Intro To Neuroinflammation Flashcards

Question

What is the chronic inflammatory response?

Answer 1

Sometimes resolution does not occur properly this is a large reason for lots of disease Due to inappropriate pro-inflammatory/anti-inflammatory balance

Answer 2

Acute as protects tissues from injury/disease

Answer 3

Rheumatoid arthritis (collagen is detected by immune cells and sees as foreign so attacks) Crones MS AD Diabetes Atherosclerosis (flaring of deposition of cholesterol in the joints) Allergies

Answer 4

- Had tolerance to normally rejected stimuli such as tissue grafts - At steady state, no innate immune cells and adaptive immune cells - No lymphatic vessels/lymph nodes - Brain is seperated from the periphery by intricate system of barriers (3 meningeal layers) - BBB has low permeability and restricted access of molecules and minimal entry of immune cells (at steady state)

Answer 5

- Blood-meningeal barrier - Blood-CSF barrier - BBB

Answer 6

- Dura mater - Arachnoid - Pia

Answer 7

- Low permeability to plasma proteins and cells due to... - Fewer caveolar (vesicles which transport molecules from the blood) - Low expression of adhesion molecules (on the endothelial cells, helps immune cells adhere to endothelia) - Tight junctions between the endothelial cells help lock the cells together

Answer 8

The brain contains resident immune cells called microglial cells Lymphatic vessels are associated with the brain The brain releases inflammatory mediators The BBB can become disrupted

Answer 9

- Derived from the myeloid/macrophage lineage - Rapidly activated in response to CNS infections/injury (first responders in the brain who can sense changes and respond to the danger and go to the site of injury to resolve the situation - Express PRR (so respond to PAMPs and DAMPs)- so can respond to pathogens too - Act as antigen presenting cells (express MHC molecules) and engulf debris and dead cells - Produce a large amount of inflammatory mediators such as cytokines and chemokines

Answer 10

- Astrocytes (supporting cells to recycle neurotransmitters): PRRs, cytokines and chemokine receptors, inflammatory mediators, involved in glial scars - Neurons: cytokines and chemokine recepotrs, inflammatory mediators, neuro-immune interactions - Oligodendrocytes (helps form the myelin and speed up impulses): express main cytokine and chemokine recepotrs, key inflammatory mediators, key in pathogenesis of MS - Endothelial cells: key sensors of periphera;/central infection as PAMPs/cytokine receptor expression, neurophil attachment and extravasation into brain tisse, chemokine production to induce peripheral acute phase response to central injury

Answer 11

- In the meninges - Connect to CNS to cervical lymph nodes - Enable cells to enter draining lymph nodes - Meninges contain innate and adaptive immune cells - Meningeal DCs survey brain parenchyma - Assist in CSF drainage

Answer 12

- Increase in caveolae (trancytosis) which is trancellular - Increase in adhesion molecules - Paracellular tight junction disruption

Answer 13

- Glial cell activation - Synthesis of inflammatory mediators - Oedema (not good as restricted room in the brain) - Expression of adhesion molecules - Invasion of immune cells - Disruption of BBB - Lymphatic system in meninges

Answer 14

- They are very sensitive to cytokines and chemokines as at a steady state these are low - When get regulation they are sensitive and can easily die

Answer 15

Inflammation Is an ancient branch of immunology existing to at least some extent in all metazoan organisms

Answer 16

- Swelling - Redness - Recruitment of leukocytes

Answer 17

When excessive or dysregulated

Answer 18

- Acute- such as tissue injury or stroke (inflammatory response then resolution) - Chronic- persistent contributing to diseases such as AD

Answer 19

- Necrotic tissue with area of infarct - Molecules that are normally intracellular are released (normally never sensed by the immune system) - They are then sensed by the immune system- these are collectively termed DAMPs - Macrophages (or microglia in the brain) sense the prescence of DAMPs as they express pattern recognition receptors which sense DAMPs - Transmits signals inside these cells which causes the production of inflammatory cytokines - The first produced is normally IL-1 and this triggers the activation of others due to pro-inflammatory actions. Recruits other inflammatory cytokines such as neutrophils - Neutrophils normally circulate in the blood and migrate into the tissue if there is an inflammatory signal. They are armed with indiscriminate weapons to kill cells. - In sterile inflammation there is no pathogen to kill, so the neurophils damage the cells - Causes a cycle of damage as more injury due to neutrophils causes more damage and the cycle continues

Answer 20

- Neutrophil - Monocyte - Basophil - Eosinophil - Natural killer cell

Answer 21

- Macrophage - Microglia (brain specific) - Mast cell - Dendritic cell

Answer 22

- Secreted inflammatory mediators - Coordinate inflammatory/immune responses - Can be pro-or anti-inflammatory - Their overproduction/dysregulation is a majorr contribution to disease

Answer 23

Immunosuppression

Answer 24

Pro-inflammatory response

Answer 25

- Lymphokines - Interleukins - Chemokines

Answer 26

Produced by lymphocytes, coordinate T-cell responses

Answer 27

Produced by mainly leukocytes. Multiple inflammatory and immunoregulatory effects

Answer 28

So called because they induce chemotaxis (cell movement). Required for recruitment of immue cells to infected/injured tissue Set up gradients for white blood cells to follow into tissue and cause inflammation

Answer 29

- Is the earliest response of innate immunity - Dependent on pattern recognition receptors on immune/host cells. - Dependent upon the recognition of conserved microbial or damage signals by the cells of the host innate immune system

Answer 30

- Host cells expressing pattern recognition receptors are cells of the innate immune system (macrophages, dendritic cells, microglia) - Come into contact with a microbial signal (PAMP) analogous to DAMP and trigger the response, signalling cascades in the immune cells - Link to signalling pathways that result in the expression of pro-inflammatory cytokines

Answer 31

Toll-Like receptors (TLR's)

Answer 32

- Membrane spanning, large family of receptors (1-13) mainly in the plasma membrane (some endosomes) - Diverse ligands and locations - Respond to PAMPs and DAMPs which are Motifs presented by pathogenic organisms present in endogenous molecules 'shielded' from the immune system until after an injury.

Answer 33

- PAMP or DAMP from the EC space bind to the Leucine rich repeat domain (LRR) which is the sensing domain on the TLR - This causes receptor dimerization and the pulling of the two domains together (TLR domain and LRR domain) - This propogates a signal inside the cell

Answer 34

(Pro-inflammatory molecules) - Bacterial endotoxin (TLR4) - Peptidoglycan (TLR2) - Flagellin (TLR5) - Single stranded viral RNA (TLR7/8) - Double stranded viral RNA (TLR9)

Answer 35

- An endogenous molecule released during cell death and modified during disease - Heat shock proteins which are IC and released when the cell dies - Uric acid crystals - ATP (when released at high levels during cell death becomes a DAMP) - DNA (when comes out of the nucleus or mitochondria into the cytoplasm, triggers the PRRs) - B-Amyloid - Some cytokines such as IL-1a (nuclear protein which isn't normally released)

Answer 36

- Pro-inflammatory gene expression - NF-kB is the major transcription factor involved (pathway)

Answer 37

- Nuclear Factor kappa-light-chain-enhancer of activated B cells - Exists in the cytoplasm as an inactive dimer (p65/p50), retained in the inactive form by binding the inhibitor Ik-Ba - Dissociation from Ik-Ba occurs after cellular stress, cytokines, TLR receptor activation

Answer 38

- PAMP/DAMP such as heat shock protein binds to the leucine rich domain of TLR4 - TLR4 will dimerize and interact with another LTR4 molecule to start signalling (propogate the signal) - This recruits adaptor proteins such as TIRAP and MyD88 - The chances of a molecule colliding is remote (due to small size) therefore, creates micro-domains or scaffolds by foeming a complex pulling other signal molecules together - Kinases are drawn to the adaptor proteins such as IRAK-4/1 (IL-1 receptor associated kinases). These phosphorylate each other propogating a signal - This comes down to the molecule TRAF6 which is an adaptor protein and further potentiated the signal to the kinase TAK1 - This activates the complex IKK which ultimately results in NF-kB activation and pro-inflammatory gene expression

Answer 39

- TNF receptor-associated factor 6 adaptor protein - Is a E3 ubiquitin ligase

Answer 40

- It catalyses the attachment of ubiquitin to a substrate such as lysine 63 to TRAF6, this is a signalling attachment - Causes the polyubiquitin chain to serve as the scaffold that allows the recruitment of the TAK1 and IKK complexes - TAK1 activates IKK by phosphorylation of its beta subunit

Answer 41

- NF-kB is normally maintained in an inactive state in the cystol yb Ik-Ba - When IKK is activated, it phosphorylates the inhibitor Ik-Ba at serines 32 and 36 subsequently triggering a ubiquitination of the inhibitor - This leads to polyubiquitination of Ik-Ba at lysine 48 - Lysine 48 targets the protein for degredation

Answer 42

- NF-kB detaches from the inhibitor and travels to the proteosome which is the molecular machinery inside the cell regulating the protein levels and causes degredation - L48 attachment is specific for degredation, so the inhibitor gets degregated - This allows NF-kB to migrate into the nucleus and induce the expression of inflammatory genes (thousands) - Most notable are the cytokines IL-1B and TNF-a

Answer 43

A small protein (76 amino acids)

Answer 44

A large complex protein complex that recognises and degrades ubiquitinated proteins. Thus regulating the concentration of certain proteins within the cell

Answer 45

- A scaffold on TRAF6 to recruit and activate TAK1 and subsequently IKK - Target Ik-Ba for degredation by the proteasome thus enabling NF0kB to migrate to the nucleus

Answer 46

When the level of the inhibitor goes down

Answer 47

- It oscillates, going in and out of the nucleus - The level of the inhibitor goes up and down - The frequency of these oscillations can determine the type of gene expressed

Answer 48

Pro-IL-1B (this product is an inactive precursor due to 'pro')

Answer 49

- Is a prototypical pro-inflammatory cytokine - Is expressed after TLR activation - One of the major damaging mediators of inflammation in the brain (when we block this, we protect the brain) - Is produced as an inactive precursor, pro-IL-1B, that requires processing by caspase-1 prior to secretion (stays inside the cell until acitvated by caspase 1) - Is secreted by a non-conventional mechanism once activated

Answer 50

- Cysteine protease - Has some catalytic activity - Produced as an inactive zymogen (pro-caspase1) - When pulled together in a scaffold, these molecules activate each other and cut IL-1B - Activation depends on cytosolic PRRs - The scafold in which caspase-1 is activated is the inflammasome

Answer 51

- A different class of PRR (not like TLRs), not membrane but cytosolic - Mainly of the NLR family - 23 members in humans (only some form inflammasomes) - The best studied is NLRP3

Answer 52

- PAMP/DAMP is the fist stimulus in the microglia on the TLR which triggers the NF-kB pathway - This induces the pro-inflammatory gene expression, get the precursor of IL-1B - NF-kB will also induce the expression of NLRP3 by the PRR - When information is received of further stress or threat due to PAMP/DAMP of necrotic cells, it will form an inflammasome - It then nucleates the oligomerisation of ASC which forms a scaffold upon which caspace 1 is recruited and activated - This cleaves directly IL-1B into an active form which is secreted from the cell - Indirectly, it causes the secretion of IL-1B's close relative IL-1a which is an unusual protein formed as a gene duplication of IL-1B - Unlike IL-1B which is an ancient protein present in all vertebrates, IL-1a is only in mammals, existing in the nucleus - When IL-1a is relesed, it is a protein pro-inflammatory DAMP

Answer 53

- Using microscopy - A lentivirus can be used to modify a macrophage genetically and add the ASC molecule (adaptor protein which forms the inflammasome). - A genetically encoded fluorescent protein called mCherry can be added. - When activate a DAMP, can visualise the formation of the inflammasome which is diffuse in the cell and behaves like a prion - Once the ASC molecule is activated, every ASC interacts with it, oligomerising - All the ASC in the cell will form a bright speck called an ASC speck - These are enormous inflammasomes up to 3 microns in which caspase 1 is activated on

Answer 54

- Took organotypic hippocampal brain slices in 3D, giving a more physiological way of studying microglia - Used IB4 to stain for microglia and ASC-citrine to stain for the inflammasome - Could see that NLRP3 inflammasome activation results in the formation of a large speck

Answer 55

- Seems that it is formed and synthesised on free ribosomes in the cytoplasm, and from here, trafficked to the inflammasome - Is then processed by caspase 1 and activated - The mature form moves to the plasma membrane and is secreted

Answer 56

- 99% of proteins, when secreted are translated into the endoplasmic reticulum - They are then bound to chaperones and modified as they pass through - They go through the golgi and are packaged into vesicles and then released from the cell by exocytosis

Answer 57

- Don't know - Could be that the infection shuts down the ER and the golgi and there needs to be an alternative method to get the stress signal out to the immune system - Or this method of IL-1B release predated eukaryogenesis, we know that prokaryotes and bacteria have protein transporters that can secrete and translocate directly across a membrane.

Answer 58

There are 3 proposed models (they are not mutually exclusive)

Answer 59

- IL-1B is packaged into lysosomes and targeted for degredation - When the cell receives an additional stimulus, these lysosomes were redirected - It was a regulated secretion of lysosomes and they can undergo exocytosis, fuse with the plasma membrane and secrete IL-1B - This was described in monocytes

Answer 60

- IL-1B goes into exosomes (small vesicles which but into endosomes forming multivesicular bodies) - These can get secreted or released as microvesicles where bits of the membrane pinch off after trafficking here, when under the plasma membrane - Could be important for trafficking of IL-1B to sites distant from the site of inflammation

Answer 61

- Results in the death of the cell - Pores in the plasma membrane form where it is released - Loss of homeostasis and the cell will die (pyroptosis) - Greatest evidence supports this method of secretion

Answer 62

- J Cell Sci (2007) - The processed, mature version of IL-1B does not stay in the cell for long, once produced, it leaves quickly - Stained IL-1B using a specific antibodies. - Stimulated with a DAMP (ATP), seeing the mature version leave the cell - Used a Western block to seperate the proteins according to size - In the lysate (contained within the cell) could see lots of the precursor and then the mature IL-1B being rapidly released - In the supernatant (outside the cell), could see, when mature IL-1B decreases inside the cell, it appears here - If, genetically, we take the sequence which codes the mature IL-1B, it gets secreted. Therefore, there is something in the pro-domain that anchors it inside the cell

Answer 63

- Cell Death and Differentiation (2016) - Transduced a macrophage (iBMDMs) with a lentivirus to tag the IL-1B with a venus tag (bright GFP) (pro-IL-1BVenus) - Also imaged a dye called propidium ionide which is a dye added to the cells which cannot enter the cell. - Only when the membrane is compromised and there is a pore in the plasma membrane will the dye enter the cell - Here it will bind to DNA in the nucleus and will become a bright fluorescent (can therefore see if pores have formed if this occurs) - Used ATP DAMP and as soon as the venus fluorescence disappears, the nucleus becomes fluorescent - Lots of venus fluorescence means lots of pro-IL-1B. But when activate due to DAMP, rapidly, pores form and the IL-1B goes out and the dye goes in at exactly the same time - This is evidence that IL-1B is secreted through a pore under these conditions

Answer 64

- It is a substrate for the enzyme caspase-1 (the same protein that cuts pro-IL-1B, also cuts the gastermin D) - Cutting the gastermin D forms pores in the plasma membrane - Once it is cleaves, the N terminus migrates to the plasma membrane and forms pores which are just wide enough for IL-1B molecules to pass through

Answer 65

- Journal of Cell Science (2017) - Oligomerises and form a pore in the membrane at the same time IL-1B is cleaved to a mature form (both by caspace-1) - Acts as a conduit for IL-1B secretion and allows it to leave the cell - Ultimately leads to the loss of cell viability and the cells will die

Answer 66

- J Cell Biol. (2020) - Lots of post-translational modifications - It get ubiquitinatd (know that deubiquitination is important for its activation) - Phosphorylation and dephosphorylation are important for its activation and inhibition of different phosphorylation sites with different kinases having different effects

Answer 67

Because there are no reagants or tools to study it The antibodies to NLRP3 which are normally used for immunocytic histochemistry to visualise the cell don't work They're very unreliable

Answer 68

- CRISPR tage the endogenous NLRP3 and knocked in to the endogenous locus on the NLRP3 gene an mScarlet protein. - When NLR3 is produced, it has this geneticelly encoded sensor in its c terminals - Can therefore localise the NLRP3 within cells and cell types - Can fate-track the NLRP3 inflammasomes - Can visualise NLRP3 induction (as an endogenous protein) - in vitro and in vivo availability

Answer 69

- Lentiviral expression can cause overexpression and overproduction of the gene - However, it is not being over-expressed here as it is an innate gene

Answer 70

- Need to consider which protein to use, fluorescent proteins are quite large, they often oligomerise - mScarlet is a monomer so does not oligomerise - Is a bright and stable fluorescence - Has reasonable maturing and folding time (typical GFP takes too long for the protein to fold within the time window of the experiement), is about 3 hour golding time.

Answer 71

- Take bone marrow-derived macrophages from mouse femur - Seperate proteins in a western block gel - In the WT mouse, can see that when add LPS (TRP activation), we induce the expression of NLRP3 - In heterozygous mice (one allele for WT NLRP3 and one for NLRP3 mScarlet), can see a mixture of the tagged and WT - Can see Homozygote for mScarlet as the tagged form- promising that we can pick up NLRP3

Answer 72

- WT is labelled for the ASC adaptor protein which forms specks - Scarlet can be seen co-localising with the aspect, so is sitting where it should - It forms inflammasomes in response to NLRP3 activating due to a stimulus (nigericin which is an ATP-like molecule with similar effects causing potassium ion efflux but is a bacterial toxin).

Answer 73

- iScience (2023) - Antibodies are large, so if have an antibody and a fluorescent label which sticjs to the protein of the target, there is an extra diameter, reducing the resolution of what can be seen - Therefore can use a nanobody which is a small fragment of the antibody which takes the fluorescence much closer to the actual molecule size and get a clearer image of what it actually looks like - We do this using STORM to see the NLRP3 in the speck - People previously beleived that there were a couple of NLRP3 molecules that oligomerize the complete prion-like oligomerisation of the adapter ASC. - However, instead, STORM nanobody suggests, that we have a dense core and a less dense periphery- showing that ASC and NLRP3 interact in a way that we do not yet understand

Answer 74

- Very low levels (barely detectable, pM range) - Low levels have very important regulatory roles for CNS homeostasis and functions (neuro-immuno modulators) - Important in communication between the brain and the periphery and vice versa

Answer 75

- Increased expression (detectable, nM range) - Role of the cytokine is to regulate neuroinflammation

Answer 76

- Classical pathway- TNF-a involving transcription of the gene and protein causing mRNA expresison and translation of the protein etc. - Non-classical pathway such as IL-1 involves intracellular storage and regulated cleavage before secretion

Answer 77

- Characterised in 1985 - Has had an array of different names since then showing the many cell types that it can act upon and the different actions depending on the cell.

Answer 78

- Yes, even though it is active - Reason for active pro-IL-1a could be that if the cell undergos necrosis, it is released and can act on the receptor

Answer 79

Ca+ Calpain from 32kDa to 17kDa (same kDa as IL-1b)

Answer 80

- icIL-1RAs - Expressed by immune cells - Causes IL-1a along the normal secretary pathway - Blocks IL-1a from binding.

Answer 81

- Anakinra - Is used in clinical settings and is sold as an anti-inflammatory for rheumatoid arthritis - Undergoing trials for stroke

Answer 82

- Traumatic brain injury (acute concussion) - Cerebral ischaemia (experimental stroke causing a lesion)

Answer 83

- Can identify which cells express IL-1B - If do this in terms of TBI or experimental stroke, can see the main cells producing IL-1B and a are microglial cells - Also other cells depening on the timing, such as endothelial cells

Answer 84

- In vitro models, culturing brain cells such as astrocytes or microglia, or together or mixed culture with neuronal cells also. - Treat the cells with bacteria such as LPS (bacteria lipopolyssacharides) - Can then visualise using immunohistochemistry- the expression of IL-1B - Can see microglia express lots of IL-1 after LPS treatment

Answer 85

- Astrocytes - Neurones - Endothelial cells (make up blood vessels)

Answer 86

- Is pro-inflammatory - Neurotoxic - Inducing neuronal cell death and brain damage

Answer 87

- Isolate cultures from the cell with a recombinant IL-1 (rather than exposing with LPS or triggering injury) - Such as astrocytic cultures, microglial cultures, neuronal cultures and neurone-astrocyte cultures

Answer 88

- Observe cellular phenotype (morphologt, adhesion, migration etc,) - Measure the expression of inflammatory mediators - Measure the activation of signalling pathways - Measure the neurotoxicity

Answer 89

- It induces astrogliosis - If treat astrocyte with IL-1 and observe overtime, the astrocyte changes morphology - The cells are normally flattened and elongated with anchorage - After treatment, they begin to retract and have loss of anchorage points. - The beta exts in the cytoskeleton causing destabalisation - This renders cells more mobile and triggers the formaiton of a glial scar

Answer 90

Focal lesion in the brain causes astrocytes to surround the lesion and form a glial scar.

Answer 91

- If increase the concentration of IL-1 and then can see the concentration dependent increase in IL-6 - However if use IL-1R antagonist, it blocks this response - Also if heat treat (boil the preparation), it inactivated the bioactive property of the molecule so stops this too

Answer 92

- Mainly act on IL-1R1 (type 1) - Needs a co-receptor, accessory protein (IL-1RAcP) - Is also a IL-1R2 however, beleive that this is a decoy-non-functional receptor due to lack of intracellular domain

Answer 93

- Has a large extracellular domain (325 aas) with 3 Ig-like domains - IL-1 binds to domain 1 and 2 - Also has an intracellular domain (213 aas) and a toll/IL-1R related (TIR) domain) similar to the TLR IC domain - Also has a transmembrane domain

Answer 94

So IL-1 can bind to both and cause intracellular signalling

Answer 95

- Binds the receptor and accessory protein - Rapid activation with different adaptor molecules causes intracellular signalling molecules - Activates 2 main pathways - The first is NF-kB and MAPK leading to the expression of inflammatory genes and the secretion of downstream inflammatory mediators - The second is the deactivation of ROCK RhoA signalling pathway

Answer 96

- Is important for cytoskeleton stabalisation - If treat with IL-1 then we block this pathway and will have destabalisation leading to a change in morphology

Answer 97

- Culture neurons with IL-1 - If add IL-1 to neurons they do not die, it is not neurotoxic

Answer 98

- Culture astrocytes with IL-1 - They do not die, IL-1 is not neurotoxic here

Answer 99

- Induces a potent neurotoxic response with a concentration dependent effect of neuronal cell death after treatment - Therefore it cannot cause damage by binding to 1 cell, it is based on different cell actions and need more than one type of cell to induce a neurotoxic response

Answer 100

- MMP-9 degrades the EC matrix - If treat cells with IL-1, it induces the expression of MMP-9 - In a co-culture, it can induce neuronal injury and this neurotoxic response can be blocked by using a MMPi (inhibitor) - The toxic response if IL-1 is driven by MMP-9

Answer 101

- Bind to the receptor and the accessory protein - This causes an intracellular response - There is no activation of NF-kB or MAPK however - Instead, there is a neuronal specific signalling pathway involving an upstream mediator - There is modulation of the NMDAR receptor in neurons - By activating this, there is calcium influx and activation of different inflammatory genes in neurons

Answer 102

- It increases the firing rate of the neurons - Due to opening the NMDA channels (requires a smaller concentraiton than in astrocytes) - May be important in neuronal plasticity

Answer 103

- Requires a much higher concentration (0.5-5nM) compared to 5-50pM in neurons - Therefore is a much slower response (gliosis) compared to depolarisation caused in the fast neuronal response

Answer 104

- Culture endothelial cells in a transwell system where these are on top (lumen cells, vessels and blood compartment) - Underneath, culture the glial cells and neuronal cells to mimic the brain compartment. - Can have circulating immune cells in the blood compartment and see the response of this system to IL-1 - Causes an increase in expression of adhesion molecules (ICAM-1 and VCAM-1) - As a consequence, neutrophils can infiltrate within the endothelial layer and find themselves within the glial, neuronal compartment. - The neutrophil migration causes necrosis and neuronal injury

Answer 105

- Generated genetic mice where the genes were targeted in a conditional cell specific manner. - Targeted the exome of the gene and then flanked this with LOXP site (WT), targeting the IL-1R1 receptor - Cross this mouse with one that expresses a creorecombinase (with the brain endothelial cells as the driver) meaning cre is only expressed in the endothelial brain cells - Cross the mice, can then generate a mouse lacking the receptor in brain endothelial cells only - Can make it inducible with tamoxifen as the trigger - When trigger this, we get decreased expression of the type 1 receptor in the brain endothelial cells - Then if induce an injury such as an experimental stroke, can treat the mouse with tamoxifen, we reduce the brain damage and an igG stain (marker for BBB dysfunction) - So, by triggering the deletion of the receptor here, we reduce the damage and dysfunction to the BBB

Answer 106

- Decrease the expression of the adhesion molecules - The complete deletion of ICAM (in brain endothelial cells) which blocks the migration of neutrophils in the brain - This therefore stops the inflammatory damage caused by the neutrophils

Answer 107

- Expression through the same PAMP/DAMP and TLR4 mechanism also causing MAPK and NF-kB IC signalling pathways - Then produces a Pro-TNFa which is 26kDa and inacive at the ER - This is then processed at the golgi and released - A TACE (converting enzyme) cleaves TNF-a into its active form (17 kDa) - There is also some membrane bound TACE triggering activation

Answer 108

- Binds to a trimer, so need three receptors together to trigger an intracellular signalling mechanism - There are 2 types- TNFR1 (p55) and TNFR2 (p75) - They have a common pathway activating NF-kB and MAPK and triggering gene expression and inflammatory mediators downstream

Answer 109

- Has a death domain and fast activated death domain - Cases activation of specific signalling mechanism here causing caspase 8, 9 and 3 activation - Leads to apoptosis (toxic response)

Answer 110

Activates kinases PI3K and Akt which are neuroprotective and cause anti-inflammatory signalling

Answer 111

- In vitro cell culture of neurons from a WT, a TNGR1 or TNFR2 KO mouse - Culture with a recombinant TNFa - If treat WT with TNFa, it will induce cell death in the dish - If repeat in TNFR1 KO, it blocks the neurotoxic pathways and stop neuronal injury - If repeat with TNFR2 KO, it blocks the neuroprotective response and increases neurotoxicity

Answer 112

- Have an autocrine effect - A vicious cycle where TNFa induces more TNFa - When starts, cannot stop and get lots of TNFa expression and a neurotoxic effect - However, is dependent on the balance of type 1 or 2 receptor activation and the area involved