ZnII(atsm) is protective in amyotrophic lateral sclerosis model mice via a copper delivery mechanism

AbstractMutations in the metalloprotein Cu,Zn-superoxide dismutase (SOD1) cause approximately 20% of familial cases of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease for which effective therapeutics do not yet exist. Transgenic rodent models based on over-expression of mutant SOD1 have been developed and these have provided opportunity to test new therapeutic strategies and to study the mechanisms of mutant SOD1 toxicity. Although the mechanisms of mutant SOD1 toxicity are yet to be fully elucidated, incorrect or incomplete metallation of SOD1 confers abnormal folding, aggregation and biochemical properties, and improving the metallation state of SOD1 provides a viable therapeutic option. The therapeutic effects of delivering copper (Cu) to mutant SOD1 have been demonstrated recently. The aim of the current study was to determine if delivery of zinc (Zn) to SOD1 was also therapeutic. To investigate this, SOD1G37R mice were treated with the metal complex diacetyl-bis(4-methylthiosemicarbazonato)zincII [ZnII(atsm)]. Treatment resulted in an improvement in locomotor function and survival of the mice. However, biochemical analysis of spinal cord tissue collected from the mice revealed that the treatment did not increase overall Zn levels in the spinal cord nor the Zn content of SOD1. In contrast, overall levels of Cu in the spinal cord were elevated in the ZnII(atsm)-treated SOD1G37R mice and the Cu content of SOD1 was also elevated. Further experiments demonstrated transmetallation of ZnII(atsm) in the presence of Cu to form the Cu-analogue CuII(atsm), indicating that the observed therapeutic effects for ZnII(atsm) in SOD1G37R mice may in fact be due to in vivo transmetallation and subsequent delivery of Cu

A CX3CRI Reporter hESC Line Facilitates Integrative Analysis of In-Vitro-Derived Microglia and Improved Microglia Identity upon Neuron-Glia Co-culture

Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.Peer reviewe

If Human Brain Organoids Are the Answer to Understanding Dementia, What Are the Questions?

Because our beliefs regarding our individuality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the "mini-brain," the "brain in a dish". At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise. One is the general challenge associated with using any stem cell-generated preparation for in vitro modelling (challenges amplified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term "dementia." We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model "dementia in a dish."The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The Australian Dementia Stem Cell Consortium has received generous start-up travel grants from the Australian NHMRC National Institute for Dementia Research. Authors have been supported by Dementia Australia Research Foundation, Yulgilbar Alzheimer’s Research Program, DHB Foundation (AP), Brain Foundation (DH, AP), the C.F. Leung Memorial Trust (AP), the University of Melbourne (AP) and Operational Infrastructure Support from the Victorian Government (DH, AP), Monash University (AG), JO and JR Wicking Trust (Equity Trustees) (ALC and AEK), University of Sydney (MV), and generous gifts from the Sinclair, Smith and Jolly families (MV). AEK is supported by a National Health and Medical Research Council (NHMRC) of Australia Boosting Dementia Research Leadership Fellowship (APP1136913). AG is supported by a NHMRC-ARC Dementia Research Development Fellowship (GNT1097461). AP is supported by an ARC Future Fellowship (FT140100047) and a NHMRC Senior Research Fellowship (1154389). LO is supported by a NHMRC of Australia Boosting Dementia Research Leadership Fellowship (APP1135720). MV is supported by a NHMRC Career Development Fellowship (APP1112813). VG is supported by Australian Research Council’s Discovery Early Career Researcher Award (DE180100775)

CuII(atsm) Attenuates Neuroinflammation

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

Cu-II(atsm) Attenuates Neuroinflammation

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 Cu-II(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 Cu-II(atsm) in reducing acute cerebrovascular inflammation caused by peripheral administration of bacterial lipopolysaccharide (LPS). Cu-II(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 Cu-II(atsm) on the neuroimmune system suggest copper complexes are potential therapeutics for the treatment of neuroinflammatory conditions.Peer reviewe

PPARβ/δ-agonist GW0742 ameliorates dysfunction in fatty acid oxidation in PSEN1ΔE9 astrocytes

Astrocytes are the gatekeepers of neuronal energy supply. In neurodegenerative diseases, bio-energetics demand increases and becomes reliant upon fatty acid oxidation as a source of energy. Defective fatty acid oxidation and mitochondrial dysfunctions correlate with hippocampal neurodegeneration and memory deficits in Alzheimer’s disease (AD), but it is unclear whether energy metabolism can be targeted to prevent or treat the disease. Here we show for the first time an impairment in fatty acid oxidation in human astrocytes derived from induced pluripotent stem cells of AD patients. The impairment was corrected by treatment with a synthetic peroxisome proliferator activated receptor delta (PPARβ/δ) agonist GW0742 which acts to regulate an array of genes governing cellular metabolism. GW0742 enhanced the expression of CPT1a, the gene encoding for a rate-limiting enzyme of fatty acid oxidation. Similarly, treatment of a mouse model of AD, the APP/PS1-mice, with GW0742 increased the expression of Cpt1a and concomitantly reversed memory deficits in a fear conditioning test. Although the GW0742-treated mice did not show altered astrocytic glial fibrillary acidic protein-immunoreactivity or reduction in amyloid beta (Aβ) load, GW0742 treatment increased hippo-campal neurogenesis and enhanced neuronal differentiation of neuronal progenitor cells. Furthermore, GW0742 prevented Aβ-induced impairment of long-term potentiation in hippocampal slices. Collectively, these data suggest that PPARβ/δ-agonism alleviates AD related deficits through increasing fatty acid oxidation in astrocytes and improves cognition in a transgenic mouse model of AD

Oral Treatment with Cu[superscript II](atsm) Increases Mutant SOD1 In Vivo but Protects Motor Neurons and Improves the Phenotype of a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

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[superscript II] [Cu[superscript 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[superscript II](atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched ⁶⁵Cu[superscript II](atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu[superscript II](atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu[superscript 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[superscript 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.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the Society for Neuroscience. The published article can be found at: http://www.jneurosci.org/

The role of strain heterogeneity in Helicobacter pylori colonisation, virulence and host adaptation

The remarkable ability of Helicobacter pylori to colonise the mammalian gastric mucosa is facilitated by conserved strategies that promote strain diversification, allowing the bacteria to continually adapt to changing conditions encountered in this harsh niche. These mechanisms include rapid mutation, highly developed recombination pathways and an abundance of genes that can be regulated by slipped-strand mispairing, which all contribute to the ability of H. pylori to elicit vastly different pathologies in individual hosts. The aims of this study were to investigate how subtle diversity between closely related H. pylori strains affects virulence and adaptation of H. pylori to new hosts. We focused our study on the progenitor and derivatives of the “gold standard” mouse-colonising H. pylori SS1 strain, and investigated the phenotypic effects of transcriptional variation and genome sequence diversity. We determined that minor transcriptional differences between in vitro passaged derivative strains of H. pylori SS1 resulted in significant alterations to the capacity of H. pylori SS1 to colonise mice. Using macroarray comparisons, we identified two vitamin B6 biosynthesis enzymes, PdxA and PdxJ, as being downregulated in low-infectivity SS1 isolates. Significantly, this report demonstrates that besides its recognized metabolic functions, vitamin B6 is required for colonisation of mice by H. pylori, which is likely to be attributable to the role of Vitamin B6 in normal flagellar gly- cosylation and assembly, as well as for motility, a key virulence factor in H. pylori. Furthermore, we determined a role for vitamin B6 salvage pathways in H. pylori colonisation and characterised a novel multifunctional pyridoxal kinase, HP0844, which is involved in vitamin B6 salvage. Our data demonstrate, for the first time, an important in vivo role for pathogen-specific de novo vitamin B6 biosynthesis. In order to investigate the impact of genetic variability on H. pylori virulence, we performed next generation sequencing to compare H. pylori strains differing in their ability to induce inflammatory responses in human gastric cell lines. For this, we used the mouse-adapted H. pylori SS1 and its progenitor, clinical isolate 10700. We identified a total of 3366 single nucleotide polymorphisms (SNPs) as having arisen during host-adaptation of H. pylori 10700 to the murine gastric mu- cosa. We hypothesised that some of these SNPs may correlate with the impaired function in H. pylori SS1 of a key H. pylori virulence factor, the cag pathogenicity island (cagPAI)-encoded type 4 secretion system (T4SS). Importantly, two of these SNPs occurred within the non-cagPAI encoded proteins, ProV and HP0958, that we demonstrate to be essential for T4SS functionality. As loss of virulence factors in vivo potentially influences disease outcome in infected hosts, these findings also provide additional virulence markers for H. pylori strain typing. Moreover, our data demonstrate that mouse adaptation of H. pylori 10700 has resulted in specific intimate interactions with the murine gastric epithelium, independent of the cagPAI, that enable H. pylori SS1 to effectively persist within the host. Lastly, we identified the involvement of an alternative nuclear factor-κB (NF-κB) sub- unit, c-Rel, in murine-specific responses to H. pylori. Taken together, our data highlight the role of genome plasticity during adaptation in vivo allowing for host selection of strains that most efficiently overcome the challenges to colonisation of individual gastric environments

The role of strain heterogeneity in Helicobacter pylori colonisation, virulence and host adaptation

The remarkable ability of Helicobacter pylori to colonise the mammalian gastric mucosa is facilitated by conserved strategies that promote strain diversification, allowing the bacteria to continually adapt to changing conditions encountered in this harsh niche. These mechanisms include rapid mutation, highly developed recombination pathways and an abundance of genes that can be regulated by slipped-strand mispairing, which all contribute to the ability of H. pylori to elicit vastly different pathologies in individual hosts. The aims of this study were to investigate how subtle diversity between closely related H. pylori strains affects virulence and adaptation of H. pylori to new hosts. We focused our study on the progenitor and derivatives of the “gold standard” mouse-colonising H. pylori SS1 strain, and investigated the phenotypic effects of transcriptional variation and genome sequence diversity. We determined that minor transcriptional differences between in vitro passaged derivative strains of H. pylori SS1 resulted in significant alterations to the capacity of H. pylori SS1 to colonise mice. Using macroarray comparisons, we identified two vitamin B6 biosynthesis enzymes, PdxA and PdxJ, as being downregulated in low-infectivity SS1 isolates. Significantly, this report demonstrates that besides its recognized metabolic functions, vitamin B6 is required for colonisation of mice by H. pylori, which is likely to be attributable to the role of Vitamin B6 in normal flagellar gly- cosylation and assembly, as well as for motility, a key virulence factor in H. pylori. Furthermore, we determined a role for vitamin B6 salvage pathways in H. pylori colonisation and characterised a novel multifunctional pyridoxal kinase, HP0844, which is involved in vitamin B6 salvage. Our data demonstrate, for the first time, an important in vivo role for pathogen-specific de novo vitamin B6 biosynthesis. In order to investigate the impact of genetic variability on H. pylori virulence, we performed next generation sequencing to compare H. pylori strains differing in their ability to induce inflammatory responses in human gastric cell lines. For this, we used the mouse-adapted H. pylori SS1 and its progenitor, clinical isolate 10700. We identified a total of 3366 single nucleotide polymorphisms (SNPs) as having arisen during host-adaptation of H. pylori 10700 to the murine gastric mu- cosa. We hypothesised that some of these SNPs may correlate with the impaired function in H. pylori SS1 of a key H. pylori virulence factor, the cag pathogenicity island (cagPAI)-encoded type 4 secretion system (T4SS). Importantly, two of these SNPs occurred within the non-cagPAI encoded proteins, ProV and HP0958, that we demonstrate to be essential for T4SS functionality. As loss of virulence factors in vivo potentially influences disease outcome in infected hosts, these findings also provide additional virulence markers for H. pylori strain typing. Moreover, our data demonstrate that mouse adaptation of H. pylori 10700 has resulted in specific intimate interactions with the murine gastric epithelium, independent of the cagPAI, that enable H. pylori SS1 to effectively persist within the host. Lastly, we identified the involvement of an alternative nuclear factor-κB (NF-κB) sub- unit, c-Rel, in murine-specific responses to H. pylori. Taken together, our data highlight the role of genome plasticity during adaptation in vivo allowing for host selection of strains that most efficiently overcome the challenges to colonisation of individual gastric environments

Neuroinflammation and Copper in Alzheimer’s Disease

Inflammation is the innate immune response to infection or tissue damage. Initiation of proinflammatory cascades in the central nervous system (CNS) occurs through recognition of danger associated molecular patterns by cognate immune receptors expressed on inflammatory cells and leads to rapid responses to remove the danger stimulus. The presence of activated microglia and astrocytes in the vicinity of amyloid plaques in the brains of Alzheimer’s disease (AD) patients and mouse models implicates inflammation as a contributor to AD pathogenesis. Activated microglia play a critical role in amyloid clearance, but chronic deregulation of CNS inflammatory pathways results in secretion of neurotoxic mediators that ultimately contribute to neurodegeneration in AD. Copper (Cu) homeostasis is profoundly affected in AD, and accumulated extracellular Cu drives Aβ aggregation, while intracellular Cu deficiency limits bioavailable Cu required for CNS functions. This review presents an overview of inflammatory events that occur in AD in response to Aβ and highlights recent advances on the role of Cu in modulation of beneficial and detrimental inflammatory responses in AD