Identification of phagocytic glial cells after lesion-induced anterograde degeneration using double-fluorescence labeling: combination of axonal tracing and lectin or immunostaining

 Retrograde and anterograde degeneration have been reported to be sufficient stimuli to activate glial cells, which, in turn, are involved in phagocytosis of degenerating material. Here we describe a double-fluorescence technique which allows for direct and simultaneous visualization of both labeled incorporated axonal debris and incorporating glial cells in the course of anterograde degeneration. Stereotaxic application of small crystals of biotinylated and tetramethylrhodamine (TRITC)-conjugated dextran amine Mini Ruby into the medial entorhinal cortex resulted in a stable rhodamine fluorescence confined to fibers and terminals in the middle molecular layer of the dentate gyrus, the stratum lacunosum-moleculare, and the crossed temporo-hippocampal pathway. Subsequent stereotaxic lesion of the entorhinal cortex induced transformation of rhodamine-fluorescent fibers and terminals into small granules. Incorporation of these granules by microglial cells [labeled by fluorescein isothiocyanate (FITC)-coupled Bandeiraea simplicifolia isolectin B₄] or astrocytes (labeled by FITC-coupled glial fibrillary acidic protein antibodies) resulted in phagocytosis-dependent labeling of these non-neuronal cells, which could be identified by double-fluorescence microscopy. Electron microscopical analysis revealed that, following lesion, the tracer remained confined to entorhinal axons which were found to be incorporated by glial cells. Our data show that TRITC- and biotin-conjugated dextran amines are versatile tracers leading to Phaseolus vulgaris leucoagglutinin-like axonal staining. Lesion-induced phagocytosis of anterogradely degenerating axons by immunocytochemically identified glial cells can be directly observed by this technique on the light and electron microscopical levels. Accepted: 8 January 1997     

Neuronal damage in autoimmune neuroinflammation mediated by the death ligand TRAIL

Here, we provide evidence for a detrimental role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in neural death in T cell-induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Clinical severity and neuronal apoptosis in brainstem motor areas were substantially reduced upon brain-specific blockade of TRAIL after induction of EAE through adoptive transfer of encephalitogenic T cells. Furthermore, TRAIL-deficient myelin-specific lymphocytes showed reduced encephalitogenicity when transferred to wild-type mice. Conversely, intracerebral delivery of TRAIL to animals with EAE increased clinical deficits, while naive mice were not susceptible to TRAIL. Using organotypic slice cultures as a model for living brain tissue, we found that neurons were susceptible to TRAIL-mediated injury induced by encephalitogenic T cells. Thus, in addition to its known immunoregulatory effects, the death ligand TRAIL contributes to neural damage in the inflamed brain.     

Phosphorylation of the growth factors bFGF, NGF and BDNF: a prerequisite for their biological activity

The aim of this work was to test whether growth factors such as basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) undergo autophosphorylation and whether this affects their biological activity. Incubation of those growth factors with [gamma-(32)P]ATP resulted in phosphorylation in vitro. The phosphate bond was resistant to alkaline pH, yet acid-labile. Addition of alkaline phosphatase resulted in time and protein dependent dephosphorylation. Concomitantly, alkaline phosphatase abolished the neuroprotective effect of those growth factors upon oxygen and glucose deprivation and upon staurosporine-induced cell death. For those studies, we were using primary cultures of cortical and hippocampal neurons from embryonic and neonatal rats. Incubation of bFGF with non-hydrolyzable ATP-gammaS resulted in phosphorylation and in neuroprotection resistant to alkaline phosphatase. We conclude that bFGF, NGF and BDNF undergo autophosphorylation on site(s) other than serine, threonine, tyrosine and/or ATP-binding, and that this binding of phosphate is essential for neuroprotection in vivo.     

Perfusion cultures require optimum respiratory ATP supply to maximize cell-specific and volumetric productivities

Perfusion processes are an emerging alternative to common fed-batch processes in the growing biopharmaceutical industry. However, the challenge of maintaining high cell-specific productivities remains. In this study, glucose limitation was applied to two perfusion steady states and compared with a third steady state without any detectable limitation. The metabolic phenotype was enhanced under glucose limitation with a decrease of 30% in glucose uptake and 75% in lactate formation. Cell-specific productivities were substantially improved by 50%. Remarkably, the productivities showed a strong correlation to respiratory adenosine triphosphate (ATP) supply. As less reduced nicotinamide adenine dinucleotide (NADH) remained in the cytosol, the ATP generation from oxidative phosphorylation was increased by almost 30%. Consequently, the efficiency of carbon metabolism and the resulting respiratory ATP supply was crucial for maintaining the highly productive cellular state. This study highlights that glucose limitation can be used for process intensification in perfusion cultures as ATP generation via respiration is significantly increased, leading to elevated productivities.     

A robust feeding strategy to maintain set‐point glucose in mammalian fed‐batch cultures when input parameters have a large error

Industrial CHO cell cultures run under fed‐batch conditions are required to be controlled in particular ranges of glucose, while glucose is constantly consumed and must be replenished by a feed. The most appropriate feeding rate is ideally stoichiometric and adaptive in nature to balance the dynamically changing rate of glucose consumption. However, high errors in biomass and glucose estimation as well as limited knowledge of the true metabolic state challenge the control strategy. In this contribution, we take these errors into account and simulate the output with uncertainty trajectories in silico in order to control glucose concentration. Other than many control strategies, which require parameter estimation, our assumptions are founded on two pillars: (i) first principles and (ii) prior knowledge about the variability of fed‐batch CHO cell culture. The algorithm was exposed to an in‐silico Design of Experiments (DoE), in which variations of parameters were changed simultaneously, such as clone‐specific behavior, precision of equipment and desired control range used. The results demonstrate that our method achieved the target of holding the glucose concentration within an acceptable range. A robust and sufficient level of control could be demonstrated even with high errors for biomass or metabolic state estimation. In a time where blockbuster drugs are queuing up for time slots of their production, this transferable control strategy that is independent of tedious establishment runs may be a decisive advantage for rapid implementation during technology transfer and scale up and decrease in campaign change over time. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:317–336, 2017     

Intrinsic Up-Regulation of 2-AG Favors an Area Specific Neuronal Survival in Different In Vitro Models of Neuronal Damage

Background

The endocannabinoid 2-arachidonoyl glycerol (2-AG) acts as a retrograde messenger and modulates synaptic signaling e. g. in the hippocampus. 2-AG also exerts neuroprotective effects under pathological situations. To better understand the mechanism beyond physiological signaling we used Organotypic Entorhino-Hippocampal Slice Cultures (OHSC) and investigated the temporal regulation of 2-AG in different cell subsets during excitotoxic lesion and dendritic lesion of long range projections in the enthorhinal cortex (EC), dentate gyrus (DG) and the cornu ammonis region 1 (CA1).

Results

2-AG levels were elevated 24 h after excitotoxic lesion in CA1 and DG (but not EC) and 24 h after perforant pathway transection (PPT) in the DG only. After PPT diacylglycerol lipase alpha (DAGL) protein, the synthesizing enzyme of 2-AG was decreased when Dagl mRNA expression and 2-AG levels were enhanced. In contrast to DAGL, the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MAGL) showed no alterations in total protein and mRNA expression after PPT in OHSC. MAGL immunoreaction underwent a redistribution after PPT and excitotoxic lesion since MAGL IR disappeared in astrocytes of lesioned OHSC. DAGL and MAGL immunoreactions were not detectable in microglia at all investigated time points. Thus, induction of the neuroprotective endocannabinoid 2-AG might be generally accomplished by down-regulation of MAGL in astrocytes after neuronal lesions.

Conclusion

Increase in 2-AG levels during secondary neuronal damage reflects a general neuroprotective mechanism since it occurred independently in both different lesion models. This intrinsic up-regulation of 2-AG is synergistically controlled by DAGL and MAGL in neurons and astrocytes and thus represents a protective system for neurons that is involved in dendritic reorganisation.     

Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides

The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies.     

DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors

DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.