During development, neural networks are established in a highly organized manner, which persists throughout life. Neurotrophins play crucial roles in the developing nervous system. Among the neurotrophins, brain-derived neurotrophic factor (BDNF) is highly conserved in gene structure and function during vertebrate evolution, and serves an important role during brain development and in synaptic plasticity. BDNF participates in the formation of appropriate synaptic connections in the brain, and disruptions in this process contribute to disorders of cognitive function. In this review, we first briefly highlight current knowledge on the expression, regulation, and secretion of BDNF. Further, we provide an overview of the possible actions of BDNF in the development of neural circuits, with an emphasis on presynaptic actions of BDNF during the structural development of central neurons. '

Insulin, synthesized by the beta cells of pancreatic islets, is of major physiological importance in metabolic homeostasis. While mature insulin consists of two polypeptide chains joined by disulphide bridges, the gene encodes for a highly conserved single chain precursor, preproinsulin [1]. In most species preproinsulin exists as a single gene, whereas in the mouse and the rat two non-allelic insulin genes are present. The human insulin gene is located on the short arm of chromosome 11 (p15.5) [2], the rat insulin I and II genes are colocalized on chromosome 1 [3] and the mouse genes are positioned on two different chromosomes, insulin I on chromosome 19 [4] and insulin II on chromosome 7 [5]. In adult islets, the nonallelic genes appear to be coordinately expressed and regulated [6, 7]. The rodent insulin II and the human genes contain three exons and two introns, whilst insulin I lacks the second intron. The organisation and structure of the insulin gene has been reviewed in detail [8]. Insulin is regulat- AbstractThe mammalian insulin gene is exclusively expressed in the beta cells of the endocrine pancreas. Two decades of intensive physiological and biochemical studies have led to the identification of regulatory sequence motifs along the insulin promoter and to the isolation of transcription factors which interact to activate gene transcription. The majority of the islet-restricted (BETA2, PDX-1, RIP3b1-Act/C1) and ubiquitous (E2A, HEB) insulin-binding proteins have been characterized. Transcriptional regulation results not only from specific combinations of these activators through DNA-protein and protein-protein interactions, but also from their relative nuclear concentrations, generating a cooperativity and transcriptional synergism unique to the insulin gene. Their DNA binding activity and their transactivating potency can be modified in response to nutrients (glucose, NEFA) or hormonal stimuli (insulin, leptin, glucagon like peptide-1, growth hormone, prolactin) through kinase-dependent signalling pathways (PI3-K, p38MAPK, PKA, CaMK) modulating their affinities for DNA and/or for each other. From the overview of the research presented, it is clear that much more study is required to fully comprehend the mechanisms involved in the regulated-expression of the insulin gene in the beta cell to prevent its impairment in diabetes. [Diabetologia (2002) 45: 309±326]

In type 2 diabetes, chronic hyperglycemia has been suggested to be detrimental to beta-cell function, causing reduced glucose-stimulated insulin secretion and disproportionately elevated proinsulin. In the present study, we investigated the effect on several beta-cell functions of prolonged in vitro exposure of human pancreatic islet cultures to high glucose concentrations. Islets exposed to high glucose levels (33 mmol/l) for 4 and 9 days showed dramatic decreases in glucose-induced insulin release and in islet insulin content, with increased proportion of proinsulin-like peptides relative to insulin. The depletion in insulin stores correlated with the reduction in insulin mRNA levels and human insulin promoter transcriptional activity. We also demonstrated that high glucose dramatically lowered the binding activity of pancreatic duodenal homeobox 1 (the glucose-sensitive transcription factor), whereas the transcription factor rat insulin promoter element 3b1 activator was less influenced and insulin enhancer factor 1 remained unaffected. Most of these beta-cell impairments were partially reversible when islets first incubated for 6 days in high glucose were transferred to normal glucose (5.5 mmol/l) concentrations for 3 days. We conclude that cultured human islets are sensitive to the deleterious effect of high glucose concentrations at multiple functional levels, and that such mechanisms may play an important role in the decreased insulin production and secretion of type 2 diabetic patients.

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The ␤ cell-specific glucose-sensitive factor (GSF), which binds the A3 motif of the rat I and human insulin promoters, is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose only in normal islets, supporting the suggested physiological role of GSF in the glucose-regulated expression of the insulin gene. GSF binding activity was observed in all insulin-producing cells. We have therefore purified this activity from the rat insulinoma RIN and found that a single polypeptide of 45 kDa was responsible for DNA binding. Its amino acid sequence, determined by microsequencing, provided direct evidence that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as PDX-1) and that, in addition to its essential roles in development and differentiation of pancreatic islets and in ␤ cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated ␤ cells. The human cDNA coding for GSF͞IPF-1 has been cloned, its cell and tissue distribution is described. Its expression in the glucagon-producing cell line ␣TC1 transactivates the wild-type human insulin promoter more efficiently than the mutated construct. It is demonstrated that high levels of ectopic GSF͞IPF-1 inhibit the expression of the human insulin gene in normal islets, but not in transformed ␤TC1 cells. These results suggest the existence of a control mechanism, such as requirement for a coactivator of GSF͞ IPF-1, which may be present in limiting amounts in normal as opposed to transformed ␤ cells.The insulin gene contains several cis-acting regulatory elements located within its 5Ј-flanking region that are recognized by trans-acting factors, some found ubiquitously, others more restricted to the ␤ cell. These interactions determine the temporal expression of the gene and its inducibility by physiological stimuli (for review see refs. 1 and 2). Important transcriptional regulatory elements have been described in the promoter regions of various insulin genes, such as the E and A boxes (3). The motifs E1 and E2, with the consensus CANNTG, were found to be implicated in tissue-specific expression of insulin by transfection studies in insulinproducing cells. These bind transcription factors of the helixloop-helix family (4, 5). However, combinations of E1 and E2 fail to confer tissue specificity in transgenic mice (6). Additional important regulatory elements containing AϩT-rich sequences are the A boxes (A1-A5; ref.3). The proximal A1 box in the rat insulin I promoter (around Ϫ80) binds a protein selectively expressed in insulin-producing cell lines, insulin promoter factor 1 (IPF-1), which is a homeodomaincontaining transactivator of the insulin gene (7,8). By gene disruption in mice, it was shown that IPF-1 is of crucial importance for normal development of the pancreas (9). The distal A3 and A4 boxes bind several homeodomain-containing proteins, such as isl-1 (10), cdx-3 and lmx-1 (11), HNF1 ␣ (12...

The PDX-1 transcription factor plays a key role in pancreas development. Although expressed in all cells at the early stages, in the adult it is mainly restricted to the beta-cell. To characterize the regulatory elements and potential transcription factors necessary for human PDX-1 gene expression in beta-cells, we constructed a series of 5' and 3' deletion fragments of the 5'-flanking region of the gene, fused to the luciferase reporter gene. In this report, we identify by transient transfections in beta- and non-beta-cells a novel beta-cell-specific distal enhancer element located between -3.7 and -3.45 kilobases. DNase I footprinting analysis revealed two protected regions, one binding the transcription factors SP1 and SP3 and the other hepatocyte nuclear factor 3beta (HNF-3beta) and HNF-1alpha. Cotransfection experiments suggest that HNF-3beta, HNF-1alpha, and SP1 are positive regulators of the herein-described human PDX-1 enhancer element. Furthermore, mutations within each motif abolished the binding of the corresponding factor(s) and dramatically impaired the enhancer activity, therefore suggesting cooperativity between these factors.

Synaptogenesis is a dynamic process that involves structural changes in developing axons and dendrites as synapses form and mature. The visual system of Xenopus laevis has been used as a model to study dynamic changes in axons and dendrites as synapses form in the living brain and the molecular mechanisms that control these processes. Brain-derived neurotrophic factor (BDNF) contributes to the establishment and refinement of visual connectivity by modulating retinal ganglion cell (RGC) axon arborization and presynaptic differentiation. Here, we have analyzed the ultrastructural organization of the Xenopus retinotectal system to understand better the maturation of this synaptic circuit and the relation between synapse ultrastructure and the structural changes in connectivity that take place in response to BDNF. Expression of yellow fluorescent protein (YFP) followed by preembedding immunoelectron microscopy was used to identify RGC axons specifically in living tadpoles. Injection of recombinant BDNF was used to alter endogenous BDNF levels acutely in the optic tectum. Our studies reveal a rapid transition from a relatively immature synaptic circuit in which retinotectal synapses are formed on developing filopodial-like processes to a circuit in which RGC axon terminals establish synapses with dendritic shafts and spines. Moreover, our studies reveal that BDNF treatment increases the number of spine synapses and docked vesicle number at YFP-identified synaptic sites within 24 hours of treatment. These fine structural changes at retinotectal synapses are consistent with the role that BDNF plays in the functional maturation of synaptic circuits and with dynamic, rapid changes in synaptic connectivity during development. INDEXING TERMSretinal ganglion cells; BDNF; YFP; immunoelectron microscopy; TrkB; Xenopus laevis Synapse formation is an important step in the establishment of functional neuronal connectivity during development. The selection of potential synaptic partners before functional synapses are formed depends not only on structural but also on functional interactions between developing axons and dendrites at nascent synaptic sites (Lohmann and Bonhoeffer, 2008). Imaging studies have revealed that filopodially mediated contacts between developing axons and dendrites precede synapse formation in the developing brain (Cohen-Cory, 2002;Lohmann and Bonhoeffer, 2008;Mumm et Wong and Wong, 2000;Yuste and Bonhoeffer, 2004). Thus, synaptogenesis is a dynamic process in which structural changes take place as synaptic circuits mature (CohenCory, 2002). Understanding the relationship between cellular and subcellular synaptic remodelling can consequently shed new light on the functional maturation of a given synaptic circuit.Because of its relative simplicity, the visual system of nonmammalian vertebrates, such as frogs and fish, has served as an accessible model for exploring the dynamics of synaptic plasticity in the living brain. Imaging and electrophysiological studies in these species have advanced our unde...