Tyrosine 394 is phosphorylated in Alzheimer's paired helical filament tau and in fetal tau with c-Abl as the candidate tyrosine kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimer's disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl) pyrazolo[3,4-d] pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimer's disease.

Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes

Despite the identification of SRY as the testis-determining gene in mammals, the genetic interactions controlling the earliest steps of male sex determination remain poorly understood. In particular, the molecular lesions underlying a high proportion of human XY gonadal dysgenesis, XX maleness and XX true hermaphroditism remain undiscovered. A number of screens have identified candidate genes whose expression is modulated during testis or ovary differentiation in mice, but these screens have used whole gonads, consisting of multiple cell types, or stages of gonadal development well beyond the time of sex determination. We describe here a novel reporter mouse line that expresses enhanced green fluorescent protein under the control of an Sf1 promoter fragment, marking Sertoli and granulosa cell precursors during the critical period of sex determination. These cells were purified from gonads of male and female transgenic embryos at 10.5 dpc (shortly after Sry transcription is activated) and 11.5 dpc (when Sox9 transcription begins), and their transcriptomes analysed using Affymetrix genome arrays. We identified 266 genes, including Dhh, Fgf9 and Ptgds, that were upregulated and 50 genes that were downregulated in 11.5 dpc male somatic gonad cells only, and 242 genes, including Fst, that were upregulated in 11.5 dpc female somatic gonad cells only. The majority of these genes are novel genes that lack identifiable homology, and several human orthologues were found to map to chromosomal loci implicated in disorders of sexual development. These genes represent an important resource with which to piece together the earliest steps of sex determination and gonad development, and provide new candidates for mutation searching in human sexual dysgenesis syndromes.

Identification of candidate genes at the corticoseptal boundary during development

Cortical midline glia are critical to the formation of the corpus callosum during development. The glial wedge is a Population of midline glia that is located at the corticoseptal boundary and expresses repulsive/growth-inhibitory molecules that guide callosal axons as they cross the midline. The glial wedge are the first cells within the cortex to express GFAP and thus may express molecules specific for glial maturation. The corticoseptal boundary is a genetically defined boundary between the cingulate cortex (dorsal telencephalon) and the septum (ventral telencephalon). The correct dorso-ventral position of this boundary is vital to the formation of both the glial wedge and the corpus callosum. Our aim was to identify genes expressed specifically within the glial wedge that might be involved in either glial differentiation, formation of the corticoseptal boundary or development of the corpus callosum. To identify such genes we have performed a differential display PCR screen comparing RNA isolated from the glial wedge with RNA isolated from control tissues such as the neocortex and septum, of embryonic day 17 mouse brains. Using 200 different combinations of primers, we identified and cloned 67 distinct gene fragments. In situ hybridization analysis confirmed the differential expression of many of the genes, and showed that clones G24F3, G39F8 and transcription factor LZIP have specific expression patterns in the telencephalon of embryonic and postnatal brains. An RNase Protection Assay (RPA) revealed that the expression of G39F8, G24173 and LZIP increase markedly in the telencephalon at E16 and continue to be expressed until at least PO, during the period when the corpus callosum is forming. (c) 2005 Elsevier B.V. All rights reserved.

Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia

Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated Vegfb knockout mice (Vegfb(-/-)). Unlike Vegfa knockout mice, which die during embryogenesis, Vegfb(-/-) mice are healthy and fertile. Despite appearing overtly normal, Vegfb(-/-) hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis. The full text of this article is available at http://www.circresaha.org.