The Proteome and Secretome of Cortical Brain Cells Infected With Herpes Simplex Virus.

Infections of the brain with herpes simplex virus type 1 (HSV-1) cause life-threatening Herpes simplex encephalitis (HSE) characterized by viral replication in neurons and neuro-inflammation including an infiltration of peripheral immune cells. HSV-1 reprograms host cells to foster its own replication and for immune evasion, but eventually the immune responses clear the infection in most patients. However, many survivors suffer from long-term neuronal damage and cannot regenerate all brain functions. HSV-1 influences the physiology of neurons, astrocytes, oligodendrocytes and microglia, and significantly changes their protein expression and secretion pattern. To characterize temporal changes upon HSV-1 infection in detail, we inoculated mixed primary cultures of the murine brain cortex, and performed quantitative mass spectrometry analyses of the cell-associated proteome and the secretome. We identified 28 differentially regulated host proteins influencing inflammasome formation and intracellular vesicle trafficking during endocytosis and secretion. The NIMA-related kinase 7 (NEK7), a critical component of the inflammasome, and ArfGap1, a regulator of endocytosis, were significantly up-regulated upon HSV-1 infection. In the secretome, we identified 71 proteins including guidance cues regulating axonal regeneration, such as semaphorin6D, which were enriched in the conditioned media of HSV-1 infected cells. Modulation of inflammasome activity and intracellular membrane traffic are critical for HSV-1 cell entry, virus assembly, and intracellular spread. Our proteome analysis provides first clues on host factors that might dampen the inflammasome response and modulate intracellular vesicle transport to promote HSV infection of the brain. Furthermore, our secretome analysis revealed a set of proteins involved in neuroregeneration that might foster neuronal repair processes to restore brain functions after clearance of an HSV-1 infection.

HSV-1 triggers paracrine fibroblast growth factor response from cortical brain cells via immediate-early protein ICP0.

BACKGROUND: Herpes simplex virus-1 (HSV-1) infections of the central nervous system (CNS) can result in HSV-1 encephalitis (HSE) which is characterized by severe brain damage and long-term disabilities. Different cell types including neurons and astrocytes become infected in the course of an HSE which leads to an activation of glial cells. Activated glial cells change their neurotrophic factor profile and modulate inflammation and repair. The superfamily of fibroblast growth factors (FGFs) is one of the largest family of neurotrophic factors comprising 22 ligands. FGFs induce pro-survival signaling in neurons and an anti-inflammatory answer in glial cells thereby providing a coordinated tissue response which favors repair over inflammation. Here, we hypothesize that FGF expression is altered in HSV-1-infected CNS cells. METHOD: We employed primary murine cortical cultures comprising a mixed cell population of astrocytes, neurons, microglia, and oligodendrocytes. Astrocyte reactivity was morphometrically monitored by an automated image analysis algorithm as well as by analyses of A1/A2 marker expression. Altered FGF expression was detected by quantitative real-time PCR and its paracrine FGF activity. In addition, HSV-1 mutants were employed to characterize viral factors important for FGF responses of infected host cells. RESULTS: Astrocytes in HSV-1-infected cortical cultures were transiently activated and became hypertrophic and expressed both A1- and A2-markers. Consistently, a number of FGFs were transiently upregulated inducing paracrine neurotrophic signaling in neighboring cells. Most prominently, FGF-4, FGF-8, FGF-9, and FGF-15 became upregulated in a switch-on like mechanism. This effect was specific for CNS cells and for a fully functional HSV-1. Moreover, the viral protein ICP0 critically mediated the FGF switch-on mechanism. CONCLUSIONS: HSV-1 uses the viral protein ICP0 for the induction of FGF-expression in CNS cells. Thus, we propose that HSV-1 triggers FGF activity in the CNS for a modulation of tissue response upon infection.

Fibroblast growth factor 23 signaling in hippocampal cells: impact on neuronal morphology and synaptic density.

Endocrine fibroblast growth factor 23 (FGF23) is predominantly secreted by osteocytes and facilitates renal phosphate excretion. However, FGF23 is also present in cerebrospinal fluid. In chronic kidney disease, FGF23 serum levels are excessively elevated and associated with learning and memory deficits. Structural plasticity of the hippocampus such as formation of new synapses or an altered dendritic arborization comprises a cellular and morphological correlate of memory formation. Therefore, we hypothesize that FGF23 alters hippocampal neuron morphology and synapses. To address this, we prepared primary murine hippocampal cultures and incubated them with recombinant FGF23 alone or together with a soluble isoform of its co-receptor α-Klotho. Neuronal expression of a fluorescent reporter allowed for a detailed evaluation of the neuronal morphology by Sholl analysis. Additionally, we evaluated synaptic density, identified by stainings, for synaptic markers. We show an enhanced number of primary neurites combined with a reduced arborization, resulting in a less complex morphology of neurons treated with FGF23. Moreover, FGF23 enhances the synaptic density in a FGF-receptor (FGF-R) dependent manner. Finally, we addressed the corresponding signaling events downstream of FGF-R employing a combination of western blots and quantitative immunofluorescence. Interestingly, FGF23 induces phospholipase Cγ activity in primary hippocampal neurons. Co-application of soluble α-Klotho leads to activation of the Akt-pathway and modifies FGF23-impact on neuronal morphology and synaptic density. Compared with other FGFs, this alternative signaling pattern is a possible reason for differential effects of FGF23 on hippocampal neurons and may thereby contribute to learning and memory deficits in chronic kidney disease patients. In this study, we show that fibroblast growth factor 23 inhibits neuronal ramification and enhances the synaptic density in primary hippocampal cultures accompanied by phospholipase Cγ-activation. Co-application of the co-receptor α-Klotho leads to an Akt-activation and further modifies neuronal morphology and number of synapses. Those effects provide a mechanistic basis for memory deficits in patients suffering from chronic kidney disease (CKD) characterized by excessively elevated FGF23 levels as well as memory deficits.

Coalition of Nuclear Receptors in the Nervous System.

A universal signaling module has been described which utilizes the nuclear form of Fibroblast growth Factor Receptor 1 (FGFR1) in a central role directing the post-mitotic development of neural cells through coordinated gene expression. In this review, we discuss in detail the current knowledge of FGFR1 nuclear interaction partners in three scenarios: (i) Engagement of FGFR1 in neuronal stem cells and regulation of neuronal differentiation; (ii) interaction with the orphan receptor Nurr1 in development of mesencephalic dopaminergic neurons; (iii) modulation of nuclear FGFR1 interactions downstream of nerve growth factor (NGF) signaling. These coalitions demonstrate the versatility of non-canonical, nuclear tyrosine kinase signaling in diverse cellular differentiation programs of neurons.

A nuclear odyssey: fibroblast growth factor-2 (FGF-2) as a regulator of nuclear homeostasis in the nervous system.

Nuclear localization of classical growth factors is a well-known phenomenon but still remains a molecular and cellular conundrum. Fibroblast growth factor-2 (FGF-2) is an excellent example of a protein which functions as an extracellular molecule involved in canonical receptor tyrosine kinase signaling as well as displaying intracellular functions. Paracrine and nuclear functions are two important sides of the same protein. FGF-2 is expressed in isoforms with different molecular weights from one mRNA species. In rodents, all of these isoforms become imported to the nucleus. In this review, we discuss structural and functional aspects of FGF-2 isoforms in the nervous system. The nuclear odyssey of FGF-2 is reflected by nuclear dynamics, localization to nuclear bodies such as nucleoli, binding to chromatin and engagement in various protein interactions. Recently discovered molecular partnerships of the isoforms shed light on their nuclear functions, thereby greatly extending our knowledge of the multifaceted functions of FGF-2.

Regulation of neuronal differentiation by proteins associated with nuclear bodies.

Nuclear bodies are large sub-nuclear structures composed of RNA and protein molecules. The Survival of Motor Neuron (SMN) protein localizes to Cajal bodies (CBs) and nuclear gems. Diminished cellular concentration of SMN is associated with the neurodegenerative disease Spinal Muscular Atrophy (SMA). How nuclear body architecture and its structural components influence neuronal differentiation remains elusive. In this study, we analyzed the effects of SMN and two of its interaction partners in cellular models of neuronal differentiation. The nuclear 23 kDa isoform of Fibroblast Growth Factor - 2 (FGF-2(23)) is one of these interacting proteins - and was previously observed to influence nuclear bodies by destabilizing nuclear gems and mobilizing SMN from Cajal bodies (CBs). Here we demonstrate that FGF-2(23) blocks SMN-promoted neurite outgrowth, and also show that SMN disrupts FGF-2(23)-dependent transcription. Our results indicate that FGF-2(23) and SMN form an inactive complex that interferes with neuronal differentiation by mutually antagonizing nuclear functions. Coilin is another nuclear SMN binding partner and a marker protein for Cajal bodies (CBs). In addition, coilin is essential for CB function in maturation of small nuclear ribonucleoprotein particles (snRNPs). The role of coilin outside of Cajal bodies and its putative impacts in tissue differentiation are poorly defined. The present study shows that protein levels of nucleoplasmic coilin outside of CBs decrease during neuronal differentiation. Overexpression of coilin has an inhibitory effect on neurite outgrowth. Furthermore, we find that nucleoplasmic coilin inhibits neurite outgrowth independent of SMN binding revealing a new function for coilin in neuronal differentiation.

Immobile survival of motoneuron (SMN) protein stored in Cajal bodies can be mobilized by protein interactions.

Reduced levels of survival of motoneuron (SMN) protein lead to spinal muscular atrophy, but it is still unknown how SMN protects motoneurons in the spinal cord against degeneration. In the nucleus, SMN is associated with two types of nuclear bodies denoted as gems and Cajal bodies (CBs). The 23 kDa isoform of fibroblast growth factor-2 (FGF-2(23)) is a nuclear protein that binds to SMN and destabilizes the SMN-Gemin2 complex. In the present study, we show that FGF-2(23) depletes SMN from CBs without affecting their general structure. FRAP analysis of SMN-EGFP in CBs demonstrated that the majority of SMN in CBs remained mobile and allowed quantification of fast, slow and immobile nuclear SMN populations. The potential for SMN release was confirmed by in vivo photoconversion of SMN-Dendra2, indicating that CBs concentrate immobile SMN that could have a specialized function in CBs. FGF-2(23) accelerated SMN release from CBs, accompanied by a conversion of immobile SMN into a mobile population. Furthermore, FGF-2(23) caused snRNP accumulation in CBs. We propose a model in which Cajal bodies store immobile SMN that can be mobilized by its nuclear interaction partner FGF-2(23), leading to U4 snRNP accumulation in CBs, indicating a role for immobile SMN in tri-snRNP assembly.

Cooperation of nuclear fibroblast growth factor receptor 1 and Nurr1 offers new interactive mechanism in postmitotic development of mesencephalic dopaminergic neurons.

Experiments in mice deficient for Nurr1 or expressing the dominant-negative FGF receptor (FGFR) identified orphan nuclear receptor Nurr1 and FGFR1 as essential factors in development of mesencephalic dopaminergic (mDA) neurons. FGFR1 affects brain cell development by two distinct mechanisms. Activation of cell surface FGFR1 by secreted FGFs stimulates proliferation of neural progenitor cells, whereas direct integrative nuclear FGFR1 signaling (INFS) is associated with an exit from the cell cycle and neuronal differentiation. Both Nurr1 and INFS activate expression of neuronal genes, such as tyrosine hydroxylase (TH), which is the rate-limiting enzyme in dopamine synthesis. Here, we show that nuclear FGFR1 and Nurr1 are expressed in the nuclei of developing TH-positive cells in the embryonic ventral midbrain. Both nuclear receptors were effectively co-immunoprecipitated from the ventral midbrain of FGF-2-deficient embryonic mice, which previously showed an increase of mDA neurons and enhanced nuclear FGFR1 accumulation. Immunoprecipitation and co-localization experiments showed the presence of Nurr1 and FGFR1 in common nuclear protein complexes. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrated the Nurr1-mediated shift of nuclear FGFR1-EGFP mobility toward a transcriptionally active population and that both Nurr1 and FGFR1 bind to a common region in the TH gene promoter. Furthermore, nuclear FGFR1 or its 23-kDa FGF-2 ligand (FGF-2(23)) enhances Nurr1-dependent activation of the TH gene promoter. Transcriptional cooperation of FGFR1 with Nurr1 was confirmed on isolated Nurr1-binding elements. The proposed INFS/Nurr1 nuclear partnership provides a novel mechanism for TH gene regulation in mDA neurons and a potential therapeutic target in neurodevelopmental and neurodegenerative disorders.