Lectures 15 & 16 - Neurodegeneration

Question 1

Define neurodegeneration

Answer 1

The loss of neurons, where neurons are dying because of disease or some other process

Question 2

Describe features of chronic neurodegeneration and example of diseases

Answer 2

Slow onset
Progressive
Last for many years

Diseases:
-Alzheimer’s Disease
-Parkinson’s Disease
-Multiple Sclerosis
-Prion Disease

Question 3

Describe features of Acute neurodegeneration and examples

Answer 3

Sudden onset
Secondary progression after initial incident
Generally patients quite healthy prior
Associated with a trigger event

Examples:
-Traumatic Brain Injury
-Stroke
-TIA (transient ischaemic attack)
-Intracranial Haemorrhage

Question 4

Describe statistics and features of strokes

Answer 4

Events of stroke are generally similar across all acute comditions (apart from traumatic brain injury)

4th most common cause of death in UK – 5% men, 8% women

152,000 strokes every year (100,000 are first strokes)

Two thirds will survive

Largest cause of severe disability (500,000+)

31% of survivors need help caring for themselves, 71% have speech impediments and 16% have to be institutionalised

1 in 53 people in the UK (1.2million people) are “stroke survivors”

Question 5

Describe the prevalence and risk of strokes

Answer 5

Incidence doubles every decade after 55

Ageing population leading to increased prevalence

Strokes more common in men but more women die

Lower risk in caucasian population compared to many other ethnic groups

Question 6

Explain the stroke mortality trend in the UK

Answer 6

Mortality reducing due to improved clinical pathways, wider access to specialist stroke wards and increased public and professional awareness (e.g. FAST campaign)

Number of cases affected by a number of modifiable risk factors:

– Hypertension (increasing/100000 population)

– Diabetes (type II increasing/100000 population)

– Hypercholestrolaemia (managed effectively by statins etc)

– Previous stroke or transient ischaemic attack

• BUT ageing population means overall prevalence is still high

Question 7

What do we mean by stroke?

Answer 7

blood supply to part of the brain has been interrupted

Question 8

What are the different types of stroke?

Answer 8

Ischaemic Stroke:
-85% of all strokes ≈ 30% Mortality
-Ischaemia – loss of blood supply
-Blood vessel that delivers blood to region of brain has been blocked by something
-signs and symptoms directly relate to which blood vessel is being blocked

Haemorrhagic Stroke:
-15% of all strokes ≈ 70% Mortality
-Artery bursts – blood leaking into brain from artery and any brain tissue that would’ve received blood from that arteries is now not receiving

Question 9

What is the most common blood vessel affected in strokes

Answer 9

Middle cerebral artery (>50%)

Question 10

What happens in a stroke where the middle cerebral artery is blocked?

Answer 10

blood flows up from outside the brain and then upwards towards the top of the brain, depending on where you block the artery will have different effects

Question 11

What is the deterioration or recovery from a stroke partially dependable on?

Answer 11

If there’s tissue that’s rescuable then there’s slight recovery, balance of this is dependent on how much blood supply there is to the surrounding tissue

Question 12

What happens to a structural lesion after a stroke

Answer 12

After a few hours and days the surrounding tissue will start to pull more blood and oxygen from other blood vessels, eventually after a week or so, you end up with a structural lesion, the core area of tissue, made up of dead neurons and astrocytes, being carried away by microglial cells

Question 13

Explain how neurodegeneration occurs in stroke

Answer 13

Stroke = loss of blood supply
Loss of blood supply = Loss of oxygen and glucose due to lack of energy store
leaidng to energy faikure (cells loose the ability to make ATP) this leads to:
-Loss of protein synthsis – decrease in protective proteins
-faulure of Na+/K+ ATPase – therefore depolarisation
-Anaerobic metabolism – decrease in pH

This depolorisation leads to calcium influx and neurotransmitter release (glutamate)

Also presents of lactate from anaerobic metabolism causes calcium influx into the cell

Calcium is toxic for a number of reasons:

Triggers activation of enzymes e.g. proteases that break down the cytoskeleton and lipases that break doen the cell membranes

Cytotoxic oedema – water drawn into cell, it swells up and can cause the cells to explode, or blockge of more blood vessles

Go intonmitochondria and produce free radicals

Other things

If unregulated these things lead to cell death

Reperfusion:

Cell death and inflammatory response created allow reprofusion

Question 14

Explain the mechanisms of neurodegeration from excitotoxicity

Answer 14

Death by overexcitation of cells, incraesed calcium therefore increased glutamate

Leading to cell death

Question 15

Explain the mechanisms of neurodegeration from free radical toxicity:

Answer 15

Increased calcium from depolarisation leads to increased free radicals, leading to cell death

Question 16

Explain the experimental evidence that showed depolarisation and glutamate release in strokes

Answer 16

done in rats

probe into the brain - measured the amount of glutamate released in ten minute time windows

injected endothelium 1 (ET1) - a vasoconstrictor into the Middle cerebral artery
blood flow through it stopped - an experimental model of stroke

the amount of glutamate that was released into the brain went up very quickly and very significantly

confirms depolarisation and glutamate release in strokes

added drug called LY377770 (blocks calcium release) therefore because there was major decrease in glutamate release when this drug present, further conforms calcium drives the glutamate release

Question 17

Explain the extra part of the experimental stroke model that showed that glutamate is toxic

Answer 17

When drug LY377770 was added, the size of lesion in the brain was greatly reduced

This drug blocks calcium release and therefore glutamate release, and so shows that glutamate is toxic

Question 18

Explain an experiment that showed that glutamate antagonists are neuroprotective

Answer 18

MK-801 is a NMDA receptor antagonist (blocks it)
glutamate can still bind but with MK-801 there calcium isn’t allowed through
experiment sh0wed if you block NMDA receptors then you reduce to nearly nothing the amount of brain tissue lost

Question 19

What does AMPA mimic the effects of?

Answer 19

Glutamate

Question 20

What happens if you inject AMPA directly into the brain?

Answer 20

It activates AMPA/Kainate receptors, causing cell death

Question 21

What happens if you inject NMDA directly into the brain

Answer 21

Cell death, caused by activation of NMDA receptors

Question 22

What happens if you inject Glutamate directly into the brain?

Answer 22

nothing happens (no cell death) due to glutamate being he endogenous neurotransmitter, brain is good at removing it, glutamate itself is not toxic

Question 23

Explain an experiment that shows the potentiation of glutamate toxicity

Answer 23

Cells exposed to a dye, that will glow orange of the cell dies.
normal untreated - cells alive
Expose cells to hypoxia for 60min(remove oxygen but leave glucose) - cells dont die
add 1mM Glutamate - cells surivie also
now together - same ampunt of glutamate and same period of hypoxia - all the cells start to light up

presence of both glutamate and lack of oxygen that makes the glutamate toxic

Question 24

Explain why glutamate becomes toxic in stroke/hypoxic conditions

Answer 24

Due to lack of glutamate transporter function
Transporters for uptake of glutamate function by co-transport with sodium, so a sodium gradient is needed for it to functin. In hypoxic conditions, this gradient isnt present. and tehfore glutamate stays in the synaptic cleft and causes signals to be sent though the brain, leading to a stroke

Question 25

What enzymes are free radicals regulated by in neurons?

Answer 25

Superoxide dismutases (Cu/ZnSOD, MnSOD) Glutathione peroxidase

Question 26

If free radicals aren't regulated, what happens?

Answer 26

React strongly with lipids, proteins and nucleic acids which results in cell death In particular bind to lipids start a chain recation that breaks down the cell membrane

Question 27

Explain how calcium overload in ischemia leads to the build up of free radicals

Answer 27

Calcium gets taken up into the mitochondria which the affets all of the enzyme processes within the mitochondria, causing the mitichomdria to depeolorise This has a number of effects: - drives production of ROS (free radicals that contain oxygen) - breakdown of xanthine - nitric oxide production these all lead to overproduction of free radicals, particularly ROS and particularly superoxide (-radical-O2minus) also interested in nitric oxide

Question 28

how is Nitric oxide formed?

Answer 28

By nitric oxide synthase (NOS)- converts arganine into -radical-NO + Citrulline + H20 Called a specific type of NOS depending on where put letter at start: Endothelial = eNOS Neuronal = nNOS Inducible = iNOS Mitochondrial = mtNOS

Question 29

Explain how the free radical nitric oxide itself isn't that toxic, and when it does become toxic

Answer 29

Can diffuse into and out of mitochondria, not that toxic becomes toxic when interacting with superoxide to form peroxynitrate which is highly toxic: -radical-NO + O2- ---> -radical-ONOO-

Question 30

Explain an experiment that shows the role free radicals play in neurotoxicity

Answer 30

neurons, incubated with a dye that glows green when free radicals are present In control cells - small amounts of free radicals inside mitochondria Remove oxygen and glucose from cells - within 5 mins lots of free radicals produced - across whole cell body, not limited to mitochondria number of neurons that die in ischemia is high block nitric oxide production by L-NAME or Block supoeroxide production by TEMPO toxicity goes down in both cases, showing you need both for free radical toxicity

Question 31

When is acute treatment used for stroke, and what are the desired outcomes?

Answer 31

used just after someone has had a stroke. Hopefully reduce neuronal degradation and early mortality. unfortunatly there are very little acute treatments available for strokes

Question 32

What drug is used to treat stroke?

Answer 32

Tissue plasminogen activator (tPA) - activates fibrinolysis to allow the body to breakdown the clot thats blocking the artery and return blood flow Edarovone (a free radical scavenger) - licensed in japan only - none of the US and UK trials showed efficacy

Question 33

What is the original trial data for the r-tPA trial (1995)

Answer 33

* 30% more likely to have minimal or no disability after 3 months than placebo * 1.7x more likely to have a better outcome than placebo * 17% mortality at 3 months vs 21% in placebo * BUT: 6.4% of r-tPA-treated patients had an intra-cerebral haemorrhage within 36 hours compared to 0.4% in placebotreated patients

Question 34

Explain features of tPA

Answer 34

* Age limited (18-79); multiple contraindications * Requires absolute certainty that there is no intracranial bleeding * Very limited time window of efficacy (within 4.5hrs of onset) * Very small percentage (12-20%) of patients actually receive r-tPA (Alteplase)

Question 35

Explain some drugs to treat stroke that made it to clinical trials but showed no efficacy

Answer 35

Selfotel - NMDA antagonist YM872 - AMPA antagonist Nimodipine - Ion channel modulator Cerovive - Free radical scavenger

Question 36

Why do so many stroke drugs not show efficacy in clinical trials/not have effect in preventing stroke

Answer 36

Most mechanisms occur within 2-3 hours of the onset of stroke Usually strke occurs in more elderly person, living alon or have a stroke in the night ad have no contact with people likely they wont get inti a clinicak managment situation within the time window that these events are occuring

Question 37

Why are outcomes of stroke drugs that didn't show efficacy in clinical trials for humans good in animal trials

Answer 37

In animals - drugs effective if you pretreat before you start stroke, or give drug immediately after, in a real world clinical situation, very difficult to get drugs into patients in this early time period

Question 38

Whats is a potentail second area for drug targets in stroke?

Answer 38

Inflammation that happens after initial excitatory period

Question 39

What has reduced the time for people to be seen for stroke?

Answer 39

FAST campaign and better treatment planning within hospitals, means time windows where drugs would be effective may be possible

Question 40

What is initial lesion expansion in stroke due to?

Answer 40

Mechanisms that occur first e.g. glutamate toxicity, free radicals ect.

Question 41

What are the changes in size of lesion after the first few hours due to?

Answer 41

Ongoing chronic inflammation, secondary processes

Question 42

Explain gene expression following ischaemia

Answer 42

1. Activation of immediate early genes (IEG) e.g. C-fos, C-jun - these genes drive transcription of other genes 2. Expression of heat shock proteins (HSP) - HSP-70, HSP-72 - stress proteins - activated in cells in times of stress - stop protein degreadation/correct protein shape 3. Expression of: -pro-inlammatory cytokines - TNF-a, IL-1b, IL-6 -Adhesion molecules - ICAM-1, P-selectins - expressed on blood vessles and used to recruit circulating inflammatory cells into tissues -Growth factors e.g NGF, BDNF, expressed prodominantly by neurons but also by astrocytes and microglia - these are survival factors for neurons Much later on - protease inhibators and remodelling factors, expressed as a way to try and grow new blood vessles and remodel the tissue thats been changed

Question 43

Explain the recruitment of inflammatory cells following ischaemia

Answer 43

Circulating inflammatory cells get recruited: - PMNs, monocytes and macrophages - quite delayed compared to a peripheral injury where you'd expect these cells to be recruited very quickly (brain much slower)

Question 44

In normal conditions of the brain, what do cytokines do?

Answer 44

There are NO cytokines expressed, they're only expressed as a response to a stroke (e.g.)

Question 45

Explain the peripheral inflammatory response

Answer 45

- expression of adhesion molecules -recruitment and activation of phagocytes -increase vascular permeability -all driven by cytokines

Question 46

Explain the CNS inflammatory response

Answer 46

-upregulation of cytokines -inflammatory response delayed by hours-days -Local activation of microglia - to start removing damaged tissue and generate scar tissue - DON'T get significant tissue swelling - due to brain being in skull - no way for it to expand, if it did it would lead to increased intercranial pressure - BAD

Question 47

Describe features of Interleukin - 1b

Answer 47

- predominant form of IL-1 in the brain - formed by activation of IL-1 converting enzyme (caspase 1) - Binds to specific IL-1 receptors (IL-1R) - associated with increased neuronal death

Question 48

How is the binding of IL-1b to IL-1Rs associated with neuronal cell death

Answer 48

increases permeability of endothelial cells upregulates adhesion molecules causes proliferation of astrocytes and microglia production of prostoglandins production of Nitrous oxides induces other cytokines e.g. TNF-a

Question 49

What is the endogenous inhibitor of IL-1R?

Answer 49

IL-1ra (IL-1 receptor antagonist)

Question 50

Explain an in vitro experiment that shows the role of IL-1

Answer 50

Cell culture: - Control conditions: Oxygen and glucose deprivation from cells to mimic ischaemic conditions Results: control - small amount of cell death adding IL-1 - significant increase in cell death adding IL-1ra - same as control IL-1ra and IL-1 - slightly lower than control, shows it was mediated through IL-1R

Question 51

Does IL-1 cause neurodegenration on ots own?

Answer 51

NO - it needs to be in ischaemic conditions e.g. stroke if you injected someone with IL-1 it would make them feel bad but no neurodegeneration would occur

Question 52

Explain an in vivo experiment that shows IL-1 causes neurodegeneration in stroke

Answer 52

- Done in mice - control conditions - middle cerebral artery occlusion control conditions cause tissue damage Increasing levels of IL-1ra = neuroprotective tells you that its blocking the effects of IL-1 thats released from the tissue if you use too much IL-1ra you get a 'rebound effect' which is suggestive of something more complex going on

Question 53

What is IL-1ra licensed clinically to treat? what may it treat in the future if clinical trials are successful?

Answer 53

Treats rheumatoid arthritis may treat stroke in future

Question 54

Explain the clinical trail undergone to see of IL-1ra could be used to treat stroke

Answer 54

patients who had a clinically defined stroke and who arrived in A&E within 6 hours of the onset of symptoms were randomly allocated to a placebo group or a 72hr infusion with IL-1ra 3 months later they were assessed: two sets of data collected: - Barthel index - modified rankin scale both measures of how independent a person is, further up on scale, more independent you are Results: proportion of most independent patients was higher for treated group had no effect at all on patients who were more severely affected by initial strokes its able ton improve quality of life of patients who had some effects of the stroke, but of you had a more serious stroke it had no effect

Question 55

Explain how in certain contexts its been seen that IL-1 is neuroprotective

Answer 55

– increase glial activation and proliferation – induce synthesis of nerve growth factor – enhance neuronal sprouting – promote neovascularisation – decrease calcium entry into neurones – enhance GABA activity * ….all of which may be neuroprotective

Question 56

Why May the certain contexts that IL-1 is neuroprotective in, not be correct?

Answer 56

could be an artefact of In vitro systems, they don't have blood circulating, context may be different in body experiments generally requires pre-treatment for several hours – IL-1 not usually increased in the brain prior to ischaemia

Question 57

What raises red flags about completly blocking IL-1?

Answer 57

IL-1 was seen in some in vitro experiments to pose neuroprotective effects dosage with toon much IL-1ra may block some of these secondary processes that are happening

Question 58

What is the link with TNF-a and neuroprotection/degeneration?

Answer 58

There is no confirmed view, literature has shown both neuroprotective and neurotoxic effects

Question 59

How is TNF-a neuroprotective (experiments)

Answer 59

TNF receptor KO mice - increase infarct (necrosis): Bruce et al 1996, Nature Med 2:788 TNF decreases glutamate toxicity in vitro: Cheng et al 1994, Neuron 12:139

Question 60

How is TNF-a neurotoxic (experiments)

Answer 60

TNF increase infarct volume - MCAO: Barone et al 1997, Stroke 28:1233 TNF binding protein decrease infarct volume Dawson et al 1996, Neurosci Lett 218:41

Question 61

Explain experimental procedures showing pre-treatment with TNF is neuroprotective in ischeamia

Answer 61

Cultured hippocampal slices exposed to experimental ischaemia, assessed 24 hrs post ischaemia Control - showed some cells dead Pretreated cells with TNF for 24 hours before inducing ischaemia - amount of cell death significantly reduced

Question 62

Explain experimental procedures showing post-treatment with TNF is neurotoxic in ischeamia

Answer 62

Cultured hippocampal slices exposed to experimental ischaemia, assessed 24 hrs post ischaemia Control - showed some cells dead if you delay addition of TNF to after the period of ischemia (and still allow the cells to recover for 24 hours) get recruitment of cells which would normally survive in this region and causes them to die.

Question 63

Is TNF itself neurotoxic in stroke?

Answer 63

no - but it can recruit cells to places where they will die - thus neurotoxicity

Question 64

Describe part of the process of TNF signalling

Answer 64

TNF Activates transcription factor NFkB which causes many gene expressions under normal situations: mitochondria are producing ROS ROS signal NFkB activation which then causes gene transcription one thingthat it drives trascriptiob of is mnSOD which detoxifies free radicals therfore negative feedback loop Under NfkB activation by TNF: increases superoxide in mitochondria (process not 100% known) which then increases the drive of trabscripton factors (p65, p50) which increases mnSOD therforeb the effect of the feedback loop is increased

Question 65

Explain why in pre-treatment with TNF, neuroprotection occurs

Answer 65

Enough time for the TNF signalling cascade to occur and therefore superoxide dismutases to be made

Question 66

Explain why in post-treatment with TNF, neurotoxicity occurs

Answer 66

ischaemia produces superoxides (ROS) addig TNF which is adding the additional burden of ROS into the cell, causing cells that would normally survive the initial ischaemia to have this extra burden of superoxide production, which is then leading to cell death

Question 67

Whats the main difference between stroke and traumatic brain injury

Answer 67

stroke affects people of older age brain injury affects predominantly under 35s

Question 68

Explain an experiment done to measure cytokine levels in traumatic head injury

Answer 68

cerebral microdialysis: Used a microdialisis probe to take out extracellular fluid frim around the injury and measure cytokines produced by the brain around the injury determined concentration of IL-1, IL-6 and NGF Outcome: measurments standardised on the glasgow outcome scale -5pt scale depending on how well you recover post-injury results: IL-1 didn't have any difference on outcome NGF - also no significant difference IL-6 - patients with high IL-6 was a predictor for a good outcome this was surprising as IL-6 is pro-inflammatory Winter et al. (2004)