Is rat an appropriate animal model to study the involvement of d-serine catabolism in schizophrenia? insights from characterization of d-amino acid oxidase.

d-Amino acid oxidase (DAAO; EC1.4.3.3) has been proposed to play a main role in the degradation of d-serine, an allosteric activator of the N-methyl-d-aspartate-type glutamate receptor in the human brain, and to be associated with the onset of schizophrenia. To prevent excessive d-serine degradation, novel drugs for schizophrenia treatment based on DAAO inhibition were designed and tested on rats. However, the properties of rat DAAO are unknown and various in\u2003vivo trials have demonstrated the effects of DAAO inhibitors on d-serine concentration in rats. In the present study, rat DAAO was efficiently expressed in Escherichia\u2003coli. The recombinant enzyme was purified as an active, 40\u2003kDa monomeric flavoenzyme showing the basic properties of the dehydrogenase-oxidase class of flavoproteins. Rat DAAO differs significantly from the human counterpart because: (a) it possesses a different substrate specificity; (b) it shows a lower kinetic efficiency, mainly as a result of a low substrate affinity; (c) it differs in affinity for the binding of classical inhibitors; (d) it is a stable monomer in the absence of an active site ligand; and (e) it interacts with the mammalian protein modulator pLG72 yielding a 3c\u2003100\u2003kDa complex in addition to the 3c\u2003200\u2003kDa one, as formed by the human DAAO. Furthermore, the concentration of endogenous d-serine in U87 glioblastoma cells was not affected by transfection with rat DAAO, whereas it was significantly decreased when expressing the human homologue. These results raise doubt on the use of the rat as a model system for testing new drugs against schizophrenia and indicate a different physiological function of DAAO in rodents and humans. Structured digital abstract \u2022 \u2002pLG72\ua0binds\u2003rDAAO\u2003by\u2003molecular sieving\u2003(View interaction)

The D-amino acid oxidase-carbon nanotubes: evaluation of cytotoxicity and biocompatibility of a potential anticancer nanosystem

The ‘enzyme prodrug therapy’ represents a promising strategy to overcome limitations of current cancer treatments by the systemic administration of prodrugs, converted by a foreign enzyme into an active anticancer compound directly in tumor sites. One example is D-amino acid oxidase (DAAO), a dimeric favoenzyme able to catalyze the oxidative deamination of D-amino acids with production of hydrogen peroxide, a reactive oxygen species (ROS), able to favor cancer cells death. A DAAO variant containing fve aminoacidic substitutions (mDAAO) was demonstrated to possess a better therapeutic efcacy under low O2 concentration than wild-type DAAO (wtDAAO). Recently, aiming to design promising nanocarriers for DAAO, multi-walled carbon nanotubes (MWCNTs) were functionalized with polyethylene glycol (PEG) to reduce their tendency to aggregation and to improve their biocompatibility. Here, wtDAAO and mDAAO were adsorbed on PEGylated MWCNTs and their activity and cytotoxicity were tested. While PEG-MWCNTs-DAAOs have shown a higher activity than pristine MWCNTs-DAAO (independently on the DAAO variant used), PEG-MWCNTs-mDAAO showed a higher cytotoxicity than PEG-MWCNTs-wtDAAO at low O2 concentration. In order to evaluate the nanocarriers’ biocompatibility, PEG-MWCNTsDAAOs were incubated in human serum and the composition of protein corona was investigated via nLC-MS/MS, aiming to characterize both soft and hard coronas. The mDAAO variant has infuenced the bio-corona composition in both number of proteins and presence of opsonins and dysopsonins: notably, the soft corona of PEG-MWCNTs-mDAAO contained less proteins and was more enriched in proteins able to inhibit the immune response than PEG-MWCNTs-wtDAAO. Considering the obtained results, the PEGylated MWCNTs conjugated with the mDAAO variant seems a promising candidate for a selective antitumor oxidative therapy: under anoxic-like conditions, this novel drug delivery system showed a remarkable cytotoxic efect controlled by the substrate addition, against diferent tumor cell lines, and a bio-corona composition devoted to prolong its blood circulation time, thus improving the drug’s biodistribution

Effective Self-Management for Early Career Researchers in the Natural and Life Sciences

Early career researchers (ECRs) are faced with a range of competing pressures in academia, making self-management key to building a successful career. The Organization for Human Brain Mapping undertook a group effort to gather helpful advice for ECRs in self-management. Keywords: ECRs; career development; early career researchers; mentoring; networking; self-managemen

Regulating levels of the neuromodulator D-serine in human brain: structural insight into pLG72 and D-amino acid oxidase interaction

The human flavoenzyme D-amino acid oxidase (hDAAO) degrades the NMDA-receptor modulator D-serine in the brain. Whereas hDAAO has been extensively characterized, little is known about its main modulator pLG72, a small protein encoded by the primate-specific gene G72 that has been associated with schizophrenia susceptibility. pLG72 interacts with neosynthesized hDAAO, promoting its inactivation and degradation. In this work we used low-resolution techniques to characterize the surface topology of the hDAAO-pLG72 complex. By using limited proteolysis coupled to mass spectrometry we could map the exposed regions in the two proteins after complex formation and highlighted an increased sensitivity to proteolysis of hDAAO in complex with pLG72. Cross-linking experiments by using bis(sulfosuccinimidyl)suberate identified the single covalent bond between T182 in hDAAO and K62 in pLG72. In order to validate the designed mode of interaction, three pLG72 variants incrementally truncated at the C-terminus, in addition to a form lacking the 71 N-terminal residues, were produced. All variants were dimeric, folded, and interacted with hDAAO. The strongest decrease in affinity for hDAAO (as well as for the hydrophobic drug chlorpromazine) was apparent for the N-terminally deleted pLG7272-153 form, which lacked K62. On the other hand, eliminating the disordered C-terminal tail yielded a more stable pLG72 protein, improved the binding to hDAAO, although giving lower enzyme inhibition. Elucidation of the mode of hDAAO-pLG72 interaction now makes it possible to design novel molecules that, by targeting the protein complex, can be therapeutically advantageous for diseases related to impairment in D-serine metabolism. This article is protected by copyright. All rights reserved

A multimodal imaging study of recognition memory in very preterm born adults

Very preterm (<32 weeks of gestation) birth is associated with structural brain alterationsand memory impairments throughout childhood and adolescence. Here, we used functional MRI(fMRI) to study the neuroanatomy of recognition memory in 49 very preterm-born adults and 50 con-trols (mean age: 30 years) during completion of a task involving visual encoding and recognition ofabstract pictures. T1-weighted and diffusion-weighted images were also collected. Bilateral hippocam-pal volumes were calculated and tractography of the fornix and cingulum was performed and assessedin terms of volume and hindrance modulated orientational anisotropy (HMOA). Online recognitionmemory task performance, assessed with A scores, was poorer in the very preterm compared with thecontrol group. Analysis of fMRI data focused on differences in neural activity between the recognitionand encoding trials. Very preterm born adults showed decreased activation in the right middle frontalgyrus and posterior cingulate cortex/precuneus and increased activation in the left inferior frontalgyrus and bilateral lateral occipital cortex (LOC) compared with controls. Hippocampi, fornix and cin-gulum volume was significantly smaller and fornix HMOA was lower in very preterm adults. Amongall the structural and functional brain metrics that showed statistically significant group differences,LOC activation was the best predictor of online task performance (P 5 0.020). In terms of associationbetween brain function and structure, LOC activation was predicted by fornix HMOA in the pretermgroup only (P 5 0.020). These results suggest that neuroanatomical alterations in very preterm bornindividuals may be underlying their poorer recognition memory performance

The cerebrospinal fluid inflammatory response to preterm birth

Background: Preterm birth is the leading risk factor for perinatal white matter injury, which can lead to motor and neuropsychiatric impairment across the life course. There is an unmet clinical need for therapeutics. White matter injury is associated with an altered inflammatory response in the brain, primarily led by microglia, and subsequent hypomyelination. However, microglia can release both damaging and trophic factors in response to injury, and a comprehensive assessment of these factors in the preterm central nervous system (CNS) has not been carried out.Method: A custom antibody array was used to assess relative levels of 50 inflammation- and myelination-associated proteins in the cerebrospinal fluid (CSF) of preterm infants in comparison to term controls.Results: Fifteen proteins differed between the groups: BDNF, BTC, C5a, FasL, Follistatin, IL-1β, IL-2, IL-4, IL-9, IL-17A, MIP-1α, MMP8, SPP1, TGFβ, and TNFβ (p &lt; 0.05). To investigate the temporal regulation of these proteins after injury, we mined a gene expression dataset of microglia isolated from a mouse model of developmental white matter injury. Microglia in the experimental model showed dynamic temporal expression of genes encoding these proteins, with an initial and sustained pro-inflammatory response followed by a delayed anti-inflammatory response, and a continuous expression of genes predicted to inhibit healthy myelination.Conclusion: Preterm CSF shows a distinct neuroinflammatory profile compared to term controls, suggestive of a complex neural environment with concurrent damaging and reparative signals. We propose that limitation of pro-inflammatory responses, which occur early after perinatal insult, may prevent expression of myelination-suppressive genes and support healthy white matter development

Assays of D-Amino Acid Oxidase Activity

D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples

One single method to produce native and Tat-fused recombinant human \u3b1-synuclein in Escherichia coli

Human \u3b1-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson's disease. Experimental evidence suggests that \u3b1-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of \u3b1-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate \u3b1-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-\u3b1-synuclein.A reliable protocol was designed to efficiently express and purify two different forms of human \u3b1-synuclein. The synthetic cDNAs encoding for the native \u3b1-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized ( 65 85\%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant \u3b1-synuclein protein forms could be purified in a single step to 65 95\% purity. Both \u3b1-synuclein recombinant proteins form fibrils and the TAT-\u3b1-synuclein is also cytotoxic in the micromolar concentration range.To further characterize the molecular mechanisms of \u3b1-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular \u3b1-synuclein for the pathogenesis and progression of Parkinson's disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant \u3b1-synuclein than previously described procedures

D-amino acid oxidase-nanoparticle system: a potential novel approach for cancer enzymatic therapy.

Aim: The authors propose a new magnetic nanoparticle–enzyme system for cancer therapy capable of targeting the enzyme and consequently decreasing the adverse effects, meanwhile improving the patient's life quality. Materials & methods: The authors have functionalized Fe3O4 nanoparticles with 3-amino-propyltriethoxysilane (APTES) and conjugated it to yeast D-amino acid oxidase (DAAO) by coupling this with glutaraldehyde. Results & conclusion: The authors have tested the Fe3O4-APTES–DAAO system on three tumor cell lines. Exposed cells show, at the electron microscope level, nanoparticles on the surface of the plasma membrane and inside endocytic vesicles. Fe3O4-APTES–DAAO caused a substantial decrease of cell viability greatly augmented when D-alanine, a DAAO substrate, was added. Fe3O4-APTES–DAAO was demonstrated to be more effective than free DAAO, confirming the validity of the system in cancer therapy. Original submitted 27 March 2012; Revised submitted 20 September 2012; Published online 5 February 201