Changes in calcineurin expression induced in the rat brain by the administration of antipsychotics.

Calcineurin (CN) was recently identified as a susceptibility gene for schizophrenia as well as showing altered RNA expression levels in the post-mortem brains of individuals with schizophrenia. CN knockout mice show a number of behaviours associated with schizophrenia, including deficits in sensorimotor gating, suggesting a link between CN and psychosis. Concurrently, we found, using genome screening techniques, that antipsychotics alter CN expression levels. Therefore, western blotting, in situ hybridization, immunocytochemistry and phosphatase assays were employed to determine what effect antipsychotics have on CN. The results indicate that clozapine, risperidone and haloperidol cause substantial reductions in the A subunit of CN but not CN B at both the RNA and protein levels in the striatum and prefrontal cortex. The changes could only be observed after repeated treatment with antipsychotics but not after acute administration. The alterations in CN protein levels were specific to antipsychotics and mediated by D2 dopamine receptor antagonism. However, despite reductions in CN protein levels, the phosphatase activity of CN was significantly elevated after treatment with antipsychotics. Collectively the results suggest that CN may be a common target for antipsychotics and that antipsychotic-induced alterations in CN may represent one of the mechanisms by which antipsychotics alleviate psychosis.

Aggregated DsRed-tagged Cx43 and over-expressed Cx43 are targeted to lysosomes in human breast cancer cells.

To investigate if either wild-type or aggregated Cx43 is abnormally targeted to lysosomes in human breast tumor cells, we examined the fate of DsRed-tagged Cx43 and over-expressed Cx43 in communication-deficient HBL-100 and MDA-MB-231 cells. DsRed-tagged Cx43 was assembled into gap junctions in control normal rat kidney cells that express endogenous Cx43 but not in Cx43-negative HBL-100 cells. However, when HBL-100 cells were engineered to coexpress wild-type Cx43 a population of DsRed-tagged Cx43 was rescued and assembled into gap junctions. Co-expression of wild-type Cx26 failed to rescue the assembly of DsRed-tagged Cx43 into gap junctions. Immunolocalization studies revealed that DsRed-tagged Cx43 was aggregated and partially localized to lysosomes. Interestingly, when human MDA-MB-231 breast tumor cells over-expressed wild-type Cx43, Cx43 protein primarily localized to lysosomes. Together, these studies provide evidence for Cx43 being targeted to lysosomes as a result of misfolding and aggregation, while in other cases, the delivery of wild-type Cx43 to lysosomes appears to be due to defects innate to the breast tumor cell type.

Autocrine hepatocyte growth factor provides a local mechanism for promoting axonal growth.

In this report, we describe a novel local mechanism necessary for optimal axonal growth that involves hepatocyte growth factor (HGF). Sympathetic neurons of the superior cervical ganglion coexpress bioactive HGF and its receptor, the Met tyrosine kinase, both in vivo and in vitro. Exogenous HGF selectively promotes the growth but not survival of cultured sympathetic neurons; the magnitude of this growth effect is similar to that observed with exogenous NGF. Conversely, HGF antibodies that inhibit endogenous HGF decrease sympathetic neuron growth but have no effect on survival. This autocrine HGF is required locally by sympathetic axons for optimal growth, as demonstrated using compartmented cultures. Thus, autocrine HGF provides a local, intrinsic mechanism for promoting neuronal growth without affecting survival, a role that may be essential during developmental axogenesis or after neuronal injury.

A critical temporal requirement for the retinoblastoma protein family during neuronal determination.

In this report, we have examined the requirement for the retinoblastoma (Rb) gene family in neuronal determination with a focus on the developing neocortex. To determine whether pRb is required for neuronal determination in vivo, we crossed the Rb-/- mice with transgenic mice expressing beta-galactosidase from the early, panneuronal Talpha1 alpha-tubulin promoter (Talpha1:nlacZ). In E12.5 Rb-/- embryos, the Talpha1:nlacZ transgene was robustly expressed throughout the developing nervous system. However, by E14. 5, there were perturbations in Talpha1:nlacZ expression throughout the nervous system, including deficits in the forebrain and retina. To more precisely define the temporal requirement for pRb in neuronal determination, we functionally ablated the pRb family in wild-type cortical progenitor cells that undergo the transition to postmitotic neurons in vitro by expression of a mutant adenovirus E1A protein. These studies revealed that induction of Talpha1:nlacZ did not require proteins of the pRb family. However, in their absence, determined, Talpha1:nlacZ-positive cortical neurons underwent apoptosis, presumably as a consequence of "mixed signals" deriving from their inability to undergo terminal mitosis. In contrast, when the pRb family was ablated in postmitotic cortical neurons, there was no effect on neuronal survival, nor did it cause the postmitotic neurons to reenter the cell cycle. Together, these studies define a critical temporal window of requirement for the pRb family; these proteins are not required for induction of neuronal gene expression or for the maintenance of postmitotic neurons, but are essential for determined neurons to exit the cell cycle and survive.

The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death.

To determine whether the p75 neurotrophin receptor (p75NTR) plays a role in naturally occurring neuronal death, we examined neonatal sympathetic neurons that express both the TrkA tyrosine kinase receptor and p75NTR. When sympathetic neuron survival is maintained with low quantities of NGF or KCl, the neurotrophin brain-derived neurotrophic factor (BDNF), which does not activate Trk receptors on sympathetic neurons, causes neuronal apoptosis and increased phosphorylation of c-jun. Function-blocking antibody studies indicate that this apoptosis is due to BDNF-mediated activation of p75NTR. To determine the physiological relevance of these culture findings, we examined sympathetic neurons in BDNF-/- and p75NTR-/- mice. In BDNF-/- mice, sympathetic neuron number is increased relative to BDNF+/+ littermates, and in p75NTR-/- mice, the normal period of sympathetic neuron death does not occur, with neuronal attrition occurring later in life. This deficit in apoptosis is intrinsic to sympathetic neurons, since cultured p75NTR-/- neurons die more slowly than do their wild-type counterparts. Together, these data indicate that p75NTR can signal to mediate apoptosis, and that this mechanism is essential for naturally occurring sympathetic neuron death.

Blocking nerve growth factor binding to the p75 neurotrophin receptor on sympathetic neurons transiently reduces trkA activation but does not affect neuronal survival.

Nerve growth factor interacts with the trkA tyrosine kinase receptor and with the p75 neurotrophin receptor. It is clear that trkA mediates most, if not all, of the stereotypical responses of sympathetic neurons to nerve growth factor but the role of the p75 neurotrophin receptor is unclear. In this study, we have asked whether a functional interaction between p75 neurotrophin receptor and trkA exists in primary sympathetic neurons by disrupting nerve growth factor binding to p75 neurotrophin receptor. Acute assays reveal that blocking antibodies directed against p75 neurotrophin receptor reduce nerve growth factor-mediated trkA tyrosine phosphorylation and reduce the amount of nerve growth factor which binds the trkA receptor. This reduction in trkA activity is relatively short-lived in vitro and blocking antibodies to p75 neurotrophin receptor do not inhibit long-term survival of nerve growth factor-dependent primary neurons. Together, these data indicate that p75 neurotrophin receptor and trkA interact within primary neurons to enhance nerve growth factor binding to the trkA receptor under conditions of acute but not chronic nerve growth factor exposure.

NGF and neurotrophin-3 both activate TrkA on sympathetic neurons but differentially regulate survival and neuritogenesis.

In this report we examine the biological and molecular basis of the control of sympathetic neuron differentiation and survival by NGF and neurotrophin-3 (NT-3). NT-3 is as efficient as NGF in mediating neuritogenesis and expression of growth-associated genes in NGF-dependent sympathetic neurons, but it is 20-40-fold less efficient in supporting their survival. Both NT-3 and NGF induce similar sustained, long-term activation of TrkA, while NGF is 10-fold more efficient than NT-3 in mediating acute, short-term TrkA activity. At similar acute levels of TrkA activation, NT-3 still mediates neuronal survival two- to threefold less well than NGF. However, a mutant NT-3 that activates TrkC, but not TrkA, is unable to support sympathetic neuron survival or neuritogenesis, indicating that NT-3-mediated TrkA activation is necessary for both of these responses. On the basis of these data, we suggest that NGF and NT-3 differentially regulate the TrkA receptor both with regard to activation time course and downstream targets, leading to selective regulation of neuritogenesis and survival. Such differential responsiveness to two ligands acting through the same Trk receptor has important implications for neurotrophin function throughout the nervous system.

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells.

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.

Adenovirus-mediated gene transfer of the tumor suppressor, p53, induces apoptosis in postmitotic neurons.

Programmed cell death is an ongoing process in both the developing and the mature nervous system. The tumor suppressor gene, p53, can induce apoptosis in a number of different cell types. Recently, the enhanced expression of p53 has been observed during acute neurological disease. To determine whether p53 overexpression could influence neuronal survival, we used a recombinant adenovirus vector carrying wild type p53 to transduce postmitotic neurons. A control consisting of the same adenovirus vector background but carrying the lacZ reporter expression cassette was used to establish working parameters for the effective genetic manipulation of sympathetic neurons. We have found that recombinant adenovirus can be used at titers sufficiently high (10 to 50 multiplicity of infection) to transduce the majority of the neuronal population without perturbing survival, electrophysiological function, or cytoarchitecture. Moreover, we demonstrate that overexpression of wild type p53 is sufficient to induce programmed cell death in neurons. The observation that p53 is capable of inducing apoptosis in postmitotic neurons has major implications for the mechanisms of cell death in the traumatized mature nervous system.

Cellular localization of gap junction mRNAs in developing rat brain.

We investigated the developmental expression and cellular resolution of connexin32 and 43 mRNA in the rat brain using in situ hybridization. Utilizing 35S-labelled probes, in situ hybridization was performed on sections of embryonic day 20 and postnatal days 3, 10, 15, 30 and adult brain. Connexin32 mRNA was first detected in brainstem nuclei at postnatal day 3 and in the midbrain at postnatal day 15. The level of this message continued to increase to postnatal day 30 where the level of message reached a plateau or slightly decreased by adulthood. The distribution of signal included the medial vestibular nucleus, dorsal paragigantocellular nucleus and fibre tracts of the midbrain and brainstem such as the cerebellar white matter, spinal trigeminal tract, and decussation of the superior cerebellar peduncle, areas containing predominantly neurons or oligodendrocytes. Connexin43 mRNA was first detected much earlier than connexin32 and was found in the leptomeniges of the E20 brain. It was found at all ages examined and distributed homogenously in the regions of the brain examined, suggesting its presence in astrocytes. The connexin43 riboprobe also hybridized strongly to the ependymal cells of the fourth ventricle and cerebral aqueduct. These results describe the cellular resolution of connexin mRNAs during development and that a differential pattern of expression exists in the various cell populations in the central nervous system.

Cortical type 2 astrocytes are not dye coupled nor do they express the major gap junction genes found in the central nervous system.

The O-2A progenitor cell first described from the rat optic nerve is a bipotential precursor of oligodendrocytes and type 2 astrocytes. Each cell expresses specific markers that distinguish them as unique cell types. O-2A progenitors cultured in high serum preferentially differentiate into type 2 astrocytes and when exposed to defined medium or low serum develop along the oligodendrocyte lineage. We analyzed the gap junction gene expression of type 2 astrocytes to determine if they are coupled to form a syncytium, like their type 1 astrocyte counterparts. Dye coupling experiments demonstrated that cortical type 2 astrocytes are not coupled, while type 1 astrocytes in the same culture dish are highly coupled. Immunocytochemistry revealed the presence of Cx43 in type 1 astrocytes but we could not detect Cx26, 32, or 43 protein in type 2 astrocytes. In situ hybridization did not detect mRNA for any of the three connexin genes in type 2 astrocytes. These data demonstrate that type 2 astrocytes do not express the major gap junction genes found in the central nervous system. The precise function of type 2 astrocytes is not known but the lack of gap junction genes expression suggests that their functions are different from the spatial buffering capacity of type 1 astrocytes.

In vivo growth of C6 glioma cells transfected with connexin43 cDNA.

In order to examine the possible role of intercellular communication via gap junctions in the control of tumor growth, we have transfected C6 glioma cells with connexin43 cDNA. We obtained several clones with variable expression of connexin43. The growth rate of these clones in culture was inversely related to the degree of expression of the transfected cDNA. To examine the growth of these transfected cells in vivo, cells were grown in spinner culture flasks to form spheroids 250-300 microns in diameter. Spheroids of nontransfected C6 cells produced large gliomas. Immunohistochemical and in situ hybridization analyses revealed relatively high levels of connexin43 protein and mRNA in the host tissue, while little of this protein was detected in the glioma. In contrast, spheroids of connexin43-transfected cells grew more slowly and exhibited elevated levels of connexin43 protein and mRNA. These findings suggest that the expression of connexin43 may be associated with the control of brain tumor growth in vivo.

Expression of gap junction genes during postnatal neural development.

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.

Regional differences in connexin32 and connexin43 messenger RNAs in rat brain.

The regional distribution of gap junction mRNAs was examined in the adult rat brain. The level of connexin43 mRNA is more abundant than connexin32, being homogeneously distributed throughout different regions of brain. In contrast, there is dramatic heterogeneity in the level of connexin32 mRNA, with the highest level in the hindbrain. These results suggest that the gap junction genes are differentially expressed in regions of the adult rat brain.

Effects of fluorescent brighteners on growth and morphology of the red alga Antithamnion kylinii.

Four fluorescent brighteners (Fluorescent Brightener 28, Fluostain 1, Fluostain II and Cellufluor) were examined with respect to their binding affinity, toxicity (their ability to stunt growth), and teratogenic effects on the red alga Antithamnion kylinii. Maximum binding occurred with FB-28 and F-II but these stains showed the greatest inhibition of growth when plants were exposed to concentrations of 0.01% for 30 min. Filaments incubated in low stain concentrations (0.0005%) showed cell abnormalities with all stain types, with FB-28 producing the most extreme deformations of both intercalary and apical cells. The experiments suggest that extensive experimentation is required to develop protocols for vital cell wall stains that minimize toxicity and maximize binding.