Prion protein expression modulates neuronal copper content

The prion protein is a copper (Cu)-binding protein. The abnormal isoform of this protein is associated with the transmissible spongiform encephalopathies or prion diseases. In prion diseases, the prion protein loses its Cu binding capacity. The effect of prion protein expression on the Cu content of the brain was investigated. Transgenic mice, either overexpressing the prion protein or expressing a mutant form lacking the Cu-binding region of the protein, were compared with wild-type mice and mice in which expression of the protein was knocked out. Age-dependent differences in Cu content of the brain were detected. Also, synaptosomal fractions from the brains of the mice showed different Cu content depending on the expression of the prion protein. Mice expressing prion protein, but without the Cu-binding domain showed reduced Cu content. Mice overexpressing the prion protein showed little difference in Cu in the brain compared with wild type but also the prion protein expressed by the mice showed a reduction in the level of Cu bound. These results confirm that prion protein expression modulates the Cu level found at the synapse and this effect is dependent on its Cu binding capacity. Loss of normal Cu binding by the prion protein altered age-related increases in metals in the brain. This may explain why many forms of human prion disease do not develop until late in life.     

Molecular aspects of disease pathogenesis in the transmissible spongiform encephalopathies

The transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of rare, fatal, and transmissible neurodegenerative diseases of mammals for which there are no known viral or bacterial etiological agents. The bovine form of these diseases, bovine spongiform encephalopathy (BSE), has crossed over into humans to cause variant Creutzfeldt-Jakob disease. As a result, BSE and the TSE diseases are now considered a significant threat to human health. Understanding the basic mechanisms of TSE pathogenesis is essential for the development of effective TSE diagnostic tests and anti-TSE therapeutic regimens. This review provides an overview of the molecular mechanisms that underlie this enigmatic group of diseases.     

Prion protein polymerisation triggered by manganese-generated prion protein seeds

Prion diseases are neurodegenerative diseases that can be transmitted between individuals. The exact cause of these diseases remains unknown. However, one of the key events associates with the disease is the aggregation of a cellular protein, the prion protein. The mechanism of this is still unclear. However, it is likely that the aggregation is trigged by a seeding mechanism in which an oligomer of the prion protein is able to catalyse polymerisation of further prion protein into larger aggregates. We have developed a model of this process using an oligomeric species generated from recombinant protein by exposure to manganese. On fractionation of the seeding species, we estimated that the smallest size the oligomer would be is an octomer. We analysed the catalytic mechanism of the seeding oligomer and its interaction with substrate. Different domains of the protein are necessary for the seeding ability of the prion protein as opposed to those required for it to form a substrate for the polymerisation reaction. Prion seeds formed from different sheep alleles are able to reproduce the characteristics of scrapie in terms of resistance to disease. However, we were also able to generate prion seed from chicken PrP a species where no prion disease is known. Our findings provide an insight into the aggregation process of the prion protein and its potential relation to disease progress.     

Cnidarian neurobiology: what does the future hold?

Cnidarians have long been recognized as occupying a unique position in nervous system evolution and, consequently, have attracted considerable attention from neurobiologists over the years. During the latter half of the 20th century, the application of a variety of electrophysiological and other methods provided us with a great deal of information about the scope and composition of the cnidarian nervous system. Here, I will briefly review what is known about cnidarian nervous systems, what remains to be found and, most importantly, discuss the status and future of the field.     

Comparison of fast and slow synaptic terminals in lobster muscle

Summary Synaptic terminals of fast (FCE) and slow (SCE) excitatory neurons were physiologically identified on separate fibres of one muscle, the closer muscle in lobster claws. The innervation by these identified fibers was demonstrated over long distances (7–21 m) by examining serial thin sections at periodic intervals. The ultrastructure of each type of innervation was consistent both qualitatively and quantitatively in two separate samples. The FCE innervation is relatively simple in having consistently small-diameter terminals each forming a single long synapse, with few synaptic vesicles, and little if any postsynaptic apparatus. The SCE innervation is more complex in having larger-diameter but more variable terminals forming several short synapses, with many synaptic vesicles and an extensive postsynaptic apparatus. These differences in the size of the synapses and the number of synaptic vesicles parallel differences in transmitter release and fatigue sensitivity characteristic of the two types of innervation. The degree of elaboration of the postsynaptic apparatus may reflect differences in the amount of transmitter taken up after release. Our data reveal for the first time in a single muscle differences between FCE and SCE innervation previously reported in different muscles and in different species.Supported by grants from NIH (NINCDS) to A.G. Humes and the late Fred Lang and from NSERC and Muscular Dystrophy Assoc. of Canada to C.K. GovindWe thank Lena Hill for her technical expertise and critical evaluation of the study, and Dr. A.G. Humes for providing research facilities     

alpha-Synuclein produces a long-lasting increase in neurotransmitter release

Wild-type alpha-synuclein, a protein of unknown function, has received much attention because of its involvement in a series of diseases that are known as synucleinopathies. We find that long-lasting potentiation of synaptic transmission between cultured hippocampal neurons is accompanied by an increase in the number of alpha-synuclein clusters. Conversely, suppression of alpha-synuclein expression through antisense nucleotide and knockout techniques blocks the potentiation, as well as the glutamate-induced increase in presynaptic functional bouton number. Consistent with these findings, alpha-synuclein introduction into the presynaptic neuron of a pair of monosynaptically connected cells causes a rapid and long-lasting enhancement of synaptic transmission, and rescues the block of potentiation in alpha-synuclein null mouse cultures. Also, we report that the application of nitric oxide (NO) increases the number of alpha-synuclein clusters, and inhibitors of NO-synthase block this increase, supporting the hypothesis that NO is involved in the enhancement of the number of alpha-synuclein clusters. Thus, alpha-synuclein is involved in synaptic plasticity by augmenting transmitter release from the presynaptic terminal.     

In vitro cell culture pO2 is significantly different from incubator pO2

Continuous noninvasive monitoring of peri‐cellular liquid phase pO₂ in adherent cultures is described. For neurons and astrocytes, this approach demonstrates that there is a significant difference between predicted and observed liquid phase pO₂. Particularly at low gas phase pO₂s, cell metabolism shifts liquid phase pO₂ significantly lower than would be predicted from the O₂ gas/air equilibrium coefficient, indicating that the cellular oxygen uptake rate exceeds the oxygen diffusion rate. The results demonstrate the need for direct pO₂ measurements at the peri‐cellular level, and question the widely adopted current practice of relying on setting the incubator gas phase level as means of controlling pericellular oxygen tension, particularly in static culture systems that are oxygen mass transfer limited. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Synapse formation and function: Insights from identified leech neurons in culture

Identified leech neurons in culture are providing novel insights to the signals underlying synapse formation and function. Identified neurons from the central nervous system of the leech can be removed individually and plated in culture, where they retain their characteristic physiological properties, grow neurites, and form specific synapses that are directly accessible by a variety of approaches. Synapses between cultured neurons can be chemical or electrical (either rectifying or not) or may not form, depending on the neuronal identities. Furthermore, the characteristics of these synapses depend on the regions of the cells that come into contact. The formation and physiology of synapses between the Retzius cell and its partners have been well characterized. Retzius cells form purely chemical, inhibitory synapses with pressuresensitive (P) cells where serotonin (5-HT) is the transmitter. Retzius cells synthesize 5-HT, which is stored in vesicles that recycle after 5-HT is secreted on stimulation. The release of 5-HT is quantal, calcium-dependent, and shows activity-dependent facilitation and depression. Anterograde and retrograde signals during synapse formation modify calcium currents, responses to 5-HT, and neurite outgrowth. The nature of these synaptogenic signals is being elucidated. For example, contact specifically with Retzius cells induces a localized selection of transmitter responses in postsynaptic P cells. This effect is signaled by tyrosine phosphorylation prior to synapse formation. © 1995 John Wiley & Sons, Inc.     

Connexins 26, 32 and 43 are expressed in virgin, pregnant and lactating mammary glands

Gap junctions (GJ) are formed by a number of homologous proteins termed connexins. Here expression of connexins Cx26, Cx32 and Cx43, was evaluated by immunofluorescence (IF) in mammary glands from virgin, pregnant and lactating rats. Cx26, Cx32 and Cx43 labeling was detected in epithelial parenchymal cells at all functional stages. Cx26 and Cx32 labeling was very low in glands from virgin animals, somewhat greater in glands from pregnant animals and significantly higher (in number and size) in lactating animals. In the last ones, Cx26 and Cx32 punctate labeling was localized to the basal and lateral membranes of alveolar epithelial cells and collecting ductules. Cx43 punctate labeling was restricted to the periphery of alveoli towards the basal pole of epithelial cells at all functional stages, and it enlarged slightly during lactation. At this localization, Cx43 may form GJ between myoepithelial cells and/or between epithelial and myoepithelial cells. Cx43 was also found to be steadily expressed in the connective tissue which surrounds and invades each parenchymal lobe, at all functional stages. At this localization, Cx43 may couple fibroblasts and/or adipose cells. IF studies in sections from lactating mice showed the same distribution of connexins. Immunoblots confirmed specificity of labeling and the presence of Cx32 and Cx43 in the mammary gland. The increase in connexin expression detected during pregnancy and lactation may be important for epithelial cell differentiation and secretion in the mammary gland.     

Microcarrier technology, present status and perspective

Only a decade after Van Wezel introduced the first product made in microcarrier cultures on industrial scale at economically acceptable costs, namely Inactivated Polio Vaccine (IPV), interest was taken in this revolutionary type of cell growth system. The basic idea was to develop a culture system with equal potentials for control of environmental culture conditions and scaling up as the systems used in industrial microbiology. Although initially only positively-charged beads were used it soon became clear that negatively-charged or amphoteric materials such as proteins or amino acids polymerized to the surface were equally useful. Eventually numerous different types of microcarrier were developed. The second generation of microcarriers consisted of macroporous beads providing increased surface area for cell attachment and growth by external and interior space. Such microcarriers offer great potential for high cell densities and enhanced productivity for certain production systems, especially recombinant CHO-cells. These carriers, which not only provide possibilities for anchorage-dependent cells but also for cells growing suspension, can be used in homogeneous bioreactors as well as in fluidized or fixed-bed systems. Despite considerable in vestments and research on the development and improvement of microcarriers one question is still open: is microcarrier technology still in its infancy or is it full-grown and is the basic idea relized? In this paper a general overview will be given of the present state of microcarrier technology and also of its perspectives.     

Modulation of differentiation in vitro

Summary Myogenic cells of the L₆ line proliferate and fuse in culture to form myotubes that actively synthesize muscle-specific proteins such as myosin. We show that the expression of the differentiated phenotype can be influenced by the electrical charges of the substratum on which the cells were grown. Negatively charged surfaces did not influence the developmental program of the cells although positively charged ones interfered with myogenesis. The interaction operates primarily by interfering with the mitotic cycle, which is slowed down, with fusion which is blocked, and with myosin synthesis, which is reduced. Our results show that growth of the cells on positively charged surfaces prevents the switching of a large fraction of the population from a proliferative state to a differentiating program. We postulate that this interference might operate through the slowdown in DNA replication. The cell culture method described represents a good model for studying the different steps involved in the differentiation of L₆ cells. This work was supported by the American Muscular Dystrophy Association.     

Signals in Stochastically Generated Neurons

To incorporate variation of neuron shape in neural models, we developed a method of generating a population of realistically shaped neurons. Parameters that characterize a neuron include soma diameters, distances to branch points, fiber diameters, and overall dendritic tree shape and size. Experimentally measured distributions provide a means of treating these morphological parameters as stochastic variables in an algorithm for production of neurons. Stochastically generated neurons shapes were used in a model of hippocampal dentate gyrus granule cells. A large part of the variation of whole neuron input resistance RN is due to variation in shape. Membrane resistivity Rm computed from RN varies accordingly. Statistics of responses to synaptic activation were computed for different dendritic shapes. Magnitude of response variation depended on synapse location, measurement site, and attribute of response.