GABA-mediated fast-hyperpolarizing inhibition depends on extrusion of chloride by the neuron-specific K-Cl cotransporter, KCC2. Here we show that sustained interictal-like activity in hippocampal slices downregulates KCC2 mRNA and protein expression in CA1 pyramidal neurons, which leads to a reduced capacity for neuronal Cl Ϫ extrusion. This effect is mediated by endogenous BDNF acting on tyrosine receptor kinase B (TrkB), with down-stream cascades involving both Shc/FRS-2 (src homology 2 domain containing transforming protein/FGF receptor substrate 2) and PLC␥ (phospholipase C␥)-cAMP response element-binding protein signaling. The plasmalemmal KCC2 has a very high rate of turnover, with a time frame that suggests a novel role for changes in KCC2 expression in diverse manifestations of neuronal plasticity. A downregulation of KCC2 may be a general early response involved in various kinds of neuronal trauma.

Pathophysiological activity and various kinds of traumatic insults are known to have deleterious long-term effects on neuronal Cl− regulation, which can lead to a suppression of fast postsynaptic GABAergic responses. Brain-derived neurotrophic factor (BDNF) increases neuronal excitability through a conjunction of mechanisms that include regulation of the efficacy of GABAergic transmission. Here, we show that exposure of rat hippocampal slice cultures and acute slices to exogenous BDNF or neurotrophin-4 produces a TrkB-mediated fall in the neuron-specific K+–Cl− cotransporter KCC2 mRNA and protein, as well as a consequent impairment in neuronal Cl− extrusion capacity. After kindling-induced seizures in vivo, the expression of KCC2 is down-regulated in the mouse hippocampus with a spatiotemporal profile complementary to the up-regulation of TrkB and BDNF. The present data demonstrate a novel mechanism whereby BDNF/TrkB signaling suppresses chloride-dependent fast GABAergic inhibition, which most likely contributes to the well-known role of TrkB-activated signaling cascades in the induction and establishment of epileptic activity.

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.

Alternative splicing plays an important role in generating proteome diversity. The polypyrimidine tract-binding protein (PTB) is a key alternative splicing factor involved in exon repression. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. We present fluorescence, NMR, and in vivo splicing data in support of a role of PTB in inducing RNA loops. We show that the RNA recognition motifs (RRMs) 3 and 4 of PTB can bind two distant pyrimidine tracts and bring their 5′ and 3′ ends in close proximity, thus looping the RNA. Efficient looping requires an intervening sequence of 15 nucleotides or longer between the pyrimidine tracts. RRM3 and RRM4 bind the 5′ and the 3′ pyrimidine tracts, respectively, in a specific directionality and work synergistically for efficient splicing repression in vivo.alternative splicing | polypyrimidine tract-binding protein | protein-RNA interactions A lternative splicing is a highly regulated biological process that plays a crucial role in generating high proteomic diversity. It has been estimated that >90% of human genes are alternatively spliced (1). Alternative splicing occurs frequently in cells, and most RNA-binding proteins that influence alternative splicing were found to be nonspliceosomal (2). The polypyrimidine tract (PPT)-binding protein (PTB) is one of the major trans-acting factors involved in splicing regulation. PTB is most often associated with its role as a splicing repressor (3-5), but it is also involved in other aspects of mRNA processing including 3′ end processing (6, 7), mRNA localization and stability (8), and internal ribosome entry site (IRES)-mediated translation (9).PTB is a 58-kDa member of the hnRNP family consisting of four RNA recognition motifs (RRMs) joined by three linkers (10, 11). PTB recognizes PPTs in the RNA target containing CU-rich elements (12, 13). The mechanism by which PTB promotes exon exclusion is poorly understood. Our NMR structure of RNAbound PTB has suggested a potential mechanism of PTB action in splicing whereby RRM3 and RRM4 bind the PPTs flanking an alternative exon and loop out the intervening RNA, thus repressing the exon (Fig. 1A) (14). The two RRM-bound PPTs appear in opposite direction as if forming a loop to exclude the intervening exon or the branched adenosine from the spliceosomal machinery. Other mechanistic models for PTB repression have proposed a direct (5) and an indirect (5, 15) competition between PTB and other splicing factors like U2AF65, corepression with Raver-1 (16) and PTB preventing exon (15) or intron definition (17). However, all proposed mechanisms are consistent with RNA looping between RRM3 and RRM4.Here, we have sought to test and characterize this suggested looping mechanism using FRET, NMR spectroscopy, and in vivo splicing assays. Results PTB34 Binds PPTs and Brings Their 5′ and 3′ Ends into Close Proximity.First, we tested the binding of RRM3 and RRM4 of PTB (PTB34, Fig. 1A) to several model RNAs using a FRET-based gel shift assay (18). We prepared a series of singl...

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Glial cell line-derived neurotrophic factor (GDNF) has potentially great clinical importance in the treatment of Parkinson's disease and several other neurodegenerative diseases, however its intracellular signaling mechanisms are poorly understood. Here we show that upon GDNF binding glycosylphosphatidylinositol (GPI)-linked GDNF receptor K K1 (GFRK K1) activates cytoplasmic Src family tyrosine kinase(s) in Ret tyrosine kinase-deficient cultured mouse dorsal root ganglion neurons and in two Ret-negative cell lines. GFRK K1-mediated Srctype kinase activation subsequently triggers phosphorylation of mitogen-activated protein kinase, cAMP response element binding protein and phospholipase CQ Q. We therefore conclude that GDNF can activate intracellular signaling pathways Retindependently via GPI-linked GFRK K1.z 1999 Federation of European Biochemical Societies.

A major event in the maturation of CNS GABAergic transmission is the qualitative change in GABA A -mediated responses from depolarizing to hyperpolarizing. In cortical regions, this is attributed to the increased expression of potassium chloride cotransporter 2b (KCC2b), the main isoform of the neuron-specific K-Cl cotransporter KCC2. We have previously shown that transcription factor early growth response 4 (Egr4) can activate the KCC2b promoter. Here we demonstrate that in immature hippocampal neurons BDNF robustly induces ERK1/2 (extracellular signal-regulated kinase 1/2)-dependent Egr4 expression and rapid Egr4-dependent activation of the KCC2b promoter. The subsequent increase in KCC2b mRNA contributes to the expression of total KCC2 protein levels. These results indicate that Egr4 is an important component in the mechanism of BDNF-dependent KCC2 gene regulation via the ERK1/2 pathway in immature neurons.

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