Increased plasmin-mediated proteolysis of L1CAM in a mouse model of idiopathic normal pressure hydrocephalus

Idiopathic normal pressure hydrocephalus (iNPH) is a common neurological disorder that is characterized by enlarged cerebral ventricles, gait difficulty, incontinence, and dementia. iNPH usually develops after the sixth decade of life in previously asymptomatic individuals. We recently reported that loss-of-function deletions in CWH43 lead to the development of iNPH in a subgroup of patients, but how this occurs is poorly understood. Here, we show that deletions in CWH43 decrease expression of the cell adhesion molecule, L1CAM, in the brains of CWH43 mutant mice and in human HeLa cells harboring a CWH43 deletion. Loss-of-function mutations in L1CAM are a common cause of severe neurodevelopmental defects that include congenital X-linked hydrocephalus. Mechanistically, we find that CWH43 deletion leads to decreased N-glycosylation of L1CAM, decreased association of L1CAM with cell membrane lipid microdomains, increased L1CAM cleavage by plasmin, and increased shedding of cleaved L1CAM in the cerebrospinal fluid. CWH43 deletion also decreased L1CAM nuclear translocation, suggesting decreased L1CAM intracellular signaling. Importantly, the increase in L1CAM cleavage occurred primarily in the ventricular and subventricular zones where brain CWH43 is most highly expressed. Thus, CWH43 deletions may contribute to adult-onset iNPH by selectively downregulating L1CAM in the ventricular and subventricular zone.

The expression of tenascin-C in neural stem/progenitor cells is stimulated by the growth factors EGF and FGF-2, but not by TGFβ1

Neural stem/progenitor cells (NSPCs) rely on internal and external cues determining their lineage decisions during brain development. The progenitor cells of the embryonic mammalian forebrain reside in the ventricular and subventricular zones of the lateral ventricles, where they proliferate, generate neurons and glial cells, and respond to external cues like growth factors. The extracellular matrix (ECM) surrounds NSPCs and influences the cell fate by providing mechanical scaffold, trophic support, and instructive signals. The ECM molecule tenascin-C (Tnc) is expressed in the proliferative zones of the developing forebrain and involved in the proliferation and maturation of NSPCs. Here, we analyzed the regulation of the Tnc gene expression by NSPCs cultivated under the influence of different growth factors. We observed that the epidermal growth factor (EGF) and the fibroblast growth factor (FGF)-2 strongly increased the expression of Tnc, whereas the transforming growth factor (TGF)β 1 had no effect on Tnc gene expression, in contrast to previous findings in cell cultures of neural and non-neural origin. The stimulation of the Tnc gene expression induced by EGF or FGF-2 was reversible and seen in constantly treated as well as short term stimulated NSPC cultures. The activation depended on the presence of the respective receptors, which was slightly different in cortical and striatal NSPC cultures. Our results confirm the influence of extracellular stimuli regulating the expression of factors that form a niche for NSPCs during embryonic forebrain development.

CAMSAP3 is required for mTORC1-dependent ependymal cell growth and lateral ventricle shaping in mouse brains

ABSTRACTMicrotubules (MTs) regulate numerous cellular processes, but their roles in brain morphogenesis are not well known. Here, we show that CAMSAP3, a non-centrosomal microtubule regulator, is important for shaping the lateral ventricles. In differentiating ependymal cells, CAMSAP3 became concentrated at the apical domains, serving to generate MT networks at these sites. Camsap3-mutated mice showed abnormally narrow lateral ventricles, in which excessive stenosis or fusion was induced, leading to a decrease of neural stem cells at the ventricular and subventricular zones. This defect was ascribed at least in part to a failure of neocortical ependymal cells to broaden their apical domain, a process necessary for expanding the ventricular cavities. mTORC1 was required for ependymal cell growth but its activity was downregulated in mutant cells. Lysosomes, which mediate mTORC1 activation, tended to be reduced at the apical regions of the mutant cells, along with disorganized apical MT networks at the corresponding sites. These findings suggest that CAMSAP3 supports mTORC1 signaling required for ependymal cell growth via MT network regulation, and, in turn, shaping of the lateral ventricles.

Diffusion basis spectrum imaging in post-hemorrhagic hydrocephalus of prematurity

ABSTRACTObjectiveThe debilitating neurological deficits of neonatal post-hemorrhagic hydrocephalus (PHH) have been linked to periventricular white matter injury. To improve understanding of the deleterious mechanisms underlying PHH-related brain injury, this study applied diffusion basis spectrum imaging (DBSI) for the first time in neonates, modeling white matter fibers to assess axonal and myelin integrity, fiber density, and extra-fiber pathologies including cellularity, edema, and inflammation. The objectives of the study were to characterize DBSI measures in key periventricular white matter tracts of PHH infants, associate those diffusion measures with ventricular size, and utilize postmortem white matter histology to compare with the MRI findings.MethodA prospective cohort of very preterm infants (n=95) underwent MRI at term equivalent age, of which 68 were controls (VPT group), 15 had high-grade intraventricular hemorrhage without hydrocephalus (IVH group), and 12 had PHH (PHH group). DBSI metrics extracted from manually segmented corpus callosum (CC), corticospinal tracts (CST), and optic radiations (OPRA) included fiber level axial diffusivity (FAD), fiber radial diffusivity (FRD), fiber fractional anisotropy (FFA), fiber fraction (FF), restricted fractions (RF), and non-restricted fractions (NRF). All measures were contrasted across groups and correlated with frontal occipital horn ratio (FOHR), a measure of ventricular size. Postmortem immunohistochemistry was performed on the CC of 10 preterm infants (five VPT, three IVH, and two PHH) and two full-term infants who died from non-neurologic causes assessing white matter intra- and extra-fiber pathologies, as well as the integrity of the adjoining ventricular and subventricular zones.ResultsExcept for FF in the CC, there were no differences in all measures between IVH and VPT infants. In the unmyelinated CC, PHH had the lowest FF, FAD, and FFA and the highest RF. In the CC, FOHR related negatively with FAD, FFA, and FF and positively with RF. In the myelinated CST, PHH had the lowest FAD, FFA, and FF and the highest FRD and RF. FOHR related negatively to FAD and FFA and positively with NRF and FRD. In the OPRA, PHH was associated with the lowest FF and the highest RF, NRF, and FAD. FOHR related positively with FAD and NRF and negatively with FF. On postmortem tissues, PHH was associated with the highest white matter cellularity counts, variable amounts of cytoplasmic vacuolation, and the lowest synaptophysin marker intensity. The adjoining ventricular and subventricular zones in PHH had poor cytoarchitecture on H&E staining and relatively increased expression of GFAP and IBA1.ConclusionsThis initial utilization of DBSI to investigate neonatal brain development and injury demonstrated that PHH was associated with diffuse periventricular white matter injury, with tract-specific microstructural patterns and severity of axonal injury, myelin injury, white matter fiber loss, hypercellularity, and inflammation. While axonal injury was present in the CST and unmyelinated CC, myelin injury occurred only in the CST. The OPRA predominantly showed inflammation with myelin preservation. White matter cellular infiltration occurred in all tracts. Postmortem immunohistochemistry confirmed the imaging findings of decreased axonal fiber density, sparser fiber architecture, and increased cellular infiltration. Larger ventricular size was associated with greater white matter disruption. Building upon these results, DBSI provides an innovative approach for investigating the complex neuropathological effects of PHH on periventricular white matter microstructure.

Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6

Different subtypes of interneurons, destined for the olfactory bulb, are continuously generated by neural stem cells located in the ventricular and subventricular zones along the lateral forebrain ventricles of mice. Neuronal identity in the olfactory bulb depends on the existence of defined microdomains of pre-determined neural stem cells along the ventricle walls. The molecular mechanisms underlying positional identity of these neural stem cells are poorly understood. Here, we show that the transcription factor Vax1 controls the production of two specific neuronal subtypes. First, it is directly necessary to generate Calbindin expressing interneurons from ventro-lateral progenitors. Second, it represses the generation of dopaminergic neurons by dorsolateral progenitors through inhibition of Pax6 expression. We present data indicating that this repression occurs, at least in part, via activation of microRNA miR-7.

CAMSAP3 is required for mTORC1-dependent ependymal cell growth and lateral ventricle shaping in mouse brains

Microtubules (MTs) regulate numerous cellular processes, but their roles in brain morphogenesis are not well known. Here we show that CAMSAP3, a non-centrosomal microtubule regulator, is important for shaping the lateral ventricles. In differentiating ependymal cells, CAMSAP3 became concentrated at the apical domains, serving to generate MT networks at these sites. Camsap3-mutated mice showed abnormally narrow lateral ventricles, in which excessive stenosis or fusion was induced, leading to a decrease of neural stem cells at the ventricular and subventricular zones. This defect was ascribed at least in part to a failure of neocortical ependymal cells to broaden their apical domain, a process necessary for expanding the ventricular cavities. mTORC1 was required for ependymal cell growth but its activity was downregulated in mutant cells. Lysosomes, which mediate mTORC1 activation, tended to be reduced at the apical regions of the mutant cells, along with disorganized apical MT networks at the corresponding sites. These findings suggest that CAMSAP3 supports mTORC1 signaling required for ependymal cell growth via MT network regulation, and, in turn, shaping of the lateral ventricles.Summary statementCAMSAP3, which mediates non-centrosomal microtubule assembly, is required for mTORC1-dependent maturation of ependymal cells at the neocortex of developing mouse brains. Loss of CAMSAP3 causes deformation of the lateral ventricles.

Regulatory interactions between Vax1, Pax6, and miR-7 regionalize the lateral Ventricular-Subventricular Zone during post-natal Olfactory Bulb neurogenesis in mice

Several subtypes of interneurons destined for the olfactory bulb are continuously generated after birth by neural stem cells located in the ventricular-subventricular zones of the lateral ventricles. Future neuronal identity depends on the positioning of pre-determined neural stem cells along the ventricle walls, which, in turn, depends on delimited expression domains of transcription factors and their cross regulatory interactions. However, mechanisms underlying positional identity of neural stem cells are still poorly understood. Here we show that the transcription factor Vax1 controls the production of two specific neuronal sub-types. First, it is directly necessary to generate Calbindin expressing interneurons from ventro-lateral progenitors. Second, it represses the generation of dopaminergic neurons by dorso-lateral progenitors through inhibiting Pax6 expression in the dorso-lateral wall. We provide evidence that this repression occurs via activation of microRNA miR-7, targeting Pax6 mRNA.

Organ Culture and Grafting of Choroid Plexus into the Ventricular CSF of Normal and Hydrocephalic HTx Rats

Choroid plexus (CP) may aid brain development and repair by secreting growth factors and neurotrophins for CSF streaming to ventricular and subventricular zones. Disrupted ventricular/subventricular zone progenitors and stem cells lead to CNS maldevelopment. Exploring models, we organ cultured the CP and transplanted fresh CP into a lateral ventricle of postnatal hydrocephalic (hyHTx) and nonhydrocephalic (nHTx) rats. After 60 days in vitro, the cultured choroid ependyma formed spherical rings with beating cilia. Cultured CP expressed endocytotic caveolin 1 and apical aquaporin 1 and absorbed horseradish peroxidase from medium. Transthyretin secretory protein was secreted by organ-cultured CP into medium throughout 60 days in vitro. Fresh CP, surviving at 1 week after lateral ventricle implantation of nHTx or hyHTx did not block CSF flow. Avascular 1-week transplants in vivo expressed caveolin 1, aquaporin 1, and transthyretin, indicating that grafted CP may secrete trophic proteins but not CSF. Our findings encourage further exploration on CP organ culture and grafting for translational strategies. Because transplanted CP, though not producing CSF, may secrete beneficial molecules for developing brain injured by hydrocephalus, we propose that upon CP removal in hydrocephalus surgery, the fractionated tissue could be transplanted back (ventricular autograft).

Spatiotemporal expansion of primary progenitor zones in the developing human cerebellum

We present histological and molecular analyses of the developing human cerebellum from 30 days after conception to 9 months after birth. Differences in developmental patterns between humans and mice include spatiotemporal expansion of both ventricular and rhombic lip primary progenitor zones to include subventricular zones containing basal progenitors. The human rhombic lip persists longer through cerebellar development than in the mouse and undergoes morphological changes to form a progenitor pool in the posterior lobule, which is not seen in other organisms, not even in the nonhuman primate the macaque. Disruptions in human rhombic lip development are associated with posterior cerebellar vermis hypoplasia and Dandy-Walker malformation. The presence of these species-specific neural progenitor populations refines our insight into human cerebellar developmental disorders.

MKP1 phosphatase is recruited by CXCL12 in glioblastoma cells and plays a role in DNA strand breaks repair

Glioblastoma (GBM) is the most frequent and aggressive primary tumor in the central nervous system. Previously, the secretion of CXCL12 in the brain subventricular zones has been shown to attract GBM cells and protect against irradiation. However, the exact molecular mechanism behind this radioprotection is still unknown. Here, we demonstrate that CXCL12 modulates the phosphorylation of MAP kinases and their regulator, the nuclear MAP kinase phosphatase 1 (MKP1). We further show that MKP1 is able to decrease GBM cell death and promote DNA repair after irradiation by regulating major apoptotic players, such as Jun-N-terminal kinase, and by stabilizing the DNA repair protein RAD51. Increases in MKP1 levels caused by different corticoid treatments should be reexamined for GBM patients, particularly during their radiotherapy sessions, in order to prevent or to delay the relapses of this tumor.