Elevated GRO-α and IL-18 in serum and brain implicate the NLRP3 inflammasome in frontotemporal dementia.

Neuroinflammation is a hallmark of frontotemporal dementia (FTD), a heterogeneous group of proteinopathies characterized by the progressive degeneration of the frontal and temporal lobes. It is marked by microglial activation and subsequent cytokine release. Although cytokine levels in FTD brain and CSF have been examined, the number of cytokines measured in each study is limited and knowledge on cytokine concentrations in FTD serum is scarce. Here, we assessed 48 cytokines in FTD serum and brain. The aim was to determine common cytokine dysregulation pathways in serum and brain in FTD. Blood samples and brain tissue samples from the superior frontal cortex (SFC) were collected from individuals diagnosed with behavioral variant FTD (bvFTD) and healthy controls, and 48 cytokines were measured using a multiplex immunological assay. The data were evaluated by principal component factor analysis to determine the contribution from different components of the variance in the cohort. Levels of a number of cytokines were altered in serum and SFC in bvFTD compared to controls, with increases in GRO-α and IL-18 in both serum and SFC. These changes could be associated with NLRP3 inflammasome activation or the NFκB pathway, which activates NLRP3. The results suggest the possible importance of the NLRP3 inflammasome in FTD. An improved understanding of the role of inflammasomes in FTD could provide valuable insights into the pathogenesis, diagnosis and treatment of FTD.

ATP-binding cassette transporter expression is widely dysregulated in frontotemporal dementia with TDP-43 inclusions.

The human brain is highly enriched in lipids and increasing evidence indicates that dysregulation of lipids in the brain is associated with neurodegeneration. ATP-binding cassette subfamily A (ABCA) transporters control the movement of lipids across cellular membranes and are implicated in a number of neurodegenerative diseases. However, very little is known about the role of ABCA transporters in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), which is a common form of younger-onset dementia. We therefore undertook a comprehensive analysis of the expression of ABCA transporters (ABCA1-13) in five key brain regions (amygdala, inferior temporal cortex, superior frontal cortex, cerebellum and parietal cortex) in FTLD-TDP and controls. We found that the expression of ABCA2, ABCA3, ABCA4, ABCA7, ABCA9, ABCA10 and ABCA13 was significantly altered in FTLD-TDP in a region-specific manner. In addition, the expression of ABCA transporters correlated specifically to different neural markers and TARDBP. These results suggest substantial dysregulation of ABCA transporters and lipid metabolism in FTLD-TDP and these changes are associated with neuroinflammation.

The Role of White Matter Dysfunction and Leukoencephalopathy/Leukodystrophy Genes in the Aetiology of Frontotemporal Dementias: Implications for Novel Approaches to Therapeutics.

Frontotemporal dementia (FTD) is a common cause of presenile dementia and is characterized by behavioural and/or language changes and progressive cognitive deficits. Genetics is an important component in the aetiology of FTD, with positive family history of dementia reported for 40% of cases. This review synthesizes current knowledge of the known major FTD genes, including C9orf72 (chromosome 9 open reading frame 72), MAPT (microtubule-associated protein tau) and GRN (granulin), and their impact on neuronal and glial pathology. Further, evidence for white matter dysfunction in the aetiology of FTD and the clinical, neuroimaging and genetic overlap between FTD and leukodystrophy/leukoencephalopathy are discussed. The review highlights the role of common variants and mutations in genes such as CSF1R (colony-stimulating factor 1 receptor), CYP27A1 (cytochrome P450 family 27 subfamily A member 1), TREM2 (triggering receptor expressed on myeloid cells 2) and TMEM106B (transmembrane protein 106B) that play an integral role in microglia and oligodendrocyte function. Finally, pharmacological and non-pharmacological approaches for enhancing remyelination are discussed in terms of future treatments of FTD.

A Nitric Oxide Storage and Transport System That Protects Activated Macrophages from Endogenous Nitric Oxide Cytotoxicity.

Nitric oxide (NO) is integral to macrophage cytotoxicity against tumors due to its ability to induce iron release from cancer cells. However, the mechanism for how activated macrophages protect themselves from endogenous NO remains unknown. We previously demonstrated by using tumor cells that glutathione S-transferase P1 (GSTP1) sequesters NO as dinitrosyl-dithiol iron complexes (DNICs) and inhibits NO-mediated iron release from cells via the transporter multidrug resistance protein 1 (MRP1/ABCC1). These prior studies also showed that MRP1 and GSTP1 protect tumor cells against NO cytotoxicity, which parallels their roles in defending cancer cells from cytotoxic drugs. Considering this, and because GSTP1 and MRP1 are up-regulated during macrophage activation, this investigation examined whether this NO storage/transport system protects macrophages against endogenous NO cytotoxicity in two well characterized macrophage cell types (J774 and RAW 264.7). MRP1 expression markedly increased upon macrophage activation, and the role of MRP1 in NO-induced 59Fe release was demonstrated by Mrp1 siRNA and the MRP1 inhibitor, MK571, which inhibited NO-mediated iron efflux. Furthermore, Mrp1 silencing increased DNIC accumulation in macrophages, indicating a role for MRP1 in transporting DNICs out of cells. In addition, macrophage 59Fe release was enhanced by silencing Gstp1, suggesting GSTP1 was responsible for DNIC binding/storage. Viability studies demonstrated that GSTP1 and MRP1 protect activated macrophages from NO cytotoxicity. This was confirmed by silencing nuclear factor-erythroid 2-related factor 2 (Nrf2), which decreased MRP1 and GSTP1 expression, concomitant with reduced 59Fe release and macrophage survival. Together, these results demonstrate a mechanism by which macrophages protect themselves against NO cytotoxicity.

Copper and conquer: copper complexes of di-2-pyridylketone thiosemicarbazones as novel anti-cancer therapeutics.

Copper is an essential trace metal required by organisms to perform a number of important biological processes. Copper readily cycles between its reduced Cu(i) and oxidised Cu(ii) states, which makes it redox active in biological systems. This redox-cycling propensity is vital for copper to act as a catalytic co-factor in enzymes. While copper is essential for normal physiology, enhanced copper levels in tumours leads to cancer progression. In particular, the stimulatory effect of copper on angiogenesis has been established in the last several decades. Additionally, it has been demonstrated that copper affects tumour growth and promotes metastasis. Based on the effects of copper on cancer progression, chelators that bind copper have been developed as anti-cancer agents. In fact, a novel class of thiosemicarbazone compounds, namely the di-2-pyridylketone thiosemicarbazones that bind copper, have shown great promise in terms of their anti-cancer activity. These agents have a unique mechanism of action, in which they form redox-active complexes with copper in the lysosomes of cancer cells. Furthermore, these agents are able to overcome P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) and act as potent anti-oncogenic agents through their ability to up-regulate the metastasis suppressor protein, N-myc downstream regulated gene-1 (NDRG1). This review provides an overview of the metabolism and regulation of copper in normal physiology, followed by a discussion of the dysregulation of copper homeostasis in cancer and the effects of copper on cancer progression. Finally, recent advances in our understanding of the mechanisms of action of anti-cancer agents targeting copper are discussed.

Roads to melanoma: Key pathways and emerging players in melanoma progression and oncogenic signaling.

Melanoma has markedly increased worldwide during the past several decades in the Caucasian population and is responsible for 80% of skin cancer deaths. Considering that metastatic melanoma is almost completely resistant to most current therapies and is linked with a poor patient prognosis, it is crucial to further investigate potential molecular targets. Major cell-autonomous drivers in the pathogenesis of this disease include the classical MAPK (i.e., RAS-RAF-MEK-ERK), WNT, and PI3K signaling pathways. These pathways play a major role in defining the progression of melanoma, and some have been the subject of recent pharmacological strategies to treat this belligerent disease. This review describes the latest advances in the understanding of melanoma progression and the major molecular pathways involved. In addition, we discuss the roles of emerging molecular players that are involved in melanoma pathogenesis, including the functional role of the melanoma tumor antigen, p97/MFI2 (melanotransferrin).

Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia.

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich ataxia (FA). This disease is due to decreased expression of the mitochondrial protein, frataxin, which leads to alterations in mitochondrial iron (Fe) metabolism. The identification of potentially toxic mitochondrial Fe deposits in FA suggests Fe plays a role in its pathogenesis. Studies using the muscle creatine kinase (MCK) conditional frataxin knockout mouse that mirrors the disease have demonstrated frataxin deletion alters cardiac Fe metabolism. Indeed, there are pronounced changes in Fe trafficking away from the cytosol to the mitochondrion, leading to a cytosolic Fe deficiency. Considering Fe deficiency can induce apoptosis and cell death, we examined the effect of dietary Fe supplementation, which led to body Fe loading and limited the cardiac hypertrophy in MCK mutants. Furthermore, this study indicates a unique effect of heart and skeletal muscle-specific frataxin deletion on systemic Fe metabolism. Namely, frataxin deletion induces a signaling mechanism to increase systemic Fe levels and Fe loading in tissues where frataxin expression is intact (i.e., liver, kidney, and spleen). Examining the mutant heart, native size-exclusion chromatography, transmission electron microscopy, Mössbauer spectroscopy, and magnetic susceptibility measurements demonstrated that in the absence of frataxin, mitochondria contained biomineral Fe aggregates, which were distinctly different from isolated mammalian ferritin molecules. These mitochondrial aggregates of Fe, phosphorus, and sulfur, probably contribute to the oxidative stress and pathology observed in the absence of frataxin.

Nitrogen monoxide (NO) storage and transport by dinitrosyl-dithiol-iron complexes: long-lived NO that is trafficked by interacting proteins.

Nitrogen monoxide (NO) markedly affects intracellular iron metabolism, and recent studies have shown that molecules traditionally involved in drug resistance, namely GST and MRP1 (multidrug resistance-associated protein 1), are critical molecular players in this process. This is mediated by interaction of these proteins with dinitrosyl-dithiol-iron complexes (Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675; Lok, H. C., Suryo Rahmanto, Y., Hawkins, C. L., Kalinowski, D. S., Morrow, C. S., Townsend, A. J., Ponka, P., and Richardson, D. R. (2012) J. Biol. Chem. 287, 607-618). These complexes are bioavailable, have a markedly longer half-life compared with free NO, and form in cells after an interaction between iron, NO, and glutathione. The generation of dinitrosyl-dithiol-iron complexes acts as a common currency for NO transport and storage by MRP1 and GST P1-1, respectively. Understanding the biological trafficking mechanisms involved in the metabolism of NO is vital for elucidating its many roles in cellular signaling and cytotoxicity and for development of new therapeutic targets.

Nitric oxide storage and transport in cells are mediated by glutathione S-transferase P1-1 and multidrug resistance protein 1 via dinitrosyl iron complexes.

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron release. We showed that NO-mediated iron efflux from cells required glutathione (GSH) (Watts, R. N., and Richardson, D. R. (2001) J. Biol. Chem. 276, 4724-4732) and that the GSH-conjugate transporter, multidrug resistance-associated protein 1 (MRP1), mediates this release potentially as a dinitrosyl-dithiol iron complex (DNIC; Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675). Recently, glutathione S-transferase P1-1 (GST P1-1) was shown to bind DNICs as dinitrosyl-diglutathionyl iron complexes. Considering this and that GSTs and MRP1 form an integrated detoxification unit with chemotherapeutics, we assessed whether these proteins coordinately regulate storage and transport of DNICs as long lived NO intermediates. Cells transfected with GSTP1 (but not GSTA1 or GSTM1) significantly decreased NO-mediated 59Fe release from cells. This NO-mediated 59Fe efflux and the effect of GST P1-1 on preventing this were observed with NO-generating agents and also in cells transfected with inducible nitric oxide synthase. Notably, 59Fe accumulated in cells within GST P1-1-containing fractions, indicating an alteration in intracellular 59Fe distribution. Furthermore, electron paramagnetic resonance studies showed that MCF7-VP cells transfected with GSTP1 contain significantly greater levels of a unique DNIC signal. These investigations indicate that GST P1-1 acts to sequester NO as DNICs, reducing their transport out of the cell by MRP1. Cell proliferation studies demonstrated the importance of the combined effect of GST P1-1 and MRP1 in protecting cells from the cytotoxic effects of NO. Thus, the DNIC storage function of GST P1-1 and ability of MRP1 to efflux DNICs are vital in protection against NO cytotoxicity.

Structure and inhibition of orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum.

Orotidine 5'-monophosphate (OMP) decarboxylase from Plasmodium falciparum (PfODCase, EC 4.1.1.23) has been overexpressed, purified, subjected to kinetic and biochemical analysis, and crystallized. The native enzyme is a homodimer with a subunit molecular mass of 38 kDa. The saturation curve for OMP as a substrate conformed to Michaelis-Menten kinetics with K m = 350 +/- 60 nM and V max = 2.70 +/- 0.10 micromol/min/mg protein. Inhibition patterns for nucleoside 5'-monophosphate analogues were linear competitive with respect to OMP with a decreasing potency of inhibition of PfODCase in the order: pyrazofurin 5'-monophosphate ( K i = 3.6 +/- 0.7 nM) > xanthosine 5'-monophosphate (XMP, K i = 4.4 +/- 0.7 nM) > 6-azauridine 5'-monophosphate (AzaUMP, K i = 12 +/- 3 nM) > allopurinol-3-riboside 5'-monophosphate ( K i = 240 +/- 20 nM). XMP is an approximately 150-fold more potent inhibitor of PfODCase compared with the human enzyme. The structure of PfODCase was solved in the absence of ligand and displays a classic TIM-barrel fold characteristic of the enzyme. Both the phosphate-binding loop and the betaalpha5-loop have conformational flexibility, which may be associated with substrate capture and product release along the reaction pathway.

The nitric oxide-iron interplay in mammalian cells: transport and storage of dinitrosyl iron complexes.

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl-dithiolato-Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl-diglutathionyl-Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.

Aromatic L-amino acids activate the calcium-sensing receptor.

The calcium-sensing receptor (CaR) is recognized as a member of class 3 of the G-protein coupled receptor superfamily. Members of this subgroup, which have large N-terminal extracellular domains, include receptors that respond specifically to the amino acid glutamate; receptors that respond to the glutamate analogue, gamma-amino butyric acid; and several receptors that act as broad-spectrum amino acid sensors. The CaR is one of these broad-spectrum amino acid sensors that, along with several other members of the subgroup, also responds to extracellular Ca2+. In this mini-review, we consider evidence that the CaR is a sensor of aromatic amino acids, that it has broad-spectrum amino acid sensing properties, that it provides an amino acid binding site in its extracellular N-terminal Venus Fly Trap domain, and that amino acids have a physiological impact on systems in which the CaR is expressed.

Activation of renal calcium and water excretion by novel physiological and pharmacological activators of the calcium-sensing receptor.

Activated Ca(2+)-sensing receptors (CaR) play key roles in the regulation of whole-body calcium metabolism by inhibiting the secretion of the key calcitropic hormone parathyroid hormone and promoting urinary calcium excretion. We have now examined the effects of intravenous administration of novel calcium receptor activators on renal function in anaesthetized female Wistar rats. The type II calcimimetic NPS R-467 and the CaR-active amino acids l-Phe and l-Ala, which act at distinct binding sites on the receptor, all activated urinary flow rate, calcium and osmolar excretion and suppressed urinary osmolality. The effects of l-Phe and NPS R-467 on urine flow rate and calcium excretion were stereoselective, consistent with the idea that these effects were mediated by calcium-sensing receptors. However, d-Phe also suppressed urinary osmolality and promoted osmolar excretion, possibly by exceeding the transport maximum in the proximal tubule. The data indicate that novel activators of CaR, including l-amino acids at physiologically relevant serum concentrations, play a significant role in the regulation of urinary calcium and water excretion.