Cortney A. Turner

The Fibroblast Growth Factor System and Mood Disorders

Recent evidence now suggests the involvement of the fibroblast growth factor (FGF) system in mood disorders. Specifically, several members of the FGF family have been shown to be dysregulated in individuals with major depression. In this review, we will introduce the FGF system in terms of structure and function during development, in adulthood, and in various regions and cell types. We will also review the FGF system as a mediator of neural plasticity. Furthermore, this review will summarize animal as well as human studies. The majority of animal studies have focused on stress, environmental enrichment, pharmacological manipulations, and the hippocampus. By contrast, human studies have focused on volumetric measurements, antidepressant literature, and, most recently, post-mortem microarray experiments. In summary, a reduced tone in the FGF system might alter brain development or remodeling and result in a predisposition or vulnerability to mood disorders, including major depression.

A New Role for FGF2 as an Endogenous Inhibitor of Anxiety

Human postmortem studies have demonstrated that fibroblast growth factor-2 (FGF2) expression is decreased in the brain of depressed individuals. It remained unclear, however, whether this is a consequence of the illness or whether FGF2 plays a primary role in the control of mood and emotions. In this series of studies, we first ask whether endogenous FGF2 expression correlates with spontaneous anxiety, a trait associated with vulnerability to severe mood disorders in humans. This is tested in two genetically distinct groups of rats selectively bred to differ dramatically in their response to novelty and anxiety-provoking conditions (HRs = low anxiety/high response to novelty vs LRs = high anxiety/low response to novelty). We demonstrate that high-anxiety LRs have significantly lower levels of hippocampal FGF2 mRNA relative to low-anxiety HRs. We then demonstrate that FGF2 expression is modifiable by environmental factors that alter anxiety—thus, environmental complexity reduces anxiety behavior and induces FGF2 expression in hippocampus, particularly in high-anxiety LRs. Finally, we directly test the role of FGF2 as an anxiolytic and show that a 3 week treatment regimen of peripherally administered FGF2 is highly effective at blunting anxiety behavior, specifically in high-anxiety LRs. This treatment is accompanied by an increase in survival of adult-born hippocampal cells, both neurons and astrocytes, most clearly in LRs. These findings implicate hippocampal FGF2 as a central integrator of genetic and environmental factors that modify anxiety, point to hippocampal neurogenesis and gliogenesis as key in this modulation, and underscore FGF2s potential as a new target for treatment of depression and anxiety disorders.

Antidepressant-like effects of intracerebroventricular FGF2 in rats

The fibroblast growth factor (FGF) system is altered in post-mortem brains of individuals with major depressive disorder (MDD), but the functional relevance of this observation remains to be elucidated. To this end, we tested whether administering agents that act on FGF receptors would have antidepressant-like effects in rodents. We microinjected either FGF2 (200 ng, i.c.v.) or the FG loop (FGL) of neural cell adhesion molecule (NCAM) (5 microg, i.c.v.) into the lateral ventricle of rats and tested them on the forced swim test. Activating FGF receptors acutely had an antidepressant-like effect in the forced swim test. Furthermore, chronic FGF2 decreased depression-like behavior as assessed by two independent tests. Finally, the FGF system itself was altered after FGF2 administration. Specifically, there was an increase in FGFR1 mRNA in the dentate gyrus 24 h post-FGF2, suggesting the potential for self-amplification of the initial signal. These results support the potential therapeutic use of FGF2 or related molecules in the treatment of MDD and point to alternate mechanisms of neuronal remodeling that may be critical in this treatment.

Fibroblast growth factor-2 (FGF2) augmentation early in life alters hippocampal development and rescues the anxiety phenotype in vulnerable animals.

Individuals with mood disorders exhibit alterations in the fibroblast growth factor system, including reduced hippocampal fibroblast growth factor-2 (FGF2). It is difficult, however, to pinpoint whether these alterations are a cause or consequence of the disorder. The present study asks whether FGF2 administered the day after birth has long-lasting effects on hippocampal development and emotionality. We show that early-life FGF2 shifts the pace of neurogenesis, with an early acceleration around weaning followed by a deceleration in adulthood. This, in turn, results in a denser dentate gyrus with more neurons. To assess the impact of early-life FGF2 on emotionality, we use rats selectively bred for differences in locomotor response to novelty. Selectively bred low-responder (bLR) rats show low levels of novelty-induced locomotion and exhibit high levels of anxiety- and depression-like behavior compared with their selectively bred high-responder counterparts. Early-life FGF2 decreased anxiety-like behavior in highly anxious bLRs without altering other behaviors and without affecting high-responder rats. Laser capture microscopy of the dentate gyrus followed by microarray analysis revealed genes that were differentially expressed in bLRs exposed to early-life FGF2 vs. vehicle-treated bLRs. Some of the differentially expressed genes that have been positively associated with anxiety were down-regulated, whereas genes that promote cell survival were up-regulated. Overall, these results show a key role for FGF2 in the developmental trajectory of the hippocampus as well as the modulation of anxiety-like behavior in adulthood, and they point to potential downstream targets for the treatment of anxiety disorders.

The fibroblast growth factor system is downregulated following social defeat

The fibroblast growth factor (FGF) system has previously been found to be altered in post-mortem brains of individuals with major depressive disorder (MDD). The present study tested whether the FGF system is altered following acute social defeat. Rats were exposed to four consecutive days of either a social defeat paradigm or novel cages. Animals were sacrificed after the last social defeat session and gene expression was assessed in the hippocampus by mRNA in situ hybridization. Molecular components of the FGF system were significantly downregulated following social defeat. Specifically, FGF2 and FGFR1 mRNA expression was decreased in various subfields of the hippocampus. Decreased tone of the FGF system following an acute social stressor is congruent with human post-mortem results of FGF system downregulation in depression. These findings suggest that modulating the FGF system may have therapeutic value in the treatment of MDD.

Short-hairpin RNA silencing of endogenous fibroblast growth factor 2 in rat hippocampus increases anxiety behavior.

BACKGROUND The fibroblast growth factor system has been implicated in the pathophysiology of mood disorders in humans and in affective behavior in animal models. However, the studies have been either correlative or involved exogenous administration of fibroblast growth factor 2 (FGF2). None of them have directly linked endogenous FGF2 to changes in emotional responses. Therefore, we began a series of studies to knockdown FGF2 by RNA interference to examine the role of brain FGF2 in emotional responsiveness. METHODS We assessed the efficacy of short-hairpin RNA (shRNA) sequences targeted to FGF2 in COS7 cells transfected with a plasmid vector containing the full-length FGF2 sequence. We then sought to assess the effects of knocking down FGF2 gene expression in vivo on behavior. We microinjected a lentiviral vector containing either a shRNA targeting FGF2 or a nonsilencing sequence bilaterally into the dentate gyrus of the rat. RESULTS In a reporter assay system, three different shRNA sequences resulted in significant FGF2 knockdown in vitro. Five weeks following a single microinjection of one of those sequences in vivo, we observed a significant decrease in FGF2 gene expression by messenger RNA in situ hybridization in the hippocampus. The FGF2 knockdown increased the time spent in the closed arms of the elevated-plus maze, a test of anxiety behavior. CONCLUSIONS The FGF2 knockdown in the hippocampus resulted in an anxiogenic effect. Together with our findings of an inverse correlation between anxiety and FGF2 expression levels, these results implicate FGF2 in the genesis and expression of anxiety disorders.

FGF2 is a target and a trigger of epigenetic mechanisms associated with differences in emotionality: partnership with H3K9me3.

Significance This study investigates the role of epigenetics as a predisposing factor that modifies inborn emotional reactivity and the vulnerability or resilience to affective disorders, such as major depression or substance abuse. It tests the hypothesis that specific modified histones contribute to basal differences in temperament that bias emotional responses to the environment. It further describes the role of a growth factor that can modify these epigenetic patterns as it alters affective behavior. Posttranslational modifications of histone tails in chromatin template can result from environmental experiences such as stress and substance abuse. However, the role of epigenetic modifications as potential predisposing factors in affective behavior is less well established. To address this question, we used our selectively bred lines of high responder (bHR) and low responder (bLR) rats that show profound and stable differences in affective responses, with bLRs being prone to anxiety- and depression-like behavior and bHRs prone to addictive behavior. We first asked whether these phenotypes are associated with basal differences in epigenetic profiles. Our results reveal broad between-group differences in basal levels of trimethylated histone protein H3 at lysine 9 (H3K9me3) in hippocampus (HC), amygdala, and nucleus accumbens. Moreover, levels of association of H3K9me3 at Glucocorticoid Receptor (GR) and Fibroblast growth Factor 2 (FGF2) promoters differ reciprocally between bHRs and bLRs in these regions, consistent with these genes’ opposing levels of expression and roles in modulating anxiety behavior. Importantly, this basal epigenetic pattern is modifiable by FGF2, a factor that modulates anxiety behavior. Thus, early-life FGF2, which decreases anxiety, altered the levels of H3K9me3 and its binding at FGF2 and GR promoters of bLRs rendering them more similar to bHRs. Conversely, knockdown of HC FGF2 altered both anxiety behavior and levels of H3K9me3 in bHRs, rendering them more bLR-like. These findings implicate FGF2 as a modifier of epigenetic mechanisms associated with emotional responsiveness, and point to H3K9me3 as a key player in the regulation of affective vulnerability.

Altered choroid plexus gene expression in major depressive disorder

Given the emergent interest in biomarkers for mood disorders, we assessed gene expression in the choroid plexus (CP), the region that produces cerebrospinal fluid (CSF), in individuals with major depressive disorder (MDD). Genes that are expressed in the CP can be secreted into the CSF and may be potential biomarker candidates. Given that we have previously shown that fibroblast growth factor family members are differentially expressed in post-mortem brain of subjects with MDD and the CP is a known source of growth factors in the brain, we posed the question whether growth factor dysregulation would be found in the CP of subjects with MDD. We performed laser capture microscopy of the CP at the level of the hippocampus in subjects with MDD and psychiatrically normal controls. We then extracted, amplified, labeled, and hybridized the cRNA to Illumina BeadChips to assess gene expression. In controls, the most highly abundant known transcript was transthyretin. Moreover, half of the 14 most highly expressed transcripts in controls encode ribosomal proteins. Using BeadStudio software, we identified 169 transcripts differentially expressed (p < 0.05) between control and MDD samples. Using pathway analysis we noted that the top network altered in subjects with MDD included multiple members of the transforming growth factor-beta (TGFβ) pathway. Quantitative real-time PCR (qRT-PCR) confirmed downregulation of several transcripts that interact with the extracellular matrix in subjects with MDD. These results suggest that there may be an altered cytoskeleton in the CP in MDD subjects that may lead to a disrupted blood-CSF-brain barrier.

NEONATAL FGF2 ALTERS COCAINE SELF-ADMINISTRATION IN THE ADULT RAT

The neurobiological bases of increased vulnerability to substance abuse remain obscure. We report here that rats that were selectively bred for greater drug-seeking behavior exhibited higher levels of FGF2 gene expression. We then asked whether a single FGF2 administration (20 ng/g, s.c.) on postnatal day 2 (PND2) can have a lifelong impact on drug-taking behavior, spatial and appetitive learning and the dopaminergic system. Indeed, early life FGF2 enhanced the acquisition of cocaine self-administration in adulthood. However, early life FGF2 did not alter spatial or operant learning in adulthood. Furthermore, early life FGF2 did not alter gene expression in the dopaminergic system in adulthood. These results suggest that elevated levels of FGF2 may lead to increased drug-taking behavior without altering learning. Thus, FGF2 may be an antecedent of vulnerability for drug-taking behavior and may provide clues to novel therapeutic approaches for the treatment of addiction.

Fibroblast growth factor 9 is a novel modulator of negative affect

Significance Molecular mechanisms mediating negative emotion and contributing to major depression remain elusive: here, we present evidence implicating fibroblast growth factor 9 (FGF9) as a key mediator. We use whole-transcriptome studies of postmortem human tissue to demonstrate that FGF9 is elevated in depression. Reverse translation animal studies demonstrate that both endogenous and exogenous FGF9 promotes anxiety- and depression-like behavior. Conversely, localized blockade of endogenous FGF9 expression decreases anxiety behavior. To our knowledge, this paper is the first description of hippocampal FGF9 function and the first evidence implicating FGF9 in negative affect. Thus, FGF9 represents a novel target for treating affective disorders. Moreover, our findings suggest that FGF2 and FGF9 work in functional opposition; we hypothesize that the balance between FGF factors may prove critical for optimal regulation of mood. Both gene expression profiling in postmortem human brain and studies using animal models have implicated the fibroblast growth factor (FGF) family in affect regulation and suggest a potential role in the pathophysiology of major depressive disorder (MDD). FGF2, the most widely characterized family member, is down-regulated in the depressed brain and plays a protective role in rodent models of affective disorders. By contrast, using three microarray analyses followed by quantitative RT-PCR confirmation, we show that FGF9 expression is up-regulated in the hippocampus of individuals with MDD, and that FGF9 expression is inversely related to the expression of FGF2. Because little is known about FGF9’s function in emotion regulation, we used animal models to shed light on its potential role in affective function. We found that chronic social defeat stress, an animal model recapitulating some aspects of MDD, leads to a significant increase in hippocampal FGF9 expression, paralleling the elevations seen in postmortem human brain tissue. Chronic intracerebroventricular administration of FGF9 increased both anxiety- and depression-like behaviors. In contrast, knocking down FGF9 expression in the dentate gyrus of the hippocampus using a lentiviral vector produced a decrease in FGF9 expression and ameliorated anxiety-like behavior. Collectively, these results suggest that high levels of hippocampal FGF9 play an important role in the development or expression of mood and anxiety disorders. We propose that the relative levels of FGF9 in relation to other members of the FGF family may prove key to understanding vulnerability or resilience in affective disorders.