BBB Structure and Neuroinflammation

What structural features make CNS endothelial cells uniquely restrictive?

Tight junctions, minimal transcytosis (high Mfsd2a), continuous basement membrane, extensive pericyte coverage, astrocyte endfeet support.

Role of tight junction proteins in BBB integrity?

Claudin-5: size-selective barrier; Occludin: stabilizes junctions; ZO-1: anchors junctions to actin cytoskeleton.

How do pericytes support BBB function?

Regulate endothelial tight junctions, vessel stability, basement membrane deposition, and capillary tone.

Role of astrocytes in BBB regulation?

Astrocytic endfeet release factors (e.g., VEGF, SHH, TGFβ) that maintain junctions, ionic balance, and vessel stability.

Paracellular vs. transcellular BBB permeability?

Paracellular = between endothelial cells; Transcellular = vesicular transport through endothelial cells.

Physiological regulators of transcytosis?

Mfsd2a suppression of caveolae, caveolin-1 expression, inflammation, metabolic state, shear stress.

How does endothelial NO support BBB health?

Promotes vasodilation, reduces leukocyte adhesion, suppresses inflammation, stabilizes tight junctions.

Why is BBB integrity essential for synaptic function?

Maintains ion homeostasis, prevents entry of neurotoxic blood components, ensures neuronal signaling stability.

Function of Mfsd2a in BBB maintenance?

Suppresses caveolar transcytosis to maintain low BBB permeability.

How does microvascular density influence BBB integrity?

Dense capillary networks reduce hypoxia and metabolic stress, preserving tight junctions and endothelial health.

Major drivers of age-related BBB breakdown?

Mitochondrial dysfunction, oxidative stress, inflammation, reduced Mfsd2a, increased transcytosis, pericyte loss.

How does endothelial senescence weaken the BBB?

Decreases tight junction proteins, increases inflammatory cytokines, disrupts cytoskeleton.

How does mitochondrial dysfunction impair BBB integrity?

Increases ROS, reduces ATP, damages junction proteins, increases permeability.

How does oxidative stress damage the BBB?

ROS disrupts tight junctions, oxidizes lipids/proteins, activates MMP-2/9.

Role of MMP-2/9 in BBB dysfunction?

Degrade tight junction proteins and basement membrane.

How does inflammaging impair BBB structure?

Chronic TNFα/IL-1β signaling disrupts tight junctions and increases endothelial activation.

Why is transcytosis increased in aged endothelium?

Reduced Mfsd2a and increased caveolin-1 expression.

Effect of pericyte loss on BBB function?

Endothelial destabilization, increased permeability, impaired vessel tone.

How does endothelial SIRT1 loss impair BBB integrity?

Increases oxidative stress, reduces mitochondrial function, destabilizes tight junction expression.

How does basement membrane remodeling affect the BBB?

Alters structural support and increases permeability to macromolecules.

How does BBB leakage trigger microglial activation?

Blood proteins (e.g., fibrinogen, albumin) activate PRRs on microglia.

Morphological characteristics of reactive microglia?

Amoeboid shape, thicker processes, retracted fine branches.

Key markers of reactive microglia?

CD68, MHCII, IBA1, F4/80.

What defines disease-associated microglia (DAMs)?

TREM2/APOE/LPL/CST7-rich signature linked to neurodegeneration.

How do blood-borne proteins activate microglia?

Fibrinogen binds microglial CD11b/CD18, induces ROS and inflammation.

How does microglial activation worsen BBB function?

Releases cytokines, ROS, and MMPs that further damage endothelial cells.

How does endothelial dysfunction trigger neuroinflammation?

Releases inflammatory cytokines, increases leukocyte adhesion, weakens junctions.

Role of astrocytes in neuroinflammation after BBB breakdown?

Release cytokines/MMPs and amplify inflammatory cascades.

How does neuroinflammation impair synaptic plasticity?

Disrupts NMDA signaling, suppresses LTP, alters hippocampal circuitry.

Why are perivascular microglia key sensors of BBB dysfunction?

They reside at the vascular interface and detect leaked proteins.

How does SIRT1 preserve tight junctions?

Deacetylates transcription factors to maintain claudin-5/occludin expression.

Role of SIRT1 in endothelial mitochondrial health?

Activates PGC-1α, promotes biogenesis, reduces mitochondrial ROS.

How does SIRT1 reduce endothelial oxidative stress?

Deacetylates NF-κB and FOXO pathways to upregulate antioxidant defenses.

How does TRF enhance SIRT1 activity?

Increases NAD⁺ availability, activates AMPK, improves metabolic state.

Why use ESKO mice in your proposal?

To test whether TRF plasma’s benefits require endothelial SIRT1.

SIRT1-independent pathways that may mediate TRF protection?

Nrf2 activation, Mfsd2a upregulation, reduced caveolin-1, anti-inflammatory metabolites.

How might TRF plasma reduce transcytosis?

By increasing Mfsd2a or decreasing caveolin-1.

What are discordant genes in rejuvenation studies?

Genes altered by aging that TRF/young plasma reverse.

How could SIRT1 activation reduce microglial activation indirectly?

Restores BBB integrity, reducing exposure to blood-derived inflammatory stimuli.

Why might TRF and young plasma show similar BBB improvements?

Both elevate anti-geronic circulating metabolites and reduce inflammation.

Why use multiple tracer sizes for BBB permeability?

To assess severity; small leaks indicate mild dysfunction, large leaks severe breakdown.

Why use two-photon microscopy for BBB studies?

Real-time, high-resolution visualization of tracer leakage deep in tissue.

How is tracer extravasation quantified?

Measure fluorescence intensity outside vessels over time.

Why is retro-orbital injection preferred?

Fast, consistent systemic delivery with low stress.

Key confounds in BBB tracer experiments?

Anesthesia effects on CBF, breathing, injection variability, laser intensity.

How does scRNA-seq identify endothelial dysfunction?

Reveals altered tight junction, transporter, mitochondrial, and inflammatory gene modules.

Role of GSEA in BBB research?

Detects coordinated changes in tight junction, oxidative stress, inflammatory pathways.

What does CellChat reveal about the BBB?

Changes in ligand-receptor signaling between endothelium, astrocytes, pericytes, microglia.

What does increased tight junction gene expression after TRF plasma imply?

Structural restoration of BBB integrity.

What demonstrates SIRT1-dependent BBB rescue?

TRF plasma restores BBB in WT but not ESKO mice + matching SIRT1-linked transcriptional signatures.