Growth factors and taurine protect against excitotoxicity by stabilizing calcium homeostasis and energy metabolism.

Taurine, brain derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF) are known to control the development of early postnatal cerebellar granule cells. This study attempted to investigate possible mechanisms of this control by determining neuronal survival, calcium homeostasis, and related calcium-mediated functions, as well as the site of action during glutamate-induced excitotoxicity in cultures of cerebellar granule cells. We report that stimulation of glutamate receptors induced a rapid increase in intracellular calcium concentrations ([Ca(2+)](i)) and a decrease in mitochondrial energy metabolism. These effects of glutamate were time- and concentration-dependent and could be specifically blocked by glutamate receptor antagonists. Taurine and bFGF but not BDNF differently regulated [Ca(2+)](i), and preserved the mitochondrial energy metabolism in the presence of glutamate. The regulation of [Ca(2+)](i) by bFGF and taurine required pretreatment of cells with these factors. Confocal microscope analysis of [Ca(2+)](i) and (45)Ca(2+) uptake studies showed that bFGF reduced the magnitude of glutamate-induced calcium uptake with no apparent regulation thereafter. Taurine, on the other hand, did not affect the level of calcium uptake induced by glutamate but rather the duration of the maximal response; this maximal response was transient and returned to basal levels approximately 10 min after glutamate receptor stimulation. We conclude from these data that bFGF and taurine prevent glutamate excitotoxicity through regulation of [Ca(2+)](i) and mitochondrial energy metabolism. Furthermore, the neuroprotective role of taurine and bFGF was enhanced by their collaboration.

Reduced number and altered morphology of microglial cells in colony stimulating factor-1-deficient osteopetrotic op/op mice.

The numerical density of microglial cells is reduced by 47% in the corpus callosum, by 37% in the parietal cortex and by 34% in the frontal cortex of mice mutant at the op locus which are totally devoid of colony stimulating factor-1 (CSF-1), the major growth factor for macrophages. Moreover, microglia in the frontal cortex of the op/op mice are smaller and have shorter cytoplasmic processes compared to control mice. Study suggests that CSF-1 plays a role in vivo in the formation and maturation of microglia and has little or no effect on perivascular cells.

Identification of a candidate human spectrin Src homology 3 domain-binding protein suggests a general mechanism of association of tyrosine kinases with the spectrin-based membrane skeleton.

Spectrin is a widely expressed protein with specific isoforms found in erythroid and nonerythroid cells. Spectrin contains an Src homology 3 (SH3) domain of unknown function. A cDNA encoding a candidate spectrin SH3 domain-binding protein was identified by interaction screening of a human brain expression library using the human erythroid spectrin (alphaI) SH3 domain as a bait. Five isoforms of the alphaI SH3 domain-binding protein mRNA were identified in human brain. Mapping of SH3 binding regions revealed the presence of two alphaI SH3 domain binding regions and one Abl-SH3 domain binding region. The gene encoding the candidate spectrin SH3 domain-binding protein has been located to human chromosome 10p11.2 --> p12. The gene belongs to a recently identified family of tyrosine kinase-binding proteins, and one of its isoforms is identical to e3B1, an eps8-binding protein (Biesova, Z., Piccoli, C., and Wong, W. T. (1997)Oncogene 14, 233-241). Overexpression of the green fluorescent protein fusion of the SH3 domain-binding protein in NIH3T3 cells resulted in cytoplasmic punctate fluorescence characteristic of the reticulovesicular system. This fluorescence pattern was similar to that obtained with the anti-human erythroid spectrin alphaI SigmaI/betaI SigmaI antibody in untransfected NIH3T3 cells; in addition, the anti-alphaI SigmaI/betaI SigmaI antibody also stained Golgi apparatus. Immunofluorescence obtained using antibodies against alphaI SigmaI/++betaI SigmaI spectrin and Abl tyrosine kinase but not against alphaII/betaII spectrin colocalized with the overexpressed green fluorescent protein-SH3-binding protein. Based on the conservation of the spectrin SH3 binding site within members of this protein family and published interactions, a general mechanism of interactions of tyrosine kinases with the spectrin-based membrane skeleton is proposed.

The regulation of phosphorylation of tau in SY5Y neuroblastoma cells: the role of protein phosphatases.

In Alzheimer disease brain the microtubule associated protein (MAP) tau is abnormally hyperphosphorylated. The role of protein phosphatases (PP) in the regulation of phosphorylation of tau was studied in undifferentiated SY5Y cells. In cells treated with 10 nM okadaic acid (OA), a PP-2A/PP-1 inhibitor, the PP-1 and -2A activities decreased by 60% and 100% respectively and the activities of MAPKs, cdc2 kinase and cdk5, but not of GSK-3, increased. OA increased the phosphorylation of tau at Thr-231/Ser-235 and Ser-3961404, but not at Ser-262/356 or Ser-199/202. An increase in tyrosinated/detyrosinated tubulin ratio, a decrease in the microtubule binding activities of tau, MAP1b and MAP2, and cell death were observed. Treatment with 1 microm taxol partially inhibited the cell death. These data suggest (1) that OA induced hyperphosphorylation of tau is probably the result of activated MAPK and cdks in addition to decreased PP-2A and PP-1 activities and (2) that in SY5Y cells the OA induced cell death is associated with a decrease in stable microtubules.

Failed cell migration and death of purkinje cells and deep nuclear neurons in the weaver cerebellum.

The mouse neurological mutant weaver has an atrophic cerebellar cortex with deficits in both Purkinje and granule cell number. Although granule cells are known to die postnatally shortly after their final cell division, the cause of the Purkinje cell deficit (cell death vs lack of production) is unknown. We report here a quantitative analysis of large cerebellar neurons of the weaver mutant during postnatal development. We explored the hypothesis that the cells of the entire cerebellar anlage were affected by the mutation by including in our study the neurons of the deep cerebellar nuclei (DCN). Our analysis reveals that in homozygous weaver mutants (1) the DCN are displaced laterally, display an abnormal anatomy, and suffer a 20-25% decrease in neuron number; (2) this numerical deficit is located in medial regions, similar to the localization of cortical deficits in both Purkinje and granule cells; (3) pyknotic figures are present in the juvenile DCN and in the Purkinje cell layer; and (4) the majority of cell death in these populations occurs not in medial regions where the numerical deficits are observed, but rather laterally where adult cell number is nearly normal. These results lead us to propose that the complete weaver phenotype includes a failure of the cell movements that lead to the fusion of the bilateral cerebellar anlage, and that this failure to migrate properly leaves some of the Purkinje cells and DCN neurons in a position where they are unable to make appropriate connections, leading to their death. In addition to implications for normal development, these observations suggest that weaver effects on the cerebellum can be unified into one consolidated model in which failure of cell movement affects all major cerebellar neurons.

Arrest of afferent axon extension by target neurons in vitro is regulated by the NMDA receptor.

Cerebellar granule neurons in vitro specifically arrest the extension of their appropriate presynaptic axons, mossy fibers. This "stop-growing signal" may be an essential step in the formation and specificity of synapses. Here, we have tested whether ionotropic glutamate receptors are involved in the stop-growing signal. When explants of basilar pontine nuclei, a mossy fiber source, were cultured on granule neurons, most pontine neurites terminated <200 microm from their explant of origin, a criterion for the stop-growing signal. In contrast, treatment with the NMDA antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) greatly increased the number of pontine neurites extending beyond 300 microm, whereas treatment with NMDA reduced the number of pontine neurites extending beyond 200 microm. A non-NMDA agonist (AMPA) and antagonist (6-cyano-7-nitroquinoxaline-2,3-dione) did not alter pontine neurite lengths. None of these agents affected neurite outgrowth from pontine explants in the absence of granule neurons, nor did any agent affect the survival of granule neurons. These results indicate that NMDA and D-AP5 specifically perturb an interaction between axons and target cells necessary for the stop-growing signal, and that NMDA receptors are critical for the development of a major cerebellar afferent system. These findings also suggest that NMDA-sensitive refinement of axon arbors during later development may involve the direct regulation of axon extension by target neurons.

Effect of K+- and kainate-mediated depolarization on survival and functional maturation of GABAergic and glutamatergic neurons in cultures of dissociated mouse cerebellum.

The effect of the depolarizing agents, an elevated potassium concentration (25 mM) or kainic acid (50 microM) on neuronal survival and differentiation was investigated in cultures of dissociated neurons from cerebella of 7-day-old mice. When maintained in the presence of an antimitotic agent such cultures consist primarily of glutamatergic and GABAergic neurons. Cell survival was monitored by measurement of DNA, and differentiation by determining uptake and depolarization coupled release of glutamate (D-aspartate as label) and GABA. The depolarizing agents were added separately or together either from the start of the culture period (7-8 days) or at day 5 in culture. The main findings are that K+ depolarization is important for differentiation of glutamatergic neurons but not for GABAergic neurons. This depolarizing signal is important during the early phase of development in culture. For glutamatergic neurons, kainate may replace K+ as a depolarizing signal whereas in case of the GABAergic neurons, kainate was toxic particularly during the late phase of development. It was further observed that the glutamatergic neurons when maintained in a medium with 5 mM K+ during the first 5 days in culture became sensitive to kainate toxicity when this amino acid was added at day 5. This was not the case when the medium contained 25 mM K+ from the start of the culture period.

Balanced interaction of growth factors and taurine regulate energy metabolism, neuronal survival, and function of cultured mouse cerebellar cells under depolarizing conditions.

The development of neuronal cells in a given cellular environment requires mechanisms that dynamically regulate the balanced interactions of multiple factors which are known to control maintenance and plasticity in function of neurons throughout constantly changing extracellular conditions. Periodic release of excitatory amino acids from both developing glial and neuronal cells into the extracellular environment and their uptake has been shown to stimulate neuronal function in concert with growth factors that control the degree of depolarization and, therefore, neuronal function. This study attempts to characterize the critical concentrations of these factors either alone or together in relation to energy metabolism, cell survival and function. We demonstrate a close correlation between energy metabolism of neuronal cells, controlled by the combination of growth-factors (beta FGF, BDNF), and glutamate-taurine as well as K+ in depolarizing concentrations (10-25 mM), during the balancing act of neuronal survival or death, and neuronal function. These functions depend on medium conditions (energy sources, ion composition), the ratio of glial cells versus neurons and cell density. Granule cell migration as a measure of developmental neuronal function was analyzed in the presence of various combinations of growth factors and taurine under various depolarizing conditions (glutamate, K+). We found that K+ concentrations > 7 mM in BME and 10% horse serum blocked migration in less than 30 min. Taurine did not prevent this effect. However, in the presence of HEPES as well as in F12-medium with HEPES, taurine restored granule cell migration. On the other hand, glutamate-or NMDA-mediated depolarization stopped migrating granule cells while NMDA antagonists extended the period of migration. Taurine amplified the stop-signal in the presence of glutamate agonists but increased the number of migrating cells in the absence of glutamate. Thus, the mechanisms of glutamate receptor mediated excitotoxicity, possibly by reducing Ca2+ influx under depolarizing conditions, but amplifies the stop-signal, Ca2+ levels may not control granule cell migration.

Increased proteolytic activity of the granule neurons may contribute to neuronal death in the weaver mouse cerebellum.

The weaver mouse mutation is a genetic defect of unknown origin that leads to impairment of cerebellar granule neuronal migration and to neuronal cell death. We investigated laminin expression and proteolytic enzyme activity in this migration-deficient mouse mutant in vivo and in vitro to search for a molecular basis for the weaver defect. The weaver cerebellum showed a general increase in immunoreactivity for laminin, for a neurite outgrowth domain of the B2 chain of laminin, and for tissue plasminogen activator compared to the normal animals. Zymographic assays and immunocytochemistry confirmed that tissue plasminogen activator was the proteolytic enzyme synthesized in excess in the weaver mouse cerebellum in vivo. When placed in culture, the weaver granule neurons survived poorly on a laminin substratum, and failed to extend long neurites, unlike the normal cerebellar granule neurons. The cultured weaver granule neurons were proteolytically overactive and secreted excessive amounts of tissue plasminogen activator, which was likely to interfere with their neurite outgrowth potential on a laminin substratum. Indeed, the weaver granule neurons but not the normal neurons degraded laminin from their culture substratum and deposited a neurite outgrowth domain of the B2 chain of laminin onto their surfaces. Electrophysiology showed that the weaver granule neurons had poor resting membrane potentials (-38 V), whereas the normal neurons had normal resting membrane potentials of (-61 V). The resting membrane potentials of the weaver granule neurons were restored to near normal (-59 V) by a protease inhibitor, aprotinin. Aprotinin also rescued the weaver granule neurons from death on a laminin substratum and promoted their neurite outgrowth to the level of the normal animals. These results indicate that increased proteolytic activity accompanied with increased synthesis of laminin, and its B2 chain, distinguish the weaver mutation from the normal animals. These molecular changes may contribute to the impairment of granule neuronal migration and to the neuronal death, characteristic of the weaver mutation.

Abnormally phosphorylated tau in SY5Y human neuroblastoma cells.

In Alzheimer disease (AD) the microtubule associated protein (MAP) tau is hyperphosphorylated at several sites. In the present study, like AD tau, tau in the human neuroblastoma SH-SY5Y was found to be hyperphosphorylated, at Ser-199/202, Thr-231, Ser-396 and Ser-404. However, in contrast to AD, the tau in SY5Y cells was not hyperphosphorylated at Ser-235 and there was only one tau isoform. Quantitative analysis revealed that approximately 80% of the SY5Y-tau was phosphorylated at Ser-199/202. The phosphorylated tau was deposited in perikarya and processes of the cells whereas most of the unphosphorylated (at Ser-199/202) tau was localized in the nucleus. Tau from the cell lysates did not bind to taxol-stabilized microtubules. In contrast, MAP1b and MAP2 from cell lysates bound to stabilized microtubules in vitro and were associated to the microtubule network in situ. Phosphorylation of tau at high levels, its inactivity with microtubules and its accumulation in SY5Y cells provide for the first time a cell model of cytoskeletal changes seen in AD.

Changes in expression of nonmuscle myosin heavy chain isoforms during muscle and nonmuscle tissue development.

Anti-human platelet myosin antibodies and two anti-peptide antibodies, anti-peptide IIA and anti-peptide IIB, which recognize macrophage-type (MIIA) and brain-type (MIIB) isoforms of nonmuscle myosin heavy chain, respectively, were used to study expression of nonmuscle myosin isoforms in various tissues of mice during development. Tissue-specific changes in the relative isoform concentrations were observed by performing immunoblots of crude myosin extracts from nonmuscle and muscle tissues. In fetal and neonatal mouse tissues, the anti-peptide IIB antibodies stained a single band, called MIIB2, while the anti-peptide IIA and anti-platelet myosin antibodies stained a band that migrated faster than MIIB2. In brain, a slower moving band, MIIB1, started to appear at 2 weeks after birth, and in the adult cerebellum it was at least as abundant as MIIB2. In thymus, MIIB2 decreased selectively shortly after birth, while in liver both MIIB2 and MIIA rapidly disappeared, but the isoform(s) detected by anti-platelet myosin antibodies (MIIApla) remained constant. The MIIB2 and MIIA as well as MIIApla found in striated muscles from fetal and neonatal mice decreased to levels that were below the limit of detection by 3 weeks of age. In cryosections of skeletal and cardiac muscles, MIIB2 was localized within the muscle cells, while MIIA and MIIApla were primarily in the blood vessels and capillaries.

Neuronal migration in cerebellar microcultures is inhibited by antibodies against a neurite outgrowth domain of laminin.

The functional role of laminin in neuronal migration was investigated by using polyclonal antibodies or their divalent (Fab')2 fragments to a neurite outgrowth promoting domain of the B2 chain of laminin in a cerebellar microculture system widely recognized as a model for neuronal migration. We show here that these antibodies or their (Fab')2 fragments totally inhibit migration of the mouse cerebellar granule cells along the glial and other neuronal cell processes. Antibodies to native laminin or other control antibodies have no inhibitory effect. Immunocytochemical analysis of the cerebellar microcultures indicates that the functional role of these antibodies may relate to the fact that the punctate deposits of laminin and its neurite outgrowth promoting domain accumulate in between the migrating neurons and the glial cells. These data provide the first direct evidence for the functional role of laminin and its neurite outgrowth domain in neuronal migration in the mammals. They further suggest that a neuronal cell surface contact with the extracellular deposits of a neurite outgrowth domain of the B2 chain of laminin may mediate neuronal-glial interactions.

The role of taurine in the survival and function of cerebellar cells in cultures of early postnatal cat.

The role of taurine and beta-alanine was analyzed in kitten cerebellar cultures. Since in contrast to mouse, cats (and primates including man) cannot synthesize sufficient taurine to maintain their body pools, we considered the cat an ideal species for the analysis of the role of taurine during early postnatal cerebellar development under controlled conditions. Unexpectedly, we found that the presence of taurine was toxic to neurons but that compounds, considered to be competitors for the beta-amino acid uptake system, support cell survival and cell function in vitro, the opposite of the results found in mice. This could be explained by the finding that only minute amounts of [3H]taurine were taken up by both cat neurons and glial cells under optimal culture conditions but that in the presence of the taurine analogues beta-alanine and guanidinoethane sulfonic acid (GES) significant amounts of taurine were found in all cell types. These differences between mouse cerebellar cells and cat cerebellar cells in vitro suggest that a re-evaluation of the mechanisms that control taurine function in cats and primates is warranted.

Progressive loss of neuronal src protein in postnatal weaver and staggerer cerebellum.

Two forms of the c-src protein-tyrosine kinase, pp60c-src, are detectable in the central nervous system. One form pp60+, appears to be exclusively expressed in neurons and is characterized by insertion of 6 amino acids compared to its non-neuronal counterpart, pp60. These 2 proteins were studied in the mutant mouse strains weaver and staggerer with postnatal loss of cerebellar granular neurons. We found a continuous postnatal decline of the neuronal form of pp60c-src, pp60+, in the cerebellum of both mutants concomitant with the degeneration of cerebellar granule cells. This indicates that granular neurons provide the main source for pp60+ in the cerebellar cortex.