DECIPHERING THE ROLE OF SULFOTRANSFERASE 4A1 IN BRAIN DEVELOPMENT AND NEURONAL FUNCTIONING

Cytosolic sulfotransferases (SULTs) are enzymes that transfer a sulfuryl group from the obligate donor PAPS (3\u2019-phosphoadenosine 5\u2019-phosphosulfate) onto a variety of exogenous and endogenous substrates (Negishi 2001). In 2000, a novel member of this family (SULT4A1) was isolated from human and rat brain (Falany 2000). To date, the exact substrate and function of SULT4A1 are not fully addressed but since it is highly conserved and expressed extensively, and almost exclusively, in the brain, it is possible that SULT4A1 may have an important role in the central nervous system. Moreover, some recent reports have associated polymorphisms in the SULT4A1 gene with susceptibility to schizophrenia (Brennan 2005; Meltzer 2008); SULT4A1 has been suggested to be associated with neurological symptoms of Phelan-McDermid Syndrome (Disciglio 2014) and altered levels of SULT4A1 protein have been observed in bipolar and Alzheimer\u2019s patients (Wang 2003; Ryan 2006). Given this background, we decided to investigate the still unknown role of SULT4A1 within neuron development and functioning. We started evaluating the physiological expression of SULT4A1 in the brain areas mainly involved in neuropsychiatric and neurodevelopmental disorders. To this purpose, we performed western blot analyses of total lysates of hippocampus, striatum, cerebral cortex and cerebellum dissected from adult mice (P60). Our results showed that SULT4A1 is highly expressed in all the analyzed areas, especially in cortex and in cerebellum. Moreover, area-specific expression of SULT4A1 appears to be similar between adult male and female mice. Considering the possible implication of SULT4A1 in the pathogenesis of neurodevelopmental disorders, a major point for our study was the evaluation of SULT4A1 expression during physiological neuronal maturation. To this purpose, we analyzed by western blot rat primary neuronal cultures at different stages of neuron maturation, which are Day-In-Vitro (DIV) 1, 7 and 14. From the results of these analyses, it was inferable that the expression of SULT4A1 appreciably rises during neuronal maturation, going from an almost undetectable level at DIV1 to an almost 4-fold greater level at DIV14 in cortical cultures. This result was confirmed by immunofluorescence (IF) staining of the same cultures where the protein showed a cytoplasmic localization and its level of expression steadily increased from DIV1 to DIV14. IF results also suggested that SULT4A1 is mainly expressed in GAD67-positive inhibitory neurons, in particular in Calbindin- and Parvalbumin-positive neurons. Therefore, to better determine SULT4A1 expression in human neurons, we obtained peripheral blood mononuclear cells (PBMCs) from control healthy individuals and reprogrammed them into induced Pluripotent Stem Cells (iPSCs). iPSC-derived neural stem cells (NSC) were differentiated into neurons for at least 50 days, time necessary to obtain MAP2-positive mature neurons. SULT4A1 expression was evaluated during neuronal maturation from NSC stage to mature neuron and the data from biochemical analysis suggested that the level of SULT4A1 protein rises during differentiation of NSCs into neurons. Considering that abnormalities in dendritic spines and neuronal arborization are some of the most consistent anatomical correlates of neurodevelopmental disorders (Hung 2008; Glausier 2013; Jiang 2013; Moyer 2015), we characterized the effect of SULT4A1 on spine dynamics and dendrite morphology: in particular, we overexpressed or silenced SULT4A1 in cortical cultures and, interestingly, we observed that both conditions altered neuronal arborization as well as spine density and morphology. Moreover, in light of the possibility that SULT4A1 polymorphisms may lead to a reduction of mRNA translatability (Brennan 2005) and to clarify the specific role of SULT4A1 in neuronal maturation and functioning, we further investigated the effects of SULT4A1 silencing. Biochemical and electrophysiological analyses of neurons infected or transfected with SULT4A1 shRNA demonstrated that SULT4A1 deficiency perturbs the composition and activity of excitatory and inhibitory synapses: indeed, we found an increase of GAD65 expression and a reduction of GluN1 levels. Interestingly, these data were in line with the electrophysiological recordings, where neurons lacking SULT4A1 displayed a slight augmentation of spontaneous inhibitory postsynaptic currents (sIPSC) frequency and a decrease of spontaneous excitatory postsynaptic currents (sEPSC) frequency

Polycyclic Aromatic Hydrocarbons (PAH) in Volcano island (Aeolian archipelago) mud utilized for therapeutic purpose

This paper examines the possible presence, distribution, nature and sources of 18 Polycyclic Aromatic Hydrocarbons (PAHs) as constituent of the muds pool collected in Vulcano Aeolian Island. PAHs are important from environmental and toxicological point of view. Analysis was performed by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM). The total concentration of Polycyclic Aromatic Hydrocarbons ranged from 112 to 154 g/Kg of dry matrix. The volcanic muds, used for therapeutic purposes, are moderately contaminated

Elongation factor-2 phosphorylation in dendrites and the regulation of dendritic mRNA translation in neurons

Neuronal activity results in long lasting changes in synaptic structure and function by regulating mRNA translation in dendrites. These activity dependent events yield the synthesis of proteins known to be important for synaptic modifications and diverse forms of synaptic plasticity. Worthy of note, there is accumulating evidence that the eukaryotic Elongation Factor 2 Kinase (eEF2K)/eukaryotic Elongation Factor 2 (eEF2) pathway may be strongly involved in this process. Upon activation, eEF2K phosphorylates and thereby inhibits eEF2, resulting in a dramatic reduction of mRNA translation. eEF2K is activated by elevated levels of calcium and binding of Calmodulin (CaM), hence its alternative name calcium/CaM-dependent protein kinase III (CaMKIII). In dendrites, this process depends on glutamate signaling and N-methyl-D-aspartate receptor (NMDAR) activation. Interestingly, it has been shown that eEF2K can be activated in dendrites by metabotropic glutamate receptor (mGluR) 1/5 signaling, as well. Therefore, neuronal activity can induce local proteomic changes at the postsynapse by altering eEF2K activity. Well-established targets of eEF2K in dendrites include brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeletal-associated protein (Arc), the alpha subunit of calcium/CaM-dependent protein kinase II (\u3b1CaMKII), and microtubule-associated protein 1B (MAP1B), all of which have well-known functions in different forms of synaptic plasticity. In this review we will give an overview of the involvement of the eEF2K/eEF2 pathway at dendrites in regulating the translation of dendritic mRNA in the context of altered NMDAR- and neuronal activity, and diverse forms of synaptic plasticity, such as metabotropic glutamate receptor-dependent-long-term depression (mGluR-LTD). For this, we draw on studies carried out both in vitro and in vivo

A manganese-rich environment supports superoxide dismutase activity in a lyme disease pathogen, Borrelia burgdorferi

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of American Society for Biochemistry and Molecular Biology for personal use, not for redistribution. The definitive version was published in Journal of Biological Chemistry 288 (2013): 8468-8478, doi:10.1074/jbc.M112.433540.The Lyme disease pathogen Borrelia burgdorferi represents a novel organism in which to study metalloprotein biology in that this spirochete has uniquely evolved with no requirement for iron. Not only is iron low, but we show here that B. burgdorferi has the capacity to accumulate remarkably high levels of manganese. This high manganese is necessary to activate the SodA superoxide dismutase (SOD) essential for virulence. Using a metalloproteomic approach, we demonstrate that a bulk of B. burgdorferi SodA directly associates with manganese and a smaller pool of inactive enzyme accumulates as apoprotein. Other metalloproteins may have similarly adapted to using manganese as co-factor including the BB0366 amino-peptidase. While B. burgdorferi SodA has evolved in a manganese-rich, iron-poor environment, the opposite is true for Mn-SODs of organisms such as E. coli and bakers’ yeast. These Mn-SODs still capture manganese in an iron-rich cell, and we tested whether the same is true for Borrelia SodA. When expressed in the iron-rich mitochondria of S. cerevisiae, B. burgdorferi SodA was inactive. Activity was only possible when cells accumulated extremely high levels of manganese that exceeded cellular iron. Moreover, there was no evidence for iron inactivation of the SOD. B. burgdorferi SodA shows strong overall homology with other members of the Mn-SOD family, but computer assisted modeling revealed some unusual features of the hydrogen bonding network near the enzyme’s active site. The unique properties of B. burgdorferi SodA may represent adaptation to expression in the manganese-rich and iron-poor environment of the spirochete

Evolution by Any Other Name: Antibiotic Resistance and Avoidance of the E-Word

The word "evolution" is rarely used in papers from medical journals describing antimicrobial resistance, which may directly impact public perception of the importance of evolutionary biology in our everyday lives

Non-invasive characterization of pleural and pericardial effusions using T1 mapping by magnetic resonance imaging

AIMS: Differentiating exudative from transudative effusions is clinically important and is currently performed via biochemical analysis of invasively obtained samples using Light's criteria. Diagnostic performance is however limited. Biochemical composition can be measured with T1 mapping using cardiovascular magnetic resonance (CMR) and hence may offer diagnostic utility for assessment of effusions. METHODS AND RESULTS: A phantom consisting of serially diluted human albumin solutions (25-200 g/L) was constructed and scanned at 1.5 T to derive the relationship between fluid T1 values and fluid albumin concentration. Native T1 values of pleural and pericardial effusions from 86 patients undergoing clinical CMR studies retrospectively analysed at four tertiary centres. Effusions were classified using Light's criteria where biochemical data was available (n = 55) or clinically in decompensated heart failure patients with presumed transudative effusions (n = 31). Fluid T1 and protein values were inversely correlated both in the phantom (r = -0.992) and clinical samples (r = -0.663, P < 0.0001). T1 values were lower in exudative compared to transudative pleural (3252 ± 207 ms vs. 3596 ± 213 ms, P < 0.0001) and pericardial (2749 ± 373 ms vs. 3337 ± 245 ms, P < 0.0001) effusions. The diagnostic accuracy of T1 mapping for detecting transudates was very good for pleural and excellent for pericardial effusions, respectively [area under the curve 0.88, (95% CI 0.764-0.996), P = 0.001, 79% sensitivity, 89% specificity, and 0.93, (95% CI 0.855-1.000), P < 0.0001, 95% sensitivity; 81% specificity]. CONCLUSION: Native T1 values of effusions measured using CMR correlate well with protein concentrations and may be helpful for discriminating between transudates and exudates. This may help focus the requirement for invasive diagnostic sampling, avoiding unnecessary intervention in patients with unequivocal transudative effusions

Discovery of Genes Essential for Heme Biosynthesis through Large-Scale Gene Expression Analysis

SummaryHeme biosynthesis consists of a series of eight enzymatic reactions that originate in mitochondria and continue in the cytosol before returning to mitochondria. Although these core enzymes are well studied, additional mitochondrial transporters and regulatory factors are predicted to be required. To discover such unknown components, we utilized a large-scale computational screen to identify mitochondrial proteins whose transcripts consistently coexpress with the core machinery of heme biosynthesis. We identified SLC25A39, SLC22A4, and TMEM14C, which are putative mitochondrial transporters, as well as C1orf69 and ISCA1, which are iron-sulfur cluster proteins. Targeted knockdowns of all five genes in zebrafish resulted in profound anemia without impacting erythroid lineage specification. Moreover, silencing of Slc25a39 in murine erythroleukemia cells impaired iron incorporation into protoporphyrin IX, and vertebrate Slc25a39 complemented an iron homeostasis defect in the orthologous yeast mtm1Δ deletion mutant. Our results advance the molecular understanding of heme biosynthesis and offer promising candidate genes for inherited anemias