Localization of BDNF and Calretinin in Olfactory Epithelium and Taste Buds of Zebrafish (Danio rerio)

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and it is involved in several fundamental functions in the central and peripheral nervous systems, and in sensory organs. BDNF regulates the chemosensory systems of mammals and is consistently expressed in those organs. In zebrafish, the key role of BDNF in the biology of the hair cells of the inner ear and lateral line system has recently been demonstrated. However, only some information is available about its occurrence in the olfactory epithelium, taste buds, and cutaneous isolated chemosensory cells. Therefore, this study was undertaken to analyze the involvement of BDNF in the chemosensory organs of zebrafish during the larval and adult stages. To identify cells displaying BDNF, we compared the cellular pattern of BDNF-displaying cells with those immunoreactive for calretinin and S100 protein. Our results demonstrate the localization of BDNF in the sensory part of the olfactory epithelium, mainly in the ciliated olfactory sensory neurons in larvae and adult zebrafish. Intense immunoreaction for BDNF was also observed in the chemosensory cells of oral and cutaneous taste buds. Moreover, a subpopulation of olfactory sensory neurons and chemosensory cells of olfactory rosette and taste bud, respectively, showed marked immunopositivity for calcium-binding protein S100 and calretinin. These results demonstrate the possible role of BDNF in the development and maintenance of olfactory sensory neurons and sensory cells in the olfactory epithelium and taste organs of zebrafish during all stages of development.     

The Zebrafish,an Outstanding Model for Biomedical Research in the Field of Melatonin and Human Diseases

The zebrafish has become an excellent model for the study of human diseases because it offers many advantages over other vertebrate animal models. The pineal gland, as well as the biological clock and circadian rhythms, are highly conserved in zebrafish, and melatonin is produced in the pineal gland and in most organs and tissues of the body. Zebrafish have several copies of the clock genes and of aanat and asmt genes, the latter involved in melatonin synthesis. As in mammals, melatonin can act through its membrane receptors, as with zebrafish, and through mechanisms that are independent of receptors. Pineal melatonin regulates peripheral clocks and the circadian rhythms of the body, such as the sleep/wake rhythm, among others. Extrapineal melatonin functions include antioxidant activity, inducing the endogenous antioxidants enzymes, scavenging activity, removing free radicals, anti-inflammatory activity through the regulation of the NF-κB/NLRP3 inflammasome pathway, and a homeostatic role in mitochondria. In this review, we introduce the utility of zebrafish to analyze the mechanisms of action of melatonin. The data here presented showed that the zebrafish is a useful model to study human diseases and that melatonin exerts beneficial effects on many pathophysiological processes involved in these diseases.     

Neurotrophin Signaling Impairment by Viral Infections in the Central Nervous System

Neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3), NT-4, and NT-5, are proteins involved in several important functions of the central nervous system. The activation of the signaling pathways of these neurotrophins, or even by their immature form, pro-neurotrophins, starts with their recognition by cellular receptors, such as tropomyosin receptor kinase (Trk) and 75 kD NT receptors (p75NTR). The Trk receptor is considered to have a high affinity for attachment to specific neurotrophins, while the p75NTR receptor has less affinity for attachment with neurotrophins. The correct functioning of these signaling pathways contributes to proper brain development, neuronal survival, and synaptic plasticity. Unbalanced levels of neurotrophins and pro-neurotrophins have been associated with neurological disorders, illustrating the importance of these molecules in the central nervous system. Furthermore, reports have indicated that viruses can alter the normal levels of neurotrophins by interfering with their signaling pathways. This work discusses the importance of neurotrophins in the central nervous system, their signaling pathways, and how viruses can affect them.     

The Impact of Physical Exercise on the Circulating Levels of BDNF and NT 4/5: A Review

(1) Background: One mechanism through which physical activity (PA) provides benefits is by triggering activity at a molecular level, where neurotrophins (NTs) are known to play an important role. However, the expression of the circulating levels of neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4/5), in response to exercise, is not fully understood. Therefore, the aim was to provide an updated overview on the neurotrophin (NT) variation levels of BDNF and NT-4/5 as a consequence of a long-term aerobic exercise intervention, and to understand and describe whether the upregulation of circulating NT levels is a result of neurotrophic factors produced and released from the brain, and/or from neurotrophic secreting peripheral organs. (2) Methods: The articles were collected from PubMed, SPORTDiscus, Web of Science, MEDLINE, and Embase. Data were analyzed through a narrative synthesis. (3) Results: 30 articles studied humans who performed training protocols that ranged from 4 to 48 weeks; 22 articles studied rodents with an intervention period that ranged from 4 to 64 weeks. (4) Conclusions: There is no unanimity between the upregulation of BDNF in humans; conversely, concerning both BDNF and NT-4/5 in animal models, the results are heterogeneous. Whilst BDNF upregulation appears to be in relative agreement, NT-4/5 seems to display contradictory and inconsistent conclusions.     

Brain Proteome and Behavioural Analysis in Wild Type,BDNF+/− and BDNF−/− Adult Zebrafish (Danio rerio) Exposed to Two Different Temperatures

Experimental evidence suggests that environmental stress conditions can alter the expression of BDNF and that the expression of this neurotrophin influences behavioural responses in mammalian models. It has been recently demonstrated that exposure to 34 °C for 21 days alters the brain proteome and behaviour in zebrafish. The aim of this work was to investigate the role of BDNF in the nervous system of adult zebrafish under control and heat treatment conditions. For this purpose, zebrafish from three different genotypes (wild type, heterozygous BDNF^+/− and knock out BDNF^−/−) were kept for 21 days at 26 °C or 34 °C and then euthanized for brain molecular analyses or subjected to behavioural tests (Y-maze test, novel tank test, light and dark test, social preference test, mirror biting test) for assessing behavioural aspects such as boldness, anxiety, social preference, aggressive behaviour, interest for the novel environment and exploration. qRT-PCR analysis showed the reduction of gene expression of BDNF and its receptors after heat treatment in wild type zebrafish. Moreover, proteomic analysis and behavioural tests showed genotype- and temperature-dependent effects on brain proteome and behavioural responding. Overall, the absent expression of BDNF in KO alters (1) the brain proteome by reducing the expression of proteins involved in synapse functioning and neurotransmitter-mediated transduction; (2) the behaviour, which can be interpreted as bolder and less anxious and (3) the cellular and behavioural response to thermal treatment.     

Damage to Olfactory Organs of Adult Zebrafish Induced by Diesel Particulate Matter

Particulate matter (PM) is an environmental hazard that is associated with various human health risks. The olfactory system is directly exposed to PM; therefore, the influence of PM exposure on olfactory function must be investigated. In this study, we propose a zebrafish olfactory model to evaluate the effects of exposure to diesel particulate matter (DPM), which was labeled Korean diesel particulate matter (KDP20). KDP20 comprises heavy metals and polycyclic aromatic hydrocarbons (PAHs). KDP20 exposed olfactory organs exhibited reduced cilia and damaged epithelium. Olfactory dysfunction was confirmed using an odor-mediated behavior test. Furthermore, the olfactory damage was analyzed using Alcian blue and anti-calretinin staining. KDP20 exposed olfactory organs exhibited histological damages, such as increased goblet cells, decreased cell density, and calretinin level. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that PAHs exposure related genes (AHR2 and CYP1A) were upregulated. Reactive oxidation stress (ROS) (CAT) and inflammation (IL-1B) related genes were upregulated. Furthermore, olfactory sensory neuron (OSN) related genes (OMP and S100) were downregulated. In conclusion, KDP20 exposure induced dysfunction of the olfactory system. Additionally, the zebrafish olfactory system exhibited a regenerative capacity with recovery conditions. Thus, this model may be used in future investigating PM-related diseases.     

Neurotrophic Factors in Experimental Cerebral Acanthamoebiasis

To date, no studies have addressed the role of neurotrophins (NTs) in Acanthamoeba spp. infections in the brain. Thus, to clarify the role of NTs in the cerebral cortex and hippocampus during experimental acanthamoebiasis in relation to the host immune status, the purpose of this study was to determine whether Acanthamoeba spp. may affect the concentration of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) in brain structures. Our results suggest that at the beginning of infection in immunocompetent hosts, BDNF and NT-3 may reflect an endogenous attempt at neuroprotection against Acanthamoeba spp. infection. We also observed a pro-inflammatory effect of NGF during acanthamoebiasis in immunosuppressed hosts. This may provide important information for understanding the development of cerebral acanthamoebiasis related to the immunological status of the host. However, the pathogenesis of brain acanthamoebiasis is still poorly understood and documented and, therefore, requires further research.     

Exposure to Zinc Sulfate Results in Differential Effects on Olfactory Sensory Neuron Subtypes in Adult Zebrafish

Zinc sulfate is a known olfactory toxicant, although its specific effects on the olfactory epithelium of zebrafish are unknown. Olfactory organs of adult zebrafish were exposed to zinc sulfate and, after 2, 3, 5, 7, 10 or 14 days, fish were processed for histological, immunohistochemical, ultrastructural, and behavioral analyses. Severe morphological disruption of the olfactory organ was observed two days following zinc sulfate exposure, including fusion of lamellae, epithelial inflammation, and significant loss of anti-calretinin labeling. Scanning electron microscopy revealed the apical surface of the sensory region was absent of ciliated structures, but microvilli were still present. Behavioral analysis showed significant loss of the ability to perceive bile salts and some fish also had no response to amino acids. Over the next several days, olfactory organ morphology, epithelial structure, and anti-calretinin labeling returned to control-like conditions, although the ability to perceive bile salts remained lost until day 14. Thus, exposure to zinc sulfate results in rapid degeneration of the olfactory organ, followed by restoration of morphology and function within two weeks. Zinc sulfate appears to have a greater effect on ciliated olfactory sensory neurons than on microvillous olfactory sensory neurons, suggesting differential effects on sensory neuron subtypes.     

The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid

Neurotrophins and their receptors are relevant factors in controlling neuroblastoma growth and progression. The histone deacetylase (HDAC) inhibitor valproic acid (VPA) has been shown to downregulate TrkB and upregulate the p75NTR/sortilin receptor complex. In the present study, we investigated the VPA effect on the expression of the neurotrophin-3 (NT-3) receptor TrkC, a favorable prognostic marker of neuroblastoma. We found that VPA induced the expression of both full-length and truncated (TrkC-T1) isoforms of TrkC in human neuroblastoma cell lines without (SH-SY5Y) and with (Kelly, BE(2)-C and IMR 32) MYCN amplification. VPA enhanced cell surface expression of the receptor and increased Akt and ERK1/2 activation by NT-3. The HDAC inhibitors entinostat, romidepsin and vorinostat also increased TrkC in SH-SY5Y, Kelly and BE(2)-C but not IMR 32 cells. TrkC upregulation by VPA involved induction of RUNX3, stimulation of ERK1/2 and JNK, and ERK1/2-mediated Egr1 expression. In SH-SY5Y cell monolayers and spheroids the exposure to NT-3 enhanced the apoptotic cascade triggered by VPA. Gene silencing of both TrkC-T1 and p75NTR prevented the NT-3 proapoptotic effect. Moreover, NT-3 enhanced p75NTR/TrkC-T1 co-immunoprecipitation. The results indicate that VPA upregulates TrkC by activating epigenetic mechanisms and signaling pathways, and sensitizes neuroblastoma cells to NT-3-induced apoptosis.      

Localization of Neurotrophin Specific Trk Receptors in Mechanosensory Systems of Killifish (Nothobranchius guentheri)

Neurotrophins (NTs) and their signal-transducing Trk receptors play a crucial role in the development and maintenance of specific neuronal subpopulations in nervous and sensory systems. NTs are supposed to regulate two sensory systems in fish, the inner ear and the lateral line system (LLS). The latter is one of the major mechanosensory systems in fish. Considering that annual fishes of the genus Nothobranchius, with their short life expectancy, have become a suitable model for aging studies and that the occurrence and distribution of neurotrophin Trk receptors have never been investigated in the inner ear and LLS of killifish (Nothobranchius guentheri), our study aimed to investigate the localization of neurotrophin-specific Trk receptors in mechanosensory systems of N. guentheri. For histological and immunohistochemical analysis, adult specimens of N. guentheri were processed using antibodies against Trk receptors and S100 protein. An intense immunoreaction for TrkA and TrkC was found in the sensory cells of the inner ear as well as in the hair cells of LLS. Moreover, also the neurons localized in the acoustic ganglia displayed a specific immunoreaction for all Trk receptors (TrkA, B, and C) analyzed. Taken together, our results demonstrate, for the first time, that neurotrophins and their specific receptors could play a pivotal role in the biology of the sensory cells of the inner ear and LLS of N. guentheri and might also be involved in the hair cells regeneration process in normal and aged conditions.     

New Evidence on the Role of D-Aspartate Metabolism in Regulating Brain and Endocrine System Physiology: From Preclinical Observations to Clinical Applications

The endogenous amino acids serine and aspartate occur at high concentrations in free D-form in mammalian organs, including the central nervous system and endocrine glands. D-serine (D-Ser) is largely localized in the forebrain structures throughout pre and postnatal life. Pharmacologically, D-Ser plays a functional role by acting as an endogenous coagonist at N-methyl-D-aspartate receptors (NMDARs). Less is known about the role of free D-aspartate (D-Asp) in mammals. Notably, D-Asp has a specific temporal pattern of occurrence. In fact, free D-Asp is abundant during prenatal life and decreases greatly after birth in concomitance with the postnatal onset of D-Asp oxidase expression, which is the only enzyme known to control endogenous levels of this molecule. Conversely, in the endocrine system, D-Asp concentrations enhance after birth during its functional development, thereby suggesting an involvement of the amino acid in the regulation of hormone biosynthesis. The substantial binding affinity for the NMDAR glutamate site has led us to investigate the in vivo implications of D-Asp on NMDAR-mediated responses. Herein we review the physiological function of free D-Asp and of its metabolizing enzyme in regulating the functions of the brain and of the neuroendocrine system based on recent genetic and pharmacological human and animal studies.     

Effects of Pituitary Adenylate Cyclase Activating Polypeptide on Cell Death

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated as a hypothalamic peptide based on its efficacy to increase adenylate cyclase (AC) activity. It has a widespread distribution throughout the body including the nervous system and peripheral organs, where PACAP exerts protective effects both in vivo and in vitro through its anti-apoptotic, anti-inflammatory, and antioxidant functions. The aim of the present paper was to review the currently available literature regarding the effects of PACAP on cell death in vitro in neural and non-neural cells. Among others, its effect on apoptosis can be detected in cerebellar granule cells against different toxic stimuli. Different neural cell types from the cerebral cortex are also prevented from cell death. PACAP also shows effects on cell death in cells belonging to the peripheral nervous system and protects both neural and non-neural cells of sensory organs. In addition, cell survival-promoting effect can be observed in different peripheral organ systems including cardiovascular, immune, respiratory, gastrointestinal, urinary, and reproductive systems. The studies summarized here indicate its noteworthy effect on cell death in different in vitro models, suggesting PACAP’s potential therapeutic usage in several pathological conditions.     

Renal and Extra Renal Manifestations in Adult Zebrafish Model of Cystinosis

Cystinosis is a rare, incurable, autosomal recessive disease caused by mutations in the CTNS gene. This gene encodes the lysosomal cystine transporter cystinosin, leading to lysosomal cystine accumulation in all cells of the body, with kidneys being the first affected organs. The current treatment with cysteamine decreases cystine accumulation, but does not reverse the proximal tubular dysfunction, glomerular injury or loss of renal function. In our previous study, we have developed a zebrafish model of cystinosis through a nonsense mutation in the CTNS gene and have shown that zebrafish larvae recapitulate the kidney phenotype described in humans. In the current study, we characterized the adult cystinosis zebrafish model and evaluated the long-term effects of the disease on kidney and extra renal organs through biochemical, histological, fertility and locomotor activity studies. We found that the adult cystinosis zebrafish presents cystine accumulation in various organs, altered kidney morphology, impaired skin pigmentation, decreased fertility, altered locomotor activity and ocular anomalies. Overall, our data indicate that the adult cystinosis zebrafish model reproduces several human phenotypes of cystinosis and may be useful for studying pathophysiology and long-term effects of novel therapies.     

Optogenetic Manipulation of Olfactory Responses in Transgenic Zebrafish: A Neurobiological and Behavioral Study

Olfaction is an important neural system for survival and fundamental behaviors such as predator avoidance, food finding, memory formation, reproduction, and social communication. However, the neural circuits and pathways associated with the olfactory system in various behaviors are not fully understood. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of neuronal circuits have created new opportunities to understand such neural circuits. Here, we generated a transgenic zebrafish to manipulate olfactory signal optically, expressing the Channelrhodopsin (ChR2) under the control of the olfactory specific promoter, omp. We observed light-induced neuronal activity of olfactory system in the transgenic fish by examining c-fos expression, and a calcium indicator suggesting that blue light stimulation caused activation of olfactory neurons in a non-invasive manner. To examine whether the photo-activation of olfactory sensory neurons affect behavior of zebrafish larvae, we devised a behavioral choice paradigm and tested how zebrafish larvae choose between two conflicting sensory cues, an aversive odor or the naturally preferred phototaxis. We found that when the conflicting cues (the preferred light and aversive odor) were presented together simultaneously, zebrafish larvae swam away from the aversive odor. However, the transgenic fish with photo-activation were insensitive to the aversive odor and exhibited olfactory desensitization upon optical stimulation of ChR2. These results show that an aversive olfactory stimulus can override phototaxis, and that olfaction is important in decision making in zebrafish. This new transgenic model will be useful for the analysis of olfaction related behaviors and for the dissection of underlying neural circuits.     

Increased Serum Levels of Brain-Derived Neurotrophic Factor Contribute to Inflammatory Responses in Patients with Rheumatoid Arthritis

The aim of this study is to investigate the role of brain-derived neurotrophic factor (BDNF) in the inflammatory responses in patients with rheumatoid arthritis (RA). Serum levels of BDNF and the precursor form of BDNF (proBDNF) from 625 RA patients and 40 controls were analyzed using enzyme-linked immunosorbent assay. Effects of BDNF on the mitogen-activated protein kinase pathway were analyzed by Western blotting. Microarray analysis was conducted to search BDNF regulated gene expression in Jurkat cells, and the differentially expressed genes were validated using T cells from patients with RA and controls. Serum BDNF levels were significantly elevated in patients with RA compared with the controls. Low serum BDNF levels were found in RA patients with anxiety or receiving biologics treatment. BDNF (20 ng/mL) enhanced the phosphorylation of ERK, JNK, and c-Jun, but suppressed the phosphorylation of p38, whereas BDNF (200 ng/mL) enhanced the phosphorylation of ERK and p38. After validation, the expression of CAMK2A, MASP2, GNG13, and MUC5AC, regulated by BDNF and one of its receptors, NGFR, was increased in RA T cells. BDNF increased the IL-2, IL-17, and IFN-γ expression in Jurkat cells and IL-2 and IFN-γ secretion in activated peripheral blood mononuclear cells.