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Mitochondrial dysfunction, the inability to efficiently utilise metabolic fuels and oxygen, contributes to pathological changes following traumatic spinal cord or traumatic brain injury (TBI). In the present study, we tested the hypothesis that succinate supplementation can improve cellular energy state under metabolically stressed conditions in a robust, reductionist in vitro model of mitochondrial dysfunction in which primary mixed glial cultures (astrocytes, microglia and oligodendrocytes) were exposed to the mitochondrial complex I inhibitor rotenone. Cellular response was determined by measuring intracellular ATP, extracellular metabolites (glucose, lactate, pyruvate), and oxygen consumption rate (OCR). Rotenone produced no significant changes in glial ATP levels. However, it induced metabolic deficits as evidenced by lactate/pyruvate ratio (LPR) elevation (a clinically-established biomarker for poor outcome in TBI) and decrease in OCR. Succinate addition partially ameliorated these metabolic deficits. We conclude that succinate can improve glial oxidative metabolism, consistent our previous findings in TBI patients’ brains. The mixed glial cellular model may be useful in developing therapeutic strategies for conditions involving mitochondrial dysfunction, such as TBI.

Although the neurotoxic effects of homocysteine have been well elucidated, the effects of homocysteine in astrocytes have received little attention until recently. Previously we have demonstrated that elevated levels of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes. However, the mechanisms behind such alterations remain unclear. As homocysteine is a key metabolite in one-carbon metabolism the present study examined if the effects of homocysteine on astrocyte function are mediated through an epigenetic mechanism. Following exposure to homocysteine for 72 h, global DNA methylation and H3K9 acetylation were examined using flow cytometric analysis. Total DNA methyltransferase activity and protein levels of DNA methyltransferase 3B were measured. Exposure to homocysteine resulted in global DNA hypomethylation (p0.05) and histone hyperacetylation (p0.05). Total DNA methyltransferase activity significantly decreased following exposure to homocysteine (from 11.5 ± 3.9 to 6.0 ± 1.7OD/h/mg protein, p0.01) which was accompanied by a significant reduction in protein levels of DNA methyltransferase 3B (p0.05). Treatment of astrocytes with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, mimicked the functional changes induced by homocysteine. In conclusion, the results demonstrate significant epigenetic modifications following exposure to homocysteine in astrocytes and these changes seem to mediate functional alterations.

Retroviral vectors are presently amongst the most widely used vectors in gene therapy clinical trials to target pathologies of different origins, such as cancers, genetic diseases or neurological disorders. This review provides an overview on the evolution of retroviral vector design and production for gene therapy applications, including state of the art developments in flexible producer cells and safe vectors. In addition, production and purification processes will be addressed, with a particular focus on the improvements undertaken to increase vector productivity and to reduce the rapid loss of infectivity, which presently represent the main challenges in retroviral vectors production for gene therapy.

J. Neurochem. (2010) 113, 735–748. Abstract Disruption of brain energy metabolism is the hallmark of cerebral ischemia, a major cause of death worldwide. Astrocytes play a key role in the regulation of brain metabolism and their vulnerability to ischemia has been described. Aiming to quantify the effects of an ischemic insult in astrocytic metabolism, primary cultures of astrocytes were subjected to 5 h of oxygen and glucose deprivation in a bioreactor. Flux distributions, before and after ischemia, were estimated by metabolic flux analysis using isotopic information and the consumption/secretion rates of relevant extracellular metabolites as constraints. During ischemia and early recovery, 30% of cell death was observed; several metabolic alterations were also identified reflecting a metabolic response by the surviving cells. In the early recovery (∼10 h), astrocytes up-regulated glucose utilization by 30% and increased the pentose phosphate pathway and tricarboxylic acid cycle fluxes by three and twofold, respectively. Additionally, a two to fivefold enhancement in branched-chain amino acids catabolism suggested the importance of anaplerotic molecules to the fast recovery of the energetic state, which was corroborated by measured cellular ATP levels. Glycolytic metabolism was predominant in the late recovery. In summary, this work demonstrates that changes in fluxes of key metabolic pathways are implicated in the recovery from ischemia in astrocytes.

Enhancing central nervous system (CNS) myelin regeneration is recognized as an important strategy to ameliorate the devastating consequences of demyelinating diseases such as multiple sclerosis. Previous findings have indicated that myelin proteins, which accumulate following demyelination, inhibit remyelination by blocking the differentiation of rat oligodendrocyte progenitor cells (OPCs) via modulation of PKCα. We therefore screened drugs for their potential to overcome this differentiation block. From our screening, tamoxifen emerges as a potent inducer of OPC differentiation in vitro. We show that the effects of tamoxifen rely on modulation of the estrogen receptors ERα, ERβ, and GPR30. Furthermore, we demonstrate that administration of tamoxifen to demyelinated rats in vivo accelerates remyelination. Tamoxifen is a well-established drug and is thus a promising candidate for a drug to regenerate myelin, as it will not require extensive safety testing. In addition, Tamoxifen plays an important role in biomedical research as an activator of inducible genetic models. Our results highlight the importance of appropriate controls when using such models.

Retrovirus vectors derived from moloney murine leukemia virus (MoMLV) were the first class of viral vectors developed for gene therapy. They have been extensively used in clinical trials, particularly in ex vivo transduction of hematopoietic stem cells. Although there is a vast experience acquired with retroviruses, their manufacturing is still a difficult task due to the low cell productivities and inherent instability of the infective virus. These viral vectors are most commonly produced using stable producer cell lines in adherent monolayer culture systems. In order to obtain high transduction efficiencies and low toxicity in clinical applications, the viral preparations should be purified, concentrated, and well characterized to attain stringent quality specifications. This chapter describes currently used protocols for manufacturing retroviruses.

Although glucose is the primary cerebral fuel, the brain is able to metabolize other substrates in hypoglycemia. Nevertheless, the metabolic consequences of this pathology at the cellular level remain largely unknown. Taking advantage of the metabolic flux analysis (MFA) methodology, this work was aimed at investigating and quantifying the effects of hypoglycemia on cerebellar neurons. After 12 hr without glucose, primary cultures were incubated with medium containing [1,6-(13)C]glucose and unlabeled glutamine, and metabolism was monitored for 30 hr. Metabolic rates of glucose, lactate, and amino acids were determined based on cell supernatant analysis and used to estimate metabolic fluxes with MFA. Percent (13)C enrichment time profiles of different keto and amino acids were measured by mass spectrometry in cell extracts and compared with the MFA results. Hypoglycemia decreased the glucose uptake rate and glycolytic metabolism by 35% whereas glutamine uptake was increased fourfold. Flux estimations fit well with data from (13)C labeling dynamics, indicating a significant activation of the pyruvate recycling (PR) pathway, accounting for 43% of the total pyruvate synthesized under control conditions and up to 71% after hypoglycemia. Increased PR appeared to be due mainly to increased glutamine oxidation given the higher label dilution observed in the hypoglycemia group. In summary, this work provides new evidence for PR as an important pathway for glutamine oxidation in cerebellar neurons, particularly after glucose deprivation.

This work evaluates the effect of purification process and storage conditions (buffer formulation and temperature) on the efficacy of Ehrlichia ruminantium (ER) elementary bodies to be used as an inactivated vaccine candidate against heartwater. In vitro assays revealed that, to avoid major losses in ER integrity and corresponding antigenic properties, a buffer with pH between 5.6 and 8 and an osmolality above 100 mOsmol/kg H2O is recommended. Amongst the tested formulations, both PBS and NaCl have shown to stabilize ER antigens at -20°C. To assess the protective properties of the different vaccine formulations, in vivo experiments were performed using a goat model. The results obtained showed that the preparation of ER antigens using a novel membrane-based purification strategy and a simple vaccine formulation (NaCl, -20°C) induced equivalent protection to the conventional vaccine based on ER antigens prepared by a multistep centrifugation methodology and stored at -20°C in PBS buffer. © 2007 Elsevier Ltd. All rights reserved.