Inflammatory Mediators in Glial and Neuronal Cell Biology Flashcards
Describe IL-1 family production
IL-1B:
1. TLR4 recognizes ligand using accessory protein MD-2. TLR signaling activated by the ligand-induced dimerization of two TLR ectodomains, which brings their cytoplasmic TIR domains close together. This allows them to interact with the TIR domains of cytoplasmic adaptor molecules, such as:
• MyD88 (myeloid differentiation factor 88)
• MAL (MyD88 adaptor-like)
• TRIF (TIR domain containing adaptor inducing IFNb)
• TRAM (TRIF related adaptor molecule)
- Adaptors recruit IRAK-1 & IRAK-4, which activate the E3 ubiquitin ligase TRAF-6
• Catalyses attachment of ubiquitin to lysine 63 of TRAF-6
• NEMO is also polyubiquitinated - Ubiquitin usually targets protein for degradation, but can also have a signaling role – here it serves as a scaffold that allows recruitment of TAK1.
- TAK1 activates IKK by phosphorylation of its beta subunit.
- IKK phosphorylates IkB at serines 32 and 36. This leads to polyubiquitination of IkB at lysine 48.
- IkB is subsequently degraded by the proteasome.
- NFkB is free to migrate to the nuclease and initiate transcription of pro-inflammatory genes, eg) IL and TNF cytokines.
In order to be activated, it has to be cleaved by the inflammasome:
1. Efflux of K+ ions from damaged cells induces dissociation of the accessory proteins from NLRP3
2. Allows activation and assembly of the pyrin domain with that of the adaptor molecule apoptosis-associated speck-like protein with a caspase recruitment domain (ASC)
3. The CARD of ASC binds to the CARD domain of pro-caspase 1, facilitating production of activate caspase 1 molecule = the inflammasome.
IL-1a:
• Same mechanism of PRR ligation can lead to the production of pro-IL1-a
• This is active, doesn’t need to be cleved.
• However, needs to be cleaved to be released → by calcium dependent calpain.
• As pro-IL-1a is biologically active, any events leading to membrane rupture or death of cells expressing pro-IL1a could result in its release and a pro-inflammatory response.
IL-1RA:
• This is a naturally occurring antagonist to IL-1
• Cells express an intracellular isoform, but also can be expressed as a soluble secreted form that goes through the ER and golgi.
How can we experimentally model brain injury and infection, and measure IL-1?
Experimental models of brain injury:
• Traumatic brain injury → hammering a brain
• Cerebral ischaemia → middle cerebral artery occlusion (results in stroke)
→ Look at IL-1 expression in these models using ICC, can see that iL-1B is expressed by microglial and vessel endothelial cells
• These cells are becoming activated in response to injury, and are secreting IL-1B.
• IL-1a and IL-1RA show the same pattern of expression, but there are temporal differences → IL-1B and IL-1a expressed first, IL-1RA expressed later.
Model of CNS infection using cell culture:
• Mixed glial culture treated with LPS to mimic infection
• Perform immunocytochemistry, can see you have strong expression of IL-1B in microglia
• Allows us to measure IL-1 in vitro
Describe the IL-1 receptor family.
- IL-1R1 → extracellular domain comprising 3 Ig-like domains, transmembrane domain and then an intracellular domain with a TIR domain.
- IL-1R2 → Has the same extracellular domain, but doesn’t have any intracellular domain. IL-1 bindng doesn’t trigger an intracellular signal, it is a decoy receptor.
- IL-1RAcP → in order to signal, upon binding of IL-1 to its receptor, you get recruitment of the IL-1 receptor accessory protein. This gives a complex allowing for full signaling.
Describe the IL-1 signalling pathway in non-neuronal cells.
- Ligand-induced dimerization of IL-1R and the IL-1R accessory proteinbrings their cytoplasmic TIR domains close together. This allows them to interact with the TIR domains of cytoplasmic adaptor molecules, such as:
• MyD88 (myeloid differentiation factor 88) - Adaptors recruit IRAK-1 & IRAK-4, which activate the E3 ubiquitin ligase TRAF-6
• Catalyses attachment of ubiquitin to lysine 63 of TRAF-6
• NEMO is also polyubiquitinated
This can have a number of different outcomes:
Activation of NFkB:
• Ubiquitin usually targets protein for degradation, but can also have a signaling role – here it serves as a scaffold that allows recruitment of TAK1.
• TAK1 activates IKK by phosphorylation of its beta subunit.
• IKK phosphorylates IkB at serines 32 and 36. This leads to polyubiquitination of IkB at lysine 48.
• IkB is subsequently degraded by the proteasome.
• NFkB is free to migrate to the nuclease and initiate transcription of pro-inflammatory genes, eg) IL-6
Activation of the MAPK cascade:
• Has similar effects to activation of NFkB, just mediated by the transcription factor c-Jun binding to AP-1
Activation of ROCK
• Signalling via RhoA, leads to the cytoskeletal destabilization we see upon astrocyte activation
Describe the 3 actions of IL-1 on astrocytes during inflammation.
IL-1 induces astrogliosis (reactive astrocytosis):
• Acts as a typical mitogenic factor for astrocytes
• Over 24 hours, after exposure to IL-1B → retraction of the cell, cytoskeleton destabilizes, allowing the cell to retract.
• Astrogliosis is an abnormal increases in the number of astrocytes due to the destruction of nearby neurons from CNS trauma, infection, ischaemic stroke or neurodegenerative disease.
Anti-inflammatory effects:
• Astrogliosis causes glial scar formation and in severe cases, inhibition of axon regeneration
− This functions to reduce the spread and persistence of inflammatory cells, decreasing tissue damage, lesion area and decreasing neuronal loss. It creates a barrier marking where the intense inflammation is needed, limiting the spread to healthy tissues.
− Reactive astrocytes defend against oxidative stress
− The repair of BBB disruption is facilitated by reactive astrocytes by their direct endfeet interaction with the blood vessel wall.
Pro-inflammatory effects: • One major hallmark of the astrocytic response to IL-1 is synthesis of a variety of secondary inflammatory mediators: − Cytokines → IL-6, TNFa − Adhesion molecules (ICAM, VCAM) − Chemokines − Prostaglandins − Neurotoxic factors such as NO and MMP
IL-1 induces IL-6 release:
• Treating astrocytes with increasing concentrations of IL-1B causes a progressive increase in the release of IL-6
• If you co-treat with IL-1RA, you get no IL-6 release
IL-1 induced neuronal death:
• We know it is neurotoxic in the brain, but what we see in culture is not so simple
• Increasing concentrations of IL-1 on neurons in culture doesn’t really see any neurotoxic effects
• The same can be said when applied to pure astrocytes → but you do get inflammatory mediator production
• However, if you mix neurons and astrocytes → increasing IL-B gives increasing neuronal death. IL-1RA is protective.
➢ Therefore likely that the inflammatory mediators produced by the astrocytes in response to IL-1 is what is causing the neurotoxicity. You need cell-cell interactions.
➢ So, IL-1 isnt directly neurotoxic – but it induces the production of mediators which are
What are the mediators?
MMP-9 mediates IL-1 neurotoxicity:
• Western blots show increasing MMP-9 induction with increasing IL-1B
• This will degraded the matrix around the neurons, leading to neurotoxicity
• Blocking MMP-9 blocks the neurotoxic response
Summary of IL-1 neurotoxicity:
• IL-1 induces reactive astrocytosis
• Expression of IL-6 by astrocytes contributes to inflammation
• Activated astrocytes migrate to site of injury – MMP-9 would facilitate this by degrading the ECM.
• Migrate to injury site and form the glial scar
• MMP-9 is also neurotoxic – kills the neurons.
Describe the IL-1 signalling pathway in neurons.
• Activation of the NFkB or MAPK pathways only happens in immune cells, eg microglia and astrocytes
• In neurons:
1. Reeceptor dimerization recruits MyD88 which then recruits a neutral sphingomyelinase (nSMase).
2. This then activates a SRC kinase, which phosphorylates and activates an NMDA receptor
3. This leads to Ca2+ influx, which activates CaMKII
4. This in turn leads to phosphorylation and activation of CREB, which acts as a transcription factor
→ So here, IL-1 is acting as a neurotransmitter
Describe the action of IL-1 on neurons.
- It is believed the above pathway is how physiological levels of IL-1 contribute to normal brain physiology - it regulates sleep, memory and LTP
- Physiological (pM) IL-1 acts to trigger neuronal firing – it triggers action potentials and enhances NMDA mediated current
- Increased levels however, trigger host-defence responses such as fever and sickness behavior, which are mediated by direct actions of IL-1 on neurons.
- In addition, pathophysiological (nM) concentrations induce hyperpolarization and inhibition of firing
→ In contrast to non-neuronal cells, IL-1 actions on neurons are therefore highly dependent on concentration and time of exposure
Describe the features of TNFR1
TNFR1 (p55):
• 55kDa
• Receptor acts as a trimer – ligand binding induces trimerisation
• Soluble TNF thought to elicit its effects primarily through TNFR1
• Expressed on most cell types
Two different signaling mechanisms:
• TNFR1 contains a cytoplasmic death domain – permits assembly of the TNFR1 signalling complex through binding of the RNF receptor associated death domain (TRADD)
• Binding of TRADD allows recruitment of other adaptor proteins:
Apoptotic pathway:
- TRADD recruits FADD
- FADD recruits caspase 8
- Caspase 8 recruits and activates caspases 3 and 9 which form the apoptosome
Gene expression pathway:
- TRADD recruits TRAF2
- TRAF2 leads to activation of the NFkB/MAPK pathways, leading to gene expression
→ Because it can activate apoptosis, activation of TNFR1 is neurotoxic
→ Unlike IL-1 where neurotoxicity is mediated through astrocytes, TNFa is directly neurotoxic
Describe the features of TNFR2
- 75kDa
- Extracellular domain the same as TNFR1, but has a larger intracellular domain
- Preferentially binds membrane TNF
Gene expression pathway:
- TRAF1 is recruited to the receptor
- TRAF1 recruits TRAF2
- TRAF2 leads to activation of the NFkB/MAPK pathways, leading to gene expression
Neuroprotective pathway:
- TRAF1 recruited to the receptor
- TRAF1 recruits TRAF2
- TRAF2 activates the PI3/Akt pathway → this is neuroprotective
→ So, TNFR2 binding believed to be primarily pro-survival signaling.
What are the roles of TNF receptors in neurotoxicity?
- If you incubate wildtype neurons with TNFa, you get potent neurotoxicity
- This would suggest that TNFR1s are preferentially activated
- However, has been found that TNFR2 can promote TNFR1 signalling by enhancing the association between solTNF and TNFR1 via a ligand passing mechanism → suggested this is the primary contribution of TNFR2 to TNFmediated signaling as opposed to direct TNFR2 signalling.
- If you incubate TNFa with TNFR1 KO neurons – no neurotoxicty. Makes sense, you have removed the receptor with the death domain.
- If you incubate TNFa with TNFR2 KO neurons, you do get neurtoxicity – again makes sense, maintaining the receptor that mediates apoptosis
→ So TNFs contribution to neurotoxicity dependent on the level at which cells express each receptor.
Describe the endogenous TNFa inhibitors.
• Like IL-1 with IL-1RA, TNFa also has endogenous inhibitors
− prostaglandins, cAMP limit TNFa production
− glucocorticoids are produced when TNFa levels are high, by activation of the HPA axis, and inhibit further TNFa production.
− IL-10 can inhibit TNFa
− TACEs cleaves tmTNF to form solTNF, and it can also cleave TNFRs to solTNFRs which can bind solTNF in the circulation
➢ Discovery of these solTNFRs led to the development of anti-TNF antibodies
➢ Treatment with these markedly reduces ischaemic or traumatic brain damage, but these rodents show greater neurological dysfunction at a later time → so although TNF contributes to neuronal injury, it could improve recovery.
Describe IL-6 receptor signalling
• Follows the same pattern of expression as TNFa – constitutive release, not maintained in the cell like IL1
• Treating cells with IL-1 has a dose dependent effect on IL-6 release
• Two types of receptor involved in the signaling
− IL-6 R (gp80)
− gp130
− Two gp80s and one gp130 come together to form a trimer
• Ligation to the receptor activates the JAK/STAT pathway
− Phosphoryltion of JAK leads to phosphorylation of STAT1 and STAT3 and the formation of STAT1/3 dimers
− These dimers translocate to the nucleus where they bind to the acute phase response element → induces the APR.
• IL-6 is used as a biomarker in patients with CNS disorders
Describe the proposed role for IL-6 in neuroinflammation.
- Expressed by astrocytes and microglia
- Mechanism is controversial – but we think it is generally more neuroprotective than neurotoxic (in IL-6 KO mice, there is more neuronal cell death)
- Has this effect because it activates astrocytes and microglia to produce IL-1RA, preventing the detrimental effects of IL-1.
- It also activates astrocytes to produce neuronal growth factor (NGF) – enhances neuron survival
