biomarker for this however)
- Changes in the responsibeness of peripheral immune cells such as dendritic cells
- Increases in aute phase protein (APPs) such as C-reactive protein (CRP) in which raised levels increase AD risk and predict the progression from MCI to AD
- Infections can make AD symptoms worse
31
What is evidence for the benefit of NSAIDs and AD?
Lower incidence of AD in patients with arthritis who chronically use NSAIDs
32
How can NSAIDs decrease AD risk and how was this observed/not observed?
- NSAIDs inhibit COX enzymes which convert arachidonic acid into PGE2 (causing neuroinflammation)
- Restrospective studies caused a 50% reduction in AD risk in chronic NSAID users
- but
- Clinical trials of NSAIDs carried out with AD patients had small positive results in small trials but no benefit in larger scale trials
33
Outline AD animal models ?
- Several mouse models of AD have mutations in genes associated with APP, PS-1 or tau (all familial)
- Display AD-related pathology and behavioural deficits
- Useful in assessing potential treatments to reduce AD pathogenesis and behaviour
- Only a model for early onset
- Will not be able to assess if inflammation plays a causal role
34
What do AD mouse models show about immune inflammatory pathway acitvation?
- Microglia and astrocyte activation especially in the hippocampus
- Increased production of cytokines, chemokines, complement proteins and acute phase proteins (APP)
- Infection (LPS) increases AD pathology and behavioural deficits
- NSAIDs reduce AD-like behavioural deficits
35
Outline the Morris water maze test in AD mice, NSAID treated mice and control mice
- WT improved and on the 7th day went straight to the platform
- The AD mouse never learned
- But when treated with an NSAID, its results were more like the WT mouse
36
Why is it suggested that NSAIDs didn't work in humans with AD?
- Perhaps used the wrong NSAID
- When given ibuprofen and others, inflammation remained unchanged
- However, when used a class of NSAIDs (fenamates), they were very effective at reducing IL-B
- Fenamates are better at reducing inflammation than COX NSAIDs
37
What does mefenamic acid do to a AD model mouse?
- In AD have lots of activated microglia
- When treat with this NSAID, these decreased back to near control
- Also improved behavioural models back to control (the novel object test) (AD mice explored familiar object)
38
What are the 7 physiological roles of amyloid?
- When the body is infected, the tissues secrete amyloid peptide to neutralise the toxicity in the virus or access a signal to attract immune cells to the site of injury
- Also to try and facilitate the signal transduction especially in the presynaptic regions
- Also affects the influx of calcium signalling for the LTP activity
- If there is a traumatic brain injury, it damages the neurons in the axons, if the neuron dies, it cannot be regenerated. So neurons try to helo themselves survuve by expressing AB o come to the injury site for repair and to improve the functioning of the axon
- In the BBB, if there is a leakage of the blood vessels, the neurons send signals to secrete AB which activates at the injury site to fill the gap preventing haemorrhage
- Will increase angiogenesis of the blood vessels (not very common in the brain however).
- When cancer cells appear due to mutation, the body has a defence mechanism to clear these by generating AB monomers which are toxic to the cancer cells, causing them to undergo apoptosis
- **So it is very good in the innate immunity, it works with the immine cells**
39
How was it studied to see if there is an association between the HSV1 infection and amyloid build up?
- Used a mouse animal model: 5xFAD which carries 5 genetic familial mutations of AD in APP1 and PC1
- Would generate a robust amyloid deposition in the brain
- After injecting the herpes simplex virus 1 (tagged with a GFP)
- The mouse hippocampus had higher deposition of amyloid plaque
- So, there is an association between the infection and the risk of AD
40
How was it assesed whether HSV1 leads to an increase in immune cell activation?
- Study the morphology of the microglia
- Using a biomarker iba1, stain and see that without the infection, the microglia has a long ramified surveying process
- When the brain is injected with the virus, the morphology becomes amoeboid, ready to engulf substances
- Co-stained with AB and HSV1
- The microglia would engulf the AB without the infection
- With the infection, the micrglia and HSV1 signals overlap meaning that the microglia is now engulfng the virus instead of the AB
- This indicates that the infection is more preferentially being egulfed by the microglia than the AB
41
What do homeostatic microglia look like?
Not high amounts, have long processes which are mobile and survey DAMPs and PAMPs
42
What do AB aggregated microglia look like?
- Microglia become amoebioid and have a larger soma in the cell body
- The come together to surround AB, trying to isolate it because it is toxic to the synapse
- It forms a vesicle barrier and secretes enzymes to degrade AB extracellulary
43
What actions do aggregated microglia take upon AB besides forming physical barriers?
- Processes the surface receptor such as TLR which can sense AB and vesicularly bind to it
- They trigger IC signalling such as phagocytosis, lysosomal degredation and the release of cytokines trying to attract more microglia to the site of AB
- They also pair up with astrocytes which clear AB through glyphatic clearance
- AB leads to the impairment of synaptic transmission. The microglia try to clear the sunapse in order to preserve the whole neuron through synaptic pruning.
44
What system causes synaptic pruning?
The complement system
45
What is the glyphatic clearing process by astrocytes?
- During sleep, the glyphatic clearing system helps to clear AB or other toxic substances
- The astrocytes take up AB and then pass to the circulation or the CSF to drain out the AB
- Therefore if have an unbalanced lifestyle (sleep) can lead to disease.
46
47
How does microglial effects on AB become chronic?
- No longer shifting to a homeostatic state but will be excessive activation and will lose phagocytosis
- Will turn on other mechanisms such as the cytokines which will cause mroe immune cells to the site
- Also the NF-KB signalling pathway and the NLRP3 inflammasome pathway
48
What is the role of NF-KB in inflammation?
- An intracellular mediator to control gene expression
- It expresses the genes that allow differentiation of T-Cells into functional T-cells such as T memory cells
- Not just information therefore but also helps increase genes leading to proliferation and cell survival
49
How was NF-KB activation observed in an AD brain?
- Used an astrocytic cell line treated with AB and stained for NF-KB signal
- With no treatment, the NF-KB stays in the cytosol
- When AB is used to treat the astrocyte, there is some nuclear staining of the NF-KB
- It represents the translocation of the activated NF-KB in the nucleus
50
How was NF-KB oberved near the AB plaques?
- Using post-mortem brain sections of 80 patients
- Can see when doing NF-KB staining, can see the plaque
- Near this region is some nuclei stained for the activation of NF-KB
- This suggests that the amyloid serves as a DAMP signal to activate the NF-KB
51
Which cells contain activated NF-KB?
Neurons
Astrocytes
Microglia
52
What occurs (downstream) to neurons in response to AB accumulation?
- Processing and protein aggregation
- AB triggers NF-KB translocation in the neuron into the neuronal nucleus
- This increases the transcription of base1
- Increasing the production of more AB
- This is the survival signal of the neuron- it generates more AB
53
What occurs (downstream) to astrocytes in response to AB accumulation?
- Activates mGluR5 transcription
- Does not produce AB
- If AB accumulates, the AB affects the glutamate uptake and the transportation
- This builds up toxicity
- Astrocytes help by up-taking excess EC glutamate through NF-KB
54
What occurs (downstream) to microglia in response to AB accumulation?
- Transcription of miRNA, APOE and proinflammatory mediators
- Sends the signal, the DAMP and the PAMP leading to the priming process
- This leads to the activation of NF-KB translocation in the nucleus, transcription of the genes causing the inflammatory response such as IL-1B and the NLRP3 inflammasome
55
56
How does NLRP3 inflammasome activation occur?
Involves several triggers and post translational modifications that coordinate to facilitate the release of IL-1B and drive an inflammatory response.
57
What did immunostaining reveal about the inflammasome mediators in AD microglia?
- Immunostained AD brain to test whether NLRP3 inflammasome mediators were expressed in the microglia
- Co-stained with ASC, NLRP3, caspase1, GSDMD (cleaved form for channel )and IL-1B
- All are present inside the microglia cells suggesting an intace inflammasome NLRP3 pathway
58
How does the inflammasome pathway play a role in AD?
- Suggests that activated NLRP3 is related to memory function
- Used a Morris Water Maze
- WT mice spend more time in the area they known the platform is in
- The AD mouse model cannot remember
- But when KO NLRP3, the memory returns suggesting that the KO of the inflammasome alleviates the AB induced memory decline
- Also is less amyloid covered in the brain region
59
How was it determined whether microglia have high or low uptake of amyloid dependent on NLRP3 activation?
- Using a surface marker of micrglia and tagging amyloid
- The AD mouse has a lower population of microglia and intracellular AB
- KO of the NLRP3, more microglia have intracellular AB suggesting that the micrglia has a higher capability of AB phagocytosis
60
How does amyloid pathology drive microglial activation and cytokine release?
- Used a primary microglial cell isolated from WT brain and treated with AB
- Also a reverse sequence of AB which means there is no aggregation (a null version)
- Only the AB treatment would lead to an increase of IL-1B expression and secretion in the primary microglia overtime
- Aggregated AB induces IL-1B release from activated micrglia in culture
61
How does aggregated AB activatethe NLRP3 assembly through caspase 1 activation?
- Used an NLRP3 KO mouse and isolated microglia from the mouse
- Contained no NLRP3 mediator
- When treated with AB, there was no IL-1B
- When ASC was KO'd there was the same response
- Suggests that the NLRP3 inflammasome pathway is not just responsible for the AB deposition but also the IL-1B release
- Therefore acts as a DAMP
62
Why-when there is no NLRP3 in the AD mouse model, the AB plaques get smaller?
- Used AB peptide with and ASC protein
- When added the ASC speck, the AB would accumulate so the peptide itself would become affregated from a higher weighted AB oligomer
- The AD mouse had a brain lysase isolate which was injected back into the mouse causing more amyloid plaques to form and aggregate
- With the addition of the ASC tag, would mean the lysate without the ASC when injected back into the mouse brain, there is no accumulation of the amyloid
- Suggesting that ASC may have a role in AB aggregation
63
How does ASC prescence affect memory function?
- Using a brain section from a transgenic mouse and the AD brain
- There is an ASC speck staining in the core region of the EC plaque suggesting it is the core protein of the amyloid plaque
- When KO ASC in amyloid mouse model, it improves the performance of the mice in the Morris water maze
- Suggesting improvement in memory function
64
What is the hypothetical model of how NLRP3 signalling leads toAD pathogenesis?
- In microglia which are still homeostatic, it sends the DAMP signal if there is AB
- This will trigger the activation of the NLRP3 inflammasome and cleave the immature IL-1B into acting and forms the gastermin D pore
- This is not only for cytokine release but it also leads to the rupture of the plasma membrane of the microglia leading to pyroptosis
- This causes the intracellular release of contents nclusing the ASC speck becomming ecxtracellular
- This causes seeding, the soluble AB comes along forming an aggregate and the amyloid plaque
- Suggests why it gets larger and larger in the brain
65
What are ways we could target the NLRP3 inflammasome pathway to treat AD?
- Could add NLRP3 molecule
- Could block the interaction between the ASC and NLRP3
- Could even block the signalling activation
66
How was it found that fenamates inhibit IL-1B release but not other NSAIDs?
- Different classes of NSAID drugs were used to see which can inhibit IL-1B secretion
- Ibuprofen has no effect in IL-1B reelase but other classes with siimilar strucutre could (fenamate)
- Mature IL-1B was reduced after treating with fenamate
67
How did fenamates act on ASC?
- Saw in a cell line that fenamate inhbits the ASC speck inflammasome formation
- Ibuprofen does not
68
Through what methodology does fenamate inihibit NLRP3 activation?
- Cells were treated in a hypotonic condition
- When cells are in this condition, VRAC (volume regulated anion channel) is blocked
- This normally regulates the cell volume by transportnig Cl ions across the membrane
- When fenamate treats the cells, the current is reduced suggesting that there is a reduction in the transportation of the chloride ion
69
What did an drug (besides a fenamate) which reduces Cl transportation do?
- SHowed similar effects
- Reduces the IL-1B suggesting that fenamate can inhibit IL-IB secretion through VRAC inhibition
70
How do fenamates affect memory in AD mice models?
- Treated the mouse model carrying 3 mutations of AD
- Implanted osmotic mini pumps which dosed mefenamic acid daily
- Tested memory after 2 weeks
- Performed the novel object test
- Without the treatment, could not recognise the novel object
- After treatment, restores their memory function
71
Where was microglial activation most identified in the brain?
- Subiculum
- CA1
- Outer cortex
72
How did fenamate affect the morphology of the microglia?
- In the AD mice, the microglia are more amoeboid compared with WT
- After treating with fenamate, the morphology becomes more ramified
- Also in the untreated AD model, there is more expression of IL-1B
- But after treatment, there is also less of this
73
Why are fenamates are potential target for drug trials in AD?
- They inhibit NLRP3 in vitro and in vivo via an effect on VRAC
- Mefenamic acid prevents cognitive deficits induced by AB
- Reverses cognitive decline in the 3xTgAD mouse model and microglial activation and the expression of IL-1B activation
74
Why can fenamates not be used in the treatment of AD and what is a potential way around this?
- They inhibit COX enzymes
- Prolonged treatments leads to stomach discomfort due to the inhibition of this
- Could find a compound similar to fenamate but without inhibiting COX
75
What is an example of a new NRP3 inhibitor?
- MCC950
- Is a very potent compound with a low IC50 (7.5nM)
- Ithas very specific binidng only to the NLRP3 inflammasome ATP motif
- It wouldn't oligomerise the molecule
- Inhibits the secretion of IL-1B but not TNFa suggesting that it only inhibits the inflammasome pathway
76
What effect does MCC950 have on cognition?
- Treated the AD mouse
- After the treatment, the memory function comes back in the novel object recognition test.
77
Why may MCC950 may not be appropriate in treating AD?
May lead to liver failure
78
What is a potential alternative route to developing new drugs to treat AD?
Drug repurposing is potentially a quicker way to get inhibitors into people
79
Why might there be other modifiable risk factors influencing NLRP3?
- 92% of people with AD have a co-morbidity
- Diabetes, CV diseases and hypertention
80
What is a factor produced in the liver in which AD patients have reduced levels?
Adiponectin (APN) in the CSF
81
What happens when APN is KO's in mice?
- They have poor function in the Morris water maxe
- Is this cognitive impairment associated with neuroinflammation?
82
What are APN's actions within inflammation?
- Is a very good anti-inflammatory molecule
- Treated microglia with an amyloid beta oligomer
- Caused activation of NF-KB in the cytosol and the nucleus
- When treated with APN, it reduced the activation level of NF-KB
- So, it inhbits the nuclear translocation of NF-KB in microglia
83
How does APN treatment influence the release of IL-1B from microglia?
- APN suppresses AB oligomer-induced IL-IB secretion
- Without activated NF-KB, there is reduced IL-1B
- There is higher IL-1B inside the microglia (promotion of phagocytosis)
84
Is APN deficiency induced cognitive decline NLRP3 dependent?
- Stained APN KO mice
- Saw higher NLRP3 expression in the microglia
- Suggests that APN went through an NLRP3 dependent pathway to supress IL-1B
85
How was a gene therapy strategy used to treat APN deficiency?
- The molecule would normally bind to different molecules leading to side effects
- The solution is using gene therapy
- Take the APN sequence under a liver-specific promoter
- Generated an adeno-associated virus to inject the APN into the mouse model
- This causes the liver (only) to express the APN virus
- It increased the APN levels but not too dramatically 50% in order to maintain a physiological level
- This treatment increased the time spent in the target area searching for the platform in the Morris water maze
- It also helped to reduce AB staining in the cortex and the hippocampus
86
What does overexpression of APN cause?
- Improved memory
- Reduced amyloid
- Reduced NLRP3 expression inside the microglia
- Reduced ASC and IL-1B in the hippocampus of the AD mouse model
87
Why is AAV gene therapy good strategy?
- Do not need to worry about the side effects due to non-specific activity unlike small molecule treatments
- Can achieve treatment through only 1 dose injection unlike recombinant which requires repeat infusion
88
What are some considerations of AD treatment?
- BBB penetration
- Toxicity
- Cost and accessibility
89
How does zinc deficiency interact with the inflammasome pathway?
- Zn deficiency is common in AD
- Would this drive cognitive impairment?
- Use the AD model, treat with defcient zinc diet
- Leads to faster development of cognitive decline
- When KO NLRP3 and treat with the same deficient diet, the memory is respored
- Suggests that deficiency works through the same NLRP3 pathway driving memory impairment in AD models
90
In addition to NLRP3 inhibitors what other treatments may be beneficial in AD?
Treating co-morbidities (such as APN by exercise) and improving nutrition (such as zinc status) could reduce inflammation contributing to conditions such as AD disease
91
What is epilepsy?
- Condition in which recurrent seizures occur
- Symptoms depend on which part of the brain is affected
- Affects people of all ages
92
Outline seizures?
- A seizure is a symptom of epilepsy
- Around 40 different types of seizure
- Is a sudden transitory and uncontrolled disruption of brain activity
- 1 in 20 will have a seizure in their lifetime
93
How many people in the UK have epilepsy?
Around 0.5 mil
94
What are the 3 seizure types?
- Focal
- Generalised
- Unknown
95
What are the 4 epilepsy types?
- Focal
- Generalised
- Combined focal and generalised
- Unknown
96
What are the different potential etiologies of epilepsy (6)?
- Structural
- Genetic such as ion channel mutation
- Infectious, rise in temp can cause
- Immune
- Metabolic
- Unknown
97
What are 5 comorbidities of epilepsy?
- Cognitive
- Psychiatric
- Motor
- Social
- Sleep
98
What percentage of epilepsy is unknown?
- 65%
- Idiopathic (no apparent cause)
- Cryptogenic (a likely cause but not identified)
99
What complex/environmental factors can lead to epilepsy?
- Abnormal brain wiring
- Chemical (neurotransmitter) imbalances (excitatory vs inhibitory)
- Ion channel dysfunction
- Abnormal connections made when attempting to repair an injury
100
What is NICE's recommended symptomatic treatment for epilepsy?
Monotherapy and then adjunctive treatment/dual therapy
101
What are 5 treatments for epilepsy?
- Carbamazapine (increase sodium channel inactivation and increase GABA)
- Ethosuximide (decrease T-type calcium channels)
- Lamotrigine (sodium channel blocker)
- Levetiracetam (blocks synaptic vesicle release)
- Sodium valproate (increase GABA)
102
What percentage of patients have some seizure control with AEDs?
70%
103
What percentage of patients are drug-refractory and what are some potential treatments for this?
- 30%
- Surgery to remove the focal point
- Deep brain stimulation
- Vagal nerve stimulation
104
What are some side effects with AEDs?
- Have to consider that patients take for life
- Memory problems
- Fatigue
- Depression
- Nausea
- Visual problems
105
Why may new drugs targetting channel pathways in epilepsy not be effective?
- Just improved molecules targetting the same pathways
- May not make headway as need a different method, a better sodium channel inhibitor won't change the overall effect
- Inflammatory drugs??
106
How can animal models of epilepsy be induced?
- Kainic acid (glutamate agonist)
- Bicuculline (GABA antagonist)
- Pilocarpine (muscarinic agonist)
- Electrical stimulation using kindling (readily stimulate to cause recurrent seizure)
107
What is a consideration for the animal models of epilepsy?
Mainly study seizures not epilepsy per se
108
How can animals have 'epilepsy' rather than seizures induced?
- Genetic absence epilepsy rat strin (GAERS)
- DBA audiogenic mice (make a noise and the mouse will have a seizure)
109
How can seizure activity be measured in both humans and animals?
- EEG
- Behaviour, for mice using Racine classification for tonic-clonic seizures
110
How can it be determined if inflammation plays a role in epilepsy?
Look for the hallmarks that can be measured to define neuroinflammation
111
How was it seen that there is an increase in inflammatory mediators in epilepsy using immunohistochemistry?
- Immunohistochemistry staining for IL-1B
- Give a kainic acid injection into the hippocampus
- See positive staining for IL-1B
- Epilepsy is one of the biggest triggers for this
112
How was it seen that there is an increase in inflammatory mediators in epilepsy using PCR?
- PCR data for proinflammatory agonist overtime IL1B, IL6, TNFa and IL1Ra
- There is an early increase in proinflammatory cytokines during a seizure/epilepsy
113
What is some clinical evidence to support the presence of inflammatory mediators in epilepsy?
- 24 hours post tonic-clonic seizure
- Looked at patient CSF using a lumbar puncture
- Increase in plasma IL-6 only in temporal lobe epilepsy
114
How was it shown that there was glial activation during epilepsy?
- Inject with pilocarpine to cause status epilepticus
- Changes in morphology of microglia (OX-42) staining
- Changes in morphology of astrocytes (GFAP) staining
- The status epilepticus changes the brain to have continuous seizures and the morphology becomes more pronounced
115
What is some clinical evidence of glial activation and IL-1B expression in epilepsy?
- Human brain tissue accessed in temporal lobe patients with hippocampal sclerosis (damage)
- Increase in IL-1B expression in endothelial and neuronal cells
- Also microglia expression of IL-1B cell types in patients with epilepsy
116
What is the evidence to show adhesion molecule action in epilepsy?
- Inject with pilocarpine
- 6 hours later, in vivo staining and fluorescence microscopy
- Imaged the surface of the brain vessels and stained for adhesion molecules
- Can see an increased expression
- When treated with diazepam can prevent the increase of VCAM
- This allowed the neurophils to become attached to the brain and migrate into the tissue
- If treated with antibodies against these molecules, the number of cells rolling reduce dramatically
- These same treatments have an anti-convulsant effect- the antibody stopped the seizures by acting against VCAM
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What effects do recombinant cytokines have on seizures?
- When treat the animal with a IL-1R antagonist, it takes linger for the animal to have a seizure and time spent in the seizure is less (anti-convulsant)
- When treat with IL-1B there is a faster onset and longer seizure activity than controls (pro-convulsant effect)
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How was IL-1 processing inhibited and what did this cause in epilepsy?
- Use kainic acid to induce seizures
- Caspase 1 inhibitor (stops the cleavage and release of IL-1B)
- Seizure onset and time is reduced
- If KO caspase 1 also see decreased activity
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What was the clinical trial of caspase 1 inhibitor VX-765 and it's results?
- 6 week randomised, double-blind, multicenter, placebo-controlled study
- Phase 2a- had safety and tolerability
- Subjects 18-64 years old with treatment-resistant partial epilepsy
- For the treatment group 20% were seizure free for a period
- But none for the placebo
- Looked like it was working
- The study was terminated due to lack of profit for the drug company
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How do DAMPs effect seizures?
- Injected HMGB1 DAMP
- Onset and seizure increased
- If KO the TLR4 receptor in which this acts, there is a longer onset and reduced time
- Shows that DAMPs are pro-convulsant
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What were the results of a combined caspase 1 inhibitor and TLR4 antagonist treatment?
- Abolishes the seizure
- Whereas carbamezipine treatment was not beneficial
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How did anakinra treatment effect an epileptic patient in 2 case studies?
- EEG seizures per hour, normally severe (20 mins of activity)
- Not responding to any medication
- Seizure episodes go away with IL-1Ra anakinra
- It is protective
- Also in another patient measured ESR (how much blood cells clot- measure of inflammation)
- Anakinra drops these markers, there is resolution of systemic inflammation and near complete resolution of seizures
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When and by who was multiple sclerosis (MS) first described?
- 1868
- Jean-Martin Charcot
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What was seen in the first description of MS?
- Was on post mortem tissues
- Saw loss of nerve fibres, neurons and their tracts
- Pockets of inflammation which was described then as swelling or oedema around the vessels
- Glial scarring or sclerosis which is where the name of the condition comes from
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What is MS?
- A slow progressive CNS disease characterised by the destruction of the myelin sheath around axons in the brain and spinal cord
- It is an immune attack against oligodendrocytes (and/or Schwann cells) within the spinal cord causing destruction of the myelin sheath and impacting the transduction signal in neurons
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What are several neurological symptoms of MS?
- Loss of sensation (touch, taste)
- Motor coordination impairment
- Vision impairment (involuntary eye movement)
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What are the 2 different pathogeneses of MS? and their prevalence?
- Periods of attack- 85%
- Primary chronic progression- 10-20%
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What are the 3 stages of MS characterised by periods of attack?
- Relapse-remitting (have remission in which functions recover fully)
- Relapsing-persistant (periods of attack but after function does not recover fully)
- Secondary progression (In both cases, they often transgress at the late stage so there are no more attacks but increased progression of the disease)
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What is the incidence of MS?
- 1 in 1000 in the UK
- Primarily affects young people between 20-40 but average age of onset for women is 28 and men is 30
- The ratio for women to men is 3:1
- Affects mainly caucasians
- Is the most common neurodegenerative disease of young adults (1 per 400)
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What are the two ways to primarily diagnose MS?
- Happens if people experience motor of sensory problems
- Use MRI
- Or CSF
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Outline how MS diagnosis occurs via MRI
- White lesions indicate pockets of inflammation- vascular oedema
- There is breakdown of the BBB and oedema around the vessels
- During the late stages there is brain atrophy with widened lateral ventricles and cortical sulci
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Outline how MS is diagnosed using CSF analysis
- Carry out a lumbar puncture
- CSF protein electrophoresis is carried out
- Shows oligoclonal IgG bands in more than 90% of MS cases
- Shows abnormal levels of immune reaction
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Outline a behavioural diagnosis technique for MS
- Visual evoked potential
- If the visual pathway is affected, expose the patient to two different stimuli
- Can then look at the response of the VEP and identify an abnormal response
- MS have delayed latency and a smaller response
- Measures the speed and strength of conduction along the optic nerve pathways
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Besides VEP, what are some other behavioural techniques for the diagnosis of MS?
- Auditory evoked potentials
- Sensory evoked potentials
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What is saltatory conduction?
- The action potential jumps from node to node
- Voltage gated Na channels are present only at the nodes of Ranvier
- Allows for fast axonal conductance
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What is the difference in conductance speed between a myelinated and unmeylinated axon?
- Unmyelinated is 0.5-10m/s
- Myelinated is 150m/s
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MS is a chronic autoimmune disease that affect what?
Any part of the central nervous system
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Where does demyelination of nerve cells that are wrapped in myelin sheath occur?
- Nerve cells in the white matter of the brain
- Axons and nerve cells in the spinal cord
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What are the primary pathological features associated with the symptoms seen in MS?
The degeneration of the myelin sheath around the acons leading to axonal degeneration
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What are the main symptoms of MS?
The disturbances of sensations and impairment of motor coordination
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What are 4 environmental risk factors for MS?
- Viruses have been implicated in MS pathogenesis, in particular: measles, rubella, mumps and herpes
- Bacterial infections
- Nutritional and dietary factors such as exposure to animals, minerals, chemical agents, metals, organic solvents and various occupational hazards
- Other environmental factors that are unknown
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What are proposed risk factors of MS?
Unknown but occurence of the disease is associated with genetic and environmental factors
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What are the mechanisms and potential causes of MS?
- Complex immune/genetic disease
- Mediated by autoimmune processes- activation of B and T cells
- Inflammation of the CNS (brain and spinal cord) white matter
- Glial scarring, axonal degeneration and neurological deficit
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In terms of inflammation and axonal damage, what occurs in MS?
Inflammation is followed by a loss of myelin sheath and axonal damage
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What is the genetic association with MS?
- With chromosome 6
- This contains a cluster of genes, with some having MHC class
- Within the MHC class of genes are more genes which code for myelin proteins
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What is the major histocompatability complex (MHC)?
- Plays a pivotal role in the execution of the immune system
- Divided into three subgroups called class 1, 2 and 3 (activate the immune system depending on the type of infection such as 1 is more viral)
- Found on antigen-presenting cells
- Anchored in the cell membrane of APCs where they display short plypeptides to T cells, that are recognised by the T cell receptors
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What occurs in the adaptive immune response in normal individuals and those with MS?
- Normally, T cells should ignore self peptides while reacting appropriately to foreign peptides
- In MS, recognition of self antigens (meylin proteins) causes an immune cell activation
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What happens with the BBB in normal individuals and in those with MS?
- The membrane that controls the passahe of substances from the blood into the CNS and vice versa
- Physical barrier between the local blood vessels and most parts of the central nervous system
- Normally, the CNS is inaccessible for T lymphocytes due to the BBB
- In MS autoreactive T cells breach the endothelial barrier and enter the brain
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What are the 10 steps of pathogenesis of MS?
1. An infection by a virus or bacteria
2. Antigen gets into the blood stream andis digested by a macrophage (APC)
3. The macrophage cell displays the antigen with an MHC molecule
4. The MHC-antigen can be recognised by special receptors on the surface of the T cell
5. The activated T cells cross the BBB (bind to adhesion molecules on endothelial cells)
6. T cells release chemicals called proteases (MMPs) which breakdown the BBB and the blood supply has uncoupling and free radicals
7. Once in the CNS, T cells encounter local APCs such as microglia which normally clear debris from the myelin sheath
8. The T cells are locally re-activated when they recognise their antigen on the surface of the local APCs (epitope-self-protein of the MBP, MOG or MAG)
9. The activated T cells secrete cytokines that stimulate the microglia and astrocyes, recruit additional inflammatory cells and induce antibody production
10. Demyelination, oligodendrocyte degeneration occurs
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What is a possible early key mediator in MS?
MMPs and free radicals
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What happens during the early stages of MS?
Circulating immune cells invade the CNS due to a leaky BBB
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What is the ceramide pathway important for?
Regulating the myelin sheath
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Why are the ceramide molecules important in MS?
- They are lipids that are metabolic and catabolic which produce different proteins
- Such as sphingosine which is pro-inflammatory
- And S1P which is anti-inflammatory
- The balance of these determines whether the signal will be pro or anti
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What is a drug that can bind to S1P?
FTY720-P (Fingolimod)
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What is a part of the ceramide pathway which makes up the myelin sheath?
Sphingomyelin which is broken by sphingolyelinase to ceramide
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What does fingolimod do?
- Blocks the recruitment of T cells from the lymph node to the circulation so less T cells
- It is also anto-inflammatory in the brain as can cross the BBB
- Causes internalisation of the SP1 receptor so T cells won't leave the lymph node as cannot recgonise
- It reduces BBB permeability and migration of macrophages by decreasing adhesion molecule expression
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What was an experiment done to see how finglimod acts on the BBB?
- An in vitro experiment culturing endothelial cells to see the function and permeability of the BBB overtime
- There is an increase in permeability unless treated with the drug or an S1P5 agonist to decrease the permeability
- So it decreased the migration of monocytes and expression of adhesion molecules
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What is the evidence that phingomyelinase is involved in recruiting T cells in MS?
- An immunihistochemistry study to show that that this is expressed in the blood vessels in MS lesions
- Showed that sphingomyelinase expression colocalises with ICAM1 expression (increases adhesion molecules)
- Sphingomyelinase KO reduces T cell infilatration using TFNa on endothelial cells. If it is not KO'd then T cell and neutrophil migration increases in the presence of TNFa
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What does sphingomyelinase do?
Is an enzyme that co-localises with an adhesion molecule to increase T cell migration
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What is natalizumab?
- Anti-VLA-4 Ab
- Targets the infiltration of T cells using an antibody against alpha 4 integrins and binds to VCAM
- It sequesters leukocytes in the blood stream, preventing crossing of the BBB into the CNS
- Acts by targeting the alpha 4 integrin contraining adhesion molecules required for migration
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What is a possible alternative therapeutic treatment for MS?
Anti integrin (in clinical trials)
