Stephen R. Salton

Antidepressant actions of the exercise-regulated gene VGF.

Exercise has many health benefits, including antidepressant actions in depressed human subjects, but the mechanisms underlying these effects have not been elucidated. We used a custom microarray to identify a previously undescribed profile of exercise-regulated genes in the mouse hippocampus, a brain region implicated in mood and antidepressant response. Pathway analysis of the regulated genes shows that exercise upregulates a neurotrophic factor signaling cascade that has been implicated in the actions of antidepressants. One of the most highly regulated target genes of exercise and of the growth factor pathway is the gene encoding the VGF nerve growth factor, a peptide precursor previously shown to influence synaptic plasticity and metabolism. We show that administration of a synthetic VGF-derived peptide produces a robust antidepressant response in mice and, conversely, that mutation of VGF in mice produces the opposite effects. The results suggest a new role for VGF and identify VGF signaling as a potential therapeutic target for antidepressant drug development.

Targeted Deletion of the Vgf Gene Indicates that the Encoded Secretory Peptide Precursor Plays a Novel Role in the Regulation of Energy Balance

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

The Extended Granin Family: Structure, Function, and Biomedical Implications

The chromogranins (chromogranin A and chromogranin B), secretogranins (secretogranin II and secretogranin III), and additional related proteins (7B2, NESP55, proSAAS, and VGF) that together comprise the granin family subserve essential roles in the regulated secretory pathway that is responsible for controlled delivery of peptides, hormones, neurotransmitters, and growth factors. Here we review the structure and function of granins and granin-derived peptides and expansive new genetic evidence, including recent single-nucleotide polymorphism mapping, genomic sequence comparisons, and analysis of transgenic and knockout mice, which together support an important and evolutionarily conserved role for these proteins in large dense-core vesicle biogenesis and regulated secretion. Recent data further indicate that their processed peptides function prominently in metabolic and glucose homeostasis, emotional behavior, pain pathways, and blood pressure modulation, suggesting future utility of granins and granin-derived peptides as novel disease biomarkers.

VGF: A Novel Role for This Neuronal and Neuroendocrine Polypeptide in the Regulation of Energy Balance ☆

Insight into the mechanisms of action of neurotrophic growth factors has been obtained through the identification and characterization of gene products that are regulated or modified at the transcriptional, translational, and/or posttranslational level in response to neurotrophin treatment. VGF (non-acronymic) was identified approximately 15 years ago as a nerve growth factor (NGF)-regulated transcript in rat PC12 pheochromocytoma cells. Subsequent studies have demonstrated that neurotrophins such as NGF and brain-derived neurotrophic factor induce vgf gene expression relatively rapidly in PC12 cells and cultured cortical neurons, respectively, in comparison to less robust regulation by epidermal growth factor (EGF) and insulin, growth factors which do not trigger the neuronal differentiation of PC12 cells. vgf gene expression is stimulated in vitro by NGF and the ras/map kinase signaling cascade through a CREB-dependent mechanism, while in vivo, VGF mRNA levels are regulated by neuronal activity, including long-term potentiation, seizure, and injury. Both the mRNA and encoded approximately 68-kDa protein (VGF) are selectively synthesized in neuroendocrine and neuronal cells. The predicted VGF sequence is rich in paired basic amino acid residues that are potential sites for proteolytic processing, and VGF undergoes regulated release from dense core secretory vesicles. Although VGF mRNA is synthesized widely, by neurons in the brain, spinal cord, and peripheral nervous system, its expression is particularly abundant in the hypothalamus. In addition, VGF peptides are found in hypophysial, adrenal medullary, gastrointestinal, and pancreatic endocrine cells, suggesting important neuroendocrine functions. Recent analysis of VGF knockout mice indeed demonstrates that VGF plays a critical role in the control of energy homeostasis. VGF knockout mice are thin, small, hypermetabolic, hyperactive, and relatively infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic pro-opiomelanocortin, neuropeptide Y, and agouti-related peptide expression. Coupled with the demonstration that VGF mRNA levels are induced in the normal mouse hypothalamic arcuate nuclei in response to fasting, important central and peripheral roles for VGF in the regulation of metabolism are suggested. Here we review previous studies of VGF in the broader context of its newly recognized role in the control of energy balance and propose several models and experimental approaches that may better define the mechanisms of action of VGF.

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family

Ayeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane–cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM–actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.

Processing, Distribution, and Function of VGF, a Neuronal and Endocrine Peptide Precursor

Abstract1. The vgf gene encodes a neuropeptide precursor with a restricted pattern of expression that is limited to a subset of neurons in the central and peripheral nervous systems and to specific populations of endocrine cells in the adenohypophysis, adrenal medulla, gastrointestinal tract, and pancreas. In responsive neurons, vgf transcription is upregulated by neurotrophins, the basis for the original identification of VGF as nerve growth factor- (NGF) inducible in PC12 cells (A. Levi, J. D. Eldridge, and B. M. Paterson, Science229:393–395, 1985).2. In this review, we shall summarize data concerning the transcriptional regulation of vgf in vitro, the structural organization of the vgf promoter as well as the transcription factors which regulate its activity.3. On the basis of in situ hybridization and immunohistochemical studies, the in vivo tissue-specific expression of VGF during differentiation and in the adult will be summarized.4. Parallel biochemical data will be reviewed, addressing the proteolytical processing of the pro-VGF precursor within the secretory compartment of neuroendocrine cells.5. Finally, analysis of the phenotype of VGF knockout mice will be discussed, implying a nonredundant role of VGF products in the regulation of energy storage and expenditure.

Comparison of RPTPζ/β, phosphacan, and trkB mRNA expression in the developing and adult rat nervous system and induction of RPTPζ/β and phosphacan mRNA following brain injury

The receptor protein tyrosine phosphatase (RPTP)ζ/β and a major isoform, phosphacan, a chondroitin sulfate proteoglycan that contains the RPTPζ/β extracellular domain but not the transmembrane and intracellular phosphatase domains, are expressed abundantly in the nervous system, primarily by astroglia. Because of similarities in the expression patterns of RPTPζ/β and the receptor tyrosine kinase TrkB, we investigated whether RNAs encoding these proteins were co-localized during development, which would suggest that these molecules might functionally interact in vivo. By in-situ hybridization, we noted extensive areas of overlap in the expression of trkB and RPTPζ/β mRNAs in the developing peripheral and central nervous systems. Analysis with a probe specific for the catalytic TrkB isoform suggested that RPTPζ/β and non-catalytic trkB mRNAs were co-expressed in particular regions of the nervous system while the catalytic trkB and RPTPζ/β transcripts were also, but to a lesser extent. RPTPζ/β and phosphacan expression were extremely similar, differing particularly in the level of expression in the ventricular and subventricular zones, hippocampus, and ependyma. Furthermore, both RPTPζ/β and phosphacan mRNAs were found in several subsets of neurons as well as astrocytes. Following CNS injury, we observed robust induction of RPTPζ/β mRNA in areas of axonal sprouting, and of both RPTPζ/β and phosphacan mRNAs in areas of glial scarring, implying that the encoded proteins and the cell adhesion molecules and extracellular matrix proteins to which they bind may contribute to recovery from injury and perhaps regulation of axonal regrowth in the nervous system.

The Neurotrophin-Inducible Gene Vgf Regulates Hippocampal Function and Behavior through a Brain-Derived Neurotrophic Factor-Dependent Mechanism

VGF is a neurotrophin-inducible, activity-regulated gene product that is expressed in CNS and PNS neurons, in which it is processed into peptides and secreted. VGF synthesis is stimulated by BDNF, a critical regulator of hippocampal development and function, and two VGF C-terminal peptides increase synaptic activity in cultured hippocampal neurons. To assess VGF function in the hippocampus, we tested heterozygous and homozygous VGF knock-out mice in two different learning tasks, assessed long-term potentiation (LTP) and depression (LTD) in hippocampal slices from VGF mutant mice, and investigated how VGF C-terminal peptides modulate synaptic plasticity. Treatment of rat hippocampal slices with the VGF-derived peptide TLQP62 resulted in transient potentiation through a mechanism that was selectively blocked by the BDNF scavenger TrkB–Fc, the Trk tyrosine kinase inhibitor K252a (100 nm), and tPA STOP, an inhibitor of tissue plasminogen activator (tPA), an enzyme involved in pro-BDNF cleavage to BDNF, but was not blocked by the NMDA receptor antagonist APV, anti-p75NTR function-blocking antiserum, or previous tetanic stimulation. Although LTP was normal in slices from VGF knock-out mice, LTD could not be induced, and VGF mutant mice were impaired in hippocampal-dependent spatial learning and contextual fear conditioning tasks. Our studies indicate that the VGF C-terminal peptide TLQP62 modulates hippocampal synaptic transmission through a BDNF-dependent mechanism and that VGF deficiency in mice impacts synaptic plasticity and memory in addition to depressive behavior.

Characterization of the 5′-end region and the first two exons of the β-protein precursor gene

Human genomic clones encoding the promoter region and the first two exons of the beta-amyloid protein precursor (beta-APP) gene were isolated. The first exon is 205 base pairs (bp) long and encodes 19 amino acids. The second exon is 168 bp long and encodes 56 amino acids. The 5-flanking sequence of the beta-APP gene was found to display promoter activity in several cell lines including PC12 cells where the highest activity was detected. The promoter region of this gene lacks the typical TATAA and CAAT boxes usually associated with eukaryotic promoters. Five copies of the GGGCGC sequence are located between positions -107 and -188 and one copy is located within the first exon of the beta-APP gene. Consensus sequences recognized by the transcription factors Sp1 and AP-1 are located upstream from the RNA start site. Palindromic sequences capable of forming stable hairpin-like structures are found around the main transcription initiation site. The structural characteristics of the beta-APP promoter indicate that multiple elements participate in the regulation of the expression of this gene.

The role of neurotrophins in major depressive disorder

Neurotrophins and other growth factors have been advanced as critical modulators of depressive behavior. Support for this model is based on analyses of knockout and transgenic mouse models, human genetic studies, and screens for gene products that are regulated by depressive behavior and/or antidepressants. Even subtle alteration in the regulated secretion of brain-derived neurotrophic factor (BDNF), for example, due to a single nucleotide polymorphism (SNP)-encoded Val-Met substitution in proBDNF that affects processing and sorting, impacts behavior and cognition. Alterations in growth factor expression result in changes in neurogenesis as well as structural changes in neuronal cytoarchitecture, including effects on dendritic length and spine density, in the hippocampus, nucleus accumbens, and prefrontal cortex. These changes have the potential to impact the plasticity and stability of synapses in the CNS, and the complex brain circuitry that regulates behavior. Here we review the role that neurotrophins play in the modulation of depressive behavior, and the downstream signaling targets they regulate that potentially mediate these behavioral pro-depressant and antidepressant effects.