Microglia - MSc Neuroscience at the Institute of ...mscneuroscience.iop.kcl.ac.uk/moodle/neurosciencemsc/lectures/A2... · 2 Microglia • Resident macrophages of the CNS • Constitute

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Microglia

Wendy Noble

[email protected]

Learning objectives

• To know the origins, morphologies and main functions of microglia

• To be able to distinguish different phenotypes of microglia

• To understand how microglia interact with neurons

• To appreciate the contribution of microglia to neuroinflammation and neurodegenerative disease

Learning objectives





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Microglia

• Resident macrophages of the

CNS

• Constitute 5-15% of total cellular

composition of the CNS

• Dynamic cells - phenotype

changes under different

conditions: effects on microglia

function

Macrophages in the CNS

Prinz and Priller, 2014

• Brain has several heterogenouspopulations of macrophages

• Have homeostatic and surveillance actions at distinct sites

The origin of microglia

Embryonic and postnatal development of microglia in mice

Prinz and Priller, 2014

• Microglia derived from erythroid/myeloid progenitor stem cells that are formed at embryonic day 7.5 (E7.5)–E8.0 in the yolk sac in mice. This is quite distinct from the origin of other macrophage populations.

• As they differentiate (A1-A2), theybegin to express many typical microglial markers, and migrate to the brain

• These microglia are thought to sustain local microglial populations

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Microglial morphology

• Ramified microglia- often found in resting state (M0 phenotype)- cells have many short fine processes that they use to monitor

the local environment


• Amoeboid microglia (M1)- Usually active microglia- commonly found in developing and damaged brain - spherical in shape, lack processes, and contain numerous

phagocytic vacuoles


• Rod cells- Commonly found in conditions such as chronic measles

infection- Markedly elongated nuclei, scanty cytoplasm and few

processes

• Multinucleated cells- Found in mycobacterial infection and around amyloid-

containing vasculature- Giant cells, multiple nuclei

• Epithelioid macrophages- Found in chronic infections such as tuberculosis and

leprosy- Many microglia cluster to form granulomas

• Dystrophic microglia- Commonly found in older age/in age-related diseases- Beading’ of microglial processes

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Main functions of microglia

Function Examples

Modulation of immuneresponses and oxidative stress

Production and secretion of anti- and pro-inflammatory cytokines, reactive oxygen species (ROS)

Pathogen recognition (innate immunity)

Microglial receptors recognise evolutionarily conserved antigens on pathogen surfaces (pathogen-/damage-associated molecular patterns e.g. LPS, -amyloid)

Antigen presentation Present pathogens bound to MHC for activation of T lymphocytes. Relevant in neuroinflammation and autoimmune disease.

Recognition of bound antibody (adaptive immune function)

Respond to antibodies bound to pathogens (opsonization). Relevant in autoimmune diseases (e.g. demyelination)

CNS development Phagocytosis (pruning of redundant synapses/neurons)Influence of microglial secretions (cytokines, neurotrophins, growth factors)

Stem cell regulation Regulation of stem cell proliferation (e.g. granule cells in hippocampus)

Lipid transport Secretion of lipoprotein particles that deliver lipids to neurons for membranes/synapse maintenance

Main functions of microglia

PhagocytosisIngestion and destruction by digestive enzymes in lysosomes of:•Neurons and other damaged cells (e.g. in neurodegenerative diseases, Wallerian degeneration, tract degeneration)•Misfolded/aggregated proteins•Synapses (during development and in disease)•Myelin (e.g. multiple sclerosis)

•Micro-organisms (e.g. abscess)•Virally infected cells (e.g. herpes encephalitis)•Erythrocytes and haemoglobin breakdown products (e.g. haemosiderin) following haemorrhage

Part 1 Summary

• Microglia are resident macrophages of the CNS

• They are dynamic cells, with alterations in morphology reflecting changes in their activation state/function

• They play many important roles in the CNS, including:- During development and disease- presentation of antigens, - induction and mediation of inflammatory states - phagocytosis of damaged cells, excess and damaged synapses,

abnormal proteins and other pathogens

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Learning objectives

Microglial phenotypes

(M0 phenotype)Originally described for peripheral macrophages – broadly apply to microglia

ramified

M1: Pro-inflammatory

M2: Anti-inflammatory

amoeboid amoeboid/ramified

Scavenging of debris, tissue modelling and repair

Promote inflammation, cause cytotoxicity, demyelination and axonal damage

Phenotyping of microglia - morphology

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Immunophenotyping of microglia

Microglial marker Function

Iba1 Ionized calcium-binding adaptor molecule 1: resting and activated microglia

CD163 Perivascular macrophage and macrophage-like microglia in areas of blood–brain barrier breakdown/Scavenger receptor for the haemoglobin–haptoglobin complex

HLA-DR Cell surface homologue of MHC II – antigen presenting function

CD68 Microglial lysosomes – phagocytosis

MSR-A Macrophage scavenger receptor-A – cell surface protein lipoprotein receptor involved in direct ligand recognition

CD64 (FcRI) High affinity receptor for IgG – role in mounting immune response

CD32 (FcRII) Low affinity receptor for IgG – phagocytosis of immune complexes and regulation of inflammation

CD16 (FcRIII) Antibody-dependent binding, uptake and killing pathogens

CD206 (Mannose R) Phagocytosis and endocytosis of endogenous and exogenous proteins

Fizz1 Inhibitor of inflammation

CD14 (TLR-4) Receptors for bacterial lipopolysaccharide, Gram-/associated with classical (M1) activation

Immunophenotyping of microglia

Resting

Activated

Phenotyping of microglia – cytokine arrays

E.g. Antibody arrays:

• Membranes pre-spotted with antibodies against a panel of cytokines

• Cytokines in sample bind to their respective antibodies

• Detection of relative signal intensities allows comparison of changes in protein amounts

controls

Increase in pro-inflamm mediators

Might suggest a change to which phenotype?

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Techniques for assessing microglial phenotype

Technique Advantages Disadvantages

Morphological analyses Easy, quick, cheap. Can use stored/fixed tissues.

Limited information on microglialfunction (except phagocytosis)

Immunohistochemistry Provides info on distribution/localisation. Can be combined with morphological assessment.

Antibodies provide limited information on phenotype.

Cytokine detection (WB, ELISA, array)

Multiplexing possible. Relatively quick. Good quality data.

Needs to be sensitive to detect small amounts. Fresh tissue needed.

Gene expression analyses

Definitive detection of microglial activation status (M1, M2 etc)

mRNA is labile. Needs fresh tissue.

Proteomics Can identify large numbers of proteins simultaneously in small sample amounts.

Not very sensitive (most existing techniques detect only few proteins of interest)

In vivo imaging Real time information aboutchanges in microglial activation

Requires live animals/patients. Expensive PET ligands (freshly synthesised).

Part 2 Summary

• Several techniques are used to distinguish between microglial phenotype, each with their own advantages and disadvantages

• The most commonly used methods are immunohistochemistry (IHC) combined with morphological assessment.

• Most accurate method is analysis of changes in gene expression pathways

• In vivo analysis allows determination of changes in mocroglial activity and localisation in real time, but resolution is poor





Learning objectives

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Neuronal on/off switches for microglia

Neuron on/off switches for microglia

CD200 – CD200RNeuronal glycoprotein CD200 activates microglial CD200R to keep microglia in resting state

1. “Resting” signals – keep microglia in non-activated surveillance state

CX3CL1 – CX3CL1R (Fractalkine R)CX3CL1R activation keeps microglia in quiescent stateAlso contributes to basal microglial surveillance (motility and dynamics of microglial processes)


CD47 – Sirp alphaCD47 (membrane protein) interacts with the myeloid inhibitory immunoreceptor SIRPα to drive a downregulatory signal that inhibits host cell phagocytosis.

2. “Don’t eat me” signals – cell does not need to be phagocytosed/cleared

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3. “Eat me” signals – this cell should be cleared by phagocytosis

1. Phosphatidylserine (phospholipidmembrane component) exposed on neurons:• When bound by opsonins milk fat

globule EGF factor 8 (MFG-E8) or growth-arrest specific protein 6 (GAS6) bind and activates vitronectin receptor (VNR)

• When bound by GAS6 or Protein S bind and activates MER receptor tyrosine kinase (MERTK)

• Binds directly to brain-specific angiogenesis inhibitor 1 (BAI1).

2. Neuron-exposed calreticulin or C1q activate low-density lipoprotein receptor related-protein (LRP).

3. C1q deposition on de-sialylatedglycoproteins converts C3 to the opsonin C3b which activates CR3 and its signalling partner, DAP12.


4: “Help” signals – this cell is injured. Often results in a toxic pro-inflammatory microglial response (P2X7)

Activation of purinoceptors (P2X, P2Y) by ATP, ADP etc leads to:• Microglial branch extension towards the

injury site. • Synthesis and release of pro-

inflammatory cytokines (e.g. IL-1beta)


5: “Survival” signals – provides neuroprotection, support or regeneration

Activation of colony stimulating factor 1 receptor (Csf1R) by Csf1 and IL-34 induces cell survival or proliferation

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Part 3 Summary

• Signalling from neurons to microglia is complex. There are five main types of signal:

• Resting• Don’t eat me• Eat me• Help• Survival

• Generally involves signal released from neurons activating a microglial receptor

• Results in different phenotypic states in microglia – inducing specific functions relevant to neighbouring cells

• High dynamism and flexibility of microglial responses essential to allow them to maintain CNS environment.

• Microglial interactions with astrocytes and neurons adds another level of complexity... Will be discussed in cytokine lecture



• To be aware of the physiological and pathological interactions of microglia with astrocytes and neurons

• To appreciate the contribution of microglia to neuroinflammation and Alzheimer’s disease

Learning objectives

Alzheimer’s disease

• AD brain is characterised by deposits of -amyloid (A) in plaques and intracellular neurofibrillary tangles of hyperphosphorylated aggregated tau

• The amyloid cascade hypothesis positions Aupstream of tau changes and neurodegeneration

• Inflammation is also a prominent feature of AD brain that accompanies Aand tau accumulations and neurodegeneration

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Microglia in Alzheimer’s disease

Heneka et al., 2015

Microglia (blue) within A plaques (brown) in AD brain

Microglia in AD brain produce the pro-inflammatory cytokine IL-1

Activated microglia (green) associate with plaques (red) in a transgenic mouse model of AD that forms plaques

Microglia in healthy brain

Heneka et al., 2014

Microglia in Alzheimer’s brain

Heneka et al., 2014

A and other damage-associated molecular pathogens activate microglia via pattern recognition receptors (PRRs)

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Microglia in Alzheimer’s brain

Heneka et al., 2014

A and other damage-associated molecular pathogens activate microglia via pattern recognition receptors (PRRs)

The NLRP3 inflammasome

Three main components:1. NLRP3: NLR family, pyrin (PYD) domain-containing 3.

2. ASC: Apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)

3. Pro-caspase-1

The NLRP3 inflammasome in microglia in AD

A

mic

rogl

ia

IL-1RNMDAR

neur

on

Altered tau phosphorylation and aggregation

Neurodegeneration

A activates microglial receptors to induce the inflammasome and NFB driven increased transcription of inflammatory mediators

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The NLRP3 inflammasome in microglia in AD

Goldmann et al., 2013

Part 4 Summary

• Activated microglia associate with Aplaques in Alzheimer’s disease (AD)

• Microglial phenotype is altered by the local environment in AD

• Microglia contain all components of the NLRP3 inflammasome

• The NLRP3 inflammasome is activated by A to drive neuroinflammation, including the increased synthesis and release of pro-inflammatory mediators.

• Knocking out components of the NLRP3 inflammasome in microglia is protective in transgenic mouse models of AD.

Recommended Reading

1. Boche D, Perry VH, Nicoll JAR (2013). Activation patterns of microglia and their identification in the human brain. Neuropath Applied Neurobiol. 39(1), 3-18.

2. Prinz M, Priller J (2014). Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease. Nat Rev Neurosci. 15, 300–31.

3. Heneka MT, Kummer MP, Latz E. (2014). Innate immune activation in neurodegenerative disease. Nat Rev Immunol. 14(7):463-77.

4. Heneka MT et al. (2015). Neuroinflammation in Alzheimer's disease.Lancet Neurol. 14(4):388-405.

Documents

Microglia - MSc Neuroscience at the Institute of ...mscneuroscience.iop.kcl.ac.uk/moodle/neurosciencemsc/lectures/A2... · 2 Microglia • Resident macrophages of the CNS • Constitute