Intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes measured with fluorescence lifetime spectroscopy.

The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease.

An impaired mitochondrial electron transport chain increases retention of the hypoxia imaging agent diacetylbis(4-methylthiosemicarbazonato)copperII.

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.

The Alzheimer's therapeutic PBT2 promotes amyloid-ß degradation and GSK3 phosphorylation via a metal chaperone activity.

Impaired metal ion homeostasis causes synaptic dysfunction and treatments for Alzheimer's disease (AD) that target metal ions have therefore been developed. The leading compound in this class of therapeutic, PBT2, improved cognition in a clinical trial with AD patients. The aim of the present study was to examine the cellular mechanism of action for PBT2. We show PBT2 induces inhibitory phosphorylation of the α- and ß-isoforms of glycogen synthase kinase 3 and that this activity is dependent on PBT2 translocating extracellular Zn and Cu into cells. This activity is supported when Aß:Zn aggregates are the source of extracellular Zn and adding PBT2 to Aß:Zn preparations promotes Aß degradation by matrix metalloprotease 2. PBT2-induced glycogen synthase kinase 3 phosphorylation appears to involve inhibition of the phosphatase calcineurin. Consistent with this, PBT2 increased phosphorylation of other calcineurin substrates, including cAMP response element binding protein and Ca²âº/calmodulin-dependent protein kinase. These data demonstrate PBT2 can decrease Aß levels by sequestering the Zn that promotes extracellular formation of protease resistant Aß:Zn aggregates, and that subsequent intracellular translocation of the Zn by PBT2 induces cellular responses with synapto-trophic potential. Intracellular translocation of Zn and Cu via the metal chaperone activity of PBT2 may be an important mechanism by which PBT2 improves cognitive function in people with AD.

An emerging role of dysfunctional axon-oligodendrocyte coupling in neurodegenerative diseases.

Myelin is made by highly specialized glial cells and enables fast axonal impulse propagation. Recent studies show that oligodendrocytes in the central nervous system are, in addition to myelination, required for the integrity and survival of axons, independent of the presence or absence of myelin itself. The underlying mechanism of this support is given by glycolytic oligodendrocytes which provide axons with energy-rich metabolites. These findings represent a paradigm shift for the physiological function of axon-associated glia, and open the intriguing possibility that oligodendrocytes are important contributors to neurodegenerative diseases in which myelinated axons are lost, such as in Alzheimer disease, amyotrophic lateral sclerosis, and multiple system atrophy. Understanding the role of axon-oligodendrocyte coupling in neurodegenerative diseases may pave the way for the development of metabolism-based therapeutic approaches.

La mielina es producida por clulas gliales altamente especializadas y permite la propagacin rpida del impulso axonal. Estudios recientes muestran que los oligodendrocitos en el sistema nervioso central son, junto con la mielinizacin, necesarios para la integridad y sobrevida de los axones, independientemente de la presencia o ausencia de mielina. El mecanismo que subyace a este soporte est dado por oligodendrocitos glicolticos que aportan a los axones metabolitos ricos en energa. Estos hallazgos representan un cambio de paradigma para la funcin fisiolgica de la gla asociada a los axones, y abre la intrigante posibilidad que los oligodendrocitos sean importantes contribuyentes a las enfermedades neurodegenerativas en que se pierden los axones mielinizados, como la Enfermedad de Alzheimer, la esclerosis lateral amiotrfica y la atrofia de mltiples sistemas. La comprensin del papel del acoplamiento del oligodendrocito con el axn en las enfermedades neurodegenerativas puede abrir la va para el desarrollo de aproximaciones teraputicas basadas en el metabolismo.

Fabrique par des cellules gliales trs spcialises, la myline permet la propagation rapide de l'influx axonal. D'aprs des tudes rcentes, les oligodendrocytes du systme nerveux central sont ncessaires, outre la mylinisation, l'intgrit et la survie des axones, indpendamment de la prsence ou non de myline. Les oligodendrocytes glycolytiques, apportant aux axones des mtabolites riches en nergie, en forment le mcanisme sous-jacent. Ces rsultats reprsentent un changement de paradigme pour la fonction physiologique de la glie associe aux axones. De manire trs intressante, les oligodendrocytes pourraient participer de faon importante aux maladies neurodgnratives dans lesquelles il y a une perte d'axones myliniss comme la maladie d'Alzheimer, la sclrose latrale amyotrophique et les atrophies multiples de systme. Comprendre le rle du couplage axone-oligodendrocytes dans les maladies neurodgnratives peut ouvrir la voie du dveloppement de traitements bass sur le mtabolisme.

Circumventing the Crabtree Effect: A method to induce lactate consumption and increase oxidative phosphorylation in cell culture.

Most cells grown in glucose-containing medium generate almost all their ATP via glycolysis despite abundant oxygen supply and functional mitochondria, a phenomenon known as the Crabtree effect. By contrast, most cells within the body rely on mitochondrial oxidative phosphorylation (OXPHOS) to generate the bulk of their energy supply. Thus, when utilising the accessibility of cell culture to elucidate fundamental elements of mitochondria in health and disease, it is advantageous to adopt culture conditions under which the cells have greater reliance upon OXPHOS for the supply of their energy needs. Substituting galactose for glucose in the culture medium can provide these conditions, but additional benefit can be gained from alternate in vitro models. Herein we describe culture conditions in which complete autonomous depletion of medium glucose induces a lactate-consuming phase marked by increased MitoTracker Deep Red staining intensity, increased expression of Kreb's cycle proteins, increased expression of electron transport chain subunits, and increased sensitivity to the OXPHOS inhibitor rotenone. We propose these culture conditions represent an alternate accessible model for the in vitro study of cellular processes and diseases involving the mitochondrion without limitations incurred via the Crabtree effect.

Immunotherapeutic approaches in prion disease: progress, challenges and potential directions.

Therapeutic trials utilizing animal models of prion disease have explored a variety of compounds and a number of approaches with varying success, including several immunotherapeutic strategies, such as passive immunization through the delivery of viruses carrying nucleic acid inserts encoding prion protein-specific immunoglobulin. Targeted, organ-specific cellular production of therapeutic proteins is a relatively unexplored approach in the treatment of neurodegeneration despite many successful experimental outcomes in animal models and human trials of other diseases of the CNS. Emphasizing studies utilizing mouse models of disease, this review outlines developments and limitations of immunological approaches to the treatment of prion diseases. In addition, the authors discuss the potential of an experimental therapeutic strategy, utilizing hybridoma cells injected directly into the CNS to establish long-term production of anti-prion antibodies in vivo within the organ associated with the greatest pathogenic change in prion disease, the brain.

Metal attenuating therapies in neurodegenerative disease.

The clinical and pathological spectrum of neurodegenerative diseases is diverse, although common to many of these disorders is the accumulation of misfolded proteins, with oxidative stress thought to be an important contributing mechanism to neuronal damage. As a corollary, transition metal ion dyshomeostasis appears to play a key pathogenic role in a number of these maladies, including the most common of neurodegenerative diseases. In this review, studies spanning a wide variety of neurodegenerative disorders are presented with their involvement of transition metals compared and contrasted, including more detailed treatise in relation to Alzheimer's disease, Parkinson's disease and prion diseases. For each of these diseases, a discussion of the evolving scientific rationale for the development of therapies aimed at ameliorating the detrimental effects of transition metal dysregulation, including results from various human trials, is then provided.