TRIP6 functions in brain ciliogenesis

TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion component. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localization along the axoneme, and its co-localization with other cilia components suggest a scaffold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis.

Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage

Intraventricular hemorrhage (IVH) represents a high risk of neonatal mortality and later neurodevelopmental impairment in prematurity. IVH is accompanied with inflammation, hemolysis, and extracellular hemoglobin (Hb) oxidation. However, microRNA (miRNA) expression in cerebrospinal fluid (CSF) of preterm infants with IVH has been unknown. Therefore, in the present study, candidate pro-inflammatory cell-free miRNAs were analyzed in CSF samples from 47 preterm infants with grade III or IV IVH vs. clinical controls (n = 14). miRNAs were quantified by RT-qPCR, normalized to “spike-in” cel-miR-39. Oxidized Hb and total heme levels were determined by spectrophotometry as well as IL-8, VCAM-1, ICAM-1, and E-selectin concentrations by ELISA. To reveal the origin of the investigated miRNAs, controlled hemolysis experiments were performed in vitro; in addition, human choroid plexus epithelial cell (HCPEpiC) cultures were treated with metHb, ferrylHb, heme, or TNF-α to replicate IVH-triggered cellular conditions. Levels of miR-223, miR-155, miR-181b, and miR-126 as well as Hb metabolites along with IL-8 were elevated in CSF after the onset of IVH vs. controls. Significant correlations were observed among the miRNAs, oxidized Hb forms, and the soluble adhesion molecules. During the post-IVH follow-up, attenuated expression of miRNAs and protein biomarkers in CSF was observed upon elimination of Hb metabolites. These miRNAs remained unaffected by a series of artificially induced hemolysis, which excluded red blood cells as their origin, while stimulation of HCPEpiCs with oxidized Hb fractions and heme resulted in increased extracellular miRNA levels in the cell culture supernatant. Overall, the hemorrhage-induced CSF miRNAs reflected inflammatory conditions as potential biomarkers in preterm IVH.

Lithium Administered to Pregnant, Lactating and Neonatal Rats: Entry into Developing Brain.

BackgroundLittle is known about the extent of drugs administered to pregnant and lactating women entering the developing brain. Lithium is one of the most commonly prescribed drugs for bipolar disorder. Here we studied transfer of lithium given to dams, into blood, brain and cerebrospinal fluid (CSF) in embryonic, postnatal and adult rats.MethodsLithium chloride in a clinically relevant dose (3.2mg/Kg body weight) was injected intraperitoneally into pregnant (E15-18) and lactating dams (birth-P16) or directly into postnatal pups (P0-P16). Acute treatment involved a single injection; chronic treatment involved twice daily injections for the duration of the experiment. Following terminal anaesthesia blood plasma, CSF and brains were collected. Lithium levels and brain distribution were measured using Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS).Results Lithium was detected in blood, CSF and brain of all fetal and postnatal pups following lithium treatment of dams. Its concentration in pups’ blood was consistently below that in maternal blood (30-35%) indicating significant protection by the placenta and breast tissue. Levels of lithium in plasma fluctuated in different treatment groups but its concentration in CSF was stable at all ages, in agreement with known stable levels of endogenous ions in CSF. Only a small non-significant increase of lithium transfer into CSF occurred following application of Na+/K+ ATPase inhibitor (Digoxin) in vivo, indicating that Na+/K+ ATPase is at most only a minor mechanism for lithium transfer across choroid plexus cells into CSF early in development. Presumably lithium transfer across choroid plexus epithelial cell is occurring predominantly via other ion channels. ConclusionsInformation obtained on the distribution of lithium in developing brain provides a basis for studying possible deleterious effects on brain development and behaviour in offspring of mothers undergoing lithium therapy.

Novel function of TRIP6, in brain ciliogenesis

TRIP6, a member of the zyxin-family of LIM domain proteins, is a focal adhesion component. trip6 deletion in the mouse revealed, unexpectedly, in view of its ubiquitous expression, a function in the brain: ependymal and choroid plexus epithelial cells were poorly developed, carrying fewer and shorter cilia, and the mice developed hydrocephalus. TRIP6 disruption, via RNAi or inhibition of its homodimerization, in a choroid plexus epithelial cell line, confirmed its function in ciliogenesis. Zyxin-family members carry numerous protein interaction domains. In common with assembly of other multiprotein complexes, ciliogenesis may be facilitated by molecular assembly factors. Super-resolution microscopy demonstrated TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localization along the axoneme, and its co-localization with other cilia components suggest a scaffold/co-transporter function for TRIP6 in cilia. This is the first discovery of a protein assembly factor essential for mammalian ciliogenesis.

Murine MPDZ-Linked Hydrocephalus is Caused by Hyperpermeability of the Choroid Plexus

ABSTRACTThough congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ. Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast-medium penetrates into the brain ventricles of mice carrying a Mpdz loss-of-function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53-fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straight-forward and concise explanation for the pathophysiology of Mpdz-linked hydrocephalus.

TNF Induces Choroid Plexus Epithelial Cell Barrier Alterations by Apoptotic and Nonapoptotic Mechanisms

The choroid plexus epithelium constitutes the structural basis of the blood-cerebrospinal fluid barrier. Since the cytokine TNF is markedly increased during inflammatory diseases in the blood and the central nervous system, we investigated by which mechanisms TNF induces barrier alteration in porcine choroid plexus epithelial cells. We found a dose-dependent decrease of transepithelial electrical resistance, increase of paracellular inulin-flux, and induction of histone-associated DNA fragmentation and caspase-3 activation after TNF stimulation. This response was strongly aggravated by the addition of cycloheximide and could partially be inhibited by the NF-B inhibitor CAPE, but most effectively by the pan-caspase-inhibitor zVAD-fmk and not by the JNK inhibitor SP600125. Partial loss of cell viability could also be attenuated by CAPE. Immunostaining showed cell condensation and nuclear binding of high-mobility group box 1 protein as a sign of apoptosis after TNF stimulation. Taken together our findings indicate that TNF compromises PCPEC barrier function by caspase and NF-B dependent mechanisms.