Transcriptional landscapes at the intersection of neuronal apoptosis and substance P-induced survival: exploring pathways and drug targets.

A change in the delicate equilibrium between apoptosis and survival regulates the neurons fate during the development of nervous system and its homeostasis in adulthood. Signaling pathways promoting or protecting from apoptosis are activated by multiple signals, including those elicited by neurotrophic factors, and depend upon specific transcriptional programs. To decipher the rescue program induced by substance P (SP) in cerebellar granule neurons, we analyzed their whole-genome expression profiles after induction of apoptosis and treatment with SP. Transcriptional pathways associated with the survival effect of SP included genes encoding for proteins that may act as pharmacological targets. Inhibition of one of these, the Myc pro-oncogene by treatment with 10058-F4, reverted in a dose-dependent manner the rescue effect of SP. In addition to elucidate the transcriptional mechanisms at the intersection of neuronal apoptosis and survival, our systems biology-based perspective paves the way towards an innovative pharmacology based on targets downstream of neurotrophic factor receptors.

Cirrhotic patients are still at risk of developing hepatocellular carcinoma despite Interferon-induced sustained virological response.

INTRODUCTION: Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and hepatocellular carcinoma (HCC). The prevalence of HCC significantly declines among patients achieving a sustained virological response (SVR) after antiviral therapy with pegylated(PEG)-interferon (IFN) and ribavirin. However, up to 5% of patients with SVR may develop HCC. PATIENTS AND METHODS: We investigated the epidemiological, clinical, biochemical and virological characteristics of a small cohort of patients with chronic hepatitis C (CHC) who developed HCC after being successfully treated with PEG-IFN-α and ribavirin. RESULTS: Between September 2000 and January 2003, 598 patients with CHC underwent a complete course of treatment with PEG-IFN-α and ribavirin; 221 out of 598 (37%) patients obtained a SVR. Throughout the 10-year post-treatment follow up, 13 of 221 ( 5.8% ) SVR patients developed HCC. All 13 patients were male and were affected with Child A liver cirrhosis; in addition, at baseline they were significantly older (p < 0.05) and had higher alpha-fetoprotein levels (p < 0.05) in comparison with those who did not develop HCC. Nine patients (69.3%) developed HCC within the first 3 years after antiviral treatment completion, one patient (7.7%) between 3 and 5 years and 3 subjects (23%) between 5 and 10 years; 12 of 13 had a solitary lesion with a mean diameter of 2.5± 0.5 cm. Eleven cases (84.6%) underwent surgical resection, one (7.7%) received liver transplantation, one (7.7%) received palliative care. CONCLUSIONS: The risk of developing HCC after achieving SVR persists in patients with HCV-related cirrhosis. As a consequence, these patients should continue to undergo long-term surveillance for HCC, in order to early detect and treat it.

[Skeletal muscle: from development to function]. / Muscolo scheletrico: dallo sviluppo alla funzione.

Aim of this minireview was to focus attention on some aspects of myogenesis, differentiation and metabolism of muscle in mammals (rodents and humans), and to summarize current knowledge on the issue referring to both our experimental contributions and those of other authors.Particular attention was directed at both the mesodermal stem cells, elements of the early embryonic period, and the regulatory genes. It was also discussed the crucial role of several growth factors, such as myogenin and bFGF, and substances of vital importance to the regulation of contraction: somatomedins [Insulin-like Growth Factors (IGF-I and IGF-II)], interleukin-15 (IL -15), transferrin (Tf), catecholamines (CA). A no secondary role in the metabolism of skeletal muscle is played by serotonin (5-HT) and its precursor [L-tryptophan (L-Tp)]. The presence of L-Tp, in the diet of growing rats, seems to be crucial for protein synthesis and for the increase in the number of mitochondria. It has also highlighted the role played by certain hormones: growth hormone (GH), thyroid hormones (T3 and T4), sex steroids on metabolism, growth and differentiation of skeletal muscle.

Dopamine D3 receptor deletion increases tissue plasminogen activator (tPA) activity in prefrontal cortex and hippocampus.

Considerable evidence indicates that dopamine (DA) influences tissue plasminogen activator (tPA)-mediated proteolytic processing of the precursor of brain-derived neurotrophic factor (proBDNF) into mature BDNF (mBDNF). However, specific roles in this process for the dopamine D3 receptor (D3R) and the underlying molecular mechanisms are yet to be fully characterized. In the present study, we hypothesized that D3R deletion could influence tPA activity in the prefrontal cortex and hippocampus. Using D3R knockout (D3(-/-)) mice, we show that receptor inactivation is associated with increased tPA expression/activity both in the prefrontal cortex and, to a greater extent, in the hippocampus. Augmented tPA expression in D3(-/-) mice correlated with increased BDNF mRNA levels, plasmin/plasminogen protein ratio and the conversion of proBDNF into mBDNF, as well as enhanced tPA and mBDNF immunoreactivity, as determined by quantitative real time polymerase chain reaction (qRT-PCR), immunoblot and immunohistochemistry. In addition, when compared to wild-type controls, D3(-/-) mice exhibited increased basal activation of the canonical cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-driven Akt/cAMP-response element-binding protein (CREB) signaling cascade, as determined by the increased Akt phosphorylation both at Thr304 and Ser473 residues, of DA and cAMP-regulated protein of 32kDa (DARPP-32) at Thr34 and a phosphorylation state-dependent inhibition of glycogen synthetase kinase-3ß (GSK-3ß) at Ser9, a substrate of Akt whose constitutive function impairs normal CREB transcriptional activity through phosphorylation at its Ser129 residue. Accordingly, CREB phosphorylation at Ser133 was significantly increased in D3(-/-) mice, whereas the GSK-3ß-dependent phosphorylation at Ser129 was diminished. Altogether, our finding reveals that mice lacking D3Rs show enhanced tPA proteolytic activity on BDNF which may involve, at least in part, a potentiated Akt/CREB signaling, possibly due to hindered GSK-3ß activity.

Epidermal growth factor receptor (EGFR) and neuregulin (Neu) activation in human airway epithelial cells exposed to nickel acetate.

Nickel compounds are potential carcinogenic agents that produce a range of biological effects, including inhibition of cell death. Because suppression of apoptosis is thought to contribute to the initiation of carcinogenesis, we investigated the effects of nickel acetate (Ni(2+)) treatment on apoptosis in two different airway epithelial cell lines (A549 and Beas-2B, respectively). Furthermore, since both the epidermal growth factor receptor (EGFR) and neuregulin (Neu) are involved in neoplastic development, mRNAs and expression levels of total and phosphorylated proteins (p-EGFR(Tyr1173) and p-Neu(Tyr1248), respectively) were also measured. We found that exposure of A549 cells to Ni(2+) resulted in significantly reduced cell viability, as well as increased apoptosis and DNA fragmentation at relatively low concentrations (0.1 and 0.5mM) after 24 and 48h. These changes were accompanied by reduced EGFR and Neu mRNAs and proteins, phosphorylated proteins as well as decreased Bcl-2 and increased BAX protein expression. Conversely, Beas-2B cells exposed to equivalent concentrations of Ni(2+) did not show evident signs of apoptosis and DNA damage, hence showing increased expression and phosphorylation of both EGFR and Neu, increased Bcl-2 and reduced BAX expression. Altogether, our finding indicate that Ni(2+) exposure differently affects apoptosis initiation either in non-tumorigenic (Beas-2B) and tumorigenic airway epithelial cells (A549), suggesting a potential involvement of EGFR/Neu receptors.

Protective effect of the dopamine D(3) receptor agonist (7-OH-PIPAT) against apoptosis in malignant peripheral nerve sheath tumor (MPNST) cells.

Emerging evidence indicates that the dopamine D(3) receptor (D(3)R) mediates protective roles both in neuronal and non-neuronal cell lines. In a previous study we proposed that neurofibromin, a large tumor suppressor protein encoded by the neurofibromatosis type 1 gene (NF1), may increase susceptibility to apoptosis after serum deprivation in malignant peripheral nerve sheath tumor (MPNST) cells, thus acting as a proapoptotic gene. In addition, it has been observed that D(3)Rs are functionally correlated to neurofibromin. In this study, we examined whether 7-OH-PIPAT, a potent dopamine D(3)R agonist, exerts an antiapoptotic role under the same culture conditions and then correlated this effect to changes in NF1 expression. Results showed that serum deprivation caused a significant reduction of cell viability (MTT assay) both after 24 and 48 h (p<0.001). Treatment with increasing concentrations of 7-OH-PIPAT (10(-9)-10(-5) M) induced a progressive increase in cell viability both after 24 and 48 h as compared to vehicle-treated cells, with significant changes at the highest concentrations tested (10(-6) and 10(-5) M). Consistently, at the latter two concentrations, a significant reduction in oligonucleosomes formation was observed, thus suggesting an antiapoptotic role of 7-OH-PIPAT. These results were confirmed by Hoechst 33254 nuclear staining. To investigate whether these effects were correlated to changes in NF1 transcript and protein expression, quantitative real-time PCR, Western blot and immunofluorescence analyses were performed. Results demonstrated that the upregulation of NF1 transcripts and protein levels induced by serum withdrawal were remarkably attenuated by 10(-6) and 10(-5) M agonist treatment within 24 h (p<0.01 and p<0.001, respectively), whereas similar effects were observed already at a lower concentration (10(-7) M) after 48 h treatment (p<0.001). In conclusion, these results suggest that D(3)R might mediate the protective response to serum deprivation in MPNST cells through the inhibition of NF1 gene expression, further underlying a subtle role of these receptors in MPNST development.

Expression profile of ErbB receptor's family in human alveolar type 2-like cell line A549 exposed to hexavalent chromium.

Occupational exposure to hexavalent chromium (Cr (VI)) compounds is associated with increased risk of pulmonary disease. In the present study we have investigated temporal expression of ErbB's receptors family in A549 cells after exposure to Cr (VI). Treatment with 10 microM or 300 microM of Na2CrO4 induced apoptotic cell death within 24h. Based on data obtained by ELISA cell death detection method and fluorescence microscopy, the concentration of 10 microM was chosen to study the expression of ErbB receptors family. Such concentration reflects a condition of acute toxicity in which cells survived up to 24h. Real time quantitative PCR has been performed to analyze the expression profiles of ErbB family genes following chromium toxicity. The expression of EGFR and ErbB2 receptors was significantly reduced after 1h and 4h of treatment while ErbB2 receptor was significantly increased and EGFR receptor returned to basal value after 24h. Instead, ErbB3 receptor was overexpressed after 1h, returned to basal level after 4h and increased its level after 24h. Exposure to chromium did not change expression level of ErbB4 receptor in A549 cell line. The present data suggests that expression changes in ErbB receptors might have a role in the carcinogenic effects induced by this pneumotoxic agent.

Down regulation of cerebellar memory related gene-1 following classical conditioning.

We have isolated and characterized the mRNA of a mouse gene named cerebellar memory related gene-1, previously found by microarray analysis to be differentially expressed following classical conditioning of the rabbit nictitating membrane response. Quantitative RT-PCR analysis showed a significant reduction in mRNA expression in cerebellar lobule HVI but not in the hippocampus of rabbits that received classical conditioning compared to control rabbits that received either unpaired stimulus presentations or were simply restrained. The mouse mRNA encodes a protein of 485 amino acids that includes different potential post-translational modification sites and five copies of the WD-repeat suggesting involvement in protein-protein interaction and regulatory function. In-situ hybridization experiments show highly localized expression of the transcript in mouse brain with the highest expression levels located in the cerebellum, hippocampus and cortex. Taken together, our results reveal a novel gene encoding a WD-repeat protein that is down-regulated in cerebellar lobule HVI as a result of learning and memory.

Gene expression profiles during long-term memory consolidation.

Changes in gene expression have been postulated to occur during long-term memory (LTM). We used high-density cDNA microarrays to assess changes in gene expression 24 h after rabbit eye blink conditioning. Paired animals were presented with a 400 ms, 1000 Hz, 82 dB tone conditioned stimulus that coterminated with a 100 ms, 60 Hz, 2 mA electrical pulse unconditioned stimulus. Unpaired animals received the same conditioned and unconditioned stimuli but presented in an explicitly unpaired manner. Differences in expression levels between paired and unpaired animals in the hippocampus and cerebellar lobule HVI, two regions activated during eye blink conditioning, indicated the involvement of novel genes as well as the participation of previously implicated genes. Patterns of gene expression were validated by in situ hybridization. Surprisingly, the data suggest that an underlying mechanism of LTM involves widespread decreased, rather than increased, gene expression. These results demonstrate the feasibility and utility of a cDNA microarray system as a tool for dissecting the molecular mechanisms of associative memory.

Regional and cellular expression of the parkin gene in the rat cerebral cortex.

A mutation in the parkin gene has been identified as the cause for an autosomal recessively inherited form of early onset Parkinson's disease. We have recently isolated the mRNA coding for the rat homologue of parkin and showed its widespread expression in the central nervous system (CNS) by in situ hybridization. In the present study we investigated the distribution of parkin in the rat cerebral cortex with a polyclonal antibody that reacts with a single approximately 52-kDa protein, corresponding to the predicted molecular mass of parkin. Conventional light microscopic studies revealed intense parkin immunoreactivity (IR) throughout the cortex. Examination of mixed cortical neuro-glial cultures by indirect immunofluorescence technique coupled to traditional epifluorescence and confocal microscopy analysis demonstrated the expression of parkin in the cytoplasm and neurites of neurons, and its absence in glial fibrillary acidic protein (GFAP)-positive astrocytes. The predominant neuronal parkin-IR and -mRNA expression was confirmed by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR), respectively, performed on highly enriched neuronal and type I astrocytes cultures. The information gathered in our study about the cellular and subcellular distribution of parkin should facilitate further research on its physiological role in the nervous system.

Cloning and distribution of the rat parkin mRNA.

We have isolated by RT-PCR and sequenced a partial cDNA coding for the rat homolog of parkin, a gene mutated in autosomal recessive juvenile parkinsonism. The 1.46 kb rat cDNA clone contains a 1376 bp coding sequence that shares strong similarity with the human parkin cDNA. RT-PCR and in situ hybridization revealed widespread expression of parkin in the rat brain and the periphery. The availability of the rat parkin cDNA and the initial elucidation of its distribution should facilitate further research on the pathophysiological role of parkin in the nervous system.

Molecular and functional characterization of pituitary adenylate cyclase-activating polypeptide (PACAP-38)/vasoactive intestinal polypeptide receptors in pancreatic beta-cells and effects of PACAP-38 on components of the insulin secretory system.

It has been previously demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) regulates insulin secretion. PACAP exerts its biological action by binding to at least three different receptor subtypes coupled to different signal transduction mechanisms. The signaling pathways underlying the insulinotropic effect of PACAP involve mainly the activation of adenylate cyclase to form cAMP, which directly and indirectly, through increased intracellular Ca2+, stimulates insulin exocytosis. In the present study we have characterized the functional and molecular expression of PACAP/vasoactive intestinal polypeptide receptors isoforms and subtypes and its isoforms in a beta-cell line and in isolated rat pancreatic islets. Although insulinoma cells express the messenger RNA encoding PAC1 (-R and -hop variants), VPAC1 and VPAC2, binding experiments indicate the preponderance of PAC1 over VPAC 1-2 receptors. We have also shown that the main signaling pathway of PACAP in beta-cells is mediated by adenylate cyclase, whereas the inositol 1,4,5-trisphosphate pathway is almost inactive. Furthermore, we have demonstrated that PACAP exerts long-term effects on beta-cells, such as transcriptional regulation of the insulin gene and genes of the glucose-sensing system (GLUT1 and hexokinase 1).

Functional and molecular expression of PACAP/VIP receptors in the rat retina.

Receptor binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), positively coupled to adenylate cyclase, have been previously described in the retina of different mammalian species. In the present study, we determined the mRNA expression of PACAP/VIP receptor variants in the rat retina and investigated their coupling to phospholipase C in addition to adenylate cyclase. The two forms of PACAP, PACAP27 and PACAP38, induced a dose-dependent (1-100 nM) increase of cAMP and [3H]inositol monophosphate levels, whereas VIP stimulated, with lower potency and efficacy, cAMP formation only. Reverse transcription-PCR analysis in the rat retina detected both type-I (PACAP-R and PACAP-HOP splice variants) and type-II (VIP-I and -2) receptor-mRNAs. These data indicate that PACAP and VIP may interact with multiple receptor subtypes and activate one (VIP) or two (PACAP) signal transduction mechanisms in the rat retina.

Cloning and expression of the programmed cell death regulator Bad in the rat brain.

The Bcl-2 family of proteins consists of both antagonists (e.g. Bcl-2) and agonists (e.g. Bax) that regulate apoptosis and compete through dimerization. In the present study we cloned the cDNA encoding the rat brain BAD, a distant member of the Bcl-2 family that was shown to promote cell death. The cloned cDNA encoded a protein of 205 amino acids, containing three putative Bcl-2 homology domains (BH1, BH2 and BH3) and no C-terminal signal-anchor sequence. The predicted amino acid sequence was identical to the Bad-cDNA recently cloned from the rat ovary with the exception of a stretch of six amino acids, thus indicating the existence of two Bad alternative splice variants or a sequence artifact in the rat ovary Bad-cDNA. Immunohistochemical analysis in the rat brain revealed the exclusive expression of Bad in the epithelial cells of the choroid plexus, a result which is consistent with a very specialized function of Bad in the brain.

Pituitary adenylate cyclase activating polypeptide prevents apoptosis in cultured cerebellar granule neurons.

The two forms of pituitary adenylate cyclase-activating polypeptide, PACAP27 and PACAP38, are two neuropeptide hormones related to the vasoactive intestinal peptide/secretin/ glucagon family of peptides. PACAP receptors that are positively coupled to adenylyl cyclase and phospholipase C have been identified in cultured cerebellar granule cells. Using the reverse transcription-polymerase chain reaction methodology, we demonstrated the expression of the PACAP-R and PACAP-R-hop mRNAs in cultured granule cells. When grown in the absence of serum or in low K+ concentrations, these neurons underwent apoptosis, a naturally occurring process characterized by cell shrinkage and internucleosomal DNA cleavage. We used these models of programmed cell death to study the relationship between PACAP receptor activation and neuronal apoptosis. Treatment with PACAP27 and PACAP38 reduced the development of apoptosis in a dose-dependent manner. The neuroprotective activity of PACAP was mimicked by high concentrations of vasoactive intestinal peptide or forskolin but not by carbamylcholine. Thus, we suggest that the activation of type I PACAP receptors may contribute to the survival of cerebellar granule neurons.

Tissue-specific and developmental expression of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors in rat brain.

The two forms of pituitary adenylate cyclase-activating polypeptide, PACAP27,and PACAP38, are novel members of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of peptides. PACAP receptors that are positively coupled to adenylate cyclase and phospholipase C have been recently identified. We examined the expression of PACAP receptors in the rat cortex, hippocampus, cerebellum and hypothalamus during postnatal development. Functional studies revealed PACAP stimulation of cAMP formation in all the brain areas examined and [3H]inositol monophosphate ([3H]InsP) accumulation only in the cerebellum and hypothalamus. Throughout development, the efficacy or PACAP in stimulating cAMP formation slightly increased in the cortex and hypothalamus and decreased in the hippocampus and cerebellum; PACAP stimulation of [3H]InsP formation decreased in the cerebellum and remained steady in the hypothalamus. The effects of PACAP27 and PACAP38 on cAMP levels and inositol phospholipid hydrolysis were dose-dependent between 1 and 100 nM. In the same brain areas, treatment with VIP increased cAMP formation at doses greater than 100nM and failed to affect [3H]InsP content, thus suggesting the existence of type-1 PACAP receptors. The reverse transcription polymerase chain reaction (RT-PCR) was used to analyse the mRNA expression of type-1 PACAP receptor splice variants. PACAP receptor gene expression in the central nervous system was regulated in a developmental- and tissue-specific manner. The PACAP-R transcript was detected in all the brain areas examined whereas PACAP-R-hop mRNA ocurred only in the cerebellum and hypothalamus. The different expression profiles and functional properties of PACAP receptors in the developing rat brain suggest an involvement of PACAP in histogenesis, maturation and neurotransmission.

Differentiation induces pituitary adenylate cyclase-activating polypeptide receptor expression in PC-12 cells.

The two forms of pituitary adenylate cyclase-activating polypeptide (PACAP), PACAP27 and PACAP38, are neuropeptide hormones related to the vasoactive intestinal peptide/secretin/glucagon family of peptides. PACAP receptors that are positively coupled to adenylyl cyclase and phospholipase C have been recently identified. We have investigated the expression of PACAP-Rs in undifferentiated and differentiated PC-12 cells. PACAP27 and PACAP38 failed to significantly increase cAMP or [3H]inositol monophosphate levels in undifferentiated PC-12 cells treated with vehicle, insulin-like growth factor I, or epidermal growth factor but greatly elevated levels after differentiation with nerve growth factor (NGF) or basic fibroblast growth factor. PACAP responsiveness increased significantly after 24 hr of NGF treatment, reaching a maximum within 4 days. At this time of differentiation, the effect of PACAP was dose dependent between 1 nM and 0.1 microM, whereas vasoactive intestinal peptide, at the maximal dose of 10 microM, slightly increased cAMP formation and failed to affect [3H]inositol monophosphate content. Radioreceptor assays, performed with 125I-PACAP27, revealed the induction of high affinity type I PACAP receptors in differentiated PC-12 cells. Using reverse transcription-polymerase chain reaction methodology, we showed the absence of type I PACAP receptor mRNAs in undifferentiated PC-12 cells and the expression of PACAP-R-hop mRNA after NGF or basic fibroblast growth factor treatment. The increased PACAP responsiveness induced by these growth factors in PC-12 cells may therefore result from the expression of the PACAP-R-hop isoform, positively coupled to both adenylyl cyclase and phospholipase C.

Biochemical changes induced by pyrphenoxone in the lens of rabbits and rats.

The xanthomatine analogue, pyrphenoxone, which is known to diminish the incidence of cataract in animals and in man, was applied in two different in vivo models of cataract induced in rabbits by tryptophan-free dietary regimen and in rats by hypergalactosemic diet. The drug was also applied at different concentrations in an in vitro model of cataract. It was found that soluble proteins and sulphurated amino acids of the lens in all in vivo and in vitro models of cataract were higher after pyrphenoxone was applied. Furthermore, the drug treatment was followed by a dose-dependent increase in reduced glutathione content in the lens of rabbits and rats. The same was found in the in vitro model of cataract. These results suggest that pyrphenoxone may act by inducing various biochemical changes that lead to a protection of lens against oxidative processes.