Mario Vallejo

Network-Level Changes in Expression of Inducible Fos-Jun Proteins in the Striatum during Chronic Cocaine Treatment and Withdrawal

Repeated exposure to psychomotor stimulants produce long-term changes in behavior ranging from addiction to behavioral sensitization. Many of these behaviors depend on the nigrostriatal system of the basal ganglia. We show here that chronic cocaine exposure not only leads to time-varying alterations in the inducibility of bZIP transcription factors in individual striatal neurons, but also to long-lasting network changes in which ensembles of striatal neurons express these proteins. These network-level adaptations suggest that the behavioral sensitization induced by repeated psychomotor stimulant exposure may reflect an enduring functional reorganization of basal ganglia circuits.

D1-class dopamine receptors influence cocaine-induced persistent expression of Fos-related proteins in striatum.

CHRONIC intermittent exposure to psychomotor stimulants induces in the striatum the expression of Fos- related proteins (Fras) that persist after the end of drug treatment. We carried out experiments to determine whether such Fras (‘chronic Fras’) require dopamine D1-class receptor function for their persistent expression in the striatum. We chronically administered cocaine to rats in a behavioral sensitization protocol and blocked D1- class receptors with SCH23390 before a final cocaine challenge. Western blotting and immunohistochemical analyses indicate that Fras persistently expressed in response to chronic treatment include proteins of two types: those that have become independent of D1-class dopamine receptor activation and those that remain dependent on D1-class receptors for their expression following drug challenge.

cAMP-Dependent Regulation of Gene Transcription by cAMP Response Element-Binding Protein and cAMP Response Element Modulator

Publisher Summary This chapter discusses cyclic AMP (cAMP)-dependent regulation of gene transcription by cAMP response element-binding protein and cAMP response element modulator. The cAMP signaling pathway is one of the most important intracellular signal transduction pathways in eukaryotic cells. In the absence of cAMP, the inactive protein kinase A (PKA) holoenzyme is a heterotetrameric protein complex consisting of two regulatory subunits (R) and two catalytic subunits (C). Two classes of R subunits, each with two isoforms, and three isoforms of the C subunits are described in the chapter. cAMP binds cooperatively to two sites on the R subunits within the holoenzyme, thereby liberating free active C subunits. Regulation of gene transcription is accomplished by the interaction of DNA-binding proteins with DNA regulatory sequences and with proteins of the general transcription machinery. Variant cAMP response elements (CREs) include asymmetrical or atypical sequences that differ from the consensus motif by single or multiple nucleotide deletions or substitutions. It is found that the interplay between the phosphorylating protein kinases and the dephosphorylating protein phosphatases is critical in determining the relative transactivation potential at any given moment.

Nuclear factor-I regulates glial fibrillary acidic protein gene expression in astrocytes differentiated from cortical precursor cells

The elucidation of the transcriptional mechanisms that regulate glial fibrillary acidic protein (GFAP) gene expression is important for the understanding of the molecular mechanisms that control astrocyte differentiation during brain development. We investigated regulatory elements located in a proximal region of the GFAP promoter, important for expression in cortical precursor cells differentiating into astrocytes. One of these elements recognizes transcription factors of the nuclear factor‐I family (NFI). We found that, in primary cultures of cortical cells, NFI occupies the GFAP promoter prior to the induction of astrocyte differentiation. In the developing cerebral cortex, the onset of expression of NFI coincides chronologically with the beginning of astrocytogenesis. Mutational analysis of the GFAP gene and transfections in primary cortical precursors show that inhibition of binding of NFI to the GFAP promoter results in decreased levels of transcriptional activity and is required for the synergistic stimulation of the GFAP promoter by the astrogenic agents, pituitary adenylate cyclase‐activating polypeptide and ciliary neurotrophic factor, which in combination enhance astrocyte differentiation to generate astrocytes with longer processes. Thus, NFI appears to be an important factor for the integration of astrogenic stimuli in the developing central nervous system.

ESSENTIAL ROLE OF PROTEIN TYROSINE PHOSPHATASE 1B IN OBESITY-INDUCED INFLAMMATION AND PERIPHERAL INSULIN RESISTANCE DURING AGING

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes (T2DM). In this study, we have evaluated the role of PTP1B in the development of aging‐associated obesity, inflammation, and peripheral insulin resistance by assessing metabolic parameters at 3 and 16 months in PTP1B−/− mice maintained on mixed genetic background (C57Bl/6J × 129Sv/J). Whereas fat mass and adipocyte size were increased in wild‐type control mice at 16 months, these parameters did not change with aging in PTP1B−/− mice. Increased levels of pro‐inflammatory cytokines, crown‐like structures, and hypoxia‐inducible factor (HIF)‐1α were observed only in adipose tissue from 16‐month‐old wild‐type mice. Similarly, islet hyperplasia and hyperinsulinemia were observed in wild‐type mice with aging‐associated obesity, but not in PTP1B−/− animals. Leanness in 16‐month‐old PTP1B−/− mice was associated with increased energy expenditure. Whole‐body insulin sensitivity decreased in 16‐month‐old control mice; however, studies with the hyperinsulinemic–euglycemic clamp revealed that PTP1B deficiency prevented this obesity‐related decreased peripheral insulin sensitivity. At a molecular level, PTP1B expression and enzymatic activity were up‐regulated in liver and muscle of 16‐month‐old wild‐type mice as were the activation of stress kinases and the expression of p53. Conversely, insulin receptor‐mediated Akt/Foxo1 signaling was attenuated in these aged control mice. Collectively, these data implicate PTP1B in the development of inflammation and insulin resistance associated with obesity during aging and suggest that inhibition of this phosphatase by therapeutic strategies might protect against age‐dependent T2DM.

Pituitary adenylate cyclase-activating polypeptide induces astrocyte differentiation of precursor cells from developing cerebral cortex

Ciliary neurotrophic factor and bone morphogenetic proteins induce astrocytogenesis in the developing rat brain by stimulating STAT- and Smad-dependent signaling, respectively. We previously found that stimulation of the cAMP-dependent signaling pathway also triggers differentiation of cerebral cortical precursor cells into astrocytes, providing an additional mechanism to promote astrocyte differentiation. In this study, we show that pituitary adenylate cyclase-activating polypeptide (PACAP), but not the related vasoactive intestinal peptide, induces astrocyte differentiation of cortical precursor cells, even after a transient exposure. Cortical precursors were found to express predominantly the short isoform of the PACAP-specific PAC1 receptor, which couples to adenylate cyclase. Consistent with this notion, we determined that exposure of cortical precursors to PACAP resulted in a dose-dependent increase in cAMP production. Pretreatment of cells with the cAMP antagonist Rp-cAMPS prevented astrocyte differentiation. Thus, PACAP acts as an extracellular signal to trigger cortical precursor cell differentiation into astrocytes via stimulation of intracellular cAMP production.

Activation of DREAM (Downstream Regulatory Element Antagonistic Modulator), a Calcium-Binding Protein, Reduces L-DOPA-Induced Dyskinesias in Mice

BACKGROUND Previous studies have implicated the cyclic adenosine monophosphate/protein kinase A pathway as well as FosB and dynorphin-B expression mediated by dopamine D1 receptor stimulation in the development of 3,4-dihydroxyphenyl-L-alanine (L-DOPA)-induced dyskinesia. The magnitude of these molecular changes correlates with the intensity of dyskinesias. The calcium-binding protein downstream regulatory element antagonistic modulator (DREAM) binds to regulatory element sites called DRE in the DNA and represses transcription of target genes such as c-fos, fos-related antigen-2 (fra-2), and prodynorphin. This repression is released by calcium and protein kinase A activation. Dominant-active DREAM transgenic mice (daDREAM) and DREAM knockout mice (DREAM(-/-)) were used to define the involvement of DREAM in dyskinesias. METHODS Dyskinesias were evaluated twice a week in mice with 6-hydroxydopamine lesions during long-term L-DOPA (25 mg/kg) treatment. The impact of DREAM on L-DOPA efficacy was evaluated using the rotarod and the cylinder test after the establishment of dyskinesia and the molecular changes by immunohistochemistry and Western blot. RESULTS In daDREAM mice, L-DOPA-induced dyskinesia was decreased throughout the entire treatment. In correlation with these behavioral results, daDREAM mice showed a decrease in FosB, phosphoacetylated histone H3, dynorphin-B, and phosphorylated glutamate receptor subunit, type 1 expression. Conversely, genetic inactivation of DREAM potentiated the intensity of dyskinesia, and DREAM(-/-) mice exhibited an increase in expression of molecular markers associated with dyskinesias. The DREAM modifications did not affect the kinetic profile or antiparkinsonian efficacy of L-DOPA therapy. CONCLUSIONS The protein DREAM decreases development of L-DOPA-induced dyskinesia in mice and reduces L-DOPA-induced expression of FosB, phosphoacetylated histone H3, and dynorphin-B in the striatum. These data suggest that therapeutic approaches that activate DREAM may be useful to alleviate L-DOPA-induced dyskinesia without interfering with the therapeutic motor effects of L-DOPA.

Experimental evidence does not support use of the "no-touch" isolation technique in colorectal cancer.

PURPOSE: The benefits of the “no-touch” isolation technique usually performed to prevent the circulation of tumor cells are not evident. The aim of this study was to determine whether conventional surgical procedures for treatment of colon cancer could provoke the circulation of tumor cells detected by a genetic technology. METHODS: Sixteen patients undergoing resection for colorectal cancer and two patients with irresectable tumors were studied. No patient showed liver or lung metastasis. With specific primers for carcinoembryonic antigen, we used reverse transcriptasepolymerase chain reaction to analyze tumor biopsy specimens and blood samples obtained from the antecubital vein before and after surgery and from the main drainage vein of the tumor when the tumor had been extracted. Peritoneal fluid was also collected in irrecsectable cases. RESULTS: Amplification of cDNA with carcinoembryonic antigen-specific primers was achieved with all tumor biopsies and samples of peritoneal fluid. In two patients carcinoembryonic antigen reverse transcriptase-polymerase chain reaction products were detected in antecubital vein blood before surgery and in one of them also after surgery. Only in one patient (Dukes C) were carcinoembryonic antigen reverse transcriptase-polymerase chain reaction products detected from the main drainage vein of the tumor. In serial dilution experiments we determined that the limit of detection of this method was ten tumor cells in 2 ml of blood. CONCLUSION: Our data suggest that the use of no-touch isolation techniques in colorectal cancer is not justified, based on lack of evidence indicating the detachment of cells from the tumor at surgery.

DREAM mediates cAMP-dependent, Ca2+-induced stimulation of GFAP gene expression and regulates cortical astrogliogenesis

In the developing mouse brain, once the generation of neurons is mostly completed during the prenatal period, precisely coordinated signals act on competent neural precursors to direct their differentiation into astrocytes, which occurs mostly after birth. Among these signals, those provided by neurotrophic cytokines and bone morphogenetic proteins appear to have a key role in triggering the neurogenic to gliogenic switch and in regulating astrocyte numbers. In addition, we have reported previously that the neurotrophic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) is able to promote astrocyte differentiation of cortical precursors via activation of a cAMP-dependent pathway. Signals acting on progenitor cells of the developing cortex to generate astrocytes activate glial fibrillary acidic protein (GFAP) gene expression, but the transcriptional mechanisms that regulate this activation are unclear. Here, we identify the previously known transcriptional repressor downstream regulatory element antagonist modulator (DREAM) as an activator of GFAP gene expression. We found that DREAM occupies specific sites on the GFAP promoter before and after differentiation is initiated by exposure of cortical progenitor cells to PACAP. PACAP raises intracellular calcium concentration via a mechanism that requires cAMP, and DREAM-mediated transactivation of the GFAP gene requires the integrity of calcium-binding domains. Cortical progenitor cells from dream−/− mice fail to express GFAP in response to PACAP. Moreover, the neonatal cortex of dream−/− mice exhibits a reduced number of astrocytes and increased number of neurons. These results identify the PACAP-cAMP-Ca2+-DREAM cascade as a new pathway to activate GFAP gene expression during astrocyte differentiation.

Developmental mechanisms of stripe patterns in rodents

Mammalian colour patterns are among the most recognizable characteristics found in nature and can have a profound impact on fitness. However, little is known about the mechanisms underlying the formation and subsequent evolution of these patterns. Here we show that, in the African striped mouse (Rhabdomys pumilio), periodic dorsal stripes result from underlying differences in melanocyte maturation, which give rise to spatial variation in hair colour. We identify the transcription factor ALX3 as a regulator of this process. In embryonic dorsal skin, patterned expression of Alx3 precedes pigment stripes and acts to directly repress Mitf, a master regulator of melanocyte differentiation, thereby giving rise to light-coloured hair. Moreover, Alx3 is upregulated in the light stripes of chipmunks, which have independently evolved a similar dorsal pattern. Our results show a previously undescribed mechanism for modulating spatial variation in hair colour and provide insights into how phenotypic novelty evolves.