Identification of a novel proliferation‐inducing determinant using lentiviral expression cloning

One of the major challenges in the post‐genome era is the correlation between genes and function or phenotype. We have pioneered a strategy for screening of cDNA libraries, which is based on sequential combination of lentiviral and oncoretroviral expression systems and can be used to identify proliferation‐modulating genes. Screening of a lentiviral expression library derived from adult human brain cDNA resulted in cloning of the potent proliferation‐inducing determinant termed pi1 (proliferation inducer 1). Transduction experiments using GFP‐expressing oncoretroviruses to target proliferation‐competent cells suggested that overexpression of pi1 initiates proliferation of human umbilical vein endothelial cells (HUVECs). Growth induction of HUVECs as well as Swiss3T3 fibroblasts was confirmed by Brd‐uridine incorporation assays, which correlated increased DNA synthesis with expression of pi1. The identified pi1 cDNA is 297 bp long and encodes a 10 kDa polypeptide. Since deregulation of proliferation control accounts for a number of today's untreatable human diseases such as neurodegenerative disorders and cancer, discovery of novel proliferation‐modulating genes is essential for developing new strategies for gene therapy and tissue engineerin

Loss of mtDNA activates astrocytes and leads to spongiotic encephalopathy

Mitochondrial dysfunction manifests as different neurological diseases, but the mechanisms underlying the clinical variability remain poorly understood. To clarify whether different brain cells have differential sensitivity to mitochondrial dysfunction, we induced mitochondrial DNA (mtDNA) depletion in either neurons or astrocytes of mice, by inactivating Twinkle (TwKO), the replicative mtDNA helicase. Here we show that astrocytes, the most abundant cerebral cell type, are chronically activated upon mtDNA loss, leading to early-onset spongiotic degeneration of brain parenchyma, microgliosis and secondary neurodegeneration. Neuronal mtDNA loss does not, however, cause symptoms until 8 months of age. Findings in astrocyte-TwKO mimic neuropathology of Alpers syndrome, infantile-onset mitochondrial spongiotic encephalopathy caused by mtDNA maintenance defects. Our evidence indicates that (1) astrocytes are dependent on mtDNA integrity; (2) mitochondrial metabolism contributes to their activation; (3) chronic astrocyte activation has devastating consequences, underlying spongiotic encephalopathy; and that (4) astrocytes are a potential target for interventions.Peer reviewe

Beta-Catenin Regulates Intercellular Signalling Networks and Cell-Type Specific Transcription in the Developing Mouse Midbrain-Rhombomere 1 Region

Peer reviewe

Identification of a novel proliferation-inducing determinant using lentiviral expression cloning

ISSN:1362-4962ISSN:0301-561

β-catenin autonomously regulates transcription in ventral neuronal progenitors.

<p>Coronal sections through midbrain of wt (a,c,e) and β-catenin^lof (b,d,f,g) embryos isolated at E10.5 (a,b), E11.5 (c,d) and E12.5 (e,f,g). Sections were co-stained with β-catenin (green), Lmx1a and TH (red) antibody. Asterisks mark β-catenin negative areas. Filled arrowheads indicate β-catenin+, Lmx1a+ cells in β-catenin^lof embryos. Cleared arrowheads point to β-catenin negative Lmx1a or TH expressing cells. V- ventricle. Tamoxifen was injected intraperitoneally into pregnant dams 48 hours prior to embryo isolation. 10 µm</p

Blockade of VEGFR3 signaling leads to functional impairment of dural lymphatic vessels without affecting autoimmune neuroinflammation

This is the author's version of the work. It is posted here by permission of the AAAS for personal use, not for redistribution. The definitive version was published in Science Immunology 8, (2023-04-14), doi: 10.1126/sciimmunol.abq0375.The recent discovery of lymphatic vessels (LVs) in the dura mater, the outermost layer of meninges around the central nervous system (CNS), has opened a possibility for the development of alternative therapeutics for CNS disorders. The vascular endothelial growth factor C (VEGF-C)/VEGF receptor 3 (VEGFR3) signaling pathway is essential for the development and maintenance of dural LVs. However, its significance in mediating dural lymphatic function in CNS autoimmunity is unclear. We show that inhibition of the VEGF-C/VEGFR3 signaling pathway using a monoclonal VEGFR3-blocking antibody, a soluble VEGF-C/D trap, or deletion of the Vegfr3 gene in adult lymphatic endothelium causes notable regression and functional impairment of dural LVs but has no effect on the development of CNS autoimmunity in mice. During autoimmune neuroinflammation, the dura mater was only minimally affected, and neuroinflammation-induced helper T (TH) cell recruitment, activation, and polarization were significantly less pronounced in the dura mater than in the CNS. In support of this notion, during autoimmune neuroinflammation, blood vascular endothelial cells in the cranial and spinal dura expressed lower levels of cell adhesion molecules and chemokines, and antigen-presenting cells (i.e., macrophages and dendritic cells) had lower expression of chemokines, MHC class II–associated molecules, and costimulatory molecules than their counterparts in the brain and spinal cord, respectively. The significantly weaker TH cell responses in the dura mater may explain why dural LVs do not contribute directly to CNS autoimmunity.Peer reviewe

De-regulation of anterior-posterior and dorso-ventral patterning upon β-catenin stabilization.

<p>Whole mount <i>in situ</i> hybridyzation of E9.5 (22–26 somites) wt (a,c,e,g,i) and β-catenin^stab (b,d,f,h,j) embryos using probes against <i>Gbx2</i> (a,b), <i>Otx2</i> (c,d), <i>Shh</i> (e,f), <i>Gli1</i> (g,h), <i>Pax3</i> (i,j). Arrows indicate changes in gene expression. In g and h, the embryos have 21–22 somites.</p

Distinct roles of the meningeal layers in CNS autoimmunity

The meninges, comprising the leptomeninges (pia and arachnoid layers) and the pachymeninx (dura layer), participate in central nervous system (CNS) autoimmunity, but their relative contributions remain unclear. Here we report on findings in animal models of CNS autoimmunity and in patients with multiple sclerosis, where, in acute and chronic disease, the leptomeninges were highly inflamed and showed structural changes, while the dura mater was only marginally affected. Although dural vessels were leakier than leptomeningeal vessels, effector T cells adhered more weakly to the dural endothelium. Furthermore, local antigen-presenting cells presented myelin and neuronal autoantigens less efficiently, and the activation of autoreactive T cells was lower in dural than leptomeningeal layers, preventing local inflammatory processes. Direct antigen application was required to evoke a local inflammatory response in the dura. Together, our data demonstrate an uneven involvement of the meningeal layers in CNS autoimmunity, in which effector T cell trafficking and activation are functionally confined to the leptomeninges, while the dura remains largely excluded from CNS autoimmune processes. This work shows a distinct involvement of the meningeal layers in CNS autoimmunity. In animal models and in patients with multiple sclerosis, the leptomeninges were highly inflamed and showed structural changes, while the dura was only marginally affected.Peer reviewe

The GDNF Target Vsnl1 Marks the Ureteric Tip

Glial cell line-derived neurotrophic factor (GDNF) is indispensable for ureteric budding and branching. If applied exogenously, GDNF promotes ectopic ureteric buds from the Wolffian duct. Although several downstream effectors of GDNF are known, the identification of early response genes is incomplete. Here, microarray screening detected several GDNF-regulated genes in the Wolffian duct, including Visinin like 1 (Vsnl1), which encodes a neuronal calcium-sensor protein. We observed renal Vsnl1 expression exclusively in the ureteric epithelium, but not in Gdnf-null kidneys. In the tissue culture of Gdnf-deficient kidney primordium, exogenous GDNF and alternative bud inducers (FGF7 and follistatin) restored Vsnl1 expression. Hence, Vsnl1 characterizes the tip of the ureteric bud epithelium regardless of the inducer. In the tips, Vsnl1 showed a mosaic expression pattern that was mutually exclusive with β-catenin transcriptional activation. Vsnl1 was downregulated in both β-catenin-stabilized and β-catenin-deficient kidneys. Moreover, in a mouse collecting duct cell line, Vsnl1 compromised β-catenin stability, suggesting a counteracting relationship between Vsnl1 and β-catenin. In summary, Vsnl1 marks ureteric bud tips in embryonic kidneys, and its mosaic pattern demonstrates a heterogeneity of cell types that may be critical for normal ureteric branching