Shh activation restores interneurons and cognitive function in newborns with intraventricular haemorrhage.

Premature infants with germinal matrix haemorrhage-intraventricular haemorrhage (GMH-IVH) suffer from neurobehavioural deficits as they enter childhood and adolescence. Yet the underlying mechanisms remain unclear. Impaired development and function of interneurons contribute to neuropsychiatric disorders. Therefore, we hypothesized that the occurrence of IVH would reduce interneuron neurogenesis in the medial ganglionic eminence and diminish the population of parvalbumin+ and somatostatin+ cortical interneurons. Because Sonic Hedgehog promotes the production of cortical interneurons, we also postulated that the activation of Sonic Hedgehog signalling might restore neurogenesis, cortical interneuron population, and neurobehavioural function in premature newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH and autopsy samples from human preterm infants. We compared premature newborns with and without IVH for intraneuronal progenitors, cortical interneurons, transcription factors regulating neurogenesis, single-cell transcriptome of medial ganglionic eminence and neurobehavioural functions. We treated premature rabbit kits with adenovirus expressing Sonic Hedgehog (Ad-Shh) or green fluorescence protein gene to determine the effect of Sonic Hedgehog activation on the interneuron production, cortical interneuron population and neurobehaviour. We discovered that IVH reduced the number of Nkx2.1+ and Dlx2+ progenitors in the medial ganglionic eminence of both humans and rabbits by attenuating their proliferation and inducing apoptosis. Moreover, IVH decreased the population of parvalbumin+ and somatostatin+ neurons in the frontal cortex of both preterm infants and kits relative to controls. Sonic Hedgehog expression and the downstream transcription factors, including Nkx2.1, Mash1, Lhx6 and Sox6, were also reduced in kits with IVH. Consistent with these findings, single-cell transcriptomic analyses of medial ganglionic eminence identified a distinct subpopulation of cells exhibiting perturbation in genes regulating neurogenesis, ciliogenesis, mitochondrial function and MAPK signalling in rabbits with IVH. More importantly, restoration of Sonic Hedgehog level by Ad-Shh treatment ameliorated neurogenesis, cortical interneuron population and neurobehavioural function in kits with IVH. Additionally, Sonic Hedgehog activation alleviated IVH-induced inflammation and several transcriptomic changes in the medial ganglionic eminence. Taken together, IVH reduced intraneuronal production and cortical interneuron population by downregulating Sonic Hedgehog signalling in both preterm rabbits and humans. Notably, activation of Sonic Hedgehog signalling restored interneuron neurogenesis, cortical interneurons and cognitive function in rabbit kits with IVH. These findings highlight disruption in cortical interneurons in IVH and identify a novel therapeutic strategy to restore cortical interneurons and cognitive function in infants with IVH. These studies can accelerate the development of new therapies to enhance the neurodevelopmental outcome of survivors with IVH.

Elevated insulin growth factor-1 in dentate gyrus induces cognitive deficits in pre-term newborns.

Prematurely born infants are deprived of maternal hormones and cared for in the stressful environment of Neonatal Intensive Care Units (NICUs). They suffer from long-lasting deficits in learning and memory. Here, we show that prematurity and associated neonatal stress disrupt dentate gyrus (DG) development and induce long-term cognitive deficits and that these effects are mediated by insulin growth factor-1 (IGF1). Nonmaternal care of premature rabbits increased the number of granule cells and interneurons and reduced neurogenesis, suggesting accelerated premature maturation of DG. However, the density of glutamatergic synapses, mature dendritic spines, and synaptic transmission were reduced in preterm kits compared with full-term controls, indicating that premature synaptic maturation was abnormal. These findings were consistent with cognitive deficits observed in premature rabbits and appeared to be driven by transcriptomic changes in the granule cells. Preterm kits displayed reduced weight, elevated serum cortisol and growth hormone, and higher IGF1 expression in the liver and DG relative to full-term controls. Importantly, blocking IGF-1 receptor in premature kits restored cognitive deficits, increased the density of glutamatergic puncta, and rescued NR2B and PSD95 levels in the DG. Hence, IGF1 inhibition alleviates prematurity-induced cognitive dysfunction and synaptic changes in the DG through modulation of NR2B and PSD95. The study identifies a novel strategy to potentially rescue DG maldevelopment and cognitive dysfunction in premature infants under stress in NICUs.

PPAR-γ activation enhances myelination and neurological recovery in premature rabbits with intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) results in periventricular inflammation, hypomyelination of the white matter, and hydrocephalus in premature infants. No effective therapy exists to prevent these disorders. Peroxisome proliferator activated receptor-γ (PPAR-γ) agonists reduce inflammation, alleviate free radical generation, and enhance microglial phagocytosis, promoting clearance of debris and red blood cells. We hypothesized that activation of PPAR-γ would enhance myelination, reduce hydrocephalus, and promote neurological recovery in newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH; autopsy brain samples from premature infants with and without IVH were analyzed. We found that IVH augmented PPAR-γ expression in microglia of both preterm human infants and rabbit kits. The treatment with PPAR-γ agonist or PPAR-γ overexpression by adenovirus delivery further elevated PPAR-γ levels in microglia, reduced proinflammatory cytokines, increased microglial phagocytosis, and improved oligodendrocyte progenitor cell (OPC) maturation in kits with IVH. Transcriptomic analyses of OPCs identified previously unrecognized PPAR-γ-induced genes for purinergic signaling, cyclic adenosine monophosphate generation, and antioxidant production, which would reprogram these progenitors toward promoting myelination. RNA-sequencing analyses of microglia revealed PPAR-γ-triggered down-regulation of several proinflammatory genes and transcripts having roles in Parkinson's disease and amyotrophic lateral sclerosis, contributing to neurological recovery in kits with IVH. Accordingly, PPAR-γ activation enhanced myelination and neurological function in kits with IVH. This also enhanced microglial phagocytosis of red blood cells but did not reduce hydrocephalus. Treatment with PPAR-γ agonist might enhance myelination and neurological recovery in premature infants with IVH.

Oestrogen treatment restores dentate gyrus development in premature newborns by IGF1 regulation.

Prematurely-born infants cared for in the neonatal units suffer from memory and learning deficits. Prematurity diminishes neurogenesis and synaptogenesis in the hippocampal dentate gyrus (DG). This dysmaturation of neurons is attributed to elevated PSD95, NMDR2A, and IGF1 levels. Since oestrogen treatment plays key roles in the development and plasticity of DG, we hypothesized that 17ß-estradiol (E2) treatment would ameliorate neurogenesis and synaptogenesis in the DG, reversing cognitive deficits in premature newborns. Additionally, E2-induced recovery would be mediated by IGF1 signalling. These hypotheses were tested in a rabbit model of prematurity and nonmaternal care, in which premature kits were gavage-fed and reared by laboratory personnel. We compared E2- and vehicle-treated preterm kits for morphological, molecular, and behavioural parameters. We also treated kits with oestrogen degrader, RAD1901, and assessed IGF1 signalling. We found that E2 treatment increased the number of Tbr2+ and DCX+ neuronal progenitors and increased the density of glutamatergic synapses in the DG. E2 treatment restored PSD95 and NMDAR2A levels and cognitive function in preterm kits. Transcriptomic analyses showed that E2 treatment contributed to recovery by influencing interactions between IGF1R and neurodegenerative, as well as glutamatergic genes. ERα expression was reduced on completion of E2 treatment at D7, followed by D30 elevation. E2-induced fluctuation in ERα levels was associated with a reciprocal elevation in IGF1/2 expression at D7 and reduction at D30. ERα degradation by RAD1901 treatment enhanced IGF1 levels, suggesting ERα inhibits IGF1 expression. E2 treatment alleviates the prematurity-induced maldevelopment of DG and cognitive dysfunctions by regulating ERα and IGF1 levels.

Reduced Hippocampal Dendrite Branching, Spine Density and Neurocognitive Function in Premature Rabbits, and Reversal with Estrogen or TrkB Agonist Treatment.

Preterm-born children suffer from neurological and behavioral disorders. Herein, we hypothesized that premature birth and non-maternal care of preterm newborns might disrupt neurobehavioral function, hippocampal dendritic arborization, and dendritic spine density. Additionally, we assessed whether 17ß-estradiol (E2) replacement or the TrkB receptor agonist, 7,8-dihydroxyflavone (DHF), would reverse compromised dendritic development and cognitive function in preterm newborns. These hypotheses were tested by comparing preterm (E28.5) rabbit kits cared and gavage-fed by laboratory personnel and term-kits reared and breast-fed by their mother doe at an equivalent postconceptional age. Neurobehavioral tests showed that both premature-birth and formula-feeding with non-maternal care led to increased anxiety behavior, poor social interaction, and lack of novelty preference compared with term-kits. Dendritic branching and number of total or mushroom dendritic spines were reduced in the CA1 field of preterm-kits compared with term controls. While CDC42 and Rac1/2/3 expression levels were lower, RhoA-activity was higher in preterm-kits compared with term controls. Both E2 and DHF treatment reversed prematurity-induced reduction in spine density, reduced total RhoA-GTPase levels, and enhanced cognitive function. Hence, prematurity and non-maternal care result in cognitive deficits, and reduced dendritic arbors and spines in CA1. E2 replacement or DHF treatment might reverse changes in dendritic spines and improve neurodevelopment in premature infants.

Disruption of Interneuron Neurogenesis in Premature Newborns and Reversal with Estrogen Treatment.

Many Preterm-born children suffer from neurobehavioral disorders. Premature birth terminates the hypoxic in utero environment and supply of maternal hormones. As the production of interneurons continues until the end of pregnancy, we hypothesized that premature birth would disrupt interneuron production and that restoration of the hypoxic milieu or estrogen treatment might reverse interneuron generation. To test these hypotheses, we compared interneuronal progenitors in the medial ganglionic eminences (MGEs), lateral ganglionic eminences (LGEs), and caudal ganglionic eminences (CGEs) between preterm-born [born on embryonic day (E) 29; examined on postnatal day (D) 3 and D7] and term-born (born on E32; examined on D0 and D4) rabbits at equivalent postconceptional ages. We found that both total and cycling Nkx2.1+, Dlx2+, and Sox2+ cells were more abundant in the MGEs of preterm rabbits at D3 compared with term rabbits at D0, but not in D7 preterm relative to D4 term pups. Total Nkx2.1+ progenitors were also more numerous in the LGEs of preterm pups at D3 compared with term rabbits at D0. Dlx2+ cells in CGEs were comparable between preterm and term pups. Simulation of hypoxia by dimethyloxalylglycine treatment did not affect the number of interneuronal progenitors. However, estrogen treatment reduced the density of total and proliferating Nkx2.1+ and Dlx2+ cells in the MGEs and enhanced Ascl1 transcription factor. Estrogen treatment also reduced Ki67, c-Myc, and phosphorylation of retinoblastoma protein, suggesting inhibition of the G1-to-S phase transition. Hence, preterm birth disrupts interneuron neurogenesis in the MGE and estrogen treatment reverses interneuron neurogenesis in preterm newborns by cell-cycle inhibition and elevation of Ascl1. We speculate that estrogen replacement might partially restore neurogenesis in human premature infants.SIGNIFICANCE STATEMENT Prematurity results in developmental delays and neurobehavioral disorders, which might be ascribed to disturbances in the development of cortical interneurons. Here, we show that preterm birth disrupts interneuron neurogenesis in the medial ganglionic eminence (MGE) and, more importantly, that estrogen treatment reverses this perturbation in the population of interneuron progenitors in the MGE. The estrogen seems to restore neurogenesis by inhibiting the cell cycle and elevating Ascl1 expression. As preterm birth causes plasma estrogen level to drop 100-fold, the estrogen replacement in preterm infants is physiological. We speculate that estrogen replacement might ameliorate disruption in production of interneurons in human premature infants.

Estrogen Treatment Reverses Prematurity-Induced Disruption in Cortical Interneuron Population.

Development of cortical interneurons continues until the end of human pregnancy. Premature birth deprives the newborns from the supply of maternal estrogen and a secure intrauterine environment. Indeed, preterm infants suffer from neurobehavioral disorders. This can result from both preterm birth and associated postnatal complications, which might disrupt recruitment and maturation of cortical interneurons. We hypothesized that interneuron subtypes, including parvalbumin-positive (PV+), somatostatin-positive (SST+), calretinin-positive (CalR+), and neuropeptide Y-positive (NPY+) interneurons, were recruited in the upper and lower cortical layers in a distinct manner with advancing gestational age. In addition, preterm birth would disrupt the heterogeneity of cortical interneurons, which might be reversed by estrogen treatment. These hypotheses were tested by analyzing autopsy samples from premature infants and evaluating the effect of estrogen supplementation in prematurely delivered rabbits. The PV+ and CalR+ neurons were abundant, whereas SST+ and NPY+ neurons were few in cortical layers of preterm human infants. Premature birth of infants reduced the density of PV+ or GAD67+ neurons and increased SST+ interneurons in the upper cortical layers. Importantly, 17 ß-estradiol treatment in preterm rabbits increased the number of PV+ neurons in the upper cortical layers relative to controls at postnatal day 14 (P14) and P21 and transiently reduced SST population at P14. Moreover, protein and mRNA levels of Arx, a key regulator of cortical interneuron maturation and migration, were higher in estrogen-treated rabbits relative to controls. Therefore, deficits in PV+ and excess of SST+ neurons in premature newborns are ameliorated by estrogen replacement, which can be attributed to elevated Arx levels. Estrogen replacement might enhance neurodevelopmental outcomes in extremely preterm infants.SIGNIFICANCE STATEMENT Premature birth often leads to neurodevelopmental delays and behavioral disorders, which may be ascribed to disturbances in the development and maturation of cortical interneurons. Here, we show that preterm birth in humans is associated with reduced population of parvalbumin-positive (PV+) neurons and an excess of somatostatin-expressing interneurons in the cerebral cortex. More importantly, 17 ß-estradiol treatment increased the number of PV+ neurons in preterm-born rabbits, which appears to be mediated by an elevation in the expression of Arx transcription factor. Hence the present study highlights prematurity-induced reduction in PV+ neurons in human infants and reversal in their population by estrogen replacement in preterm rabbits. Because preterm birth drops plasma estrogen level 100-fold, estrogen replacement in extremely preterm infants might improve their developmental outcome and minimize neurobehavioral disorders.

Glycogen synthase kinase-3ß inhibition enhances myelination in preterm newborns with intraventricular hemorrhage, but not recombinant Wnt3A.

Intraventricular hemorrhage (IVH) in preterm infants results in reduced proliferation and maturation of oligodendrocyte progenitor cells (OPCs), and survivors exhibit reduced myelination and neurological deficits. Wnt signaling regulates OPC maturation and myelination in a context dependent manner. Herein, we hypothesized that the occurrence of IVH would downregulate Wnt signaling, and that activating Wnt signaling by GSK-3ß inhibition or Wnt3A recombinant human protein (rh-Wnt3A) treatment might promote maturation of OPCs, myelination of the white matter, and neurological recovery in premature rabbits with IVH. These hypotheses were tested in autopsy samples from preterm infants and in a rabbit model of IVH. Induction of IVH reduced expressions of activated ß-catenin, TCF-4, and Axin2 transcription factors in preterm newborns. Both AR-A014418 (ARA) and Wnt-3A treatment activated Wnt signaling. GSK-3ß inhibition by intramuscular ARA treatment accelerated maturation of OPCs, myelination, and neurological recovery in preterm rabbits with IVH compared to vehicle controls. In contrast, intracerebroventricular rh-Wnt3A treatment failed to enhance myelination and neurological function in rabbits with IVH. ARA treatment reduced microglia infiltration and IL1ß expression in rabbits with IVH relative to controls, whereas Wnt3A treatment elevated TNFα, IL1ß, and IL6 expression without affecting microglia density. GSK-3ß inhibition downregulated, while rh-Wnt3A treatment upregulated Notch signaling; and none of the two treatments affected the Sonic-Hedgehog pathway. The administration of ARA or rh-Wnt3A did not affect gliosis. The data suggest that GSK-3ß inhibition promoted myelination by suppressing inflammation and Notch signaling; and Wnt3A treatment failed to enhance myelination because of its pro-inflammatory activity and synergy with Notch signaling. GSK-3ß inhibitors might improve the neurological outcome of preterm infants with IVH.

Structural basis for suppression of hypernegative DNA supercoiling by E. coli topoisomerase I.

Escherichia coli topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. A full length structure of E. coli topoisomerase I reported here shows how the C-terminal domains bind single-stranded DNA (ssDNA) to recognize the accumulation of negative supercoils in duplex DNA. These C-terminal domains of E. coli topoisomerase I are known to interact with RNA polymerase, and two flexible linkers within the C-terminal domains may assist in the movement of the ssDNA for the rapid removal of transcription driven negative supercoils. The structure has also unveiled for the first time how the 4-Cys zinc ribbon domain and zinc ribbon-like domain bind ssDNA with primarily π-stacking interactions. This novel structure, in combination with new biochemical data, provides important insights into the mechanism of genome regulation by type IA topoisomerases that is essential for life, as well as the structures of homologous type IA TOP3α and TOP3ß from higher eukaryotes that also have multiple 4-Cys zinc ribbon domains required for their physiological functions.

Antimicrobial Susceptibility and SOS-Dependent Increase in Mutation Frequency Are Impacted by Escherichia coli Topoisomerase I C-Terminal Point Mutation.

Topoisomerase functions are required in all organisms for many vital cellular processes, including transcription elongation. The C terminus domains (CTD) of Escherichia coli topoisomerase I interact directly with RNA polymerase to remove transcription-driven negative supercoiling behind the RNA polymerase complex. This interaction prevents inhibition of transcription elongation from hypernegative supercoiling and R-loop accumulation. The physiological function of bacterial topoisomerase I in transcription is especially important for a rapid network response to an antibiotic challenge. In this study, Escherichia coli with a topA66 single nucleotide deletion mutation, which results in a frameshift in the TopA CTD, was shown to exhibit increased sensitivity to trimethoprim and quinolone antimicrobials. The topoisomerase I-RNA polymerase interaction and the SOS response to the antimicrobial agents were found to be significantly reduced by this topA66 mutation. Consequently, the mutation frequency measured by rifampin selection following SOS induction was diminished in the topA66 mutant. The increased antibiotic sensitivity for the topA66 mutant can be reversed by the expression of recombinant E. coli topoisomerase I but not by the expression of recombinant Mycobacterium tuberculosis topoisomerase I that has a nonhomologous CTD even though the recombinant M. tuberculosis topoisomerase I can restore most of the plasmid DNA linking number deficiency caused by the topA66 mutation. Direct interactions of E. coli topoisomerase I as part of transcription complexes are likely to be required for the rapid network response to an antibiotic challenge. Inhibitors of bacterial topoisomerase I functions and interactions may sensitize pathogens to antibiotic treatment and limit the mutagenic response.

A surface plasmon resonance study of the intermolecular interaction between Escherichia coli topoisomerase I and pBAD/Thio supercoiled plasmid DNA.

To date, the bacterial DNA topoisomerases are one of the major target biomolecules for the discovery of new antibacterial drugs. DNA topoisomerase regulates the topological state of DNA, which is very important for replication, transcription and recombination. The relaxation of negatively supercoiled DNA is catalyzed by bacterial DNA topoisomerase I (topoI) and this reaction requires Mg(2+). In this report, we first quantitatively studied the intermolecular interactions between Escherichia coli topoisomerase I (EctopoI) and pBAD/Thio supercoiled plasmid DNA using surface plasmon resonance (SPR) technique. The equilibrium dissociation constant (Kd) for EctopoI-pBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg(2+) on the catalysis of EctopoI-pBAD/Thio reaction. A slightly higher equilibrium dissociation constant (~15 nM) was obtained for Mg(2+) coordinated EctopoI (Mg(2+)EctopoI)-pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (kd) for Mg(2+)EctopoI-pBAD/Thio interactions (~0.043 s(-1)), compared to EctopoI-pBAD/Thio interactions (~0.017 s(-1)). These results suggest that enzyme turnover during plasmid DNA relaxation is enhanced due to the presence of Mg(2+) and furthers the understanding of importance of the Mg(2+) ion for bacterial topoisomerase I catalytic activity.

Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I.

DNA topoisomerases control DNA topology by breaking and rejoining DNA strands via covalent complexes with cleaved DNA substrate as catalytic intermediates. Here we report the structure of Escherichia coli topoisomerase I catalytic domain (residues 2-695) in covalent complex with a cleaved single-stranded oligonucleotide substrate, refined to 2.3-Å resolution. The enzyme-substrate intermediate formed after strand cleavage was captured due to the presence of the D111N mutation. This structure of the covalent topoisomerase-DNA intermediate, previously elusive for type IA topoisomerases, shows distinct conformational changes from the structure of the enzyme without bound DNA and provides detailed understanding of the covalent catalysis required for strand cleavage to take place. The portion of cleaved DNA 5' to the site of cleavage is anchored tightly with extensive noncovalent protein-DNA interactions as predicted by the "enzyme-bridged" model. Distortion of the scissile strand at the -4 position 5' to the cleavage site allows specific selectivity of a cytosine base in the binding pocket. Many antibacterial and anticancer drugs initiate cell killing by trapping the covalent complexes formed by topoisomerases. We have demonstrated in previous mutagenesis studies that accumulation of the covalent complex of bacterial topoisomerase I is bactericidal. This structure of the covalent intermediate provides the basis for the design of novel antibiotics that can trap the enzyme after formation of the covalent complex.

The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining.

The strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA and was hypothesized to be required for positioning of the scissile phosphate for DNA cleavage to take place. Mutants of recombinant Yersinia pestis topoisomerase I with hydrophobic substitutions at this position were found in genetic screening to exhibit a dominant lethal phenotype, resulting in drastic loss in Escherichia coli viability when overexpressed. In depth biochemical analysis of E. coli topoisomerase I with the corresponding Arg-321 mutation showed that DNA cleavage can still take place in the absence of this arginine function if Mg(2+) is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These new experimental results demonstrate that catalysis of DNA rejoining by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3'-OH end to result in DNA rejoining. Small molecules that can interfere or distort the enzyme-DNA interactions required for DNA rejoining by bacterial type IA topoisomerases could be developed into novel antibacterial drugs.

3,4-dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I.

OBJECTIVES: Antibiotic resistance in bacterial pathogens is a serious clinical problem. Novel targets are needed to combat increasing drug resistance in Escherichia coli. Our objective is to demonstrate that 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2yl]-1H-benzimidazole (DMA) inhibits E. coli DNA topoisomerase I more strongly than human topoisomerase I. In addition, DMA is non-toxic to mammalian cells at antibiotic dosage level. METHODS: In the present study, we have established DMA as an antibacterial compound by determining MICs, post-antibiotic effects (PAEs) and MBCs for different standard as well as clinical strains of E. coli. We have described the differential catalytic inhibitory mechanism of bis-benzimidazole, DMA, for human and E. coli topoisomerase I and topoisomerase II by performing different assays, including relaxation assays, cleavage-religation assays, DNA unwinding assays, ethidium bromide displacement assays, decatenation assays and DNA gyrase supercoiling assays. RESULTS: DMA significantly inhibited bacterial growth at a very low concentration, but did not affect human cell viability at higher concentrations. Activity assays showed that it preferentially targeted E. coli topoisomerase I over human topoisomerase I, topoisomerase II and gyrase. Cleavage-religation assays confirmed DMA as a poison inhibitor of E. coli topoisomerase I. This study illuminates new properties of DMA, which may be further modified to develop an efficient topoisomerase inhibitor that is selective towards bacterial topoisomerase I. CONCLUSIONS: This is the first report of a bis-benzimidazole acting as an E. coli topoisomerase I inhibitor. DMA is a safe, non-cytotoxic molecule to human cells at concentrations that are needed for antibacterial activity.

Recovery of the brain after intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) remains a major complication of prematurity, worldwide. The severity of IVH is variable, ranging from a tiny germinal matrix bleed to a moderate-to-large ventricular hemorrhage or periventricular hemorrhagic infarction. Survivors with IVH often suffer from hydrocephalus and white matter injury. There is no tangible treatment to prevent post-hemorrhagic cerebral palsy, cognitive deficits, or hydrocephalus in these infants. White matter injury is attributed to blood-induced damage to axons and maturing oligodendrocyte precursors, resulting in reduced myelination and axonal loss. Hydrocephalus results from obstructed CSF circulation by blood clots, increased CSF production, and reduced CSF absorption by lymphatics and arachnoid villi. Several strategies to promote neurological recovery have shown promise in animal models, including the elimination of blood and blood products, alleviating cerebral inflammation and oxidative stress, as well as promoting survival and maturation of oligodendrocyte precursors. The present review integrates novel mechanisms of brain injury in IVH and the imminent therapies to alleviate post-hemorrhagic white matter injury and hydrocephalus in the survivors with IVH.

Topoisomerase I function during Escherichia coli response to antibiotics and stress enhances cell killing from stabilization of its cleavage complex.

OBJECTIVES: To explore the role of topoisomerase I in gene activation and increased RecA levels during the bacterial SOS response, as well as the effect of antibiotic treatment and stress challenge on cell killing initiated by trapped topoisomerase I cleavage complex. METHODS: A mutant Escherichia coli strain with a ΔtopA mutation was used to investigate the role of topoisomerase I function in the SOS response to trimethoprim and mitomycin C. Induction of the recA and dinD1 promoters was measured using luciferase reporters of these promoters fused to luxCDABE. An increase in the RecA level following trimethoprim treatment was quantified directly by western blotting. The effect of stress challenge from trimethoprim and acidified nitrite treatments on cell killing by topoisomerase I cleavage complex accumulation was measured by the decrease in viability following induction of recombinant mutant topoisomerase I that forms a stabilized cleavage complex. RESULTS: Topoisomerase I function was found to be required for efficient transcriptional activation of the recA and dinD1 promoters during the E. coli SOS response to trimethoprim and mitomycin C. The role of topoisomerase I in the SOS response was confirmed with quantitative western blot analysis of RecA following trimethoprim treatment. The bactericidal effect from topoisomerase I cleavage complex accumulation was shown to be enhanced by stress challenge from trimethoprim and acidified nitrite. CONCLUSIONS: Bacterial topoisomerase I function is actively involved in the SOS response to antibiotics and stress challenge. Cell killing initiated by the topoisomerase I cleavage complex would be enhanced by antibiotics and the host response. These findings provide further support for bacterial topoisomerase I as a therapeutic target.

The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli: application for identification of potential FRET-dye labeling sites.

BACKGROUND: Mycobacterium tuberculosis topoisomerase I (MtTOP1) and Escherichia coli topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex. RESULTS: We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive topA function of E. coli strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in E. coli. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS) assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate. CONCLUSIONS: The DNA relaxation and cleavage complex accumulation of M. tuberculosis topoisomerase I can be measured with genetic assays in E. coli, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.

Mutation adjacent to the active site tyrosine can enhance DNA cleavage and cell killing by the TOPRIM Gly to Ser mutant of bacterial topoisomerase I.

The TOPRIM DXDXXG residues of type IA and II topoisomerases are involved in Mg(II) binding and the cleavage-rejoining of DNA. Mutation of the strictly conserved glycine to serine in Yersinia pestis and Escherichia coli topoisomerase I results in bacterial cell killing due to inhibition of DNA religation after DNA cleavage. In this study, all other substitutions at the TOPRIM glycine of Y. pestis topoisomerase I were examined. While the Gly to Ala substitution allowed both DNA cleavage and religation, other mutations abolished DNA cleavage. DNA cleavage activity retained by the Gly to Ser mutant could be significantly enhanced by a second mutation of the methionine residue adjacent to the active site tyrosine. Induction of mutant topoisomerase with both the TOPRIM glycine and active site region methionine mutations resulted in up to 40-fold higher cell killing rate when compared with the single TOPRIM Gly to Ser mutant. Bacterial type IA topoisomerases are potential targets for discovery of novel antibiotics. These results suggest that compounds that interact simultaneously with the TOPRIM motif and the molecular surface around the active site tyrosine could be highly efficient topoisomerase poisons through both enhancement of DNA cleavage and inhibition of DNA rejoining.

Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region.

Among bacterial topoisomerase I enzymes, a conserved methionine residue is found at the active site next to the nucleophilic tyrosine. Substitution of this methionine residue with arginine in recombinant Yersinia pestis topoisomerase I (YTOP) was the only substitution at this position found to induce the SOS response in Escherichia coli. Overexpression of the M326R mutant YTOP resulted in approximately 4 log loss of viability. Biochemical analysis of purified Y. pestis and E. coli mutant topoisomerase I showed that the Met to Arg substitution affected the DNA religation step of the catalytic cycle. The introduction of an additional positive charge into the active site region of the mutant E. coli topoisomerase I activity shifted the pH for optimal activity and decreased the Mg(2+) binding affinity. This study demonstrated that a substitution outside the TOPRIM motif, which binds Mg(2+)directly, can nonetheless inhibit Mg(2+) binding and DNA religation by the enzyme, increasing the accumulation of covalent cleavage complex, with bactericidal consequence. Small molecules that can inhibit Mg(2+) dependent religation by bacterial topoisomerase I specifically could be developed into useful new antibacterial compounds. This approach would be similar to the inhibition of divalent ion dependent strand transfer by HIV integrase in antiviral therapy.