David A. Talmage

Cysteine-rich domain isoforms of the Neuregulin-1 gene are required for maintenance of peripheral synapses

Neuregulin-1 (NRG-1) signaling has been implicated in inductive interactions between pre- and postsynaptic partners during synaptogenesis. We used gene targeting to selectively disrupt cysteine-rich domain-(CRD-) containing NRG-1 isoforms. In CRD-NRG-1-/-mice, peripheral projections defasciculated and displayed aberrant branching patterns within their targets. Motor nerve terminals were transiently associated with broad bands of postsynaptic ACh receptor (AChR) clusters. Initially, Schwann cell precursors accompanied peripheral projections, but later, Schwann cells were absent from axons in the periphery. Following initial stages of synapse formation, sensory and motor nerves withdrew and degenerated. Our data demonstrate the essential role of CRD-NRG-1-mediated signaling for coordinating nerve, target, and Schwann cell interactions in the normal maintenance of peripheral synapses, and ultimately in the survival of CRD-NRG-1-expressing neurons.

Integration of endocannabinoid and leptin signaling in an appetite-related neural circuit.

Recently developed therapeutics for obesity, targeted against cannabinoid receptors, result in decreased appetite and sustained weight loss. Prior studies have demonstrated CB1 receptors (CB1Rs) and leptin modulation of cannabinoid synthesis in hypothalamic neurons. Here, we show that depolarization of perifornical lateral hypothalamus (LH) neurons elicits a CB1R-mediated suppression of inhibition in local circuits thought to be involved in appetite and natural reward. The depolarization-induced decrease in inhibitory tone to LH neurons is blocked by leptin. Leptin inhibits voltage-gated calcium channels in LH neurons via the activation of janus kinase 2 (JAK2) and of mitogen-activated protein kinase (MAPK). Leptin-deficient mice are characterized by both an increase in steady-state voltage-gated calcium currents in LH neurons and a CB1R-mediated depolarization-induced suppression of inhibition that is 6-fold longer than that in littermate controls. Our data provide direct electrophysiological support for the involvement of endocannabinoids and leptin as modulators of hypothalamic circuits underlying motivational aspects of feeding behavior.

Phosphorylation of middle T by pp60c-src: A switch for binding of phosphatidylinositol 3-kinase and optimal tumorigenesis

Substitution of phenylalanine for tyrosine 315 of the polyoma virus middle T (mT) protein lowers the incidence and limits the spectrum of tumors induced following inoculation of the virus into newborn mice. This substitution removes the major site of phosphorylation by pp60c-src without altering the ability of mT to associate with or to activate pp60c-src. The mutant mT fails to show binding of a phosphatidylinositol 3-kinase (Ptdlns 3-kinase) activity that is normally present in wild-type mT complexes. Furthermore, an anti-peptide antiserum that specifically recognizes mT lacking phosphate at tyrosine 315 precipitates binary (mT-pp60c-src) but not ternary (mT-pp60c-src-Ptdlns 3-kinase) complexes from wild-type infected cell extracts. Reprecipitation with either anti-pp60c-src or anti-mT serum brings down ternary complexes containing mT phosphorylated on tyrosine 315. Phosphorylation of mT by pp60c-src in vivo is therefore a critical event for binding of Ptdlns 3-kinase and for expression of the full tumorigenic potential of the virus.

Retinoic acid induced growth arrest of human breast carcinoma cells requires protein kinase Cα expression and activity

Retinoic acid inhibits proliferation of hormone‐dependent, but not hormone‐independent breast cancer cells. Retinoic acid‐induced changes in cellular proliferation and differentiation are associated with disturbances in growth factor signaling and frequently with changes in protein kinase C expression. PKCδ, ϵ, and ζ are expressed in both hormone‐dependent (T‐47D) and hormone‐independent (MDA‐MB‐231) cell lines. Retinoic acid arrested T‐47D proliferation, induced PKCα expression and concomitantly repressed PKCζ expression. The changes in PKCα and PKCζ reflect retinoic acid‐induced changes in mRNA. In contrast, retinoic acid had no effect on growth, or PKC expression in MDA‐MB‐231 cells. Growth arrest and the induction of PKCα, but not the reduction in PKCζ, resulted from selective activation of RARα. In total, these results support an important role for PKCα in mediating the anti‐proliferative action of retinoids on human breast carcinoma cells. J. Cell. Physiol. 172:306–313, 1997.

Separation of Host Range from Transformation Functions of the hr-t Gene of Polyomavirus

hr-t mutants of polyomavirus are defective in virus growth as well as in cell transformation, and have genetic alterations that invariably affect both the middle and small T proteins. We have examined the growth properties of three site-directed mutants that either eliminate or alter the middle T without affecting the small T protein. Mutant 808A encodes large and small T proteins but no middle T; it grew poorly in NIH 3T3 cells. In contrast, mutants 1387T and 1178T which express altered middle T along with normal large and small T proteins grew nearly as well as wild-type virus. Thus, although the altered middle T proteins encoded by 1387T and 1178T are defective for cell transformation, they retained the ability to induce expression of a cellular permissivity factor(s) required for virus production. At the biochemical level, the induction of permissivity by middle T was manifested primarily in terms of phosphorylation of VP1 on threonine and in efficient encapsidation of viral DNA to form infectious virus. The natural role of middle T involves regulation of phosphorylation events, and can be enacted, at least in part, independently of interactions with pp60c-src.

Cholinergic circuits and signaling in the pathophysiology of schizophrenia.

Central cholinergic signaling has long been associated with aspects of memory, motivation, and mood, each affected functions in neuropsychiatric disorders such as schizophrenia. In this chapter, we review evidence related to the core hypothesis that dysregulation of central cholinergic signaling contributes to the pathophysiology of schizophrenia. Although central cholinergic circuits are resistant to simplification-particularly when one tries to parse the contributions of various classes of cholinergic receptors to disease related phenomena--the potential role of ACh signaling in Schizophrenia pathophysiology deserves careful consideration for prospective therapeutics. The established role of cholinergic circuits in attentional tuning is considered along with recent work on how the patterning of cholinergic activity may modulate corticostriatal circuits affected in schizophrenia.

Heterogeneity in state and expression of viral DNA in polyoma virus-induced tumors of the mouse

We have examined the state and expression of polyoma viral DNA in representative epithelial and mesenchymal tumors, using a combination of biochemical and in situ methods. Results showed wide variations among tumor types and also in different regions within individual tumors, with respect to copy number of viral DNA, presence or absence of deletions, and expression of early and late viral proteins. Epithelial tumors showed the greatest heterogeneity. High copy free viral DNA, frequently with deletions, was found in all such tumors. A portion of free viral DNA was recoverable as transcriptionally active minichromosomes. Three distinct subpopulations of cells were distinguished by in situ analyses. Type 1 cells showed high copy free viral DNA and expressed the major viral capsid protein VP1; these cells appeared to be at various stages of productive (lytic) viral infection. Some productively infected cells were able to undergo mitosis; in a portion of these cells, VP1 was found in close association with the mitotic spindle. Type 2 cells contained high copy free DNA but did not express VP1; by some unknown mechanism, these cells manifest a post-replication block to late gene expression and lytic infection. Type 3 cells contained only low copy, presumably integrated, viral DNA and expressed no VP1; they thus resemble cells transformed in vitro by the virus. Epithelial tumors contained variable mixtures of these subpopulations, while mesenchymal tumors were composed of Type 3 cells only. Differences in virus-cell interactions are discussed in terms of their possible implications in tumor development.

Retinoic acid inhibits transformation by preventing phosphatidylinositol 3-kinase dependent activation of the c-fos promoter.

Retinoic acid inhibits transformation of cells by polyoma virus middle T oncoprotein. Inhibition of transformation results from a retinoic acid-dependent failure of cells to fully express the c-fos proto-oncogene. Retinoic acid prevents transactivation of the c-fos promoter by disrupting signaling between tyrosine kinases at the plasma membrane and trans-acting factors at the c-fos promoter. We used complementary genetic, biochemical and molecular approaches to demonstrate that: (1) phosphatidylinositol 3-kinase signaling is the principle mechanism of polyoma virus middle T oncoprotein activation of c-fos expression; (2) middle T/phosphatidylinositol 3-kinase transactivation of the c-fos promoter and transformation of cells requires activation of both the small GTP-binding protein Rac and Jun N-terminal kinase; (3) retinoic acid inhibits activation of Jun N-terminal kinase, thereby preventing c-fos transactivation and transformation; and (4) middle T activation of c-fos transcription requires both the serum response element and the promoter proximal cyclic AMP response element. These studies identify a novel target through which retinoids prevent oncogenic transformation.

Apolipoprotein E Inhibits Serum-stimulated Cell Proliferation and Enhances Serum-independent Cell Proliferation

Independently of its role in lipid homeostasis, apolipoprotein E (apoE) inhibits cell proliferation. We compared the effects of apoE added to media (exogenous apoE) with the effects of stably expressed apoE (endogenous apoE) on cell proliferation. Exogenous and endogenous apoE increased population doubling times by 30–50% over a period of 14 days by prolonging the G1 phase of the cell cycle. Exogenous and endogenous apoE also decreased serum-stimulated DNA synthesis by 30–50%. However, apoE did not cause cell cycle arrest; both apoE-treated and control cells achieved equivalent saturation densities at 14 days. Further analyses demonstrated that exogenous and endogenous apoE prevented activation of MAPK but not induction of c-fosexpression in response to serum growth factors. Endogenous (but not exogenous) apoE altered serum concentration-dependent effects on proliferation. Whereas control (non-apoE-expressing) cell numbers increased with increasing serum concentrations (1.6-fold for every 2-fold increase in serum), apoE-expressing cell numbers did not differ as serum levels were raised from 2.5 to 10%. In addition, in low serum (0.1%), apoE-expressing cells had elevated DNA synthesis levels compared with control cells. We conclude that apoE does not simply inhibit cell proliferation; rather, the presence of apoE alters the response to and requirement for serum mitogens.

Polyomavirus middle T antigen induces ribosomal protein S6 phosphorylation through pp60c-src-dependent and -independent pathways.

Phosphorylation of ribosomal protein S6 is elevated in polyomavirus-infected cells. This elevation results only in part from activation of S6 kinase activity. These effects appear to reflect independent activities of wild-type middle T antigen. Hr-t mutant NG59, encoding a defective middle T protein, and mutant Py808A, encoding no middle T protein, were unable to induce S6 kinase activity or elevate S6 phosphorylation. Two other site-directed mutants encoding altered middle T proteins did elevate S6 phosphorylation while only weakly stimulating S6 kinase activity. These results suggest at least two independent pathways leading to elevation of S6 phosphorylation. One pathway leads to induction of S6 kinase activity following activation of pp60c-src by transformation-competent middle T antigen. Another pathway operates independently of S6 kinase induction and can be regulated by transformation-defective middle T mutants such as Py1387T. This mutant, encoding a truncated middle T protein that failed to associate with the plasma membrane and to activate pp60c-src, caused increased levels of S6 phosphorylation without detectably increasing S6 kinase activity. The ability of mutants such as Py1387T to induce S6 phosphorylation correlated with their ability to increase phosphorylation of VP1, an event linked to maturation of infectious virions.