Lecture 13: Microglia and Ependymal Cells

Question 1

microglia can be a

Answer 1

friend and a foe

Question 2

ependymal cells are important …

Answer 2

important defence mechanism in looking after our internal environment

Question 3

Microglia features

Answer 3

small cells, highly branched,
each normally own domain (15-30μm)
constitute between 5-20% of all glial cell
important for surveying and reacting to the internal environment

Question 4

Identifying microglia

Answer 4

Difficult to identify
Immuno-cytochemical identification
Iba1 - actin binding proteins therefore good for marking microglia

Question 5

Functions of microglia

Answer 5

homeostasis – routinely monitor extracellular environment
activity dependent synapse elimination - can manage synapses and if they need to be removed they can do that
phagocytosis of surplus neural precursor cells
defence function -> normal (-> exacerbate inflammation) (can go wrong and this can cause disease)
many roles in disease processes

Have different functions depending on the stage of life, region of the CNS and the environment (whether it is health or disease) ultimately they act by sensing and regulating the environment of the CNS, they eliminate certain structures such as pathogens, dead cells and protein aggregates and others and can secrete cytokines and neurotrophic factors for this purpose and for other immune functions too, during life they can contribute to neurogenesis, neuronal circuit shaping, vascular shaping, and homeostasis

Question 6

Distribution of microglia

Answer 6

varies dependent on region
greater in grey matter
around synapses
with astrocytes

Question 7

Microglial morphology - embryonic development

Answer 7

Invade CNS in late embryonic development
́development from haemopoietic cells of bone marrow
́Myeloid origin (not ectodermal) ́

Question 8

Microglial morphology - in adult

Answer 8

In adult
́Variable shape – rearrangements of actin cytoskeleton ́Iba1– shape changes (protein that contributes to the shape change)
́Can divide - can divide quite a lot but they can also slowly renew themselves, can be around for a long time (2 decades) but also can replicate in response to an immune or toxin attack as well
́Renew slowly at a median rate of 28% per year, and some microglia last for more than two decades.

Question 9

Microglial morphology

Answer 9

Invade CNS in late embryonic development
́development from haemopoietic cells of bone marrow
́Myeloid origin (not ectodermal) ́

In adult
́Variable shape – rearrangements of actin cytoskeleton ́Iba1– shape changes
́Can divide
́Renew slowly at a median rate of 28% per year, and some microglia last for more than two decades.

Question 10

Microglial origin

Answer 10

Not derived from the same embryonic lines as neurons and astrocytes, they actually share the same origin as macrophages and other haemopoetic cells so really they originate from those cells that are scavenging CNS for plaque, damaged neurons and infectious agents as well

develop from haemopoetic cells of bone marrow - myeloid origin rather than ectodermal

Question 11

Ultrastructure of microglia

Answer 11

elongated nuclei “bean” shaped with peripheral heterochromatin
́ scattered cisternae of rough endoplasmic reticulum and Golgi complexes at both poles
́ a microglial cell is adjacent to a neuron there is usually a thin astrocytic process (arrow)

Has dark cytoplasm and it has granules of rER and Golgi complexes at both poles so its nuclei takes on a bean like shape

Question 12

Two states of microglia (remember form depends on the function)

Answer 12

resting state and activated state

from resting go through morphological changes to become activated, structure becomes quite different

Question 13

Resting state of microglia overall function

Answer 13

(not moving)
but
Ramified (branched) processes survey the microenvironment (Surveying microglia) - can survey all the time to see if they need to react to something that is in the brain
Maintain a constant level of available microglia to rapidly detect and fight infection - guardians of the brain

Question 14

IBA1

Answer 14

actin binding protein, used to identify microglia

Question 15

Activated state of microglia overall function

Answer 15

Activated
Ameoboid
free movement throughout the neural tissue =scavenging,
phagocytose debris,
Development/pruning

Question 16

why are we interested in microglial function?

Answer 16

Resting = Surveillant
́sensing the condition of the extracellular milieu
́synaptic pruning (prune if required) and developmental apoptosis, neurogenesis (influences neurogenesis)
assessing synapses

́Detect injury
́Receptors in the microglial cell membrane
́initiate the process of active response

Response to injury
́synthesis and release of chemokines – attract other microglia (can make chemokine etc so it can elicit a response)
Proliferation or entry of monocytes via BBB (often faulty in disease)
Become motile, apoptosis, phagocytic

activity is wide and varied in response to toxicity

Question 17

resting = surveillant …

Answer 17

Resting = Surveillant
́sensing the condition of the extracellular milieu
́synaptic pruning (prune if required) and developmental apoptosis, neurogenesis (influences neurogenesis)
assessing synapses

Question 18

Microglia - detect injury

Answer 18

́Detect injury
́Receptors in the microglial cell membrane
́initiate the process of active response

Question 19

Microglia - response to injury

Answer 19

Response to injury
́synthesis and release of chemokines – attract other microglia (can make chemokine etc so it can elicit a response)
Proliferation or entry of monocytes via BBB (often faulty in disease)
Become motile, apoptosis, phagocytic

Question 20

By the resting (surveillant) microglia the entire volume of the brain is examined every

Answer 20

4 to 5 hours

Question 21

Four main themes of function for microglia …

Answer 21

1 - neural development - really important in programmed cell death
2 - homeostasis of synapses/synaptic interactions - can also survey, monitor and prune presynaptic terminals and dendritic spines to maintain homeostasis of the synapses, phagocytosis, synaptic plasticity
3 - adult neurogenesis - includes phagocytosis adult newborn cells
4 - neurological and psychiatric disorders - associated with these disorders if the physiological functions have been affected in these microglia

Question 22

Microglia actively

Answer 22

survey and shape neuronal circuit structure and function

Question 23

Microglia and neural development

Answer 23

Cytokines/inflammation ….
inflammatory molecules
cytokines
apoptosis can release inflammatory molecules cytokines so that they can assist in synaptic pruning and apoptosis

Growth factors …
Growth factors
promote synaptic plasticity and neurogenesis
the same microglia have the capacity to release growth factors which likely promote synaptic plasticity

Question 24

Microglia and homeostasis of synapses

Answer 24

microglia affect basal neurotransmission and synaptic plasticity i.e. long term potentiation

• Microglia - sense defunct synapses and phagocytose them in normal brain
• Via fractalkine
• Synaptic pruning by microglia is essential for the remodeling of synaptic circuits synaptic plasticity

In the adult brain we have these very soluble factors such as BDNF and TNFalpha that can be released by microglia which affects the basal transmission and plasticity and they can also act directly on astrocytes and affect it as well
In addition, have fractalkine signalling via the soluble fractalkine which are released by neurons and microglia have fractalkine receptors which can modulate the microglia and synapse interactions which can affect LTP behaviour in the mature CNS, the neurons can talk to the microglia and the microglia can also talk to the neurons and affect synaptic plasticity and basal neurotransmission

Microglia are important for basal level functioning in the brain

Question 25

Microglia and neurogenesis

Answer 25

microglia regulation of neurogenesis during lifespan Two regions of the brain where we make new neurons as adults and one is the subventricukqr zone near the lateral ventricle and the other is the subgranular zone which is in the denture gyrus of the hippocampus Neuro genesis doesn’t replace all the neurons in the brain, only a few that become new neurons, figuring out how to up regrulate theses in neurodegenerative disorders Important regulation of neuro genesis over age as well, When your have the first* neuronal stem cells hey can phagocytise not very good cells (spoptotic and dysfunctional progenitors), can support progenitor migration, synaptic maintenance, secretion of tropic factors Over time, disease processes such as Parkinson’s or Alzheimer’s and other modulators factors come into play such as signalling factors, Eli genetic factors, genetic factors and environmental factors Pathologically activated microglia, has a huge effect on neurogenesis Can do the opposite of healthy - can impair phagocytosis of apoptosis and dysfunctional progenitors, dysfunctional progenitors migration or altered, failure of synaptic maintenance, secretion of cytotoxic factors

Question 26

The good and the bad of microglia

Answer 26

injury to the brain .... | Proliferation and hypertrophy -> GOOD or BAD

Question 27

Good response by microglia to local injury - how does it start?

Answer 27

microglia branches quickly hone in on the injury site and then the microglia surround it and form a barrier to protect it, first response to injury that is quire fast so that healing can start

Question 28

Good response by microglia to local injury - PRRs and TLRs

Answer 28

Microglia actively survey the environment with pattern recognition receptors (PRRs) - recognise pathogen associated patterns , molecular patterns of pathogens TLR: Toll-like receptors = type I transmembrane receptors with extracellular domain Extracellular side of TLR-recognition of the microbial product The extracellular side of the TLR recognises the microbial product which sends a message to the cytoplasmic side of the toll like receptor so there it can recruit signal,ing molecules and alter kinase activation/transcription factors and finally it can modify gene expression and can tailor the immune response to the pathogen of origin (reacts to what the pathogen is) therefore it is important for inflammation/defence Cytoplasmic side of TLR - TIR domain - recruit signalling molecules - alter kinase activation / transcription factors - > Modify gene expression Tailors immune response to the specific pathogen Inflammation / defense

Question 29

PRRs =

Answer 29

pattern recognition receptors

Question 30

TLRs =

Answer 30

toll-like receptors

Question 31

TLR2 and TLR4

Answer 31

bacterial lipids

Question 32

TLR3

Answer 32

viral DNA

Question 33

TLR9

Answer 33

Bacterial DNA

Question 34

ROS

Answer 34

reactive oxygen species = neurotoxic

Question 35

Microglial activation is good but can become over activated ...

Answer 35

Ligand recognized – internalized – eliminated (good) BUT ́Microglial can become overactivated (BAD) ́produce cytotoxic factors = neurotoxic ́Superoxide, nitric oxide, tumour necrosis factor-α (some are good and actually protect the environment but it about the over activation and overproduction of these factors that makes them bad) ́Causes of overactivation -not well understood ́Environmental toxins - pesticide (environmental toxins such as pesticides can actually activate our microglia) ́Neurodegenerative disease -> many microglia (many are over activated) ́Alzheimer’s disease – microglial activation increases as disease progresses

Question 36

Microglia actively survey the environment with pattern recognition receptors (PRRs) - TLR activated - inflammation/defense over exaggerated/overactivated....

Answer 36

Inflammation/defense (over exaggeration causes the problem) = too much ROS (microglial apoptotic cell death or - too much pro inflammatory gene expression - > overactivity ROS extracellular - > neurotoxicity ROS can actually be good for signalling but it is the over activation that causes the problems

Question 37

ROS leads to

Answer 37

neurodegeneration

Question 38

Toxins can cause neurotoxicity through activation of ___________ -> increased _______

Answer 38

NADPH oxidase | ROS (reactive oxygen species)

Question 39

Toxins examples can cause neurotoxicity through activation of NADPH oxidase -> increased ROS

Answer 39

Lipopolysaccahride (E coli), paraquat (chemical herbicide), MPTP (synthetic heroin -> acute Parkinsonian symptoms), amyloid beta, thrombin etc.

Question 40

NADPH oxidase activation to produce ROS is activated in....

Answer 40

NADPH oxidase activation to produce ROS is activated in; Alzheimer’s disease Parkinson’s disease Involved in the neural damage in response to cerebral vascular dysfunction (experimentally - Ischaemic stroke is reduced in mice lacking functional NADPH oxidase)

Question 41

becoming activated microglia with ROS

Answer 41

activated microglia when they start becoming activated, if there is inflammation we can have kinase activation, transcription factor activation and intracellular ROS concentration increases need to limit the level of response in the microglia because if there are too many microglia they can get damaged from the ROS as well

Question 42

Microglia can act on other cells ... astrocytes...

Answer 42

Microglia -> astrocyte dysfunction -> damage activation of microglia release cytokines etc ``` alter astrocyte behaviour impaired glutamate uptake activation of postsynaptic inotropic glut receptors Ca2+ influx Excitotoxicity - neuron death ``` Environment is important at cellular level Remember in disease - many things are happening at once Astrocytes are important in mopping up glutamate (another neurotransmitter at synapse), what can happen with microglia is that the production of the ROS in the microglia these can inhibit the glutamate transporters that are in the astrocytes so you have glutamate that is not being mopped up by astrocytes and they are in the extracellular environment in the synapse which will cause a lot of problems with the neurons because it going the bind to extracellular and extrasynaltic receptors which potentially causes increase in calcium concentration in the neurons causing neurotoxicity and killing them - misbehaving microglia can directly affect astrocytes which can then directly affect neurons because of the extra amount of glutamate around and causing that extra toxicity to be around the neurons and therefore you are not going to get the prime depolarisation either

Question 43

Environmental activation of microglia - air pollution

Answer 43

Air pollution - pervasive and harmful environmental toxicants modern world,

Question 44

Environmental activation of microglia - ASD

Answer 44

Air pollution - pervasive and harmful environmental toxicants modern world, ́ Large scale epidemiological studies - prenatal air pollution exposure - increased risk of neurodevelopmental disorders – autism spectrum disorder (ASD). - link between environmental toxins, microglia and potentially ASD as well

Question 45

Environmental activation of microglia - diesel exhaust particles (DEP)

Answer 45

́ Diesel exhaust particles (DEP) = primary toxic component of air pollution -> activate microglia in vitro and in vivo in adult rodents Impact of gestational exposure to DEP on microglial morphology - developing brains, male and female mice. ́ increased inflammatory cytokine protein ́ altered the morphology of microglia, = activation or a delay in maturation, only male mice

Question 46

Microglia and neurons overlap in juvenile offspring prenatally exposed to DEP

Answer 46

increased overlap between microglia and neurons in juvenile offspring prenatally exposed to DEP overlap affects how the neuron works volume of microglia is bigger with exposure to DEP particles compared to normal Male overlap between microglia and neuron is bigger in the exposed vs in the not exposed so they could say that the exposure of the environmental toxin prenatal could affect male mice does affect mice developmentally

Question 47

Microglial can become over activated and cause neurotoxicity

Answer 47

Have a toxic trigger and then release neuro toxic factors and have this direct neuro toxic insult and the neuron responds and you may get this microglia activated from this neuron and then you get a self-perpetuating neurotoxicity cycle happen with these neurons

Question 48

PET images - AD vs normal patient

Answer 48

more microglial activation in AD

Question 49

Targeting microglia could be a potential therapy for AD

Answer 49

Since we know it is toxic Tangles of tau protein form inside neurons as Alzheimer’s disease develops. The tangles attract immune cells known as microglia. The protein APOE collects inside microglia, helping the immune cells transform into their activated form. Activated microglia attach and injure neurons, causing brain damage and leading to cognitive decline If you can eliminate the microglia if they are in this activated form then you can potentially inhibit the death of the neurons

Question 50

DAM - disease microglia

Answer 50

a unique microglia type associated with restricting development of AD How they found Found a disease microglia -initially have an early stage change in the microglia cells by gene expression and then have a decrease in the immune checkpoints (immune response genes are changing) to cause it to increase phagocytosis and can see this change in the microglia as the genes progress

Question 51

DAM - how they were found

Answer 51

How they were found... Wildtype vs AD model Single cell RNA sequencing of all immune cells - sequenced all the single cells of the immune cells within the mouse models and looked specifically at the microglia Found a disease microglia -initially have an early stage change in the microglia cells by gene expression and then have a decrease in the immune checkpoints (immune response genes are changing) to cause it to increase phagocytosis and can see this change in the microglia as the genes progress

Question 52

Gene expression profiles of microglia reveal how microglia...

Answer 52

...progressively switch states with disease homeostatic microglia - stage 1 DAM - stage 2 DAM

Question 53

Hypothetical dual role for disease associated microglia (DAM) in the procession of disease

Answer 53

if we can understand the gene expression and understand that there is this progression in the changes of microglia through the activated state then we can actually target this microglia

Question 54

microglia can produce ...

Answer 54

cytokines (both pro-inflammatory and anti- inflammatory cytokines) Also: growth factors, chemokines and neurotrophins.

Question 55

Ependymal cells line

Answer 55

the fluid filled cavity of the brain which is the ventricular system, line the ventricular system of the CNS Ependymal cells are within the ventricle and choroid plexus makes the cerebrospinal fluid (protective from impact), ependymal cells are lining the ventricle where you have the choroid plexus as well and they have cilia which can move cilia, form a bit of a barrier between the CSF and the brain exchanges

Question 56

Ependymal cell shape

Answer 56

cuboidal -columnar shape apical microvilli, cila contain intermediate filaments

Question 57

Function of ependymal cells

Answer 57

Ependymal epithelium: interface between brain and CSF ́Brain-CSF barrier and filter - filtration barrier ́Inflammatory response - can release factors and protect the cells that surround them ́A regulator of osmotic pressure ́ Control the concentrations of regulatory peptides - ensure that their are not too many, regulation control of CSF flow - can move the CSF through the brain by the cilia (effective stroke pushes fluid and then recovery stroke puts it back in the position to do an effective stroke again) Trophic and metabolic support ́In specialized locations secrete cerebrospinal fluid ́Function as neural stem cells, (self-renewal and multipotency)? - increased cell proliferation and neurogenesis in the adult human Huntington's disease brain - The demonstration of endogenous stem/progenitor cells in the hippocampus and the subependymal layer (SEL) of the basal ganglia in the adult mammalian brain has raised the exciting possibility that these undifferentiated cells may be able to generate neurons for cell replacement in neuro- degenerative diseases such as HD.

Question 58

'Brain-washing'

Answer 58

The brain nightly rinses itself though csf so a good night sleep is essential for brain health which might help to clear the brain of the toxic waste, rhythmic waves of blood and csf