Pharmacology and Therapeutics - Research Publications

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    Regular Physical Exercise Modulates Iron Homeostasis in the 5xFAD Mouse Model of Alzheimer's Disease
    Belaya, I ; Kucharikova, N ; Gorova, V ; Kysenius, K ; Hare, DJ ; Crouch, PJ ; Malm, T ; Atalay, M ; White, AR ; Liddell, JR ; Kanninen, KM (MDPI, 2021-08-01)
    Dysregulation of brain iron metabolism is one of the pathological features of aging and Alzheimer's disease (AD), a neurodegenerative disease characterized by progressive memory loss and cognitive impairment. While physical inactivity is one of the risk factors for AD and regular exercise improves cognitive function and reduces pathology associated with AD, the underlying mechanisms remain unclear. The purpose of the study is to explore the effect of regular physical exercise on modulation of iron homeostasis in the brain and periphery of the 5xFAD mouse model of AD. By using inductively coupled plasma mass spectrometry and a variety of biochemical techniques, we measured total iron content and level of proteins essential in iron homeostasis in the brain and skeletal muscles of sedentary and exercised mice. Long-term voluntary running induced redistribution of iron resulted in altered iron metabolism and trafficking in the brain and increased iron content in skeletal muscle. Exercise reduced levels of cortical hepcidin, a key regulator of iron homeostasis, coupled with interleukin-6 (IL-6) decrease in cortex and plasma. We propose that regular exercise induces a reduction of hepcidin in the brain, possibly via the IL-6/STAT3/JAK1 pathway. These findings indicate that regular exercise modulates iron homeostasis in both wild-type and AD mice.
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    Copper-ATSM as a Treatment for ALS: Support from Mutant SOD1 Models and Beyond
    Nikseresht, S ; Hilton, JBW ; Kysenius, K ; Liddell, JR ; Crouch, PJ (MDPI, 2020-11-01)
    The blood-brain barrier permeant, copper-containing compound, CuII(atsm), has successfully progressed from fundamental research outcomes in the laboratory through to phase 2/3 clinical assessment in patients with the highly aggressive and fatal neurodegenerative condition of amyotrophic lateral sclerosis (ALS). The most compelling outcomes to date to indicate potential for disease-modification have come from pre-clinical studies utilising mouse models that involve transgenic expression of mutated superoxide dismutase 1 (SOD1). Mutant SOD1 mice provide a very robust mammalian model of ALS with high validity, but mutations in SOD1 account for only a small percentage of ALS cases in the clinic, with the preponderant amount of cases being sporadic and of unknown aetiology. As per other putative drugs for ALS developed and tested primarily in mutant SOD1 mice, this raises important questions about the pertinence of CuII(atsm) to broader clinical translation. This review highlights some of the challenges associated with the clinical translation of new treatment options for ALS. It then provides a brief account of pre-clinical outcomes for CuII(atsm) in SOD1 mouse models of ALS, followed by an outline of additional studies which report positive outcomes for CuII(atsm) when assessed in cell and mouse models of neurodegeneration which do not involve mutant SOD1. Clinical evidence for CuII(atsm) selectively targeting affected regions of the CNS in patients is also presented. Overall, this review summarises the existing evidence which indicates why clinical relevance of CuII(atsm) likely extends beyond the context of cases of ALS caused by mutant SOD1.
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    Targeting Nrf2 to Suppress Ferroptosis and Mitochondrial Dysfunction in Neurodegeneration
    Abdalkader, M ; Lampinen, R ; Kanninen, KM ; Malm, TM ; Liddell, JR (FRONTIERS MEDIA SA, 2018-07-10)
    Ferroptosis is a newly described form of regulated cell death, distinct from apoptosis, necroptosis and other forms of cell death. Ferroptosis is induced by disruption of glutathione synthesis or inhibition of glutathione peroxidase 4, exacerbated by iron, and prevented by radical scavengers such as ferrostatin-1, liproxstatin-1, and endogenous vitamin E. Ferroptosis terminates with mitochondrial dysfunction and toxic lipid peroxidation. Although conclusive identification of ferroptosis in vivo is challenging, several salient and very well established features of neurodegenerative diseases are consistent with ferroptosis, including lipid peroxidation, mitochondrial disruption and iron dysregulation. Accordingly, interest in the role of ferroptosis in neurodegeneration is escalating and specific evidence is rapidly emerging. One aspect that has thus far received little attention is the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). This transcription factor regulates hundreds of genes, of which many are either directly or indirectly involved in modulating ferroptosis, including metabolism of glutathione, iron and lipids, and mitochondrial function. This potentially positions Nrf2 as a key deterministic component modulating the onset and outcomes of ferroptotic stress. The minimal direct evidence currently available is consistent with this and indicates that Nrf2 may be critical for protection against ferroptosis. In contrast, abundant evidence demonstrates that enhancing Nrf2 signaling is potently neuroprotective in models of neurodegeneration, although the exact mechanism by which this is achieved is unclear. Further studies are required to determine to extent to which the neuroprotective effects of Nrf2 activation involve the prevention of ferroptosis.
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    Cu-II(atsm) Attenuates Neuroinflammation
    Choo, XY ; Liddell, JR ; Huuskonen, MT ; Grubman, A ; Moujalled, D ; Roberts, J ; Kysenius, K ; Patten, L ; Quek, H ; Oikari, LE ; Duncan, C ; James, SA ; McInnes, LE ; Hayne, DJ ; Donnelly, PS ; Pollari, E ; Vahatalo, S ; Lejavova, K ; Kettunen, M ; Malm, T ; Koistinaho, J ; White, AR ; Kanninen, KM (FRONTIERS MEDIA SA, 2018-09-24)
    Background: Neuroinflammation and biometal dyshomeostasis are key pathological features of several neurodegenerative diseases, including Alzheimer's disease (AD). Inflammation and biometals are linked at the molecular level through regulation of metal buffering proteins such as the metallothioneins. Even though the molecular connections between metals and inflammation have been demonstrated, little information exists on the effect of copper modulation on brain inflammation. Methods: We demonstrate the immunomodulatory potential of the copper bis(thiosemicarbazone) complex CuII(atsm) in an neuroinflammatory model in vivo and describe its anti-inflammatory effects on microglia and astrocytes in vitro. Results: By using a sophisticated in vivo magnetic resonance imaging (MRI) approach, we report the efficacy of CuII(atsm) in reducing acute cerebrovascular inflammation caused by peripheral administration of bacterial lipopolysaccharide (LPS). CuII(atsm) also induced anti-inflammatory outcomes in primary microglia [significant reductions in nitric oxide (NO), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor (TNF)] and astrocytes [significantly reduced NO, MCP-1, and interleukin 6 (IL-6)] in vitro. These anti-inflammatory actions were associated with increased cellular copper levels and increased the neuroprotective protein metallothionein-1 (MT1) in microglia and astrocytes. Conclusion: The beneficial effects of CuII(atsm) on the neuroimmune system suggest copper complexes are potential therapeutics for the treatment of neuroinflammatory conditions.
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    Targeting mitochondrial metal dyshomeostasis for the treatment of neurodegeneration
    Liddell, JR (FUTURE MEDICINE LTD, 2015-08-01)
    Mitochondrial impairment and metal dyshomeostasis are suggested to be associated with many neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Friedreich's ataxia. Treatments aimed at restoring metal homeostasis are highly effective in models of these diseases, and clinical trials hold promise. However, in general, the effect of these treatments on mitochondrial metal homeostasis is unclear, and the contribution of mitochondrial metal dyshomeostasis to disease pathogenesis requires further investigation. This review describes the role of metals in mitochondria in health, how mitochondrial metals are disrupted in neurodegenerative diseases, and potential therapeutics aimed at restoring mitochondrial metal homeostasis and function.
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    C-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress
    Meyerowitz, J ; Parker, SJ ; Vella, LJ ; Ng, DCH ; Price, KA ; Liddell, JR ; Caragounis, A ; Li, Q-X ; Masters, CL ; Nonaka, T ; Hasegawa, M ; Bogoyevitch, MA ; Kanninen, KM ; Crouch, PJ ; White, AR (BIOMED CENTRAL LTD, 2011-08-08)
    BACKGROUND: TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing. RESULTS: We found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK). JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs. CONCLUSIONS: Our studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.
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    An impaired mitochondrial electron transport chain increases retention of the hypoxia imaging agent diacetylbis(4-methylthiosemicarbazonato)copper(II)
    Donnelly, PS ; Liddell, JR ; Lim, S ; Paterson, BM ; Cater, MA ; Savva, MS ; Mot, AI ; James, JL ; Trounce, IA ; White, AR ; Crouch, PJ (NATL ACAD SCIENCES, 2012-01-03)
    Radiolabeled diacetylbis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] is an effective positron-emission tomography imaging agent for myocardial ischemia, hypoxic tumors, and brain disorders with regionalized oxidative stress, such as mitochondrial myopathy, encephalopathy, and lactic acidosis with stroke-like episodes (MELAS) and Parkinson's disease. An excessively elevated reductive state is common to these conditions and has been proposed as an important mechanism affecting cellular retention of Cu from Cu(II)(atsm). However, data from whole-cell models to demonstrate this mechanism have not yet been provided. The present study used a unique cell culture model, mitochondrial xenocybrids, to provide whole-cell mechanistic data on cellular retention of Cu from Cu(II)(atsm). Genetic incompatibility between nuclear and mitochondrial encoded subunits of the mitochondrial electron transport chain (ETC) in xenocybrid cells compromises normal function of the ETC. As a consequence of this impairment to the ETC we show xenocybrid cells upregulate glycolytic ATP production and accumulate NADH. Compared to control cells the xenocybrid cells retained more Cu after being treated with Cu(II)(atsm). By transfecting the cells with a metal-responsive element reporter construct the increase in Cu retention was shown to involve a Cu(II)(atsm)-induced increase in intracellular bioavailable Cu specifically within the xenocybrid cells. Parallel experiments using cells grown under hypoxic conditions confirmed that a compromised ETC and elevated NADH levels contribute to increased cellular retention of Cu from Cu(II)(atsm). Using these cell culture models our data demonstrate that compromised ETC function, due to the absence of O(2) as the terminal electron acceptor or dysfunction of individual components of the ETC, is an important determinant in driving the intracellular dissociation of Cu(II)(atsm) that increases cellular retention of the Cu.
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    Inhibition of TDP-43 Accumulation by Bis(thiosemicarbazonato)-Copper Complexes
    Parker, SJ ; Meyerowitz, J ; James, JL ; Liddell, JR ; Nonaka, T ; Hasegawa, M ; Kanninen, KM ; Lim, S ; Paterson, BM ; Donnelly, PS ; Crouch, PJ ; White, AR ; Kahle, PJ (PUBLIC LIBRARY SCIENCE, 2012-08-03)
    Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable bis(thiosemicarbazonato)copper(II) complexes (Cu(II)(btsc)s) can modulate kinase activity and induce neuroprotective effects. In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the Cu(II)(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The Cu(II)(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either Cu(II)(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. Cu(II)(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, Cu(II)(atsm) reduced accumulation of C-terminal (219-414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that Cu(II)(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.
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    The challenges of using a copper fluorescent sensor (CS1) to track intracellular distributions of copper in neuronal and glial cells
    Price, KA ; Hickey, JL ; Xiao, Z ; Wedd, AG ; James, SA ; Liddell, JR ; Crouch, PJ ; White, AR ; Donnelly, PS (ROYAL SOC CHEMISTRY, 2012-01-01)
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    Lipophilic adamantyl- or deferasirox-based conjugates of desferrioxamine B have enhanced neuroprotective capacity: implications for Parkinson disease
    Liddell, JR ; Obando, D ; Liu, J ; Ganio, G ; Volitakis, I ; San Mok, S ; Crouch, PJ ; White, AR ; Codd, R (ELSEVIER SCIENCE INC, 2013-07-01)
    Parkinson disease (PD) is a neurodegenerative disease characterized by death of dopaminergic neurons in the substantia nigra region of the brain. Iron content is also elevated in this region in PD and is implicated in the pathobiology of the disease. Desferrioxamine B (DFOB) is a high-affinity iron chelator and has shown efficacy in animal models of Parkinson disease. The high water solubility of DFOB, however, attenuates its ability to enter the brain. In this study, we have conjugated DFOB to derivatives of adamantane or the clinical iron chelator deferasirox to produce lipophilic compounds designed to increase the bioavailability of DFOB to brain cells. We found that the novel compounds are highly effective in preventing iron-mediated paraquat and hydrogen peroxide toxicity in neuronal-like BE2-M17 dopaminergic cells, primary neurons, and iron-loaded or glutathione-depleted primary astrocytes. The compounds also alleviated paraquat toxicity in BE2-M17 cells that express the PD-causing A30P mutation of α-synuclein. This protection was ∼66-fold more potent than DFOB alone and also more effective than other cell-permeative metal chelators, clioquinol and phenanthroline. These results demonstrate that increasing the bioavailability of DFOB through the conjugation of lipophilic fragments greatly enhances its protective capacity. These novel compounds have potential as therapeutics for the treatment of PD and other conditions of Fe dyshomeostasis.