RESUMO
Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. In mouse development, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a unique perspective of BBB TJs and open new directions for understanding TJ functionality in biological barriers, ultimately enabling restoration in disease or modulation for drug delivery.
Assuntos
Barreira Hematoencefálica/citologia , Microscopia/métodos , Junções Íntimas/fisiologia , Animais , Camundongos , Camundongos Endogâmicos ICR , Microscopia/classificaçãoRESUMO
Congenital central hypoventilation syndrome (CCHS) is a rare life-threatening condition affecting the autonomic nervous system that usually presents shortly after birth as hypoventilation or central apnea during sleep. In the majority of cases, heterozygous polyalanine expansion mutations within the third exon of the paired-like homeobox 2B (PHOX2B) gene underlie CCHS. Here, we report the generation of two induced pluripotent stem cell (iPSC) lines from two identical twins with a heterozygous PHOX2B expansion mutation (+5 alanine residues). Both generated lines highly express pluripotency markers, can differentiate into the three germ layers, retain the disease-causing mutation and display normal karyotypes.