A dual-omics approach on the effects of fibroblast growth factor-2 (FGF-2) on ventral tegmental area dopaminergic neurons in response to alcohol consumption in mice.

Harmful alcohol consumption is a major socioeconomic burden to the health system, as it can be the cause of mortality of heavy alcohol drinkers. The dopaminergic (DAergic) system is thought to play an important role in the pathogenesis of alcohol drinking behaviour; however, its exact role remains elusive. Fibroblast growth factor 2 (FGF-2), a neurotrophic factor, associated with both the DAergic system and alcohol consumption, may play an important role in DAergic neuroadaptations during alcohol abuse. Within this study, we aimed to clarify the role of endogenous FGF-2 on the DAergic system and whether there is a possible link to alcohol consumption. We found that lack of FGF-2 reduces the alcohol intake of mice. Transcriptome analysis of DAergic neurons revealed that FGF-2 knockout (FGF-2 KO) shifts the molecular fingerprint of midbrain dopaminergic (mDA) neurons to DA subtypes of the ventral tegmental area (VTA). In line with this, proteomic changes predominantly appear also in the VTA. Interestingly, these changes led to an altered regulation of the FGF-2 signalling cascades and DAergic pathways in a region-specific manner, which was only marginally affected by voluntary alcohol consumption. Thus, lack of FGF-2 not only affects the gene expression but also the proteome of specific brain regions of mDA neurons. Our study provides new insights into the neuroadaptations of the DAergic system during alcohol abuse and, therefore, comprises novel targets for future pharmacological interventions.

Chronic Voluntary Alcohol Consumption Alters Promoter Methylation and Expression of Fgf-2 and Fgfr1.

Alcohol abuse accounts for 3.3 million deaths annually, rendering it a global health issue. Recently, fibroblast growth factor 2 (FGF-2) and its target, fibroblast growth factor receptor 1 (FGFR1), were discovered to positively regulate alcohol-drinking behaviors in mice. We tested whether alcohol intake and withdrawal alter DNA methylation of Fgf-2 and Fgfr1 and if there is a correlation regarding mRNA expression of these genes. Blood and brain tissues of mice receiving alcohol intermittently over a six-week period were analyzed using direct bisulfite sequencing and qRT-PCR analysis. Assessment of Fgf-2 and Fgfr1 promoter methylation revealed changes in the methylation of cytosines in the alcohol group compared with the control group. Moreover, we showed that the altered cytosines coincided with binding motives of several transcription factors. We also found that Fgf-2 and Fgfr1 gene expression was significantly decreased in alcohol-receiving mice compared with control littermates, and that this effect was specifically detected in the dorsomedial striatum, a brain region involved in the circuitry of the reward system. Overall, our data showed alcohol-induced alterations in both mRNA expression and methylation pattern of Fgf-2 and Fgfr1. Furthermore, these alterations showed a reward system regional specificity, therefore, resembling potential targets for future pharmacological interventions.

FGF2 is an endogenous regulator of alcohol reward and consumption.

Alcohol use disorder (AUD) is a chronic, relapsing disorder, characterized by escalating alcohol drinking and loss of control, with very limited available treatments. We recently reported that the expression of fibroblast growth factor 2 (Fgf2) is increased in the striatum of rodents following long-term excessive alcohol drinking and that the systemic or intra-striatal administration of recombinant FGF2 increases alcohol consumption. Here, we set out to determine whether the endogenous FGF2 plays a role in alcohol drinking and reward, by testing the behavioural phenotype of Fgf2 knockout mice. We found that Fgf2 deficiency resulted in decreased alcohol consumption when tested in two-bottle choice procedures with various alcohol concentrations. Importantly, these effects were specific for alcohol, as a natural reward (sucrose) or water consumption was not affected by Fgf2 deficiency. In addition, Fgf2 knockout mice failed to show alcohol-conditioned place preference (CPP) but showed normal fear conditioning, suggesting that deletion of the growth factor reduces alcohol's rewarding properties. Finally, Fgf2 knockout mice took longer to recover from the loss of righting reflex and showed higher blood alcohol concentrations when challenged with an intoxicating alcohol dose, suggesting that their ethanol metabolism might be affected. Together, our results show that the endogenous FGF2 plays a critical role in alcohol drinking and reward and indicate that FGF2 is a positive regulator of alcohol-drinking behaviours. Our findings suggest that FGF2 is a potential biomarker for problem alcohol drinking and is a potential target for pharmacotherapy development for AUD.

What Can We Learn from FGF-2 Isoform-Specific Mouse Mutants? Differential Insights into FGF-2 Physiology In Vivo.

Fibroblast growth factor 2 (FGF-2), ubiquitously expressed in humans and mice, is functionally involved in cell growth, migration and maturation in vitro and in vivo. Based on the same mRNA, an 18-kilo Dalton (kDa) FGF-2 isoform named FGF-2 low molecular weight (FGF-2LMW) isoform is translated in humans and rodents. Additionally, two larger isoforms weighing 21 and 22 kDa also exist, summarized as the FGF-2 high molecular weight (FGF-2HMW) isoform. Meanwhile, the human FGF-2HMW comprises a 22, 23, 24 and 34 kDa protein. Independent studies verified a specific intracellular localization, mode of action and tissue-specific spatiotemporal expression of the FGF-2 isoforms, increasing the complexity of their physiological and pathophysiological roles. In order to analyze their spectrum of effects, FGF-2LMW knock out (ko) and FGF-2HMWko mice have been generated, as well as mice specifically overexpressing either FGF-2LMW or FGF-2HMW. So far, the development and functionality of the cardiovascular system, bone formation and regeneration as well as their impact on the central nervous system including disease models of neurodegeneration, have been examined. This review provides a summary of the studies characterizing the in vivo effects modulated by the FGF-2 isoforms and, thus, offers a comprehensive overview of its actions in the aforementioned organ systems.

Characterizing the multiple roles of FGF-2 in SOD1G93A ALS mice in vivo and in vitro.

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 G93A -dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.

Expression of the axon-guidance protein receptor Neuropilin 1 is increased in the spinal cord and decreased in muscle of a mouse model of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a degenerative motor neuron disorder. It is supposed that ALS is at least in part an axonopathy. Neuropilin 1 is an important receptor of the axon repellent Semaphorin 3A and a co-receptor of vascular endothelial growth factor. It is probably involved in neuronal and axonal de-/regeneration and might be of high relevance for ALS pathogenesis and/or disease progression. To elucidate whether the expression of either Neuropilin1 or Semaphorin3A is altered in ALS we investigated these proteins in human brain, spinal cord and muscle tissue of ALS-patients and controls as well as transgenic SOD1G93A and control mice. Neuropilin1 and Semaphorin3A gene and protein expression were assessed by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. Groups were compared using either Student t-test or Mann-Whitney U test. We observed a consistent increase of Neuropilin1 expression in the spinal cord and decrease of Neuropilin1 and Semaphorin3A in muscle tissue of transgenic SOD1G93A mice at the mRNA and protein level. Previous studies have shown that damage of neurons physiologically causes Neuropilin1 and Semaphorin3A increase in the central nervous system and decrease in the peripheral nervous system. Our results indicate that this also occurs in ALS. Pharmacological modulation of expression and function of axon repellents could be a promising future therapeutic option in ALS.

Isoform-selective as opposed to complete depletion of fibroblast growth factor 2 (FGF-2) has no major impact on survival and gene expression in SOD1G93A amyotrophic lateral sclerosis mice.

We have previously shown that total knockout of fibroblast growth factor-2 (FGF-2) results in prolonged survival and improved motor performance in superoxide dismutase 1 (SOD1G93A ) mutant mice, the most widely used animal model of the fatal adult onset motor neuron disease amyotrophic lateral sclerosis (ALS). Moreover, we found differential expression of growth factors in SOD1G93A mice, with distinct regulation patterns of FGF-2 in spinal cord and muscle tissue. Within the present study we aimed to characterize FGF-2-isoform specific effects on survival, motor performance as well as gene expression patterns predominantly in muscle tissue by generating double mutant SOD1G93A FGF-2 high molecular weight- and SOD1G93A FGF-2 low molecular weight-knockout mice. While isoform specific depletion was not beneficial regarding survival or motor performance of double mutant mice, we found isoform-dependent differential gene expression of epidermal growth factor (EGF) in the muscle of SOD1G93A FGF-2 low molecular weight knockout mice compared to single mutant SOD1G93A mice. This significant downregulation of EGF in the muscle tissue of double mutant SOD1G93A FGF-2 low molecular weight knockout mice implies that FGF-2 low molecular weight knockout (or the presence of the FGF-2 high molecular weight isoform) selectively impacts EGF gene expression in ALS muscle tissue.

4-Aminopyridine Induced Activity Rescues Hypoexcitable Motor Neurons from Amyotrophic Lateral Sclerosis Patient-Derived Induced Pluripotent Stem Cells.

Despite decades of research on amyotrophic lateral sclerosis (ALS), there is only one approved drug, which minimally extends patient survival. Here, we investigated pathophysiological mechanisms underlying ALS using motor neurons (MNs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying mutations in FUS or SOD1. Patient-derived MNs were less active and excitable compared to healthy controls, due to reduced Na(+) /K(+) ratios in both ALS groups accompanied by elevated potassium channel (FUS) and attenuated sodium channel expression levels (FUS, SOD1). ALS iPSC-derived MNs showed elevated endoplasmic reticulum stress (ER) levels and increased caspase activation. Treatment with the FDA approved drug 4-Aminopyridine (4AP) restored ion-channel imbalances, increased neuronal activity levels and decreased ER stress and caspase activation. This study provides novel pathophysiological data, including a mechanistic explanation for the observed hypoexcitability in patient-derived MNs and a new therapeutic strategy to provide neuroprotection in MNs affected by ALS. Stem Cells 2016;34:1563-1575.

Altered Immunomodulatory Responses in the CX3CL1/CX3CR1 Axis Mediated by hMSCs in an Early In Vitro SOD1G93A Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron (MN) disease characterized by progressive MN loss and muscular atrophy resulting in rapidly progressive paralysis and respiratory failure. Human mesenchymal stem/stromal cell (hMSC)-based therapy has been suggested to prolong MN survival via secretion of growth factors and modulation of cytokines/chemokines. We investigated the effects of hMSCs and a hMSC-conditioned medium (CM) on Cu/Zn superoxidase dismutase 1G93A (SOD1G93A) transgenic primary MNs. We found that co-culture of hMSCs and MNs resulted in slightly higher MN numbers, but did not protect against staurosporine (STS)-induced toxicity, implying marginal direct trophic effects of hMSCs. Aiming to elucidate the crosstalk between hMSCs and MNs in vitro, we found high levels of vascular endothelial growth factor (VEGF) and C-X3-C motif chemokine 1 (CX3CL1) in the hMSC secretome. Co-culture of hMSCs and MNs resulted in altered gene expression of growth factors and cytokines/chemokines in both MNs and hMSCs. hMSCs showed upregulation of CX3CL1 and its receptor CX3CR1 and downregulation of interleukin-1 ß (IL1ß) and interleukin-8 (IL8) when co-cultured with SOD1G93A MNs. MNs, on the other hand, showed upregulation of growth factors as well as CX3CR1 upon hMSC co-culture. Our results indicate that hMSCs only provide moderate trophic support to MNs by growth factor gene regulation and may mediate anti-inflammatory responses through the CX3CL1/CX3CR1 axis, but also increase expression of pro-inflammatory cytokines, which limits their therapeutic potential.

SENP7 deSUMOylase-governed transcriptional program coordinates sarcomere assembly and is targeted in muscle atrophy.

Disorganization of the basic contractile unit of muscle cells, i.e., the sarcomeres, leads to suboptimal force generation and is a hallmark of muscle atrophy. Here, we demonstrate that the nuclear role of SENP7 deSUMOylase is pivotal for sarcomere organization. SENP7 expression is temporally upregulated in mature muscle cells and directly regulates transcription of the myosin heavy chain (MyHC-IId) gene. We identify SENP7-dependent deSUMOylation of flightless-1 (Fli-I) as a signal for Fli-I association with scaffold attachment factor b1 (Safb1). SENP7 deficiency leads to higher Fli-I SUMOylation and lower chromatin residency of Safb1, thus generating transcriptionally incompetent chromatin conformation on MyHC-IId. Consequently, lower expression of MyHC-IId causes sarcomere disorganization and disrupted muscle cell contraction. Remarkably, cachexia signaling impedes the SENP7-governed transcriptional program, leading to muscle atrophy, with profound loss of motor protein MyHC-IId. We propose a SENP7-driven distinct transcription program as paramount for muscle cell function, which was found targeted in cachexia.

Regulation of SETD7 Methyltransferase by SENP3 Is Crucial for Sarcomere Organization and Cachexia.

Precise assembly of the sarcomere, a force-generating unit in striated muscles, is critical for muscle contraction. Defective sarcomere organization is linked to myopathies and cachexia. The molecular mechanisms concerning sarcomere assembly are poorly understood. Here, we report that the SUMO-specific isopeptidase SENP3 determines sarcomere assembly by specifically regulating the sarcomeric contractile myosin heavy-chain gene MyHC-II. The contractile ability of mature muscle cells is severely compromised in SENP3-depleted cells. Mechanistically, SENP3 is associated with the SETD7 histone methyltransferase and deSUMOylates SETD7. By recruiting SETD7 to MyHC-II, SENP3 promotes association of SETD7 with transcriptionally active RNA polymerase II and precludes the opposing methyltransferase Suv39h1. Strikingly, SENP3 is degraded in cachexia, characterized by dramatic loss of sarcomeric protein, particularly MyHC-II. SENP3 regulation of SETD7 is impaired in cachexia, leading to perturbed MyHC-II expression and disorganized sarcomeres. Our findings reveal an unanticipated role of SENP3 in sarcomere assembly and cachexia.

Analysis of the therapeutic potential of different administration routes and frequencies of human mesenchymal stromal cells in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Cellular therapy represents a novel option for the treatment of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Its major aim is the generation of a protective environment for degenerating motor neurons. Mesenchymal stromal cells secrete different growth factors and have antiapoptotic and immunomodulatory properties. They can easily and safely be isolated from human bone marrow and are therefore considered promising therapeutic candidates. In the present study, we compared intraventricular application of human mesenchymal stromal cells (hMSCs) versus single and repeated intraspinal injections in the mutant SOD1G93A transgenic ALS mouse model. We observed significant reduction of lifespan of animals treated by intraventricular hMSC injection compared with the vehicle treated control group, accompanied by changes in weight, general condition, and behavioural assessments. A potential explanation for these rather surprising deleterious effects lies in increased microgliosis detected in the hMSC treated animals. Repeated intraspinal injection at two time points resulted in a slight but not significant increase in survival and significant improvement of motor performance although no hMSC-induced changes of motor neuron numbers, astrogliosis, and microgliosis were detected. Quantitative real time polymerase chain reaction showed reduced expression of endothelial growth factor in animals having received hMSCs twice compared with the vehicle treated control group. hMSCs were detectable at the injection site at Day 20 after injection into the spinal cord but no longer at Day 70. Intraspinal injection of hMSCs may therefore be a more promising option for the treatment of ALS than intraventricular injection and repeated injections might be necessary to obtain substantial therapeutic benefit.