Pharmacology and Therapeutics - Research Publications

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    Iron overload and impaired iron handling contribute to the dystrophic pathology in models of Duchenne muscular dystrophy
    Alves, FM ; Kysenius, K ; Caldow, MK ; Hardee, JP ; Chung, JD ; Trieu, J ; Hare, DJ ; Crouch, PJ ; Ayton, S ; Bush, A ; Lynch, GS ; Koopman, R (WILEY, 2022-06)
    BACKGROUND: Oxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity. METHODS: We assessed iron distribution and total elemental iron using LA-ICP-MS on skeletal muscle cross-sections of 8-week-old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin-null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron-enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron- and mitochondria-related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed. RESULTS: We observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS-1, the rate-limiting enzyme for haem synthesis (dko -44%, P < 0.05), and the haem-containing protein myoglobin (dko -54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (-30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem-containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology. CONCLUSIONS: Muscles from dystrophic mice have increased iron levels and dysregulated iron-related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem-containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem-containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.
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    An integrated mass spectrometry imaging and digital pathology workflow for objective detection of colorectal tumours by unique atomic signatures
    Paul, B ; Kysenius, K ; Hilton, JB ; Jones, MWM ; Hutchinson, RW ; Buchanan, DD ; Rosty, C ; Fryer, F ; Bush, A ; Hergt, JM ; Woodhead, JD ; Bishop, DP ; Doble, PA ; Hill, MM ; Crouch, PJ ; Hare, DJ (ROYAL SOC CHEMISTRY, 2021-08-14)
    Tumours are abnormal growths of cells that reproduce by redirecting essential nutrients and resources from surrounding tissue. Changes to cell metabolism that trigger the growth of tumours are reflected in subtle differences between the chemical composition of healthy and malignant cells. We used LA-ICP-MS imaging to investigate whether these chemical differences can be used to spatially identify tumours and support detection of primary colorectal tumours in anatomical pathology. First, we generated quantitative LA-ICP-MS images of three colorectal surgical resections with case-matched normal intestinal wall tissue and used this data in a Monte Carlo optimisation experiment to develop an algorithm that can classify pixels as tumour positive or negative. Blinded testing and interrogation of LA-ICP-MS images with micrographs of haematoxylin and eosin stained and Ki67 immunolabelled sections revealed Monte Carlo optimisation accurately identified primary tumour cells, as well as returning false positive pixels in areas of high cell proliferation. We analysed an additional 11 surgical resections of primary colorectal tumours and re-developed our image processing method to include a random forest regression machine learning model to correctly identify heterogenous tumours and exclude false positive pixels in images of non-malignant tissue. Our final model used over 1.6 billion calculations to correctly discern healthy cells from various types and stages of invasive colorectal tumours. The imaging mass spectrometry and data analysis methods described, developed in partnership with clinical cancer researchers, have the potential to further support cancer detection as part of a comprehensive digital pathology approach to cancer care through validation of a new chemical biomarker of tumour cells.
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    Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)
    Hare, DJ ; Kysenius, K ; Paul, B ; Knauer, B ; Hutchinson, RW ; O'Connor, C ; Fryer, F ; Hennessey, TP ; Bush, AI ; Crouch, PJ ; Doble, PA (JOURNAL OF VISUALIZED EXPERIMENTS, 2017-01-01)
    Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.
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    Oral Treatment with CuII(atsm) Increases Mutant SOD1 In Vivo but Protects Motor Neurons and Improves the Phenotype of a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis
    Roberts, BR ; Lim, NKH ; McAllum, EJ ; Donnelly, PS ; Hare, DJ ; Doble, PA ; Turner, BJ ; Price, KA ; Lim, SC ; Paterson, BM ; Hickey, JL ; Rhoads, TW ; Williams, JR ; Kanninen, KM ; Hung, LW ; Liddell, JR ; Grubman, A ; Monty, J-F ; Llanos, RM ; Kramer, DR ; Mercer, JFB ; Bush, AI ; Masters, CL ; Duce, JA ; Li, Q-X ; Beckman, JS ; Barnham, KJ ; White, AR ; Crouch, PJ (SOC NEUROSCIENCE, 2014-06-04)
    Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1.