Gulay Filiz

Increasing Cu bioavailability inhibits Aβ oligomers and tau phosphorylation

Cognitive decline in Alzheimers disease (AD) involves pathological accumulation of synaptotoxic amyloid-β (Aβ) oligomers and hyperphosphorylated tau. Because recent evidence indicates that glycogen synthase kinase 3β (GSK3β) activity regulates these neurotoxic pathways, we developed an AD therapeutic strategy to target GSK3β. The strategy involves the use of copper-bis(thiosemicarbazonoto) complexes to increase intracellular copper bioavailability and inhibit GSK3β through activation of an Akt signaling pathway. Our lead compound CuII(gtsm) significantly inhibited GSK3β in the brains of APP/PS1 transgenic AD model mice. CuII(gtsm) also decreased the abundance of Aβ trimers and phosphorylated tau, and restored performance of AD mice in the Y-maze test to levels expected for cognitively normal animals. Improvement in the Y-maze correlated directly with decreased Aβ trimer levels. This study demonstrates that increasing intracellular copper bioavailability can restore cognitive function by inhibiting the accumulation of neurotoxic Aβ trimers and phosphorylated tau.

Metal Ionophore Treatment Restores Dendritic Spine Density and Synaptic Protein Levels in a Mouse Model of Alzheimer's Disease

We have previously demonstrated that brief treatment of APP transgenic mice with metal ionophores (PBT2, Prana Biotechnology) rapidly and markedly improves learning and memory. To understand the potential mechanisms of action underlying this phenomenon we examined hippocampal dendritic spine density, and the levels of key proteins involved in learning and memory, in young (4 months) and old (14 months) female Tg2576 mice following brief (11 days) oral treatment with PBT2 (30 mg/kg/d). Transgenic mice exhibited deficits in spine density compared to littermate controls that were significantly rescued by PBT2 treatment in both the young (+17%, p<0.001) and old (+32%, p<0.001) animals. There was no effect of PBT2 on spine density in the control animals. In the transgenic animals, PBT2 treatment also resulted in significant increases in brain levels of CamKII (+57%, p = 0.005), spinophilin (+37%, p = 0.04), NMDAR1A (+126%, p = 0.02), NMDAR2A (+70%, p = 0.05), pro-BDNF (+19%, p = 0.02) and BDNF (+19%, p = 0.04). While PBT2-treatment did not significantly alter neurite-length in vivo, it did increase neurite outgrowth (+200%, p = 0.006) in cultured cells, and this was abolished by co-incubation with the transition metal chelator, diamsar. These data suggest that PBT2 may affect multiple aspects of snaptic health/efficacy. In Alzheimers disease therefore, PBT2 may restore the uptake of physiological metal ions trapped within extracellular β-amyloid aggregates that then induce biochemical and anatomical changes to improve cognitive function.

Restored degradation of the Alzheimer's amyloid-β peptide by targeting amyloid formation

Accumulation of neurotoxic amyloid‐β (Aβ) is central to the pathology of Alzheimer’s disease (AD). Elucidating the mechanisms of Aβ accumulation will therefore expedite the development of Aβ‐targeting AD therapeutics. We examined activity of an Aβ‐degrading protease (matrix metalloprotease 2) to investigate whether biochemical factors consistent with conditions in the AD brain contribute to Aβ accumulation by altering Aβ sensitivity to proteolytic degradation. An Aβ amino acid mutation found in familial AD, Aβ interactions with zinc (Zn), and increased Aβ hydrophobicity all strongly prevented Aβ degradation. Consistent to all of these factors is the promotion of specific Aβ aggregates where the protease cleavage site, confirmed by mass spectrometry, is inaccessible within an amyloid structure. These data indicate decreased degradation due to amyloid formation initiates Aβ accumulation by preventing normal protease activity. Zn also prevented Aβ degradation by the proteases neprilysin and insulin degrading enzyme. Treating Zn‐induced Aβ amyloid with the metal‐protein attenuating compound clioquinol reversed amyloid formation and restored the peptide’s sensitivity to degradation by matrix metalloprotease 2. This provides new data indicating that therapeutic compounds designed to modulate Aβ‐metal interactions can inhibit Aβ accumulation by restoring the catalytic potential of Aβ‐degrading proteases.

Differential modulation of Alzheimer's disease amyloid β-peptide accumulation by diverse classes of metal ligands

Biometals have an important role in AD (Alzheimers disease) and metal ligands have been investigated as potential therapeutic agents for treatment of AD. In recent studies the 8HQ (8-hydroxyquinoline) derivative CQ (clioquinol) has shown promising results in animal models and small clinical trials; however, the actual mode of action in vivo is still being investigated. We previously reported that CQ-metal complexes up-regulated MMP (matrix metalloprotease) activity in vitro by activating PI3K (phosphoinositide 3-kinase) and JNK (c-jun N-terminal kinase), and that the increased MMP activity resulted in enhanced degradation of secreted Abeta (amyloid beta) peptide. In the present study, we have further investigated the biochemical mechanisms by which metal ligands affect Abeta metabolism. To achieve this, we measured the effects of diverse metal ligands on cellular metal uptake and secreted Abeta levels in cell culture. We report that different classes of metal ligands including 8HQ and phenanthroline derivatives and the sulfur compound PDTC (pyrrolidine dithiocarbamate) elevated cellular metal levels (copper and zinc), and resulted in substantial loss of secreted Abeta. Generally, the ability to inhibit Abeta levels correlated with a higher lipid solubility of the ligands and their capacity to increase metal uptake. However, we also identified several ligands that potently inhibited Abeta levels while only inducing minimal change to cellular metal levels. Metal ligands that inhibited Abeta levels [e.g. CQ, 8HQ, NC (neocuproine), 1,10-phenanthroline and PDTC] induced metal-dependent activation of PI3K and JNK, resulting in JNK-mediated up-regulation of metalloprotease activity and subsequent loss of secreted Abeta. The findings in the present study show that diverse metal ligands with high lipid solubility can elevate cellular metal levels resulting in metalloprotease-dependent inhibition of Abeta. Given that a structurally diverse array of ligands was assessed, the results are consistent with the effects being due to metal transport rather than the chelating ligand interacting directly with a receptor.

The role of metals in modulating metalloprotease activity in the AD brain.

Biometals such as copper and zinc have an important role in Alzheimer’s disease (AD). Accumulating evidence indicates that copper homeostasis is altered in AD brain with elevated extracellular and low intracellular copper levels. Studies in animals and cell cultures have suggested that increasing intracellular copper can ameliorate AD-like pathology including amyloid deposition and tau phosphorylation. Modulating copper homeostasis can also improve cognitive function in animal models of AD. Treatments are now being developed that may result in redistribution of copper within the brain. Metal ligands such as clioquinol (CQ), DP-109 or pyrrolidine dithiocarbamate (PDTC) have shown promising results in animal models of AD, however, the actual mode of action in vivo has not been fully determined. We previously reported that CQ-metal complexes were able to increase intracellular copper levels in vitro. This resulted in stimulation of phosphoinositol-3-kinase activity and mitogen activated protein kinases (MAPK). Increased kinase activity resulted in up-regulated matrix metalloprotease (MMP2 and MMP3) activity resulting in enhanced degradation of secreted Aβ. These findings are consistent with previous studies reporting metal-mediated activation of MAPKs and MMPs. How this activation occurs is unknown but evidence suggests that copper may be able to activate membrane receptors such as the epidermal growth factor receptor (EGFR) and result in downstream activation of MAPK pathways. This has been supported by studies showing metal-mediated activation of EGFR through ligand-independent processes in a number of cell-types. Our initial studies reveal that copper complexes can in fact activate EGFR. However, further studies are necessary to determine if metal complexes such as CQ-copper induce up-regulation of Aβ-degrading MMP activity through this mechanism. Elucidation of this pathway may have important implications for the development of metal ligand based therapeutics for treatment of AD and other neurodegenerative disorders.

Selective CREB-dependent cyclin expression mediated by the PI3K and MAPK pathways supports glioma cell proliferation

The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials.

Zinc induces depletion and aggregation of endogenous TDP-43.

Ubiquitinated neuronal aggregates containing TDP-43 are pathological hallmarks in the spectrum of frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS). In affected neurons, TDP-43 undergoes C-terminal fragmentation, phosphorylation, and ubiquitination and forms aggregates in the cytoplasm or nucleus. Although in vitro studies have been able to recapitulate these features using transfected cell culture models, little is known about the biochemical mechanisms that underlie pathological changes to endogenous TDP-43. As altered metal ion homeostasis and increased oxidative stress are central features of neurodegeneration, including FTLD and ALS, we sought to determine the affects of these factors on endogenous TDP-43 metabolism in mammalian cells. Treatment of SY5Y neuronal-like cells expressing endogenous TDP-43 with zinc (Zn) induced depletion of TDP-43 expression and formation of inclusions that were TDP-43 positive. TDP-43 was also detected in the cytosol of Zn-affected cells but this was not aggregated. No evidence of C-terminal fragmentation, phosphorylation, or ubiquitination was observed. The depletion and aggregation of TDP-43 were associated with the specific action of Zn but were not seen with copper, iron, or H(2)O(2). These studies describe for the first time specific induction of endogenous TDP-43 aggregation in neuronal-like cells and suggest that specific Zn-associated processes could affect TDP-43 metabolism in neurodegenerative diseases.

Sustained activation of glial cell epidermal growth factor receptor by bis(thiosemicarbazonato) metal complexes is associated with inhibition of protein tyrosine phosphatase activity.

Bis(thiosemicarbazonato) metal complexes (M(II)(btsc)) have demonstrated potential neuroprotective activity in cell and animal models of Alzheimers disease (AD). Metal complexes can activate the epidermal growth factor receptor (EGFR), leading to inhibition of amyloid peptide accumulation in neuronal cells. As glial cells also have an important role in modulating neuronal health and survival in AD, we examined the effect of M(II)(btsc) on activity of EGFR in an astroglial cell line. Our findings reveal potent activation of glial EGFR by glyoxalbis(N(4)-methylthiosemicarbazonato)Cu(II)] (Cu(II)(gtsm)). Activation of EGFR by Cu(II)(gtsm) involved phosphorylation of multiple tyrosine residues and was mediated by a cognate ligand-independent process involving M(II)(btsc) inhibition of protein tyrosine phosphatase (PTP) activity. EGFR activation resulted in release of growth factors and cytokines with potential modulatory effects on neuronal function. These studies provide an important insight into the mechanism of action of a neuroprotective M(II)(btsc) and provide a basis for future studies into this novel approach to AD therapy.

Clioquinol Promotes Cancer Cell Toxicity through Tumor Necrosis Factor α Release from Macrophages

Copper has an important role in cancer growth, angiogenesis, and metastasis. Previous studies have shown that cell-permeable metal ligands, including clioquinol (CQ) and pyrrolidine dithiocarbamate, inhibit cancer cell growth in cell culture and in vivo. The mechanism of action has not been fully determined but may involve metal-mediated inhibition of cancer cell proteasome activity. However, these studies do not fully account for the ability of cell-permeable metal ligands to inhibit cancer cell growth without affecting normal cells. In this study, we examined the effect of CQ on macrophage-mediated inhibition of HeLa cancer cell growth in vitro. When CQ was added to RAW 264.7 macrophage-HeLa cell cocultures, a substantial increase in HeLa cell toxicity was observed compared with CQ treatment of HeLa cells cultured alone. Transfer of conditioned medium from CQ-treated macrophages to HeLa cells also induced HeLa cell toxicity, demonstrating the role of secreted factors in the macrophage-mediated effect. Further investigation revealed that CQ induced copper-dependent activation of macrophages and release of tumor necrosis factor (TNF) α.In studies with recombinant TNFα, we showed that the level of TNFα released by CQ-treated macrophages was sufficient to induce HeLa cell toxicity. Moreover, the toxic effect of conditioned medium from CQ-treated macrophages could be prevented by addition of neutralizing antibodies to TNFα. These studies demonstrate that CQ can induce cancer cell toxicity through metal-dependent release of TNFα from macrophages. Our results may help to explain the targeted inhibition of tumor growth in vivo by CQ.

Activation of epidermal growth factor receptor by metal-ligand complexes decreases levels of extracellular amyloid beta peptide

The epidermal growth factor receptor is a receptor tyrosine kinase expressed in a range of tissues and cell-types. Activation of the epidermal growth factor receptor by a number of ligands induces downstream signalling that modulates critical cell functions including growth, survival and differentiation. Abnormal epidermal growth factor receptor expression and activation is also involved in a number of cancers. In addition to its cognate ligands, the epidermal growth factor receptor can be activated by metals such as zinc (Zn) and copper (Cu). Due to the important role of these metals in a number of diseases including neurodegenerative disorders, therapeutic approaches are being developed based on the use of lipid permeable metal-complexing molecules. While these agents are showing promising results in animal models and clinical trials, little is known about the effects of metal-ligand complexes on cell signalling pathways. In this study, we investigated the effects of clioquinol (CQ)-metal complexes on activation of epidermal growth factor receptor. We show here that CQ-Cu complexes induced potent epidermal growth factor receptor phosphorylation resulting in downstream activation of extracellular signal-regulated kinase. Similar levels of epidermal growth factor receptor activation were observed with alternative lipid permeable metal-ligands including neocuproine and pyrrolidine dithiocarbamate. We found that CQ-Cu complexes induced a significant reduction in the level of extracellular Abeta1-40 in cell culture. Inhibition of epidermal growth factor receptor activation by PD153035 blocked extracellular signal-regulated kinase phosphorylation and restored Abeta1-40 levels. Activation of the epidermal growth factor receptor by CQ-Cu was mediated through up-regulation of src kinase activity by a cognate ligand-independent process involving membrane integrins. These findings provide the first evidence that metal-ligand complexes can activate the epidermal growth factor receptor with potentially neuroprotective effects.