Lecture 5: Hematopoietic Progenitors and Microglia

Question 1

What are some important roles of microglia?

Answer 1

• Immune surveillance
  -Maintain neuronal homeostasis
• Turnover of myelin
  -Respond to brain injuries
  -Clear unwanted extracellular aggregates
  -Regulate synaptic plasticity
  -Influence the blood brain barrier function.
  Also help maintain synaptic plasticity (helps us forget memories we don’t need).

Question 2

What happens if there are too many synapses pruned vs if there aren’t enough?

Answer 2

It is thought that with schizophrenia there were too many synapses that haven’t been pruned.
With the AD, too many synapses were pruned.

Question 3

What role does microglia play in AD?

Answer 3

Microglia degrades beta amyloid (beta amyloid increases the chances of developing AD, so microglia helps lower the chances of AD).

Question 4

Where does microglia come from?

Answer 4

Microglia arises from early hematopoietic cells (mesoderm) within the yolk sac that migrates into the developing brain.Once microglia occupy the brain they self-renew to maintain their numbers.

Question 5

How often is microglia replaced?

Answer 5

Gradual replacement every 4 years in humans.

Question 6

___ protocols have been developed that aim to mimic developmental ontogeny (development of a single individual)

Answer 6

1)iPS microglia (iMG)

Question 7

How were ing protocols developed?

Answer 7

This was done by knocking out genes in mice and finding growth factors that were important for growing microglia.

Question 8

Are iPS-microglia really microglia?

Answer 8

-Microglial markers- specific proteins for microglia (marked via antibodies).
-Cytokine production (done where immune system is stimulated)
- Directed migration- Chemokines (proteins) that immune cells respond to.
-Phagocytosis of CNS substrates- They are the clean up crew that secretes ADP when the cell dies.
-Microglial transcriptomic signature: on an mRNA level they differ here and the type of signature helps show that iPS microglia are microglia.
- Interactions with other cells or pathology

Question 9

More than 96% of iPS microglia co-express the microglial enriched proteins _____ and ____

Answer 9

1) P2RY12
2) TREM2
These markers are the first clue that this is microglia.
It is a highly pure population which is advantageous for regenerative therapies.
They are highly expressed in the microglia and nowhere else really.

Question 10

iPS microglia secrete ____ and ___ in response to inflammatory stimuli

Answer 10

1) cytokines
2) chemokines

Question 11

iPS microglia can phagocytose relevant brain-derived substrates such as ____ and ____

Answer 11

1) beta-amyloid
2) synapses

Question 12

iPS microglia can migrate toward a ____ cue

Answer 12

1) chemoattractant
ADP which can be released from dying cells is a strong chemoattractant cue for microglia.
Testing this is done by injecting ADP which mimics cell death into the host and seeing directionally where the microglia go.

Question 13

iPS microglia exhibit __ release in response to appropriate stimuli

Answer 13

calcium
ADP induces microglia chemotaxis by triggering calcium release (green flash) from endoplasmic reticulum.

Question 14

How is the iPS microglia sequenced?

Answer 14

Whole transcriptome RNA sequencing analysis of IPS microglia
Look at RNA expression, all MRNA in one set of cells vs another.
The closer the dots to each other, the more similar the micoglia.

Question 15

What can we do with microglia?

Answer 15

Examine the effects of drugs on microglial function (make drugs to change them, ex: find drugs to change the amount of synapses).
Modify genes and see how microglial behavior changes
See how microglia interact with other cell types.
Try transplanting them

Question 16

Describe how we can examine the effects of drugs on microglial function.

Answer 16

Do an assay of synpatic phagocytsosis.
Each color represents microglia: see how the drugs increase or decrease the amount of synapses eaten.
Perform screenings to find drugs that alter a given microglial function in a desireable way.

Question 17

Describe how modifying genes can show how microglia behavior changes

Answer 17

Loss of function mutations in the microglial gene TREM2 increases AD risk 3 fold.
We can use CRISPR to knockout TREM2 in IPS cells and then see how loss of TREM2 effects phagocytosis of an AD associated pathological protein.

Question 18

See how interactions with neurons effect microglia

Answer 18

Genetic changes associated with microglia

Question 19

Can we transplant microglia?

Answer 19

Yes!
MITRG mice are a special immune deficent mouse strain that was developed to improve xenotransplantation of human blood cells.
Growth factors include Rag2KO, il2ryKO, hCSF1, hCSF2, hTPO
hCSF1 is a critical growth factor for microglia
But it differs between mice vs. humans.
Mice are sed to help human cells survive in this experiment.

Question 20

Transplantation into MITRG allows ___ microglial engraftment

Answer 20

long term
more than 6 months

Question 21

iPS microglial transplantation into adult MITRG mice leads to relatively limited number of engrafted microglia.
Why is that?

Answer 21

Because the adult mouse brain is already full of mouse microglia
Microglia have a niche aka a certain # of microglia and it is good at maintaining the correct amount.

Question 22

What happens if we transplant human microglial progenitors into a postnatal day 1 (P1) mice?

Answer 22

In this case, there is more room for the human microglia to grow.
You get more of a even split of the mouse cells and human cells
80% of the microglia within the targeted forebrain regions are human.
Engrafted human microglia exhibit context-dependent differences in morphology, distribution, and marker expression.

Question 23

Do transplanted human microglia also function appropriately within the mouse brain?

Answer 23

To test this, we can use a “cranial window” combined with a two photon microscope to image microglia movement in a live brain.
GFP expressing human microglia within the rodent brain exhibits homeostatic surveillance.
Cell body isn’t moving but processes more.
When causing injury ADP releases.
Human microglial response to laser ablation follows similar kinetics as murine microglia.

Question 24

Can we also use these approaches to study microglial function during disease?

Answer 24

Yes,
IPS microglia migrate toward and interact with AD amyloid plaques and exhibit pathology induced changes in protein expression.

Question 25

What happens if we transplant TREM2 knockout microglia?

Answer 25

Loss of function mutations in the microglial gene TREM2 increases AD risk 3 fold.
TREM2 KO microglia do not properly sense and migrate toward plaques.

Question 26

What are the conclusions of lecture 5?

Answer 26

iPS-microglia mimic all of the important functional and genetic features of human brain microglia
We can use IPS microglia to study how drugs or genes effect function and how they interact with neurons in vitro.
We can transplant them into specialized immune deficient mice to study human microglial function in vivo in both normal and diseased settings.
Transplantation of IPS microglia may also be developed for therapeutic applications (potentially).