Dmitri Chilov

A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, and the permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two known receptors Flt1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2). The Flt4 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. In this study, we have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human prostatic carcinoma cells. While VEGF-C is homologous to other members of the VEGF/platelet derived growth factor (PDGF) family, its C-terminal half contains extra cysteine-rich motifs characteristic of a protein component of silk produced by the larval salivary glands of the midge, Chironomus tentans. VEGF-C is proteolytically processed, binds Flt4, which we rename as VEGFR-3 and induces tyrosine autophosphorylation of VEGFR-3 and VEGFR-2. In addition, VEGF-C stimulated the migration of bovine capillary endothelial cells in collagen gel. VEGF-C is thus a novel regulator of endothelia, and its effects may extend beyond the lymphatic system, where Flt4 is expressed.

Genomic Organization of the Mouse and Human Genes for Vascular Endothelial Growth Factor B (VEGF-B) and Characterization of a Second Splice Isoform

A second isoform and the genomic structures of mouse and human vascular endothelial growth factor B are described. Both genes consist of seven coding exons and span about 4 kilobases of DNA. The two identified isoforms of vascular endothelial growth factor B are generated by alternative splicing where different splice acceptor sites in exon 6 introduce a frameshift and a partial use of different but overlapping reading frames. Consequently, the COOH-terminal domains in the two isoforms show no resemblance. Mouse and human cDNA clones for the novel isoform of vascular endothelial growth factor B encoded a secreted protein of 186 amino acid residues. Expression in transfected cells generated a protein of 25 kDa which upon secretion was modified by O-linked glycosylation and displayed a molecular mass of 32 kDa under reducing conditions. The protein was expressed as a disulfide-linked homodimer, and heterodimers were generated when coexpressed with vascular endothelial growth factor. The entirely different COOH-terminal domains in the two isoforms of vascular endothelial growth factor B imply that some functional properties of the two proteins are distinct.

Genomic Organization of Human and Mouse Genes for Vascular Endothelial Growth Factor C

We report here the cloning and characterization of human and mouse genes for vascular endothelial growth factor C (VEGF-C), a newly isolated member of the vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) family. Both VEGF-C genes comprise over 40 kilobase pairs of genomic DNA and consist of seven exons, all containing coding sequences. The VEGF homology domain of VEGF-C is encoded by exons 3 and 4. Exons 5 and 7 encode cysteine-rich motifs of the type C6C10CRC, and exon 6 encodes additional C10CXCXC motifs typical of a silk protein. A putative alternatively spliced rare RNA form lacking exon 4 was identified in human fibrosarcoma cells, and a major transcription start site was located in the human VEGF-C gene 523 base pairs upstream of the translation initiation codon. The upstream promoter sequences contain conserved putative binding sites for Sp-1, AP-2, and NF-κB transcription factors but no TATA box, and they show promoter activity when transfected into cells. The VEGF-C gene structure is thus assembled from exons encoding propeptides and distinct cysteine-rich domains in addition to the VEGF homology domain, and it shows both similarities and distinct differences in comparison with other members of the VEGF/PDGF gene family.

Phosphorylated β-catenin localizes to centrosomes of neuronal progenitors and is required for cell polarity and neurogenesis in developing midbrain.

β-catenin has well-established functions in cell growth and differentiation as part of the Wnt signaling pathway and in regulation of cellular adhesion with E-cadherin. Here we studied its significance in midbrain development by temporally controlled deletion of β-catenin allowing simultaneous analysis of complete (β-cat-null) and partial (β-cat-low) loss-of-function phenotypes in progenitor cells. β-cat-null cells did not contain centrosomes or a microtubule network and were unpolarized forming delaminated bulges. β-cat-low cells displayed defects in the orientation of the mitotic spindle, increased asymmetric cell divisions and premature differentiation in absence of alterations in polarity or adhesion. The spindle defect was associated with decreased centrosomal S33/S34/T41 phosphorylated β-catenin (p-β-cat) and centrosomal and microtubule defects. Interestingly, neural progenitor cells in mice expressing only unphosphorylatable β-catenin share several phenotypes with β-catenin loss-of-function mice with defects in microtubules and polarity. The results demonstrate a novel function for p-β-cat in maintaining neuroepithelial integrity and suggest that centrosomal p-ß-cat is required to maintain symmetric cleavages and polarity in neural progenitors.

Development and plasticity of meningeal lymphatic vessels

The recent discovery of meningeal lymphatic vessels (LVs) has raised interest in their possible involvement in neuropathological processes, yet little is known about their development or maintenance. We show here that meningeal LVs develop postnatally, appearing first around the foramina in the basal parts of the skull and spinal canal, sprouting along the blood vessels and cranial and spinal nerves to various parts of the meninges surrounding the central nervous system (CNS). VEGF-C, expressed mainly in vascular smooth muscle cells, and VEGFR3 in lymphatic endothelial cells were essential for their development, whereas VEGF-D deletion had no effect. Surprisingly, in adult mice, the LVs showed regression after VEGF-C or VEGFR3 deletion, administration of the tyrosine kinase inhibitor sunitinib, or expression of VEGF-C/D trap, which also compromised the lymphatic drainage function. Conversely, an excess of VEGF-C induced meningeal lymphangiogenesis. The plasticity and regenerative potential of meningeal LVs should allow manipulation of cerebrospinal fluid drainage and neuropathological processes in the CNS.

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

β-catenin is a multifunctional protein involved in both signalling by secreted factors of Wnt family and regulation of the cellular architecture. We show that β-catenin stabilization in mouse midbrain-rhombomere1 region leads to robust up-regulation of several Wnt signalling target genes, including Fgf8. Suggestive of direct transcriptional regulation of the Fgf8 gene, β-catenin stabilization resulted in Fgf8 up-regulation also in other tissues, specifically in the ventral limb ectoderm. Interestingly, stabilization of β-catenin rapidly caused down-regulation of the expression of Wnt1 itself, suggesting a negative feedback loop. The changes in signal molecule expression were concomitant with deregulation of anterior-posterior and dorso-ventral patterning. The transcriptional regulatory functions of β-catenin were confirmed by β-catenin loss-of-function experiments. Temporally controlled inactivation of β-catenin revealed a cell-autonomous role for β-catenin in the maintenance of cell-type specific gene expression in the progenitors of midbrain dopaminergic neurons. These results highlight the role of β-catenin in establishment of neuroectodermal signalling centers, promoting region-specific gene expression and regulation of cell fate determination.

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.

Editing activity for eliminating mischarged tRNAs is essential in mammalian mitochondria

Abstract Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.

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.Astrocytes in the brain are metabolically dynamic. Here, Ignatenko, Chilov and colleagues delete mitochondrial DNA (mtDNA) in a cell type specific manner, and show that inactivation of mtDNA helicase Twinkle in astrocytes leads to spongiotic encephalopathy.