PubTransformer

A site to transform Pubmed publications into these bibliographic reference formats: ADS, BibTeX, EndNote, ISI used by the Web of Knowledge, RIS, MEDLINE, Microsoft's Word 2007 XML.

Michael Orford - Top 30 Publications

Mechanisms of action for the medium-chain triglyceride ketogenic diet in neurological and metabolic disorders.

High-fat, low-carbohydrate diets, known as ketogenic diets, have been used as a non-pharmacological treatment for refractory epilepsy. A key mechanism of this treatment is thought to be the generation of ketones, which provide brain cells (neurons and astrocytes) with an energy source that is more efficient than glucose, resulting in beneficial downstream metabolic changes, such as increasing adenosine levels, which might have effects on seizure control. However, some studies have challenged the central role of ketones because medium-chain fatty acids, which are part of a commonly used variation of the diet (the medium-chain triglyceride ketogenic diet), have been shown to directly inhibit AMPA receptors (glutamate receptors), and to change cell energetics through mitochondrial biogenesis. Through these mechanisms, medium-chain fatty acids rather than ketones are likely to block seizure onset and raise seizure threshold. The mechanisms underlying the ketogenic diet might also have roles in other disorders, such as preventing neurodegeneration in Alzheimer's disease, the proliferation and spread of cancer, and insulin resistance in type 2 diabetes. Analysing medium-chain fatty acids in future ketogenic diet studies will provide further insights into their importance in modified forms of the diet. Moreover, the results of these studies could facilitate the development of new pharmacological and dietary therapies for epilepsy and other disorders.

Oxidative Stress: Mechanistic Insights into Inherited Mitochondrial Disorders and Parkinson's Disease.

Oxidative stress arises when cellular antioxidant defences become overwhelmed by a surplus generation of reactive oxygen species (ROS). Once this occurs, many cellular biomolecules such as DNA, lipids, and proteins become susceptible to free radical-induced oxidative damage, and this may consequently lead to cellular and ultimately tissue and organ dysfunction. Mitochondria, as well as being a source of ROS, are vulnerable to oxidative stress-induced damage with a number of key biomolecules being the target of oxidative damage by free radicals, including membrane phospholipids, respiratory chain complexes, proteins, and mitochondrial DNA (mt DNA). As a result, a deficit in cellular energy status may occur along with increased electron leakage and partial reduction of oxygen. This in turn may lead to a further increase in ROS production. Oxidative damage to certain mitochondrial biomolecules has been associated with, and implicated in the pathophysiology of a number of diseases. It is the purpose of this review to discuss the impact of such oxidative stress and subsequent damage by reviewing our current knowledge of the pathophysiology of several inherited mitochondrial disorders together with our understanding of perturbations observed in the more commonly acquired neurodegenerative disorders such as Parkinson's disease (PD). Furthermore, the potential use and feasibility of antioxidant therapies as an adjunct to lower the accumulation of damaging oxidative species and hence slow disease progression will also be discussed.

Neuronal decanoic acid oxidation is markedly lower than that of octanoic acid: A mechanistic insight into the medium-chain triglyceride ketogenic diet.

The medium-chain triglyceride (MCT) ketogenic diet contains both octanoic (C8) and decanoic (C10) acids. The diet is an effective treatment for pharmacoresistant epilepsy. Although the exact mechanism for its efficacy is not known, it is emerging that C10, but not C8, interacts with targets that can explain antiseizure effects, for example, peroxisome proliferator-activated receptor-γ (eliciting mitochondrial biogenesis and increased antioxidant status) and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. For such effects to occur, significant concentrations of C10 are likely to be required in the brain.

Sox1 is required for the specification of a novel p2-derived interneuron subtype in the mouse ventral spinal cord.

During mouse development, the ventral spinal cord becomes organized into five progenitor domains that express different combinations of transcription factors and generate different subsets of neurons and glia. One of these domains, known as the p2 domain, generates two subtypes of interneurons, V2a and V2b. Here we have used genetic fate mapping and loss-of-function analysis to show that the transcription factor Sox1 is expressed in, and is required for, a third type of p2-derived interneuron, which we named V2c. These are close relatives of V2b interneurons, and, in the absence of Sox1, they switch to the V2b fate. In addition, we show that late-born V2a and V2b interneurons are heterogeneous, and subsets of these cells express the transcription factor Pax6. Our data demonstrate that interneuron diversification in the p2 domain is more complex than previously thought and directly implicate Sox1 in this process.

SOX1 links the function of neural patterning and Notch signalling in the ventral spinal cord during the neuron-glial fate switch.

During neural development the transition from neurogenesis to gliogenesis, known as the neuron-glial (Nu/G) fate switch, requires the coordinated function of patterning factors, pro-glial factors and Notch signalling. How this process is coordinated in the embryonic spinal cord is poorly understood. Here, we demonstrate that during the N/G fate switch in the ventral spinal cord (vSC) SOX1 links the function of neural patterning and Notch signalling. We show that, SOX1 expression in the vSC is regulated by PAX6, NKX2.2 and Notch signalling in a domain-specific manner. We further show that SOX1 regulates the expression of Hes1 and that loss of Sox1 leads to enhanced production of oligodendrocyte precursors from the pMN. Finally, we show that Notch signalling functions upstream of SOX1 during this fate switch and is independently required for the acquisition of the glial fate perse by regulating Nuclear Factor I A expression in a PAX6/SOX1/HES1/HES5-independent manner. These data integrate functional roles of neural patterning factors, Notch signalling and SOX1 during gliogenesis.

Generation of an ABCG2(GFPn-puro) transgenic line--a tool to study ABCG2 expression in mice.

The ATP-binding cassette (ABC) transporter 2 (ABCG2) is expressed by stem cells in many organs and in stem cells of solid tumors. These cells are isolated based on the side population (SP) phenotype, a Hoechst 3342 dye efflux property believed to be conferred by ABCG2. Because of the limitations of this approach we generated transgenic mice that express Nuclear GFP (GFPn) coupled to the Puromycin-resistance gene, under the control of ABCG2 promoter/enhancer sequences. We show that ABCG2 is expressed in neural progenitors of the developing forebrain and spinal cord and in embryonic and adult endothelial cells of the brain. Using the neurosphere assay, we isolated tripotent ABCG2-expressing neural stem cells from embryonic mouse brain. This transgenic line is a powerful tool for studying the expression of ABCG2 in many tissues and for performing functional studies in different experimental settings.

Spatially distinct functions of PAX6 and NKX2.2 during gliogenesis in the ventral spinal cord.

During ventral spinal cord (vSC) development, the p3 and pMN progenitor domain boundary is thought to be maintained by cross-repressive interactions between NKX2.2 and PAX6. Using loss-of-function analysis during the neuron-glial fate switch we show that the identity of the p3 domain is not maintained by the repressive function of NKX2.2 on Pax6 expression, even in the absence of NKX2.9. We further show that NKX2.2 is necessary to induce the expression of Slit1 and Sulfatase 1 (Sulf1) in the vSC in a PAX6-independent manner. Conversely, we show that PAX6 regulates Sulf1/Slit1 expression in the vSC in an NKX2.2/NKX6.1-independent manner. Consequently, deregulation of Sulf1 expression in Pax6-mutant embryos has stage-specific implications on neural patterning, associated with enhancement of Sonic Hedgehog activity. On the other hand, deregulation of Slit1 expression in gliogenic neural progenitors leads to changes in Astrocyte subtype identity. These data provide important insights into specific functions of PAX6 and NKX2.2 during glial cell specification that have so far remained largely unexplored.

Modification of human bacterial artificial chromosome clones for functional studies and therapeutic applications.

Cellular genomic reporter assays for screening and evaluation of inducers of fetal hemoglobin.

Reactivation of fetal hemoglobin (HbF) expression using pharmacological agents represents a potential strategy for the therapy of beta-thalassemia, sickle cell disease, HbE and other beta-hemoglobinopathies. However, the drugs currently available have low efficacy and specificity and are associated with high toxicity. We describe the development of stable cellular genomic reporter assays (GRAs) based on the green fluorescence protein (EGFP) gene under the Ggamma-globin promoter in the intact human beta-globin locus. We show that human erythroleukemic cell lines stably transfected with a Ggamma-EGFP beta-globin locus construct can maintain a uniform basal level of EGFP expression over long periods of continuous culture and that induction of EGFP expression parallels the induction of the endogenous globin genes. We compared the EGFP-induction potency of a number of chemotherapeutic agents, including histone deacetylase inhibitors and DNA-binding agents. We show that hydroxyurea and butyrate result in moderate levels of induction (70-80%) but with an additive inductive effect. Among the DNA-binding agents tested, cisplatin was the most potent inducer of HbF expression, (442+/-32%), a level which is comparable to hemin (764+/-145%). These results indicate that cellular GRAs containing Ggamma-EGFP-modified beta-globin locus constructs can be used to develop novel inducers of HbF synthesis for the therapy of beta-hemoglobinopathies.

Targeted modification of a human beta-globin locus BAC clone using GET Recombination and an I-Scei counterselection cassette.

There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in any genomic locus, using a human beta-globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences. A counterselection cassette, consisting of an inducible I-SceI gene, its recognition site, and an antibiotic resistance gene, is inserted into the targeted region using GET Recombination. A PCR fragment carrying the modification but no selectable marker replaces the counterselection cassette in a second round of GET Recombination. The unique I-SceI site in the counterselection cassette is cut by I-SceI endonuclease, strongly selecting against nonrecombinant clones and yielding up to 30% correct recombinants.

Insertion of modifications in the beta-globin locus using GET recombination with single-stranded oligonucleotides and denatured PCR fragments.

We describe the use of the GET recombination system with oligonucleotides or single-stranded polymerase chain reaction (PCR) fragments to insert modifications in the human beta-globin locus without counterselection. The method involves recombination between oligonucleotides or denatured PCR fragments and homologous sequences in the beta-globin gene in a clone of 205-kb bacterial artificial chromosome (BAC), based on the inducible expression of the recE, recT, and gam genes. In this method, oligonucleotides or denatured PCR fragments are electroporated directly into cells carrying both the globin BAC and the pGETrec plasmid, after induction of the GET recombination system. Recombinant BAC clones are identified by PCR, using allele-specific amplification for the mutated sequences. We have used this approach to insert a unique restriction site as well as a common thalassemia mutation (stop codon 39, C-->T) into the human beta-globin locus. We have observed the frequency of recombinant clones to be as high as 1 in 100-200 clones. Therefore, this approach provides a simple and efficient method for introducing point mutations and other fine modifications into BACs, and should greatly facilitate the use of BACs for functional studies and therapeutic applications.

Insertion of common mutations into the human beta-globin locus using GET Recombination and an EcoRI endonuclease counterselection cassette.

A large number of mutations have been described in the human beta-globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human beta-globin locus. We report here the use of the GET Recombination system with an EcoRI/Kan(R) counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG-->AAG mutation and the codon 41-42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human beta-globin locus. The counterselection cassette was first inserted into the target sequence in the beta-globin gene, and then a PCR fragment carrying the required modification was used to replace it. Efficient counterselection depends upon the tight regulation of the highly toxic EcoRI endonuclease gene by expression of lacI(q). Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones. The technique can be performed on any known gene sequence using current BAC technology, allowing identification and comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders.

Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human beta -globin locus in erythropoietic cells.

Reactivation of fetal hemoglobin genes has been proposed as a potential therapeutic procedure in patients with beta-thalassemia, sickle cell disease, or other beta-hemoglobinopathies. In vitro model systems based on small plasmid globin gene constructs have previously been used in human and mouse erythroleukemic cell lines to study the molecular mechanisms regulating the expression of the fetal human globin genes and their reactivation by a variety of pharmacologic agents. These studies have led to great insights in globin gene regulation and the identification of a number of potential inducers of fetal hemoglobin. In this study we describe the development of enhanced green fluorescence protein (EGFP) reporter systems based on bacterial artificial chromosomes (EBACs) to monitor the activity of the epsilon-, (G)gamma-, (A)gamma-, delta-, and beta-globin genes in the beta-globin locus. Additionally, we demonstrate that transfection of erythroleukemia cells with our EBACs is greatly enhanced by expression of EBNA1, which also facilitates episomal maintenance of our constructs in human cells. Our studies in human cells have shown physiologically relevant differences in the expression of each of the globin genes and also demonstrate that hemin is a potent inducer of EGFP expression from EGFP-modified epsilon-, (G)gamma-, and (A)gamma-globin constructs. In contrast, the EGFP-modified delta- and beta-globin constructs consistently produced much lower levels of EGFP expression on hemin induction, mirroring the in vivo ontogeny. The EGFP-modified beta-globin eukaryotic BAC (EBAC) vector system can thus be used in erythroleukemia cells to evaluate induction of the epsilon- and gamma-globin genes from the intact human beta-globin locus.