Browsing by Author "Middleton, RJ"

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    The 18 kDa translocator protein, microglia and neuroinflammation
    (Wiley, 2014-10-26) Liu, GJ; Middleton, RJ; Hatty, CR; Kam, WWY; Chan, RHY; Pham, TQ; Harrison-Brown, M; Dodson, E; Veale, K; Banati, RB
    The 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is expressed in the injured brain. It has become known as an imaging marker of “neuroinflammation” indicating active disease, and is best interpreted as a nondiagnostic biomarker and disease staging tool that refers to histopathology rather than disease etiology. The therapeutic potential of TSPO as a drug target is mostly based on the understanding that it is an outer mitochondrial membrane protein required for the translocation of cholesterol, which thus regulates the rate of steroid synthesis. This pivotal role together with the evolutionary conservation of TSPO has underpinned the belief that any loss or mutation of TSPO should be associated with significant physiological deficits or be outright incompatible with life. However, against prediction, full Tspo knockout mice are viable and across their lifespan do not show the phenotype expected if cholesterol transport and steroid synthesis were significantly impaired. Thus, the “translocation” function of TSPO remains to be better substantiated. Here, we discuss the literature before and after the introduction of the new nomenclature for TSPO and review some of the newer findings. In light of the controversy surrounding the function of TSPO, we emphasize the continued importance of identifying compounds with confirmed selectivity and suggest that TSPO expression is analyzed within specific disease contexts rather than merely equated with the reified concept of “neuroinflammation.” © 2014 The Authors
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    Cellular sources and regional variations in the expression of the neuroinflammatory marker translocator protein (TSPO) in the normal brain
    (Multidisciplinary Digital Publishing Institute (MDPI), 2018-09-11) Betlazar, C; Harrison-Brown, M; Middleton, RJ; Banati, RB; Liu, GJ
    The inducible expression of the mitochondrial translocator protein 18 kDa (TSPO) by activated microglia is a prominent, regular feature of acute and chronic-progressive brain pathology. This expression is also the rationale for the continual development of new TSPO binding molecules for the diagnosis of “neuroinflammation” by molecular imaging. However, there is in the normal brain an ill-defined, low-level constitutive expression of TSPO. Taking advantage of healthy TSPO knockout mouse brain tissue to validate TSPO antibody specificity, this study uses immunohistochemistry to determine the regional distribution and cellular sources of TSPO in the normal mouse brain. Fluorescence microscopy revealed punctate TSPO immunostaining in vascular endothelial cells throughout the brain. In the olfactory nerve layers and glomeruli of the olfactory bulb, choroid plexus and ependymal layers, we confirm constitutive TSPO expression levels similar to peripheral organs, while some low TSPO expression is present in regions of known neurogenesis, as well as cerebellar Purkinje cells. The distributed-sparse expression of TSPO in endothelial mitochondria throughout the normal brain can be expected to give rise to a low baseline signal in TSPO molecular imaging studies. Finally, our study emphasises the need for valid and methodologically robust verification of the selectivity of TSPO ligands through the use of TSPO knockout tissues. © 2018 The Authors
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    Control of neuroinflammation through radiation-induced microglial changes
    (MDPI, 2021-09-10) Boyd, A; Byrne, S; Middleton, RJ; Banati, RB; Liu, GJ
    Microglia, the innate immune cells of the central nervous system, play a pivotal role in the modulation of neuroinflammation. Neuroinflammation has been implicated in many diseases of the CNS, including Alzheimer’s disease and Parkinson’s disease. It is well documented that microglial activation, initiated by a variety of stressors, can trigger a potentially destructive neuroinflammatory response via the release of pro-inflammatory molecules, and reactive oxygen and nitrogen species. However, the potential anti-inflammatory and neuroprotective effects that microglia are also thought to exhibit have been under-investigated. The application of ionising radiation at different doses and dose schedules may reveal novel methods for the control of microglial response to stressors, potentially highlighting avenues for treatment of neuroinflammation associated CNS disorders, such as Alzheimer’s disease and Parkinson’s disease. There remains a need to characterise the response of microglia to radiation, particularly low dose ionising radiation. © The Authors - Open Access
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    Epigenetic silencing of the human 18 kDa translocator protein in a T cell leukemia cell line
    (Mary Ann Liebert, Inc. publishers, 2017-02-01) Middleton, RJ; Kam, WWY; Liu, GJ; Banati, RB
    The mitochondrial membrane 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is constitutively expressed in most organs, most abundantly in hormonal tissue and cells of mononuclear phagocyte lineage, while in the brain, TSPO expression is induced in the wake of injury, inflammation, and neurodegeneration. Increased TSPO expression is also prominent in several cancerous tissues where it appears to correlate with the degree of malignancy. Currently, TSPO is thus actively investigated as a generic biomarker for disease activity and a therapeutic target for a wide range of diseases. In this study, we report a Jurkat human T cell leukemia cell line that has only trace expression of TSPO mRNA. Through the use of bisulphite genomic sequencing, we show that the Jurkat TSPO promoter is highly methylated except for CpG sites that are adjacent to the transcription start site. Control measurements in HEK-293, HeLa, and U87-MG cells with high TSPO mRNA expression showed low levels of TSPO promoter methylation. Demethylation with 5-aza-2'-deoxycytidine (5-aza-dC) caused a dose-dependent increase in TSPO mRNA with a corresponding demethylation of the TSPO promoter in Jurkat cells. Treating HeLa and U87-MG cells with 5-aza-dC caused no change in the level of TSPO mRNA. These observations confirm the epigenetic regulation of TSPO and suggest it to be a more common mechanism by which the differential expression of TSPO in various cell types and in health and disease may be explained. ©2017 Mary Ann Liebert, Inc.
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    Functional gains in energy and cell metabolism after TSPO gene insertion
    (Taylor & Francis, 2017-02-02) Liu, GJ; Middleton, RJ; Kam, WWY; Chin, DY; Hatty, CR; Chan, RHY; Banati, RB
    Recent loss-of-function studies in tissue-specific as well as global Tspo (Translocator Protein 18 kDa) knockout mice have not confirmed its long assumed indispensability for the translocation of cholesterol across the mitochondrial inter-membrane space, a rate-limiting step in steroid biosynthesis. Instead, recent studies in global Tspo knockout mice indicate that TSPO may play a more fundamental role in cellular bioenergetics, which may include the indirect down-stream regulation of transport or metabolic functions. To examine whether overexpression of the TSPO protein alters the cellular bioenergetic profile, Jurkat cells with low to absent endogenous expression were transfected with a TSPO construct to create a stable cell line with de novo expression of exogenous TSPO protein. Expression of TSPO was confirmed by RT-qPCR, radioligand binding with [3H]PK11195 and immunocytochemistry with a TSPO antibody. We demonstrate that TSPO gene insertion causes increased transcription of genes involved in the mitochondrial electron transport chain. Furthermore, TSPO insertion increased mitochondrial ATP production as well as cell excitability, reflected in a decrease in patch clamp recorded rectified K channel currents. These functional changes were accompanied by an increase in cell proliferation and motility, which were inhibited by PK11195, a selective ligand for TSPO. We suggest that TSPO may serve a range of functions that can be viewed as downstream regulatory effects of its primary, evolutionary conserved role in cell metabolism and energy production. © 2017 ANSTO
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    Gd-TPP-DOTA reduces cell viability in cancer cells via synchrotron radiotherapy
    (Australian National University, 2021-08-24) Middleton, RJ; Howell, NR; Livio, E; Wyatt, NA; Chacon, A; Fraser, BH; Barnes, M; Cameron, M; Rendina, LM; Häusermann, D; Lerch, MLF; Safavi-Naeini, M
    High-Z elements have been proposed as radiosensitisers in X-ray photon radiotherapy due to their emission of multiple high-LET photo- and Auger electrons following X-ray irradiation. Gadolinium is a particularly attractive candidate radiosensitiser, since it can also be used as an MRI contrast agent. In this study, we report on the efficacy of Gd-triphenylphosphonium salt-DOTA (Gd(III)-TPP-DOTA) for synchrotron microbeam radiation therapy dose enhancement. The compound utilises the mitochondrial targeting moiety triphenylphosphonium (TPP) to accumulate Gd in the inner mitochondrial membrane. Experiments were conducted using the dynamic mode option at hutch 2B of the Imaging and Medical Beamline at the Australian Synchrotron. Human glioblastoma multiforme cells (T98G cell line) were cultured to 80-90% confluence in T12.5 flasks. Approximately 24 hours prior to irradiation, the cultures were either treated with a 500 μM solution of Gd(III)DOTA-TPP or a vehicle control. Spatial dose distribution of synchrotron broad beam (BB) and single/multiple microbeams were measured using a micron-scale X-Tream dosimetry system and Gafchromic films in air and at 2 cm depth in solid water (same depth as the monolayer of cells in T12.5 flasks). A total of 96 flasks were irradiated, with doses of 0, 1, 2, 3, 4, 5, 10 and 16 Gy delivered in valley (MRT) or uniformly (BB). Post irradiation, each flask was re-seeded into 7 x 96 well-plates to perform the resazurin cell proliferation assay up to 7 days after irradiation. Our preliminary analysis indicates that for cells irradiated by 3 Gy of BB or MRT radiation, the addition of Gd(III)DOTA-TPP results in a reduction in viable cell mass by 24.25% and 25.79%, respectively, compared with untreated flasks. © The Authors
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    Green fluorescent protein alters the transcriptional regulation of human mitochondrial genes after gamma irradiation
    (Springer/Plenum Publishers, 2013-07-01) Kam, WWY; Middleton, RJ; Lake, V; Banati, RB
    Green fluorescent proteins (GFP), extensively used as reporters in biological and imaging studies, are assumed to be mostly biologically inert. Here, we test the assumption in regard to the transcriptional regulation of 18 mitochondrially encoded genes in GFP expressing human T-cell line (JURKAT cells) exposed to gamma radiation. Using quantitative polymerase chain reaction, we demonstrate that wild type and GFP expressing JURKAT cells have different baseline mitochondrial transcript expression (10 out of the 18 tested genes) and after a single dose of radiation (100 Gy) show a significantly different transcriptional regulation of their mitochondrial genes. While in wild type cells, ten of the tested genes are up-regulated in response to radiation exposure, GFP expressing cells show less transcriptional regulation with a small down-regulation in five genes. Our results indicate that the presence of GFP in the cytoplasm can alter the cellular response to ionizing radiation.© 2013, Springer.
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    Guwiyang Wurra–‘Fire Mouse’: a global gene knockout model for TSPO/PBR drug development, loss-of-function and mechanisms of compensation studies
    (Portland Press Limited, 2015-08-03) Middleton, RJ; Liu, GJ; Banati, RB
    The highly conserved 18-kDa translocator protein (TSPO) or peripheral benzodiazepine receptor (PBR), is being investigated as a diagnostic and therapeutic target for disease conditions ranging from inflammation to neurodegeneration and behavioural illnesses. Many functions have been attributed to TSPO/PBR including a role in the mitochondrial permeability transition pore (MPTP), steroidogenesis and energy metabolism. In this review, we detail the recent developments in determining the physiological role of TSPO/PBR, specifically based on data obtained from the recently generated Tspo knockout mouse models. In addition to defining the role of TSPO/PBR, we also describe the value of Tspo knockout mice in determining the selectivity, specificity and presence of any off-target effects of TSPO/PBR ligands. © 2015 Authors
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    IFN-γ-induced signal-on fluorescence aptasensors: from hybridization chain reaction amplification to 3D optical fiber sensing interface towards a deployable device for cytokine sensing
    (Royal Society of Chemistry, 2019-04-29) Zhang, FY; Deng, F; Liu, GJ; Middleton, RJ; Inglis, DW; Anwer, A; Wang, S; Liu, GZ
    Interferon-gamma (IFN-γ), a proinflammatory cytokine, has been used as an early indicator of multiple infectious diseases or tumors. In order to explore the detection capability of a commonly used anti-IFN-γ aptamer, a simple target induced strand-displacement aptasensing strategy was tested by introducing three different complementary strands and two different signal/quencher pairs. The Texas red/BHQ2-based sensor showed the best affinity constant (Kd) of 21.87 ng mL−1. It was found that the strand-displacement aptasensing strategy was impacted by the complementary position and length of the complementary strands. Additionally, the hybridization chain reaction (HCR) amplification strategy was introduced, yielding a 12-fold improved sensitivity of 0.45 ng mL−1. In order to further explore the sensing platform for spatially localized cytokine detection, the Texas red/BHQ2-based strand-displacement aptasensor was successfully fabricated on the 3D optical fiber surface to achieve a deployable sensing device for monitoring IFN-γ based on the fluorescence spots counting strategy. Finally, the three developed aptasensing strategies (strand-displacement strategy, HCR amplification strategy, 3D optical fiber aptasensor) were applied for detection of IFN-γ secreted by PBMCs with comparable results to those of ELISA. The deployable 3D optical fiber aptasensor with the superior sensitivity is potential to be used for detection of spatially localized IFN-γ in vivo. © 2019 The Royal Society of Chemistry
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    The impact of high and low dose ionising radiation on the central nervous system
    (Elsevier B.V., 2016-10-09) Betlazar, C; Middleton, RJ; Banati, RB; Liu, GJ
    Responses of the central nervous system (CNS) to stressors and injuries, such as ionising radiation, are modulated by the concomitant responses of the brains innate immune effector cells, microglia. Exposure to high doses of ionising radiation in brain tissue leads to the expression and release of biochemical mediators of ‘neuroinflammation’, such as pro-inflammatory cytokines and reactive oxygen species (ROS), leading to tissue destruction. Contrastingly, low dose ionising radiation may reduce vulnerability to subsequent exposure of ionising radiation, largely through the stimulation of adaptive responses, such as antioxidant defences. These disparate responses may be reflective of non-linear differential microglial activation at low and high doses, manifesting as an anti-inflammatory or pro-inflammatory functional state. Biomarkers of pathology in the brain, such as the mitochondrial Translocator Protein 18 kDa (TSPO), have facilitated in vivo characterisation of microglial activation and ‘neuroinflammation’ in many pathological states of the CNS, though the exact function of TSPO in these responses remains elusive. Based on the known responsiveness of TSPO expression to a wide range of noxious stimuli, we discuss TSPO as a potential biomarker of radiation-induced effects. © 2016 The Authors Published by Elsevier B.V.
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    Knockout of TSPO delays and reduces amyloid, Tau, astrocytosis and behavioral dysfunctions in Alzheimer’s disease
    (Cold Spring Harbor Laboratory, 2022-03-28) Ceyzériat, K; Meyer, L; Boutjelda, F; Tsartsalis, S; Amossé, Q; Middleton, RJ; Liu, GJ; Banati, RB; Zilli, T; Garibotto, V; Millet, P; Tournier, BB
    The 18kDa translocator protein (TSPO) is up-regulated in glial cells in neurodegenerative diseases. In Alzheimer’s disease (AD) animal models, TSPO is first overexpressed in astrocytes and then in microglia. However, the precise role of TSPO in the onset and progression of pathology and symptoms characteristic of the disease remains unknown. Here, we report that in the absence of TSPO in 3xTgAD mice the expected disease onset is significantly delayed and a reduction is seen in the hippocampal load of poorly and highly aggregated forms of Tau (−44% and −82%, respectively) and Aβ42 (−25% and −95%, respectively), at 9 months of age. In addition, the astrocyte reactivity was decreased in 3xTgAD.TSPO−/− mice with a reduction in the morphologic complexity and the size of astrocytes in the dorso-dorsal hippocampus and the hilus. Functionally, the absence of TSPO ameliorated the cognitive consequences of adeno-associated virus-induced Tau over-expression in the hippocampus. This suggests that TSPO plays an important role in the active disease progression of AD. TSPO-inhibiting drugs thus merit further exploration as to their potential to reduce the rate of neurodegenerative disease progression. © 2022 The Authors
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    Long-term diazepam treatment enhances microglial spine engulfment and impairs cognitive performance via the mitochondrial 18 kDa translocator protein (TSPO)
    (Springer Nature, 2022-02-28) Shi, Y; Cui, M; Ochs, K; Brendel, M; Strübing, FL; Briel, N; Eckenweber, F; Zou, CY; Banati, RB; Liu, GJ; Middleton, RJ; Rupprecht, R; Rudolph, U; Zeilhofer, HU; Rammes, G; Herms, J; Dorostkar, MM
    Benzodiazepines are widely administered drugs to treat anxiety and insomnia. In addition to tolerance development and abuse liability, their chronic use may cause cognitive impairment and increase the risk for dementia. However, the mechanism by which benzodiazepines might contribute to persistent cognitive decline remains unknown. Here we report that diazepam, a widely prescribed benzodiazepine, impairs the structural plasticity of dendritic spines, causing cognitive impairment in mice. Diazepam induces these deficits via the mitochondrial 18 kDa translocator protein (TSPO), rather than classical γ-aminobutyric acid type A receptors, which alters microglial morphology, and phagocytosis of synaptic material. Collectively, our findings demonstrate a mechanism by which TSPO ligands alter synaptic plasticity and, as a consequence, cause cognitive impairment. © 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.
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    Microgravity × radiation: a space mechanobiology approach toward cardiovascular function and disease
    (Frontiers Media S.A., 2021-10-29) Basirun, C; Ferlazzo, ML; Howell, NR; Liu, GJ; Middleton, RJ; Martinac, B; Narayanan, SA; Poole, K; Gentile, C; Chou, J
    In recent years, there has been an increasing interest in space exploration, supported by the accelerated technological advancements in the field. This has led to a new potential environment that humans could be exposed to in the very near future, and therefore an increasing request to evaluate the impact this may have on our body, including health risks associated with this endeavor. A critical component in regulating the human pathophysiology is represented by the cardiovascular system, which may be heavily affected in these extreme environments of microgravity and radiation. This mini review aims to identify the impact of microgravity and radiation on the cardiovascular system. Being able to understand the effect that comes with deep space explorations, including that of microgravity and space radiation, may also allow us to get a deeper understanding of the heart and ultimately our own basic physiological processes. This information may unlock new factors to consider with space exploration whilst simultaneously increasing our knowledge of the cardiovascular system and potentially associated diseases. © 2021 Basirun, Ferlazzo, Howell, Liu, Middleton, Martinac, Narayanan, Poole, Gentile and Chou.
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    Mitochondrial translocator protein (TSPO) expression in the brain after whole body gamma irradiation
    (Frontier Media S.A., 2021-10-25) Betlazar, C; Middleton, RJ; Howell, NR; Storer, B; Davis, E; Davies, JB; Banati, RB; Liu, GJ
    The brain’s early response to low dose ionizing radiation, as may be encountered during diagnostic procedures and space exploration, is not yet fully characterized. In the brain parenchyma, the mitochondrial translocator protein (TSPO) is constitutively expressed at low levels by endothelial cells, and can therefore be used to assess the integrity of the brain’s vasculature. At the same time, the inducible expression of TSPO in activated microglia, the brain’s intrinsic immune cells, is a regularly observed early indicator of subtle or incipient brain pathology. Here, we explored the use of TSPO as a biomarker of brain tissue injury following whole body irradiation. Post-radiation responses were measured in C57BL/6 wild type (Tspo+/+) and TSPO knockout (Tspo–/–) mice 48 h after single whole body gamma irradiations with low doses 0, 0.01, and 0.1 Gy and a high dose of 2 Gy. Additionally, post-radiation responses of primary microglial cell cultures were measured at 1, 4, 24, and 48 h at an irradiation dose range of 0 Gy-2 Gy. TSPO mRNA and protein expression in the brain showed a decreased trend after 0.01 Gy relative to sham-irradiated controls, but remained unchanged after higher doses. Immunohistochemistry confirmed subtle decreases in TSPO expression after 0.01 Gy in vascular endothelial cells of the hippocampal region and in ependymal cells, with no detectable changes following higher doses. Cytokine concentrations in plasma after whole body irradiation showed differential changes in IL-6 and IL-10 with some variations between Tspo–/– and Tspo+/+ animals. The in vitro measurements of TSPO in primary microglial cell cultures showed a significant reduction 1 h after low dose irradiation (0.01 Gy). In summary, acute low and high doses of gamma irradiation up to 2 Gy reduced TSPO expression in the brain’s vascular compartment without de novo induction of TSPO expression in parenchymal microglia, while TSPO expression in directly irradiated, isolated, and thus highly activated microglia, too, was reduced after low dose irradiation. The potential link between TSPO, its role in mitochondrial energy metabolism and the selective radiation sensitivity, notably of cells with constitutive TSPO expression such as vascular endothelial cells, merits further exploration. © The Authors - Open Access
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    Oldest directly dated remains of sheep in China
    (Springer Nature, 2014-11-24) Dodson, JR; Dodson, E; Banati, RB; Li, XQ; Atahan, P; Hu, SM; Middleton, RJ; Zhou, XY; Nan, S
    The origins of domesticated sheep (Ovis sp.) in China remain unknown. Previous workers have speculated that sheep may have been present in China up to 7000 years ago, however many claims are based on associations with archaeological material rather than independent dates on sheep material. Here we present 7 radiocarbon dates on sheep bone from Inner Mongolia, Ningxia and Shaanxi provinces. DNA analysis on one of the bones confirms it is Ovis sp. The oldest ages are about 4700 to 4400 BCE and are thus the oldest objectively dated Ovis material in eastern Asia. The graphitisised bone collagen had δ13C values indicating some millet was represented in the diet. This probably indicates sheep were in a domestic setting where millet was grown. The younger samples had δ13C values indicating that even more millet was in the diet, and this was likely related to changes in foddering practices. © 2014, Macmillan Publishers Limited
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    Positron emission tomography and functional characterization of a complete PBR/TSPO knockout
    (Springer Nature, 2014-11-19) Banati, RB; Middleton, RJ; Chan, RHY; Hatty, CR; Wai-Ying Kam, W; Quin, C; Graeber, MB; Parmar, A; Zahra, D; Callaghan, PD; Fok, S; Howell, NR; Grégoire, MC; Szabo, A; Pham, TQ; Davis, E; Liu, GJ
    The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer’s disease to anxiety. Here we show that global C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from GuwiyangWurraTSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of GuwiyangWurraTSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs. Copyright © 2014, Springer Nature
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    Selective, high-contrast detection of syngeneic glioblastoma in vivo
    (Springer Nature, 2020-06-19) Banati, RB; Wilcox, P; Xu, R; Yin, G; Si, E; Son, ET; Shimizu, M; Holsinger, RMD; Parmar, A; Zahra, D; Arthur, A; Middleton, RJ; Liu, GJ; Charil, A; Graeber, MB
    Glioblastoma is a highly malignant, largely therapy-resistant brain tumour. Deep infiltration of brain tissue by neoplastic cells represents the key problem of diffuse glioma. Much current research focuses on the molecular makeup of the visible tumour mass rather than the cellular interactions in the surrounding brain tissue infiltrated by the invasive glioma cells that cause the tumour’s ultimately lethal outcome. Diagnostic neuroimaging that enables the direct in vivo observation of the tumour infiltration zone and the local host tissue responses at a preclinical stage are important for the development of more effective glioma treatments. Here, we report an animal model that allows high-contrast imaging of wild-type glioma cells by positron emission tomography (PET) using [18 F]PBR111, a selective radioligand for the mitochondrial 18 kDa Translocator Protein (TSPO), in the Tspo−/− mouse strain (C57BL/6-Tspotm1GuMu(GuwiyangWurra)). The high selectivity of [18 F]PBR111 for the TSPO combined with the exclusive expression of TSPO in glioma cells infiltrating into null-background host tissue free of any TSPO expression, makes it possible, for the first time, to unequivocally and with uniquely high biological contrast identify peri-tumoral glioma cell invasion at preclinical stages in vivo. Comparison of the in vivo imaging signal from wild-type glioma cells in a null background with the signal in a wild-type host tissue, where the tumour induces the expected TSPO expression in the host’s glial cells, illustrates the substantial extent of the peritumoral host response to the growing tumour. The syngeneic tumour (TSPO+/+) in null background (TSPO−/−) model is thus well suited to study the interaction of the tumour front with the peri-tumoral tissue, and the experimental evaluation of new therapeutic approaches targeting the invasive behaviour of glioblastoma. © 2020, The Author(s).
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    SP-103 - Scandium-47 and lutetium-177 radiolabelling and stability studies of 1st and 2nd generation DOTA-triphenylphosphonium ligands – potential radionuclide theranostics for treatment of glioblastoma multi-forme
    (Elsevier, 2021-05-17) Wyatt, NA; Hogan, L; Pellegrini, PA; Roberts, MP; Hall, A; Smith, N; Hemzal, E; Hill, L; Howell, NR; Middleton, RJ; Safavi-Naeini, M; Rendina, LM; Fraser, BH
    Scandium-47 has emerged as a promising radioisotope for targeted radionuclide tumor therapy. This is due, to a significant extent, from the combination of low energy / short range β- emission, the availability of a “perfect theranostic pair” with Sc-44 for companion PET imaging, the potential to form highly stable radiometal complexes, and the availability of suitable γ emissions for companion SPECT imaging. Sc-47 also has a shorter half-life (3.35 d) than the chemically similar Lu-177 (6.7 d) which is significant given recent in vitro research that suggests longer lived isotopes require more initial radioactivity to have the same effect upon cell viability [3]. The shorter half-life of Sc-47 also suggests it may be more suitable for smaller biological vectors (with shorter biological half-lives) such as small molecules and low MW peptides. One area of clinical treatment where Sc-47 can have impact and where improvements in patient outcomes and survival rates remain stubbornly low is glioblastoma multiforme (GBM). GBM is the most common and aggressive form of malignant brain tumor and represents around 60% of all adult brain tumors with a global incidence of <10 per 100,000 persons. The prognosis for GBM patients is poor with a -ear survival rate of 37%, 5 year rate of 5% and a median survival time of 10 months. The current standard of treatment is resection of the tumor followed by radiation therapy and chemotherapy. Given this poor prognosis there is a clear and unmet need for improved classes of treatment. Although significant progress has been made towards bringing GBM targeted radionuclide therapies to the clinic, the efforts to date have not included utilizing Sc-44/ Sc-47. Given this we are developing and evaluating Sc-44/Sc-47 and Lu-177/Ga-68 radiolabelled triphenylphosphonium (TPP) functionalised DOTA ligands (1st and 2nd generation) as potential theranostics for GBM. Described herein is our work on comparing the radiolabelling efficiency (Sc-47 vs. Lu-177) and stability studies (PBS pH 7.4, rat plasma) for our 1st and 2nd generation DOTA-TPP ligands. The presence of an additional carbonyl group in the 2nd generation DOTATPP ligand was anticipated to increase the number of donor atoms around the radiometal and affect radiolabelling reaction conditions and, more importantly, increase radiometal complex stability. Copyright © 2021 Elsevier Inc.
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    Space radiation and individual radiosensitivity - ANSTO CAS & Human Health in air beam experiments
    (Australian Nuclear Science and Technology Organisation, 2021-11-26) Ferlazzo, ML; Howell, NR; Liu, GJ; Zahra, D; Middleton, RJ; Foray, N
    Radiation exposure is a major limiting factor for long duration manned space flights. Radiation protection standards are based on the assumption that individuals are equally resistant to ionizing radiation. However, for over a century, there is evidence that humans do not respond equally to radiation. Particularly, the studies of secondary effects post-radiotherapy have shown a great variability among individuals. More specifically, large discrepancies among astronauts after the same flight were observed. Recently, from a collection of hundreds of fibroblast cell lines derived from patients suffering from genetic disease or post-radiotherapy radiosensitivity, we have shown that the delay in the nucleoshuttling of the ATM protein may cause a lack of double strand break (DSB) recognition, incomplete DSB repair and radiosensitivity. Interestingly, the model of the ATM nucleoshuttling was shown to be relevant not only for low-dose and repeated exposures, but also for high-LET particles, which renders this model compatible with space radiation exposure scenarios. Lastly, this model could lead to a novel approach for radiation protection, consisting of interventions to accelerate ATM nucleoshuttling. Such an approach may help in developing efficient countermeasures that could assist with manned space flights. In 2019-2021, teams from ANSTO CAS and Human Health have been collaborating to adapt the ANTARES beamline for in air irradiation of living matter and study the effects of secondary radiation produced by interraction of cosmic and galactic rays with spacecraft shielding. DNA repair and mitochondrial activity processes will be studied. © 2021 The Authors
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    Subcellular distribution of the 18 kDa translocator protein and transcript variant PBR-S in human cells
    (Elsevier, 2017-05-20) Liu, GJ; Middleton, RJ; Banati, RB
    Despite continued interest in the 18 kDa translocator protein (PBR/TSPO) as a biomarker and a therapeutic target for a range of diseases, its functional role, such as in the steroid synthesis pathway and energy metabolism has either become contentious or remains to be described more precisely. The PBR/TSPO gene consists of four exons, while a shorter isoform termed PBR-S lacks exon 2. The PBR-S 102-codon open reading frame differs to that of PBR/TSPO, resulting in a protein that is completely unrelated to PBR/TSPO. To our knowledge, PBR-S protein has never been described and has no known or proposed function. To obtain possible clues on the role of this uncharacterised protein, we compared the subcellular distribution of PBR-S to that of PBR/TSPO. By expressing fluorescently tagged PBR/TSPO, we confirmed that full-length PBR/TSPO co-localises with mitochondria in HeLa, HEK-293, MDA-MB-231, BJ and U87-MG human cell lines. Unlike the strictly mitochondrial localisation of PBR/TSPO, PBR-S has a punctate distribution throughout the cytosol that co-localises with lysosomes in HeLa, HEK-293, MDA-MB-231, BJ and U87-MG cells. In summary, within the cell lines examined we confirm mitochondria rather than occasionally reported other localisations, such as the cell nucleus, to be the only site where PBR/TSPO resides. Due to the lack of any shared protein sequences and the different subcellular locations, we suggest that the previously uncharacterised PBR-S protein variant of the PBR/TSPO gene is likely to serve a different yet to be discovered function compared to PBR/TSPO. © 2017 Elsevier B.V