Glia 2

Radial Glia and Neurogenesis

• Radial glia can be seen as common progenitor cells:

  • For both neuronal and glial lineages.

  • Evidence from development supports their ability to give rise to both neurons and glia.

• Two models of cell production:

  • Neuroepithelium stem-like cell: Produces only neuronal progenitors.

  • Radial glia: A master progenitor that produces mature neuronal subtypes and glia.

• Evidence primarily comes from adult studies.

• Cajal's Error:

  • Cajal incorrectly stated that once development ends, growth and regeneration cease, and the adult brain is fixed and immutable.

  • He believed there was no regeneration of neurons or other brain cells.

Adult Neurogenesis

• Neurogenesis occurs in at least two sites in the adult brain:

  • Dentate gyrus of the hippocampus: Important for memory consolidation.

  • Subventricular zone of the lateral ventricle: Gives rise to neuronal progenitors that migrate to the olfactory bulb (important for replenishing neurons due to constant turnover related to sensation).

• Dentate Gyrus:

  • Stem cells in the dentate gyrus have radial glia-like morphology and phenotype.

  • Express markers seen in radial glia during development.

  • Can give rise to neuronal precursors, which become granule cells (neurons in the granule layer), and astrocytes.

Aging and Neurogenesis

• Neurogenic potential decreases with aging.

• Study Example:

  • Mice at 2 months, 8 months, and 24 months of age.

  • Stem cells marked with a reporter mouse expressing fluorescent protein under the Nestin promoter (expressed in stem cells).

  • The number of stem cells decreases rapidly with age.

  • Once stem cells exhaust their ability to proliferate, they convert into astrocytes (the default fate choice).

  • Suggests a universal system for radial glia to astrocyte transition.

Reprogramming Adult Cells

• Hypothesis: With the right factors, adult brain cells can be reprogrammed to convert into other cell types.

• Based on understanding key transcription factors that determine cell fate.

• Examples of successful reprogramming:

  • Neurons to astrocytes, to glia, and back to neurons.

  • Transitions done in different brain regions.

  • Transitions from one cell type to another and back again.

• Implication: Cells retain the ability to revert to a primordial progenitor state, suggesting a common origin for many cell types.

Microglia: Introduction

• Focus shift to microglia: A specific type of glial cell.

  • Unlike "glia," which is an umbrella term, microglia refers to a particular cell type.

  • Overview of microglia history, development, and recent research areas.

Key Concepts About Microglia

• Microglia are macrophages:

  • Not just close cousins, but actual macrophages.

  • Derived from the same developmental pathway as other macrophages that colonize organs in the body.

  • They are specialized macrophages residing in the brain, exposed to unique signals.

• Microglia are one of several immune-capable populations in the brain:

  • Recent research (last 5 years) shows other immune cells reside primarily in interfaces of the brain (meninges, etc.).

  • These cells can interact with the rest of the brain.

Immune Populations in the Brain

• Technique: CyTOF (time of flight mass cytometry):

  • Stains cells with antibodies for specific markers, coupled with heavy metals for quantification.

  • Provides rich data on cell populations.

• Experiment:

  • Dissociate brain tissue (parenchyma, meninges, choroid plexus).

  • Stain with a panel of antibodies (30-40).

  • Analyze using CyTOF.

• Data Visualization:

  • Each dot represents a cell, proximity indicates similarity in marker expression.

  • Analysis revealed resident myeloid cells (microglia) and other immune cells like B cells, T cells, and dendritic cells.

  • Non-microglia immune cells were primarily located in the meninges and choroid plexus.

• Quantification:

  • Microglia make up the majority (78%) of immune cells in the brain, but a significant portion are other immune cells.

Heterogeneity of Microglia

• Even within the microglia population, there is a degree of heterogeneity.

• Microglia subpopulations:

  • Likely different states associated with different events.

• Analogy to astrocytes:

  • Astrocytes are also likely a mix of many populations.

Macrophages Associated with Brain Borders

• Beyond microglia (in the parenchyma), there are macrophages associated with brain borders:

  • Perivascular macrophages (associated with blood vessels).

  • Choroid plexus macrophages.

  • Meningeal macrophages.

• Research focus:

  • Understanding their functions and different populations.

Single-Cell Sequencing of Macrophages

• Method: Single-cell RNA sequencing:

  • Sequence mRNA content of individual cells.

  • Cluster cells based on transcriptome similarity.

• Results:

  • Distinct populations of microglia and perivascular macrophages.

  • Circulating monocytes are very different from brain-resident macrophages.

  • Perivascular macrophages and microglia show some similarity, suggesting that residing in the brain confers unifying properties.

Open Questions Regarding Macrophages

• Many questions remain unanswered regarding the functions of border-associated macrophages.

• Potential functions (related to their location):

  • Choroid plexus macrophages may be involved in CSF flow or production.

  • Perivascular macrophages may sense blood components.

  • Providing mechanistic evidence to validate the hypothesised functions is difficult.

Microglia: Detailed Characteristics

• Characterized by del Rio Hortega around 1920.

• Morphology:

  • Highly ramified cells with a characteristic mosaic distribution (they occupy territories that do not overlap, covering the entire brain parenchyma).

  • Processes constantly move and scan territories to detect disturbances and debris for removal.

• Differences between gray and white matter:

  • Density and morphology differ (polarized in white matter to adapt to axons).

• Macrophages:

  • Equipped with molecules to sense and react to changes (immune challenges, molecules damaging to neurons).

Functions of Microglia in the Steady State

• Phagocytosis:

  • Can phagocytose dying cells, dead cells, and synaptic elements.

  • May prune synapses during development (removing supernumerary synapses).

• Modulation of Synaptic Activity:

  • May directly modulate activity through neurotransmitters.

• Immune Communication:

  • Communicate with the rest of the immune system through systemic circulation.

  • Sense and react to inflammatory molecules entering the brain.

History of Microglia Research

• Early Identification:

  • Robertson described a different glial cell type called "stabchenzellen" (rod-like cells) due to elongated nuclei.

• Role in Neurological Conditions:

  • Babeş noticed reactive cells in brains exposed to rabies virus and attributed an immunological role to them. - changiing morphology

• Naming and Development:

  • Del Rio Hortega named them microglia and explored their development.

Early Theories of Microglia Development

• Initial Belief:

  • Microglia develop from the mesoderm (like other immune cells).

• Del Rio Hortega's Model:

  • Macrophages circulating in newborns enter the brain from specific sites around white matter structures.

  • These macrophages colonize the brain and form the foundation of microglia.

Re-evaluation of Microglia Origin

• Challenging the Initial Hypothesis:

  • Late 1980s, researchers began to question whether perivascular macrophages truly derive from circulating monocytes.

Productivity in Neuroscience Research

• Trends in Scientific Productivity:

  • Neurons: Steady progression since 1945.

  • Astrocytes and Oligodendrocytes: Research picked up in the 60s/70s, then stabilized.

  • Microglia: Research started in the late 80s and has rapidly increased, now exceeding other brain cell types.

Fountain Model of Microglia Development

• Fetal macrophages in the perinatal brain, located at the borders.

• Cells enter from circulation into the white matter (corpus callosum).

• Colonize adjacent structures.

• Form the adult mosaic distribution.

Modern Understanding of Microglia Development

• Current Model: Microglia are derived from the yolk sac.

  • Yolk sac is a non-embryonic structure with stem cells.

  • Erythro-myeloid progenitors (EMPs) migrate into the embryo early in development (E7-8 in mice, week 5-6 post-conception in humans).

  • EMPs enter circulation and colonize the brain.

Blood-Brain Barrier Development

• The blood-brain barrier (BBB) starts to form around embryonic day 12.

• After BBB closes, it prevents traffic of cells into the brain.

Role of the Fetal Liver

• The yolk sac seeds the fetal liver.

• The liver becomes the main hematopoietic organ of the embryo.

• Cells from the fetal liver colonize other organs and establish tissue-resident macrophage populations.

• Brain Microglia:
  Migrate directly from Yolk sac and do not relay from fetal liver.
  Other tissue resident macrophage are initially generated from the fetal liver cells.

Microglia in Adult Life

• Adult Hematopoiesis: Occurs in the bone marrow, generating monocytes that replace macrophages in other organs.

• Brain: Sustained by self-renewal.

• Injury Note: Upon injury or BBB disruption, monocytes may enter the brain and differentiate into microglia-like cells.

Stepwise Development of Microglia

• EMPs colonize the brain.

• Differentiate into pre-macrophages.

• Pre-macrophages develop into fully mature microglia.

  • The key step for microglia differentiation involves the environment in which they exists.
    EMPs that commits to A1 cells. A1 cells are pre-macrophages. A2 cells develop into pre-Microglia.
    It goes in three steps:

• Step 1: EMP to Pre-macrophage (A1) = A Intrinsic program via specific transcription factors getting moulated based on high expression of PU.1 TF

• Step 2: Pre-macrophage to Microglia Progenitor(A2): High expression of CX3CR1 on the Surface
  Pre-macrophages express markers typical of tissue-resident macrophages. The full conversion requires the right stimuli that lead to the expression of specific transcription factors. Full conversion is instructed by the environment. Exposure to interleukin 34 and TGF beta drives upregulation of specific transcription factors, converting the cell to fully mature microglia. SAL1 unique to microglia use as marker.

Heterogeneity of Microglia Populations

• Key question: How does developmental origin affect microglia in the steady state in the adult brain?

• Microglia Are Not Equal: Cells from different regions, ages, sexes, etc., have different profiles and phenotypes, causing a degree of heterogeneity.

Regional Heterogeneity of Microglia

• Sequencing Experiment:

  • Isolate microglia from different anatomical regions (cerebellum, striatum, cortex, hippocampus).

  • Compare their transcriptional activity.

• Result:
  Microglia from different regions had different gene expression patterns.
  There were groups of Genes that upregulated/downregulated depending on the source area.

Comparison to Other Macrophages

• Experiment: Compare microglia from different brain regions to peritoneal macrophages and bone marrow macrophages.

• Results:

  • Microglia from different brain regions clustered together, but were distinct from non-brain macrophages.

  • Brain-resident microglia are very different from macrophages found elsewhere, not just small, region-based differences.

Single-Cell Sequencing of Microglia in Heterogeneity Development

• Single Cell Sequencing studies done in 2019s
  
  Method: Single-cell sequencing of microglia to determine cell diversity, it accounts for sex and age
  

• Use of a t-SNE plot to examine cell and genetic marker expression in cell clusters.
  * Results: There are clusters depend on age.
  Embyronic(unique cluster in early development)
  Post-natal (distinct)
  adult versus aging (clusters differ).
  some were uniquely clustered in injured brain.
  

• Each cluster is dependent on age, depends on external factors, and is based on function. It is important to consider this.
  

Microglia density and Age relationship

• There isn't as drastic of change in microglia density with aging in test with sample mouse brains.
  
  density stays the same across white and gray brain matter from young to old.

• The next question that arrises is that well then did all our microglia just live on and stay alive since the beginning of development? Are they extremely long live?
  Or are they die and are constantly being replaced?

  • Tested using Mice with some samples dosed with an analog nucleotide (BrdU) which is incorporated in DNA without damaging duplicating cells.

• The experiment shows that number of cells doubles from the analog nucleotide.
  This shows cells cycling and proliferation.
  After 5 days the population resumes to the orginal state due to
  A process of proliferation coupled to a process of apoptosis in order to keep that density balanced.
  This is also show with the live-imaging to analysis the movement of the cells
  

  • If you now plot the movement and life cycle in the brain on day 1. Plot the cell in gray, movement is in blue and cells that died are colored red. This shows that life cycle and turnover are fast and cells are in constent turnover.

In Summary

• Very diverse and complex Immune system inbrain. There are key functions in brain development.
  
  Homeostatsia is important and required. This derives from the yolk sac, wich is different from other classical theories and populaitons. Macrophages Do not repair the microglia in adulthood. It's actually a constant turnover cycle.