Selective intracellular release of copper and zinc ions from bis(thiosemicarbazonato) complexes reduces levels of Alzheimer disease amyloid-β peptide

Copper and zinc play important roles in Alzheimer disease pathology with recent reports describing potential therapeutics based on modulation of metal bioavailability. We examined the ability of a range of metal bis(thiosemicarbazonato) complexes (MII(btsc), where M = Cu II or ZnII) to increase intracellular metal levels in Chinese hamster ovary cells overexpressing amyloid precursor protein (APP-CHO) and the subsequent effect on extracellular levels of amyloid-β peptide (Aβ). The CuII(btsc) complexes were engineered to be either stable to both a change in oxidation state and dissociation of metal or susceptible to intracellular reduction and dissociation of metal. Treatment of APP-CHO cells with stable complexes resulted in elevated levels of intracellular copper with no effect on the detected levels of Aβ. Treatment with complexes susceptible to intracellular reduction increased intracellular copper levels but also resulted in a dose-dependent reduction in the levels of monomeric Aβ. Treatment with less stable ZnII(btsc) complexes increased intracellular zinc levels with a subsequent dose-dependent depletion of monomeric Aβ levels. The increased levels of intracellular bioavailable copper and zinc initiated a signaling cascade involving activation of phosphoinositol 3-kinase and c-Jun N-terminal kinase. Inhibition of these enzymes prevented Aβ depletion induced by the MII(btsc) complexes. Inhibition of metalloproteases also partially restored Aβ levels, implicating metal-driven metalloprotease activation in the extracellular monomeric Aβ depletion. However, a role for alternative metal-induced Aβ metabolism has not been ruled out. These studies demonstrate that M II(btsc) complexes have potential for Alzheimer disease therapy

Degradation of the Alzheimer disease amyloid β-peptide by metal-dependent up-regulation of metalloprotease activity

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid β-peptide (Aβ) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu2+ or Zn2+ resulted in an ∼85-90% reduction of secreted Aβ-(1-40) and Aβ-(1-42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted Aβ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu2+. MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu2+ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of Aβ-(1-40). Our findings identify an alternative mechanism of action for CQ in the reduction of Aβ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients

Mitochondrial Oxidative Stress Causes Hyperphosphorylation of Tau

Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD): tau phosphorylation, and ß-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2) die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau) in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576) with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Aß load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD

Overexpression of Aβ is associated with acceleration of onset of motor impairment and superoxide dismutase 1 aggregation in an amyotrophic lateral sclerosis mouse model

10.1111/j.1474-9726.2006.00200.xAging Cell52153-165ACGE

Serum matrix metalloproteinase-9 activity is dysregulated with disease progression in the mutant SOD1 transgenic mice

Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neurodegenerative disorder characterized by progressive deterioration of motor neurons in the spinal cord, brainstem, and cerebral cortex. Matrix metalloproteinase-9 (MMP-9) is proposed to be a biomarker for ALS due to a potential pathological role in the disease. However, despite numerous studies, it is still unclear whether there is a direct correlation between MMP-9 expression in serum and progression of disease. Therefore, we used a TgSOD1 G93A mouse with a low transgene copy number. This model shows slow disease progression analogous to human ALS and provides a useful model to study biomarker expression at different stages of disease. Using zymography, we found that serum MMP-9 activity was significantly elevated in animals showing early signs of disease when compared to the younger, pre-symptomatic animals. This was followed by a decrease in MMP-9 activity in TgSOD1 G93A mice with end-stage disease. These results were confirmed in serum of a high copy number strain of TgSOD1 G93A mice with rapid progression. MMP-9 expression was changed accordingly in spinal motor neurons, glia and neuropil, suggesting a spinal cord contribution to blood MMP-9 activity. Serum MMP-2 activity followed a similar profile as the MMP-9 in these two models. These data indicate that circulating MMP-9 is altered throughout the course of disease progression in mice. Further studies in human ALS may validate the suitability of serum MMP-9 activity as a biomarker for early stage disease.7 page(s

Mitochondrial Oxidative Stress Causes Hyperphosphorylation of Tau

Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer’s disease (AD): tau phosphorylation, and ß-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2) die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau) in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a wellcharacterized mouse model of AD (Tg2576) with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Aß load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. Thes

In vitro characterization of Pittsburgh compound-B binding to Lewy bodies.

Dementia with Lewy bodies (DLB) is pathologically characterized by the presence of alpha-synuclein-containing Lewy bodies within the neocortical, limbic, and paralimbic regions. Like Alzheimer's disease (AD), Abeta plaques are also present in most DLB cases. The contribution of Abeta to the development of DLB is unclear. [11C]-Pittsburgh compound B ([11C]-PIB) is a thioflavin-T derivative that has allowed in vivo Abeta burden to be quantified using positron emission tomography (PET). [11C]-PIB PET studies have shown similar high cortical [11C]-PIB binding in AD and DLB subjects. To establish the potential binding of PIB to alpha-synuclein in DLB patients, we characterized the in vitro binding of PIB to recombinant human alpha-synuclein and DLB brain homogenates. Analysis of the in vitro binding studies indicated that [3H]-PIB binds to alpha-synuclein fibrils but with lower affinity than that demonstrated/reported for Abeta(1-42) fibrils. Furthermore, [3H]-PIB was observed to bind to Abeta plaque-containing DLB brain homogenates but failed to bind to DLB homogenates that were Abeta plaque-free ("pure DLB"). Positive PIB fluorescence staining of DLB brain sections colocalized with immunoreactive Abeta plaques but failed to stain Lewy bodies. Moreover, image quantification analysis suggested that given the small size and low density of Lewy bodies within the brains of DLB subjects, any contribution of Lewy bodies to the [11C]-PIB PET signal would be negligible. These studies indicate that PIB retention observed within the cortical gray matter regions of DLB subjects in [11C]-PIB PET studies is largely attributable to PIB binding to Abeta plaques and not Lewy bodies

APP vs. copper correlation.

<p>Levels of APP and Copper in the soluble and pellet phases of brain homogenate from untreated Tg2576 and Tg2576:<i>sod2</i> crossed mice (pooled) were compared graphically and found to correlate strongly (* p<0.02).</p

Chronic exposure to U18666A is associated with oxidative stress in cultured murine cortical neurons

Findings that antioxidant treatment may be beneficial in Alzheimer\u27s disease indicate that oxidative stress is an important factor in its pathogenesis. Studies have also suggested that cholesterol imbalance in the brain might be related to the development of neurological disorders. Previously, we have reported that U18666A, a cholesterol transport-inhibiting agent, leads to apoptosis and intracellular cholesterol accumulation in primary cortical neurons. In this study, we found that neuronal apoptosis mediated by U18666A is associated with oxidative stress in the treated cortical neurons. Cortical neurons treated with U18666A also showed decreased secretion and increased intraneuronal accumulation of β-amyloid. The association of neuronal apoptosis with oxidative stress and Aβ accumulation may provide clues to the pathogenesis of Alzheimer\u27s disease, as well as the role oxidative stress plays in other neurodegenerative diseases