Post-Subfunctionalization Functions of HIF-1αA and HIF-1αB in Cyprinid Fish: Fine-Tuning Mitophagy and Apoptosis Regulation Under Hypoxic Stress.

Hypoxia-inducible factor 1 (HIF-1) is a crucial transcriptional factor that can restore oxygen balance in the body by regulating multiple vital activities. Two HIF-1α copies were retained in cyprinid fish after experiencing a teleost-specific genome duplication. How the "divergent collaboration" of HIF-1αA and HIF-1αB proceeds in regulating mitophagy and apoptosis under hypoxic stress in cells of cyprinid fish remains unclear. In this study, zebrafish HIF-1αA/B expression plasmids were constructed and transfected into the epithelioma papulosum cyprini cells and were subjected to hypoxic stress. HIF-1αA induced apoptosis through promoting ROS generation and mitochondrial depolarization when cells were subjected to oxygen deficiency. Conversely, HIF-1αB was primarily responsible for mitophagy induction, prompting ATP production to mitigate apoptosis. HIF-1αA did not induce mitophagy in the mitochondria and lysosomes co-localization assay but it was involved in the regulation of different mitophagy pathways. Over-expression of HIF-1αA increased the expression of bnip3, fundc1, Beclin1, and foxo3, suggesting it has a dual role in mitochondrial autophagy and cell death. Each duplicated copy also experienced functional divergence and target shifting in the regulation of complexes in the mitochondrial electron transport chain (ETC). Our findings shed light on the post-subfunctionalization function of HIF-1αA and HIF-1αB in zebrafish to fine-tune regulation of mitophagy and apoptosis following hypoxia exposure.

In Parkinson's patient-derived dopamine neurons, the triplication of α-synuclein locus induces distinctive firing pattern by impeding D2 receptor autoinhibition.

Pathophysiological changes in dopamine neurons precede their demise and contribute to the early phases of Parkinson's disease (PD). Intracellular pathological inclusions of the protein α-synuclein within dopaminergic neurons are a cardinal feature of PD, but the mechanisms by which α-synuclein contributes to dopaminergic neuron vulnerability remain unknown. The inaccessibility to diseased tissue has been a limitation in studying progression of pathophysiology prior to degeneration of dopamine neurons. To address these issues, we differentiated induced pluripotent stem cells (iPSCs) from a PD patient carrying the α-synuclein triplication mutation (AST) and an unaffected first-degree relative (NAS) into dopaminergic neurons. In human-like dopamine neurons α-synuclein overexpression reduced the functional availability of D2 receptors, resulting in a stark dysregulation in firing activity, dopamine release, and neuronal morphology. We back-translated these findings into primary mouse neurons overexpressing α-synuclein and found a similar phenotype, supporting the causal role for α-synuclein. Importantly, application of D2 receptor agonist, quinpirole, restored the altered firing activity of AST-derived dopaminergic neurons to normal levels. These results provide novel insights into the pre-degenerative pathophysiological neuro-phenotype induced by α-synuclein overexpression and introduce a potential mechanism for the long-established clinical efficacy of D2 receptor agonists in the treatment of PD.

Transgenerational hypocortisolism and behavioral disruption are induced by the antidepressant fluoxetine in male zebrafish Danio rerio.

The global prevalence of depression is high during childbearing. Due to the associated risks to the mother and baby, the selective serotonin reuptake inhibitor fluoxetine (FLX) is often the first line of treatment. Given that FLX readily crosses the placenta, a fetus may be susceptible to the disruptive effects of FLX during this highly plastic stage of development. Here, we demonstrate that a 6-day FLX exposure to a fetus-relevant concentration at a critical developmental stage suppresses cortisol levels in the adult zebrafish (F0). This effect persists for three consecutive generations in the unexposed descendants (F1 to F3) without diminution and is more pronounced in males. We also show that the in vivo cortisol response of the interrenal (fish "adrenal") to an i.p. injection of adrenocorticotropic hormone was also reduced in the males from the F0 and F3 FLX lineages. Transcriptomic profiling of the whole kidney containing the interrenal cells revealed that early FLX exposure significantly modified numerous pathways closely associated with cortisol synthesis in the male adults from the F0 and F3 generations. We also show that the low cortisol levels are linked to significantly reduced exploratory behaviors in adult males from the F0 to F2 FLX lineages. This may be a cause for concern given the high prescription rates of FLX to pregnant women and the potential long-term negative impacts on humans exposed to these therapeutic drugs.

Part B: Morphometric and transcriptomic responses to sub-chronic exposure to the polycyclic aromatic hydrocarbon phenanthrene in the fathead minnow (Pimephales promelas).

Phenanthrene is a tricyclic polycyclic aromatic hydrocarbon and environmental contaminant found in high concentrations around urban catchments and in the vicinity of oil extraction activities. Fish exposed to phenanthrene can exhibit altered reproductive hormone profiles and/or differences within gonadosomatic index and altered gamete proportions, but the mechanisms underlying these changes are not fully understood. In this study, we conducted a sub-chronic bioassay and measured transcriptional responses in the liver, the major tissue involved in generating lipids for oocyte growth. Adult male and female fathead minnow (Pimephales promelas) were exposed to an average measured concentration of 202 µg phenanthrene/L for a 7 week period. Condition factor was reduced in both males and females, while female fish also showed decreased gonadosomatic index relative to control females. In females exposed to phenanthrene, perinucleolar proportions were increased ∼1.9-fold relative to the control group whereas the proportions of vitellogenic oocytes decreased ∼8.8 fold. In males exposed to phenanthrene, spermatogonia proportions were increased ∼2.3 fold in testicular tissues compared to control fish. Thus, gametes were at an earlier stage of maturation in phenanthrene-treated fish compared to controls. However, no differences were detected in the production of 17ß-estradiol or testosterone from the gonad in either sex. Catalase activity was also assessed in the liver as a measure of oxidative stress and this biomarker did not change in activity in either sex. In addition to endpoints in the ovary, the female hepatic transcriptome was measured, as this tissue produces lipids for oocyte maturation. Transcriptomic responses to phenanthrene exposure suggested a reduction in vitellogenin mRNA, and lipid metabolism and immune system pathways. Comparisons of hepatic transcriptome responses with Part A (72 h phenanthrene exposure) showed that energy homeostasis pathways were consistently altered following phenanthrene exposure over multiple durations and concentrations. We suggest that altered energy homeostasis may be adversely affecting reproductive efforts, as impaired reproduction has been observed in other studies investigating polycyclic aromatic hydrocarbons.

Secretoneurin A Directly Regulates the Proteome of Goldfish Radial Glial Cells In Vitro.

Radial glial cells (RGCs) are the main macroglia in the teleost brain and have established roles in neurogenesis and neurosteroidogenesis. They are the only brain cell type expressing aromatase B (cyp19a1b), the enzyme that synthesizes estrogens from androgen precursors. There are few studies on the regulation of RGC functions, but our previous investigations demonstrated that dopamine stimulates cyp19a1b expression in goldfish RGCs, while secretoneurin A (SNa) inhibits the expression of this enzyme. Here, we determine the range of proteins and cellular processes responsive to SNa treatments in these steroidogenic cells. The focus here is on SNa, because this peptide is derived from selective processing of secretogranin II in magnocellular cells embedded within the RGC-rich preoptic nucleus. Primary cultures of RGCs were treated (24 h) with 10, 100, or 1,000 nM SNa. By using isobaric tagging for relative and absolute quantitation and a Hybrid Quadrupole Obritrap Mass Spectrometry system, a total of 1,363 unique proteins were identified in RGCs, and 609 proteins were significantly regulated by SNa at one or more concentrations. Proteins that showed differential expression with all three concentrations of SNa included H1 histone, glutamyl-prolyl-tRNA synthetase, Rho GDP dissociation inhibitor γ, vimentin A2, and small nuclear ribonucleoprotein-associated protein. At 10, 100, and 1,000 nM SNa, there were 5, 195, and 489 proteins that were downregulated, respectively, whereas the number of upregulated proteins were 72, 44, and 51, respectively. Subnetwork enrichment analysis of differentially regulated proteins revealed that processes such as actin organization, cytoskeleton organization and biogenesis, apoptosis, mRNA processing, RNA splicing, translation, cell growth, and proliferation are regulated by SNa based on the proteomic response. Moreover, we observed that, at the low concentration of SNa, there was an increase in the abundance of proteins involved in cell growth, proliferation, and migration, whereas higher concentration of SNa appeared to downregulate proteins involved in these processes, indicating a dose-dependent proteome response. At the highest concentration of SNa, proteins linked to the etiology of diseases of the central nervous system (brain injuries, Alzheimer disease, Parkinson's disease, cerebral infraction, brain ischemia) were also differentially regulated. These data implicate SNa in the control of cell proliferation and neurogenesis.

Dieldrin Augments mTOR Signaling and Regulates Genes Associated with Cardiovascular Disease in the Adult Zebrafish Heart (Danio rerio).

Dieldrin is a legacy organochlorine pesticide that is persistent in the environment, despite being discontinued from use in North America since the 1970s. Some epidemiologic studies suggest that exposure to dieldrin is associated with increased risks of neurodegenerative disease and breast cancer by inducing inflammatory responses in tissues as well as oxidative stress. However, the direct effects of organochlorine pesticides on the heart have not been adequately addressed to date given that these chemicals are detectable in human serum and are environmentally persistent; thus, individuals may show latent adverse effects in the cardiovascular system due to long-term, low-dose exposure over time. Our objective was to determine whether low-level exposure to dieldrin at an environmentally relevant dose results in aberrant molecular signaling in the vertebrate heart. Using transcriptomic profiling and immunoblotting, we determined the global gene and targeted protein expression response to dieldrin treatment and show that dieldrin affects gene networks in the heart that are associated with processes related to cardiovascular disease, specifically cardiac arrest and ventricular fibrillation. We report that genes regulating inflammatory responses, a significant risk factor for cardiovascular disease, are upregulated by dieldrin whereas transcripts related to lysosomal function are significantly downregulated. To verify these findings, proteins in these pathways were examined with immunoblotting, and our results demonstrate that dieldrin constitutively activates Akt/mTOR signaling and downregulates lysosomal genes, participating in autophagy. Our data demonstrate that dieldrin induces genes associated with cardiovascular dysfunction and compromised lysosomal physiology, thereby identifying a novel mechanism for pesticide-induced cardiotoxicity.

Sex- and tissue-specific effects of waterborne estrogen on estrogen receptor subtypes and E2-mediated gene expression in the reproductive axis of goldfish.

This research examined the gene expression profile of three goldfish estrogen receptor (ER) subtypes in multiple tissues in relation to mRNA levels of aromatase B and vitellogenin (VTG) following waterborne estrogen exposures. The protocol consisted of: i) adult male goldfish in late gonadal recrudescence exposed to 1 nM 17beta-estradiol (E2); ii) adult male and female goldfish in early sexual regression exposed to 1 nM E2 for 3, 6, 12 and 24h; and, iii) sexually mature, adult male goldfish exposed to 0.3 nM 17alpha-ethynylestradiol (EE2) for 24h. Liver produced the most consistent response with up-regulation of ERalpha in sexually regressed, mature and recrudescing males and in sexually regressed females. The dose and length of exposure, reproductive state and sex affected the auto-regulation of ERbeta1 by E2. ERbeta2 was not affected in any experiments suggesting it may not be auto-regulated by E2. Aromatase B and VTG gene expression were affected by E2, but also by other experimental conditions. EE2 induced liver ERalpha and VTG mRNA levels indicating that high environmental EE2 levels induce E2-mediated gene expression in a model teleost. These studies reveal a more complicated action of estrogenic compounds that has important implications on estrogenic endocrine disruptors in teleosts.