Abstract: Co-localization of dopamine D1 (D1R) and D3 receptors (D3R) in specific neuronal populations in the striatum and nucleus accumbens suggests that their cross-talk in the regulation of rewarding mechanisms and emotional and cognitive processes and in the development of motor dysfunctions might involve direct interactions. This paper summarizes recent data showing that D1R and D3R form a receptor heteromer in the striatum. A unique characteristic of this receptor complex is a synergistic interaction by which D3R stimulation increases D1R agonist affinity, allows a stronger stimulatory coupling of the D1R to the cAMP system and potentiates D1R-mediated behaviour. The putative role of the D1R-D3R heteromer in the development of motor dysfunctions is also discussed.
Abstract: The pre-synaptic protein alpha-synuclein is the main component of Lewy bodies and Lewy neurites, the defining neuropathological characteristics of Parkinson's disease and dementia with Lewy bodies. Mutations in the alpha-synuclein gene cause familial forms of Parkinson's disease and dementia with Lewy bodies. We previously described a transgenic mouse line expressing truncated human alpha-synuclein(1-120) that develops alpha-synuclein aggregates, striatal dopamine deficiency and reduced locomotion, similar to Parkinson's disease. We now show that in the striatum of these mice, as in Parkinson's disease, synaptic accumulation of alpha-synuclein is accompanied by an age-dependent redistribution of the synaptic SNARE proteins SNAP-25, syntaxin-1 and synaptobrevin-2, as well as by an age-dependent reduction in dopamine release. Furthermore, the release of FM1-43 dye from PC12 cells expressing either human full-length alpha-synuclein(1-140) or truncated alpha-synuclein(1-120) was reduced. These findings reveal a novel gain of toxic function of alpha-synuclein at the synapse, which may be an early event in the pathogenesis of Parkinson's disease.
Abstract: The prostate is one of the most abundant sources of nerve growth factor (NGF) in different species, including humans. NGF and its receptors are implicated in the control of prostate cell proliferation and apoptosis and it can either support or suppress cell growth. The co-expression of both NGF receptors, p75(NGFR) and tropomyosin-related kinase A (trkA), represents a crucial condition for the antiproliferative effect of NGF; indeed, p75(NGFR) is progressively lost during prostate tumorigenesis and its disappearance represents a malignancy marker of prostate adenocarcinoma (PCa). Interestingly, a dysregulation of NGF signal transduction was found in a number of human tumors. This review summarizes the current knowledge on the role of NGF and its receptors in prostate and in PCa. Conclusions bring to the hypothesis that the NGF network could be a candidate for future pharmacological manipulation in the PCa therapy: in particular the re-expression of p75(NTR) and/or the negative modulation of trkA could represent a target to induce apoptosis and to reduce proliferation and invasiveness of PCa.
Abstract: Diverse nuclear factor-kappaB subunits mediate opposite effects of extracellular signals on neuron survival. While RelA is activated by neurotoxic agents, c-Rel drives neuroprotective effects. In brain ischaemia RelA and p50 factors rapidly activate, but how they associate with c-Rel to form active dimers and contribute to the changes in diverse dimer activation for neuron susceptibility is unknown. We show that in both cortical neurons exposed to oxygen glucose deprivation (OGD) and mice subjected to brain ischaemia, activation of p50/RelA was associated with inhibition of c-Rel/RelA dimer and no change p50/c-Rel. Targeting c-Rel and RelA expression revealed that c-Rel dimers reduced while p50/RelA enhanced neuronal susceptibility to anoxia. Activation of p50/RelA complex is known to induce the pro-apoptotic Bim and Noxa genes. We now show that c-Rel-containing dimers, p50/c-Rel and RelA/c-Rel, but not p50/RelA, promoted Bcl-xL transcription. Accordingly, the OGD exposure induced Bim, but reduced Bcl-xL promoter activity and decreased the content of endogenous Bcl-xL protein. These findings demonstrate that within the same neuronal cell, the balance between activation of p50/RelA and c-Rel-containing complexes fine tunes the threshold of neuron vulnerability to the ischaemic insult. Selective targeting of different dimers will unravel new approaches to limit ischaemia-associated apoptosis.
Abstract: Evidence indicate that it is difficult to obtain specific antibodies to G protein-coupled receptors and different technical difficulties may allow the generation of antibodies that lack specificity. We conducted experiments to validate the specificity of commercially available antibodies raised against dopamine (DA) receptors hD(1), hD(4), and hD(5) using a transfection approach: we studied whether, in HEK 293 cells selectively transfected with the various cloned subtypes, each antibody generates bands only in cells expressing its cognate receptor but not in those expressing the other DA receptors. Our results demonstrated that hD(1) and hD(4) receptor antibodies recognize not only their respective epitope, but also other DA receptor subtypes, while for the hD(5) receptor detection, we observed a signal only in the lane loaded with hD(5)-transfected HEK 293 cells, although with a lack of purity. Therefore, we recommend caution on the use of commercially available DA receptor antibodies.
Abstract: Nuclear factor-kappaB (NF-kappaB) has been proposed to serve a dual function as a regulator of neuron survival in pathological conditions associated with neurodegeneration. NF-kappaB is a transcription family of factors comprising five different proteins, namely p50, RelA/p65, c-Rel, RelB and p52, which can combine differently to form active dimers in response to external stimuli. Recent research shows that diverse NF-kappaB dimers lead to cell death or cell survival in neurons exposed to ischemic injury. While the p50/p65 dimer participates in the pathogenesis of post-ischemic injury by inducing pro-apoptotic gene expression, c-Rel-containing dimers increase neuron resistance to ischemia by inducing anti-apoptotic gene transcription. We present, in this report, the latest findings and consider the therapeutic potential of targeting different NF-kappaB dimers to limit ischemia-associated neurodegeneration.
Abstract: BACKGROUND AND PURPOSE: Leptin is an adipose hormone endowed with angiopoietic, neurotrophic, and neuroprotective properties. We tested the hypothesis that leptin might act as an endogenous mediator of recovery after ischemic stroke and investigated whether nuclear transcription factors kappaB activation is involved in leptin-mediated neuroprotection. METHODS: The antiapoptotic effects of leptin were evaluated in cultured mouse cortical neurons from wild-type or NF-kappaB/c-Rel(-/-) mice exposed to oxygen-glucose deprivation. Wild-type, c-Rel(-/-) and leptin-deficient ob/ob mice were subjected to permanent middle cerebral artery occlusion. Leptin production was measured in brains from wild-type mice with quantitative reverse transcriptase-polymerase chain reaction and immunostaining. Mice received a leptin bolus (20 microg/g) intraperitoneally at the onset of ischemia. RESULTS: Leptin treatment activated the nuclear translocation of nuclear transcription factors kappaB dimers containing the c-Rel subunit, induced the expression of the antiapoptotic c-Rel target gene Bcl-xL in both control and oxygen-glucose deprivation conditions, and counteracted the oxygen-glucose deprivation-mediated apoptotic death of cultured cortical neurons. Leptin-mediated Bcl-xL induction and neuroprotection against oxygen-glucose deprivation were hampered in cortical neurons from c-Rel(-/-) mice. Leptin mRNA was induced and the protein was detectable in microglia/macrophage cells from the ischemic penumbra of wild-type mice subjected to permanent middle cerebral artery occlusion. Ob/ob mice were more susceptible than wild-type mice to the permanent middle cerebral artery occlusion injury. Leptin injection significantly reduced the permanent middle cerebral artery occlusion-mediated cortical damage in wild-type and ob/ob mice, but not in c-Rel(-/-) mice. CONCLUSIONS: Leptin acts as an endogenous mediator of neuroprotection during cerebral ischemia. Exogenous leptin administration protects against ischemic neuronal injury in vitro and in vivo in a c-Rel-dependent manner.
Abstract: AIMS: Evidence indicates that dopamine (DA) and DA receptors play a role in the central nervous system (CNS) control of micturition; however, while the central DAergic role in the micturition physiology has been extensively investigated, the expression and the function of DA receptors in the urinary tract are still under investigation. Here, we studied the distribution of DA receptor subtypes in different parts of the human male urinary tract. METHODS: Fragments were collected from 34 men. The mRNAs encoding DA receptors were assessed by RT-PCR, followed by densitometric analysis. Adenylyl cyclase (AC) activity was evaluated using a commercially available RIA kit. Statistical analysis was carried out using one-way ANOVA, with the Bonferroni's post hoc test. RESULTS: Results obtained indicated that RT-PCR products of D(1), D(4), and D(5) subtypes were obtained in each part studied, while no signal was observed for the D(2) and D(3) receptor subtypes. The pharmacological characterization demonstrated that the expressed DA receptors were linked to AC. CONCLUSIONS: DA receptors were expressed throughout the human male urinary tract, from the ureter to the prostatic urethra. In particular, we observed a distinctive DA receptor subtype distribution, with evidence of the presence of mRNA encoding both subtypes of the D(1)-like DA receptor family (D(1) and D(5)), while the D(4) receptors were the only expressed subtype of the D(2)-like family. These results suggested that DAergic drugs used for the treatment of a number of diseases may influence the micturition physiology not only in the CNS, but at the peripheral level as well.
Abstract: Nuclear factor-kappaB (NF-kappaB) is a dimeric transcription factor composed of five members, p50, RelA/p65, c-Rel, RelB, and p52 that can diversely combine to form the active transcriptional dimer. NF-kappaB controls the expression of genes that regulate a broad range of biological processes in the central nervous system such as synaptic plasticity, neurogenesis, and differentiation. Although NF-kappaB is essential for neuron survival and its activation may protect neurons against oxidative-stresses or ischemia-induced neurodegeneration, NF-kappaB activation can contribute to inflammatory reactions and apoptotic cell death after brain injury and stroke. It was proposed that the death or survival of neurons might depend on the cell type and the timing of NF-kappaB activation. We here discuss recent evidence suggesting that within the same neuronal cell, activation of diverse NF-kappaB dimers drives opposite effects on neuronal survival. Unbalanced activation of NF-kappaB p50/RelA dimer over c-Rel-containing complexes contributes to cell death secondary to the ischemic insult. While p50/RelA acts as transcriptional inducer of Bcl-2 family proapoptotic Bim and Noxa genes, c-Rel dimers specifically promote transcription of antiapototic Bcl-xL gene. Changes in the nuclear content of c-Rel dimers strongly affect the threshold of neuron vulnerability to ischemic insult and agents, likewise leptin, activating a NF-kappaB/c-Rel-dependent transcription elicit neuroprotection in animal models of brain ischemia.
Abstract: After denervation of adult rat abdominal muscles, the postsynaptic apparatus of neuromuscular junctions (NMJs) retains its original architecture and clustering of acetylcholine receptors (AChRs). When descending fibers of the spinal cord are surgically diverted to this muscle by a nerve grafting procedure, supraspinal glutamatergic neurons can innervate muscle fibers and restore motor function; the newly formed NMJs switch from a cholinergic to a glutamatergic-type synapse. We show here that regenerating nerve endings contact the fibers in an area occupied by cholinergic endplates. These NMJs are morphologically indistinguishable from those in controls, but they differ in the subunit composition of AChRs. Moreover, by immunofluorescence and immunoelectron microscopy, new NMJs express glutamatergic synapse markers. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 partially colocalizes with AChRs, and vesicular glutamate transporter 2 is localized in the presynaptic compartment. Immunoprecipitation analysis of membranes from reinnervated muscle showed that AMPA receptor subunits GluR1 and GluR2 coimmunoprecipitate with rapsyn, the AChR-anchoring protein at the NMJ. Taken together, these results indicate that cholinergic endplates can be targeted by new glutamatergic projections and that the clustering of AMPA receptors occurs there.
Abstract: Colocalization of dopamine D1 (D1R) and D3 receptors (D3R) in specific neuronal populations suggests that their functional cross-talk might involve direct interactions. Here we report that the D1R coimmunoprecipitates with the D3R from striatal protein preparations, suggesting that they are clustered together in this region. Using bioluminescence resonance energy transfer (BRET(2)), we further suggest the existence of a physical interaction between D1R and D3R. Tagged D1R and D3R cotransfected in human embryonic kidney (HEK) 293 cells generated a significant BRET(2) signal that was insensitive to agonist stimulation, suggesting that they form a constitutive heterodimer. D1R and D3R regulate adenylyl cyclase (AC) in opposite ways. In HEK 293 cells coexpressing D1R and D3R, dopamine stimulated AC with higher potency and displaced [3H]R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) binding with higher affinity than in cells expressing the D1R. In HEK 293 cells individually expressing D1R or D3R, agonist stimulation induces internalization of D1R but not of D3R. Heterodimerization with D3R abolishes agonist-induced D1R cytoplasmic sequestration induced by selective D1R agonists and enables internalization of the D1R/D3R complex in response to the paired stimulation of both D1R and D3R. This mechanism involves beta-arrestin binding because it was blocked by mutant beta-arrestinV53D. These data suggest that as a result of dimerization, the D3R is switched to the desensitization mechanisms typical of the D1R. These data give a novel insight into how D1R and D3R may function in an integrated way, providing a molecular mechanism by which to converge D1R- and D3R-related dysfunctions.
Abstract: A dysregulation of the nerve growth factor (NGF)-mediated control of prostate cell growth is associated with the malignant progression of prostate epithelial cells. Exogenous NGF induced in prostate cancer (PCa) cell lines DU145 and PC3 the expression of p75(NGFR), accompanied by a reduction of the cell malignancy. The aim of this study was to analyze the profile of NGF-regulated genes the PCa cell line DU145 by using the cDNA microarray technique. NGF treatment of DU145 cells decreased the expression of 52 known genes, while the expression of 40 known genes was increased. NGF treatment of the DU145 cell line modified the expression profile of clusters of genes involved in invasion and metastasis, in cell proliferation and apoptosis, inflammation, cell metabolism and transcriptional activity. Interestingly, NGF induced the same pattern of gene modifications in both PCa cell lines. Data presented here may help to identify gene/proteins that dispose to PCa progression and to assess future markers that could allow the development of new clinic diagnostic and therapeutical approaches.
Abstract: Progressive degeneration and intraneuronal Lewy bodies made of filamentous alpha-synuclein (alpha-syn) in dopaminergic cells of the nigrostriatal system are characteristics of Parkinson's disease (PD). Glucose uptake is reduced in some of the brain regions affected by PD neurodegenerative changes. Defects in mitochondrial activity in the substantia nigra have been observed in the brain of patients affected by PD and substantia nigra lesions can induce the onset of a secondary parkinsonism. Thus, energy starvation and consequently metabolic impairment to dopaminergic neurons may be related to the onset of PD. On this line, we evaluated the effect of nutrient starvation, reproduced 'in vitro' by glucose deprivation (GD), in primary mesecephalic neuronal cultures and dopaminergic-differentiated SH-SY5Y cells, to evaluate if decreased glucose support to dopaminergic cells can lead to mitochondrial damage, neurodegeneration and alpha-syn misfolding. Furthermore, we investigated the effect of dopamine (DA) treatment in the presence of a DA-uptake inhibitor or of the D(2)/D(3) receptor (D(2)R/D(3)R) agonist quinpirole on GD-treated cells, to evaluate the efficacy of these therapeutic compounds. We found that GD induced the formation of fibrillary aggregated alpha-syn inclusions containing the DA transporter in dopaminergic cells. These alterations were accompanied by dopaminergic cell death and were exacerbated by DA overload. Conversely, the block of DA uptake and D(2)R/D(3)R agonist treatment exerted neuroprotective effects. These data indicate that glucose starvation is likely involved in the induction of PD-related pathological changes in dopaminergic neurons. These changes may be counteracted by the block of DA uptake and by dopaminergic agonist treatment.
Abstract: The aim of our study was to investigate the direct effects of atypical antipsychotics on muscle cell functions in order to ascertain the diabetic liability of these drugs. We investigated the effects of olanzapine, clozapine and alpha-methyl-5-hydroxytryptamine on basal glucose uptake and glucose uptake in response to insulin using in vitro cultures of mouse skeletal muscle satellite cells (C2C12). We extended our study to the effects of these compounds on cell proliferation, survival and differentiation into myotubes and on the growth of differentiated myotubes. Olanzapine and alpha-methyl-5-HT stimulated 2-deoxyglucose uptake in C2C12 myoblasts in a dose-dependent manner (minimal effective dose: 2 microM olanzapine and 10 microM alpha-methyl-5-HT). The treatment with clozapine had no effect on glucose transport. Insulin and olanzapine increased the plasma membrane (PM) abundance of glucose transporter GLUT4. We investigated whether protein kinase Akt (PKB) and AMP-dependent kinase may participate in mediating olanzapine effects on glucose transport. Clozapine and olanzapine did not induce DNA laddering in differentiating myoblasts and differentiated myotubes and did not affect myotube growth. Olanzapine-induced glucose disposal in vitro is consistent with the acute lowering of plasma glucose/insulin concentrations that occurs in rats before olanzapine-induced overeating [Albaugh, V.L., Henry, C.R., Bello, N.T., Hajnal, A., Lynch, S.L., Halle, B., Lynch, C.J., 2006. Hormonal and metabolic effects of olanzapine and clozapine related to body weight in rodents. Obesity 14, 36-50].
Abstract: AIMS: Muscarinic receptor subtypes expressed in the human urinary bladder mucosa were characterized, investigating whether there were gender-dependent differences and if aging could induce changes in their expression. METHODS: The study was carried out on 34 subjects, 22 men and 12 women, divided in four groups, based on gender and age. Gene expression was evaluated by quantitative RT-PCR. The Western blot was performed using the 4-12% NuPAGE Bis-Tris Gel System. RESULTS: The molecular expression of each subtype of the M(1) receptor family was observed and it was not influenced either by gender or age. M(2) receptor family transcripts revealed that both M(2) and M(4) were detected and that the M(2) transcripts were modified by both gender and age. Indeed, M(2) mRNA was lower in old rather than adult men (P < 0.05), but higher in rather old than adult women (P < 0.05). Further, adult men expressed more M(2) mRNA than adult women (P < 0.05), while the opposite was detected in old age (P < 0.05). The Western blot followed by quantification confirmed that the mRNAs were translated into proteins, and that the M(2) subtype showed similar modifications found at molecular level. DISCUSSION: The selective modification of M(2) receptors observed at the urinary bladder mucosa levels indicates that this anatomical structure could play an active role in the pathophysiology of micturition and supports evidence suggesting an effect of antimuscarinic drugs at this level. Whether these results may influence the age-dependent development of micturition disorders remains to be determined.
Abstract: Clinical improvements in Parkinson's disease produced by dopamine D3 receptor-preferring agonists have been related to their neuroprotective actions and, more recently, to their neuroregenerative properties. However, it is unclear whether dopamine agonists produce their neurotrophic effects by acting directly on receptors expressed by the mesencephalic dopaminergic neurons or indirectly on receptors expressed by astrocytes, via release of neurotrophic factors. In this study, we investigated the effects of the dopamine D3 receptor-preferring agonists quinpirole and 7-hydroxy-N,N-di-propyl-2-aminotetralin (7-OH-DPAT), as well as of the indirect agonist amphetamine, on dopaminergic neurons identified by tyrosine hydroxylase immunoreactivity (TH-IR). Experiments were performed on neuronal-enriched primary cultures containing less than 0.5% of astrocytes prepared from the mouse embryo mesencephalon. After 3 days of incubation, both quinpirole (1-10 microm) and 7-OH-DPAT (5-500 nm) dose-dependently increased the maximal dendrite length (P < 0.001), number of primary dendrites (P < 0.01) and [3H]dopamine uptake (P < 0.01) of TH-IR-positive mesencephalic neurons. Similar effects were observed with 10 microm amphetamine. All neurotrophic effects were blocked by the unselective D2/D3 receptor antagonist sulpiride (5 microm) and by the selective D3 receptor antagonist SB-277011-A at a low dose (50 nm). Quinpirole and 7-OH-DPAT also increased the phosphorylation of extracellular signal-regulated kinase (ERK) within minutes, an effect blocked by pretreatment with SB-277011-A. Inhibition of the D2/D3 receptor signalling pathway to ERK was obtained with PD98059, GF109203 or LY294002, resulting in blockade of neurotrophic effects. These data suggest that dopamine agonists increase dendritic arborizations of mesencephalic dopaminergic neurons via a direct effect on D2/D3 receptors, preferentially involving D3 receptor-dependent neurotransmission.
Abstract: Nuclear factor-kappaB (NF-kappaB) is a transcriptional regulator of neuron survival eliciting diverse effects according to the specific composition of its active dimer. While p50/p65 mediates neurodegenerative events, c-Rel-containing dimers promote cell survival. Stimulation of metabotropic glutamate receptors type 5 (mGlu5) reduces neuron vulnerability to amyloid-beta through activation of anti-apoptotic, c-Rel-dependent transcription of Bcl-X(L) pathway. We here evaluated the protective activity of mGlu5 agonists in dopaminergic SK-N-SH cells exposed to 1-methyl-4-phenylpyridinium (MPP(+)), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causing parkinsonism in experimental animals. MPP(+) produced a concentration-dependent cell loss. Activation of mGlu5 receptors by CHPG (1 mM) and 3HPG (50 microM) abolished the toxic effect produced by 3 microM MPP(+). The neuroprotection was associated with activation of NF-kappaB p65/c-Rel dimer and reduction of p50/p65. These effects were prevented by the mGlu5 receptor antagonist MPEP (5 microM). It is suggested that mGlu5 receptor agonists through activation of a c-Rel-dependent anti-apoptotic pathway can rescue dopaminergic cell from mitochondrial toxicity.
Abstract: Chronic L-dopa administration is associated with development of dyskinesias. The molecular mechanisms of these side-effects, however, remain elusive. Dopamine (DA) receptors interact with other receptors to form highly organized complexes where their activity is finely tuned by several proteins. The DA D1R forms a heteromeric complex with the NMDA receptor (NMDAR) and this interaction influences the trafficking of both receptors. Using the 6-hydroxydopamine rat model of Parkinson's disease, we report a correlation between the development of L-dopa-induced dyskinesias and changes in synaptic D1R/NMDAR complexes.
Abstract: Among the diverse mechanisms involved in the pathophysiology of post-ischemic and post-traumatic injuries, excitotoxicity and nuclear factor-kappaB (NF-kappaB) activation through induction of IkappaB kinase (IKK) complex have a primary role. We investigated the effects of the selective inhibitor of the IKK2 subunit, the anilinopyrimidine derivative AS602868, on excitotoxic injury produced in rat organotypic hippocampal slices and cerebellar primary neurons. Brief exposure to N-methyl-D-aspartate (NMDA) induces astrocyte reactivity, neuron cell death and oligodendrocyte degeneration in hippocampal slices. Application of AS602868 elicited a long-lasting protection of both neurons and oligodendrocytes. Maximal effect was observed with prolonged application of the compound after NMDA exposure. Neuroprotection was also evident in primary cultures of cerebellar granule cells starting from 20 nM concentration. AS602868-elicited neuroprotection correlated with inhibition of NF-kappaB activity. Our results suggest that AS602868 may prove to be a valuable approach in treating neurodegeneration and demyelination associated with cerebral trauma and ischemia.
Abstract: Clinical and laboratory findings suggest that cannabinoids and their receptors are implicated in schizophrenia. The role of cannabinoids in schizophrenia remains however poorly understood, as data are often contradictory. The primary aim of this study was to investigate whether the cannabinoid CB1 receptor antagonists rimonabant and AM251 are able to reverse deficits of sensorimotor gating induced by phencyclidine and to mimic the 'atypical' antipsychotic profile of clozapine. The prepulse inhibition (PPI) of the startle reflex was used to measure deficits of sensorimotor gating. PPI-disruptive effects of phencyclidine and their antagonism by rimonabant, AM251, and clozapine were studied in rats. The effects of rimonabant were carefully examined taking into account dose ranges, vehicle, and route of administration. We also examined the ability of rimonabant to reduce the PPI-disruptive effects of dizocilpine and apomorphine. Rimonabant as well as AM251 significantly counteracted the phencyclidine-disruptive model of PPI, comparable to the restoring effect of clozapine; no augmentation effect was observed with rimonabant and clozapine as cotreatment. Rimonabant also significantly attenuated the PPI disruptive effects of dizocilpine and apomorphine. Taken together, our results indicate that CB1 receptor antagonists do produce 'atypical' antipsychotic profile mimicking that of clozapine in the phencyclidine disruption of sensorimotor gating. Our findings further suggest that CB1 receptor antagonism may be involved in restoring disturbed interactions between the activity of the endocannabinoid system and glutamate neurotransmitter system implied in schizophrenia.
Abstract: Regulation of D2 receptor (D2R) expression is crucial in the function of dopaminergic systems. Because alterations of D2R expression may contribute to the development of different disorders, it is important to elucidate the mechanisms regulating D2R gene transcription. We report the characterization of two putative nuclear factor-kappaB (NF-kappaB) motifs, referred to as D2-kappaB sites, in the human D2R promoter, and demonstrate that they bind NF-kappaB subunits and stimulate D2R promoter activity. D2-kappaB sites show different degrees of conservation and specificity, when compared with canonical kB sites. The D2-kappaB1 site (from -407 to -398) is highly conserved and binds p50/p65 and p50/c-Rel complexes, whereas D2-kappaB2 (from -513 to -504) is more degenerated and only binds p50/p65 heterodimers. Activation of D2-kappaB sites in COS-7 cells expressing a luciferase reporter vector containing the D2R promoter resulted in increased transcriptional activity. Site-directed mutagenesis of each D2-kappaB site differentially modified D2R promoter activity. In particular, mutation of the D2-kappaB1 motif did not affect D2R promoter response to p50/c-Rel complexes, whereas inactivation of the D2-kappaB2 site decreased it. Mutations of either D2-kappaB1 or D2-kappaB2 sites attenuated the D2R promoter transcriptional efficiency induced by p50/p65 complexes. Thus, D2R transcription mediated by p50/c-Rel is supported mainly by the D2-kappaB2 site, whereas both sites are necessary to support the full transcriptional activity mediated by p50/p65 complexes. A correlation was found between NF-kappaB activity and D2R expression in the pituitary and pituitary-derived cells but not in the striatum, suggesting that NF-kappaB regulation of D2R expression could be a pituitary-specific mechanism.
Abstract: BACKGROUND/AIMS: To our knowledge, a suitable animal model to investigate how atypical antipsychotics may induce diabetes in patients has not received much attention. METHODS: We investigated the effects of acute as well as subchronic administration of clozapine on food intake, body weight gain, glucose tolerance and insulin secretion in response to glucose in Sprague-Dawley rats. We then evaluated the effects of clozapine on corticosterone secretion and 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) and phosphoenolpyruvate carboxykinase (PEPCK) expression in the liver. We investigated the in vitro effects of clozapine on glucose uptake and development of differentiated myotubes in skeletal muscle cell (C2C12) cultures. RESULTS: Clozapine administration caused hyperglycemia (p < 0.05) in female rats. In male rats, the increase of plasma glucose levels after clozapine injection was not statistically significant. The increase of plasma insulin concentrations and the intraperitoneal glucose tolerance test results proved that clozapine reduced insulin sensitivity in female rats. These endocrine and metabolic effects of clozapine were not related to changes in feeding behavior of fat accumulation. We observed a stimulatory effect of clozapine on corticosterone (p < 0.01) secretion in both female and male rats. Chronic clozapine administration upregulated PEPCK and 11beta-HSD-1 expression in rat liver. Clozapine did not inhibit basal and insulin-induced glucose transport in murine myotubes but it was able to antagonize the stimulatory effect of alpha-methyl-5-hydroxytryptamine on glucose uptake. CONCLUSION: Clozapine induces sex-related alterations of glucose homeostasis and insulin sensitivity in rodents. We discussed the possible contribution of clozapine-induced activation of HPA and clozapine antagonistic activity at peripheral 5-HT(2A) receptors to the observed metabolic alterations.
Abstract: We originally suggested that inhibition of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) death pathway could be taken into consideration as a potential therapeutic strategy for Alzheimer's disease (AD). However, because the critical role of TRAIL in immune surveillance, the neutralization of TRAIL protein by an antibody to prevent its binding to death receptors is definitely a risky approach. Here, we demonstrated that the blockade of the TRAIL death receptor DR5 with a specific antibody completely prevented amyloid beta peptide (A beta) neurotoxicity in both neuronal cell line and primary cortical neurons. DR5 was demonstrated to be a key factor in TRAIL death pathway. In fact, whereas TRAIL expression was enhanced dose-dependently by concentrations of beta amyloid ranging from 10 nM to 1 microM, only the highest toxic dose of A beta (25 microM) induced the increased expression of DR5 and neuronal cell death. In addition, the increased expression of DR5 receptor after beta amyloid treatment was sustained by p53 transcriptional activity, as demonstrated by the data showing that the p53 inhibitor Pifithrin alpha prevented both beta amyloid-induced DR5 induction and cell death. These data suggest a sequential activation of p53 and DR5 upon beta amyloid exposure. Further insight into the key role of DR5 in AD was suggested by data showing a significant increase of DR5 receptor in cortical slices of AD brain. Thus, these findings may give intracellular TRAIL pathway a role in AD pathophysiology, making DR5 receptor a possible candidate as a pharmacological target.
Abstract: Reelin (RELN) is a key molecule for the regulation of neuronal migration in the developing CNS. The reeler mice, which have spontaneous autosomal recessive mutation in the RELN gene, reveal multiple defects in brain development. Morphological, neurochemical and behavioral alterations have been detected in heterozygous reeler (HR) mice, suggesting that not only the presence, but also the level of RELN influences brain development. Several studies implicate an involvement of RELN in the pathophysiology of neuropsychiatric disorders in which an alteration of the cholinergic cortical pathways is implicated as well. Thus, we decided to investigate whether the basal forebrain (BF) cholinergic system is altered in HR mice by examining cholinergic markers at the level of both cell body and nerve terminals. In septal and rostral, but not caudal, basal forebrain region, HR mice exhibited a significant reduction in the number of choline acetyltransferase (ChAT) immunoreactive (ir) cell bodies compared with control mice. Instead, an increase in ChAT ir neurons was detected in lateral striatum. This suggests that an alteration in ChAT ir cell migration which leads to a redistribution of cholinergic neurons in subcortical forebrain regions occurs in HR mice. The reduction of ChAT ir neurons in the BF was paralleled by an alteration of cortical cholinergic nerve terminals. In particular, the HR mice presented a marked reduction of acetylcholinesterase (AChE) staining accompanied by a small reduction of cortical thickness in the rostral dorsomedial cortex, while the density of AChE staining was not altered in the lateral and ventral cortices. Present results show that the cholinergic basalo-cortical system is markedly, though selectively, impaired in HR mice. Rostral sub-regions of the BF and rostro-medial cortical areas show significant decreases of cholinergic neurons and innervation, respectively.
Abstract: Mutations in the presenilin genes account for the majority of familial Alzheimer disease (FAD) cases. In the present report we demonstrated that the FAD-linked presenilin 2 mutations (PS2 M239I and T122R) alter cystatin C trafficking in mouse primary neurons reducing secretion of its glycosylated form. These mutations showed a different impact on cystatin C: PS2 T122R had a much stronger effect determining a dramatic intracellular accumulation of cystatin C (native and glycosylated), followed by a reduction in the secretion of both forms. Several experimental evidences suggest that cystatin C exerts a protective role in the brain and favors stem cells proliferation. Confocal imaging showed that the effect of PS2 T122R mutation was a massive recruitment of cystatin C into the neuronal processes, in the presence of an intact cytoskeletal structure. The consequent reduction in the cystatin C extracellular levels might result in a failure of neuroregeneration. Understanding the interplay of PS2 and cystatin C in the pathogenesis of AD might highlight new therapeutic prospective.
Abstract: Recent findings indicate that skin-derived precursor cells (SKPs) of mouse dermis can differentiate in cells with neuronal-like morphology. However, direct evidence supporting the establishment of functional phenotype is missing. In the present study, SKP cells were obtained using published in vitro techniques and studied at 14- to 21-day differentiation, when neuronal-like morphology was observed. The experiment was repeated 39 times. Co-cultures with cortical astrocytes were also used to enhance the process of neural differentiation. Expression of GAP43 and light-chain MAP-2(c) but not markers of dendritic and synaptic terminal differentiation such as heavy-chain MAP-2(ab) and synapsin were observed. Voltage-clamp electrophysiology recordings showed potassium currents, but neither action potentials generation nor electrotonic response to exogenous administration of nicotine or kainic acid. These observations suggest that mouse SKPs do not differentiate into mature functional neurons, at least using the published methodologies for in vitro differentiation.
Abstract: Glutamate-mediated mechanisms are related to the motor complications of L-DOPA therapy in Parkinson's disease (PD). In striatal postsynaptic densities (PSD), the dopamine D1 receptor (D1R) is part of an oligomeric complex with the glutamate N-methyl-D-aspartate receptor (NMDAR), determining the strength of corticostriatal transmission. We studied D1R/NMDAR complex alterations induced by L-DOPA in the 6-hydroxydopamine-lesioned rat model of PD. L-DOPA-treated hemiparkinsonian rats were determined to be dyskinetic or nondyskinetic based on behavioral testing. D1R/NMDAR assemblies containing NR1-C2 and NR2B subunits were decreased in the PSD of lesioned striatum. Short-term L-DOPA administration improved akinesia and restored the synaptic abundance of D1R, NR1-C2 and NR2B. Prolonged L-DOPA treatment also normalized synaptic D1R/NMDAR complexes in nondyskinetic rats, but remarkably reduced them in the dyskinetic group without changing their interaction. This decrease involved NR1-C2, NR1-C2', NR2A, and NR2B subunits. The composition of residual synaptic D1R/NMDAR complexes in dyskinetic rats may thus be different from that observed in lesioned rats, suggesting that expression of different motor dysfunctions might be related to the receptor profile at corticostriatal synapses. The levels of D1R/NMDAR complexes were unchanged in total striatal membrane proteins, suggesting that the decrease of these species in the PSD is likely to reflect an altered receptor trafficking. In human embryonic kidney 293 cells expressing the D1R/NMDAR, complex costimulation of both D1R and NMDAR, but not individual receptor activation, promoted internalization, suggesting that development of dyskinesias might be related to agonist-mediated down-regulation of the D1R/NMDAR complex at corticostriatal synapses.
Abstract: The nuclear transcription factors kappaB (NF-kappaB) function as key regulators of physiological processes in the central nervous system. Aberrant regulation of NF-kappaB can underlie neurological disorders associated with neurodegeneration. A large number of studies have reported a dual role of NF-kappaB in regulating neuron survival in pathological conditions. A recent progress in understanding the mechanisms responsible for opposite effects elicited by NF-kappaB in brain dysfunctions arises from the identification of diverse NF-kappaB complexes specifically involved in the mechanism of neuronal cell death or cell survival. We here discuss the latest findings and consider the therapeutic potential of targeting distinct NF-kappaB complexes for the treatment of neurodegenerative disorders and memory dysfunctions.
Abstract: We examined the effects of the adipose hormone leptin on the development of mouse cortical neurons. Treatment of neonatal and adult mice with intraperitoneal leptin (5 mg/kg) induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in pyriform and entorhinal cortex neurons. Stimulation of cultured embryonic cortical neurons with leptin evoked Janus kinase 2 and ERK1/2 phosphorylation and activated the downstream effector 90-kDa ribosomal protein S6 kinase. Moreover, leptin elicited the phosphorylation of the phosphatidylinositol 3-kinase effector Akt and evoked Ser-9 phosphorylation of glycogen synthase kinase-3beta (GSK3beta), an event inactivating this kinase. Leptin-mediated GSK3beta phosphorylation was prevented by the MEK/ERK inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, or the protein kinase C inhibitor GF109203X. Exposure of cortical neurons to leptin also induced Ser-41 phosphorylation of the neuronal growth-associated protein GAP-43, an effect prevented by LY294002 and GF109203X but not by PD98059. Ser-41-GAP-43 phosphorylation is usually high in expanding axonal growth cones. Neurons exposed to 100 ng/ml leptin for 72 h displayed reduced rate of growth cone collapse, a shift of growth cone size distribution toward higher values, and a 4-fold increase in mean growth cone surface area compared with control cultures. The leptin-induced growth cone spreading was hampered in cortical neurons from Lepr(db/db) mice lacking functional leptin receptors; it was associated with localized Ser-9-GSK3beta phosphorylation and mimicked by the GSK3beta inhibitor SB216763. At concentrations preventing GSK3beta phosphorylation, PD98059, LY294002, or GF109203X reversed the leptin-induced growth cone surface enlargement. We concluded that the leptin-mediated regulation of growth cone morphogenesis in cortical neurons relies on upstream regulators of GSK3beta activity.
Abstract: Acetylcholine is the specific chemical code of spinal nerve terminal transmission at the mammalian neuromuscular junction (NMJ), whereas nicotinic acetylcholine receptors inserted into the membrane of muscle fibres mediate signalling for the muscle response. Glutamate has a primary role in neuromuscular transmission of organisms that are phylogenetically distant from mammals, the invertebrates, including insect and molluscs. Recent research has shown that diverting descending glutamatergic fibres in the spinal cord to rat skeletal muscle by means of a peripheral nerve graft causes the cholinergic synapse to switch to the glutamatergic type. These data demonstrate that under appropriate surgical manipulation supraspinal neurons can directly target muscle fibres and specify the postsynaptic receptors to achieve a functional glutamatergic NMJ.
Abstract: Beta-amyloid (Abeta) peptides are key proteins in the pathophysiology of Alzheimer's disease (AD). While Abeta42 aggregates very rapidly to form early diffuse plaques, supplemental Abeta40 deposition is required to form mature neuritic plaques. We here investigated the role of nuclear factor-kappaB (NF-kappaB) pathway in Abeta40-mediated neuronal damage and amyloid pathology. In rat primary neurons and human postmitotic neuronal cells, the Abeta peptide induced a dose-dependent neuronal death, reduced the levels of the anti-apoptotic protein Bcl-XL, enhanced the cytosolic release of cytochrome c, and elicited the intracellular accumulation and secretion of Abeta42 oligomers. Moreover, Abeta40 activated the NF-kappaB pathway by selectively inducing the nuclear translocation of p65 and p50 subunits, and promoted an apoptotic profile of gene expression. As inhibitors of the NF-kappaB pathway, we tested the capability of a double-stranded kappaB decoy oligonucleotide, the anti-inflammatory drug aspirin and the selective IkappaB kinase 2 inhibitor, AS602868, to modify the Abeta40-mediated effects. These treatments, transiently applied before Abeta exposure, completely inhibited p50/p65 nuclear translocation and neuronal damage. The kappaB decoy also inhibited the Abeta-induced release of cytochrome c, restored the levels of Bcl-XL, and prevented intraneuronal accumulation and secretion of Abeta42. These results open up interesting perspectives on the development of novel strategies targeting out NF-kappaB p50/p65 dimers for pharmacological intervention in AD.
Abstract: An increasing amount of evidence suggests that the family of nuclear factor kappaB (NF-kappaB) transcription factors plays an important role in synaptic plasticity and long-term memory formation. The present study investigated the regulation of NF-kappaB family members p50, p65/RelA, and c-Rel in the hippocampus in response to metabotropic glutamate receptor (mGluR) signaling. Activation of group I metabotropic glutamate receptors (GpI-mGluRs) with the agonist (S)-3,5-dihydroxyphenylglycine (DHPG) resulted in a time-dependent increase in DNA binding activity of p50, p65, and c-Rel in area CA1 of the hippocampus. An antagonist of mGluR5, 2-Methyl-6-(phenylethynyl)pyridine, inhibited the DHPG-induced activation of NF-kappaB, whereas an antagonist of mGluR1, (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid, did not. Using a series of inhibitors, we investigated the signaling pathways necessary for DHPG-induced activation of NF-kappaB and found that they included the phosphatidyl inositol 3-kinase, protein kinase C, mitogen-activated protein kinase kinase, and p38-mitogen-activated protein kinase pathways. To determine the functional significance of mGluR-induced regulation of NF-kappaB, we measured long-term depression (LTD) of Schaffer-collateral synapses in the hippocampus of c-Rel knock-out mice. Early phase LTD was normal in c-rel(-/-) mice. However, late-phase LTD (>90 min) was impaired in c-rel(-/-) mice. The observations of this deficit in hippocampal synaptic plasticity prompted us to further investigate long-term memory formation in c-rel(-/-) mice. c-rel(-/-) mice exhibited impaired performance in a long-term passive avoidance task, providing additional evidence for c-Rel in long-term memory formation. These results demonstrate that the NF-kappaB transcription factor family is regulated by GpI-mGluRs in the hippocampus and that the c-Rel transcription factor is necessary for long-term maintenance of LTD and formation of long-term memory.
Abstract: The transcription factor nuclear factor kappaB (NF-kappaB) is well known for its antiapoptotic action. However, in some disorders, such as cerebral ischemia, a proapoptotic function of NF-kappaB has been demonstrated. To analyze which subunit of NF-kappaB is functional in cerebral ischemia, we induced focal cerebral ischemia in mice with a germline deletion of the p52 or c-Rel gene or with a conditional deletion of RelA in the brain. Only RelA deficiency reduced infarct size. Interestingly, expression of the proapoptotic BH3 (Bcl-2 homology domain 3)-only genes Bim and Noxa in cerebral ischemia depended on RelA and the upstream kinase IKK (IkappaB kinase). RelA stimulated Bim and Noxa gene transcription in primary cortical neurons and bound to the promoter of both genes. Thus, the deleterious function in cerebral ischemia is specific for the NF-kappaB subunit RelA and may be mediated through Bim and Noxa.
Abstract: Dopamine and glutamate have been shown to extensively interact in the striatum, nucleus accumbens, hippocampus and prefrontal cortex, to regulate different physiological functions, including locomotor activity, positive reinforcement, attention and working memory. Although dysfunctions of dopamine transmission have long been identified as critical determinants of neurological and neuropsychiatric disorders, such as Parkinson's disease and schizophrenia, there is now increasing evidence that concurrent alterations of dopamine and glutamate function may play a central role in the pathophysiology of these diseases. Thus, defining the characteristics of dopamine-glutamate interactions may be crucial to identify alternative molecular targets for the development of novel pharmacological tools. At the postsynaptic level, interactions between the dopamine D1 and the glutamate NMDA receptors appear to be particularly relevant. Different mechanisms are involved in this interactions: 1) D1R-dependent, second messenger-mediated phosphorylation of NMDAR subunits; 2) coordinated regulation of receptor trafficking at synaptic sites; 3) formation of an heteromeric D1/NMDA receptor complex. In this paper we review the molecular mechanisms, functional implications and pharmacological significance of D1R/NMDAR interaction via direct protein-protein oligomerization.
Abstract: NF-kappaB is a nuclear transcription factor involved in the control of fundamental cellular functions including cell survival. Among the many target genes of this factor, both pro- and anti-apoptotic genes have been described. To evaluate the contribution of NF-kappaB activation to excitotoxic insult, we analysed the effect of IkappaBalpha (IkappaBalpha) phosphorylation blockade on glutamate-induced toxicity in adult mouse hippocampal slices. By using immunocytochemical and EMSA techniques, we found that (i) acute exposure of hippocampal slices to NMDA induced nuclear translocation of NF-kappaB, (ii) NMDA-mediated activation of NF-kappaB was prevented by BAY 11-7082, an inhibitor of IkappaBalpha phosphorylation and degradation, and (iii) BAY 11-7082-mediated inhibition of NF-kappaB activation was associated with neuroprotection.
Abstract: AIM: Several studies have proposed a role for alpha1 adrenoceptors (ARs) in ureteral physiology, indicating that they are present in the ureter; however, few studies have been done to identify alpha1 AR subtypes present in this area. Thus, this study was carried out to characterize the alpha1 AR subtype gene and protein expression in proximal, medial, and distal region of the human ureter. METHODS: Molecular characterization of alpha1 AR subtypes were analyzed by semi-quantitative RT-PCR. alpha1 AR protein expression was studied by saturation binding curves and by competition binding curves with selective antagonists. Analysis of data was performed using the GraphPad PRISM 4 software. RESULTS: Analysis of saturation binding curves revealed a heterogeneous distribution of alpha1 AR binding sites, the B(max) for the distal ureter was indeed 52.5 +/- 5.4 fmol/mg prot, while a lower similar density of alpha1 ARs was demonstrated in the medial (25.2 +/- 1.7 fmol/mg prot) and proximal (23.4 +/- 0.4 fmol/mg prot) ureters. Molecular and pharmacological characterization of alpha1 AR subtypes indicated that each receptor was present, although with differences in terms of the amount expressed. CONCLUSIONS: Human ureter was endowed with each alpha1 AR subtype, although alpha1D and alpha1A ARs were prevalent over alpha1B ARs. Radioligand binding results revealed that there were no significant differences in the K(d) between ureteral regions, while a heterogeneous distribution of alpha1 AR binding sites was detected, with the highest density of alpha1 ARs in the distal ureter and a lower similar density in the medial and proximal ureters.
Abstract: Acetylcholine is the main neurotransmitter at the mammalian neuromuscular junction (NMJ) where nicotinic acetylcholine receptors mediate the signaling between nerve terminals and muscle fibers. We show that under glutamatergic transmission, rat NMJ switches from cholinergic type synapse to glutamatergic synapse. Connecting skeletal muscle to the lateral white matter of the spinal cord by grafting the distal stump of the transected motor nerve produced functional muscle reinnervation. The restored neuromuscular activity became resistant to common curare blockers but sensitive to the glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist. Analysis of the regenerated nerve disclosed new glutamatergic axons and the disappearance of cholinergic fibers. Many axons belonged to the supraspinal neurons located in the red nucleus and the brainstem nuclei. Finally, the innervated muscle displayed high expression and clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits glutamate receptors 1 and 2. Our data suggest that supraspinal neurons can target skeletal muscle, which retains the plasticity to generate functional glutamatergic NMJ.
Abstract: Opposite effects of nuclear factor-kappaB (NF-kappaB) on neuron survival rely on activation of diverse NF-kappaB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1beta. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-beta (Abeta), a peptide implicated in Alzheimer's disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Abeta toxicity depends on c-Rel activation. Abeta peptide induced NF-kappaB factors p50 and p65. The mGlu5 agonists activated c-Rel, besides p50 and p65, and the expression of manganese superoxide dismutase (MnSOD) and Bcl-X(L). Targeting c-Rel expression by RNA interference suppressed the induction of both antiapoptotic genes. Targeting c-Rel or Bcl-X(L) prevented the prosurvival effect of mGlu5 agonists. Conversely, c-Rel overexpression or TAT-Bcl-X(L) addition rescued neurons from Abeta toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Rel-dependent antiapoptotic pathway responsible for neuroprotection against Abeta peptide.
Abstract: NF-kappaB is a nuclear transcription factor involved in the control of fundamental cellular functions including regulation of cell survival. We investigated NF-kappaB activation induced by two opposing modulators of cell viability: IL-1beta and glutamate. We found that IL-1beta activated p50, p65 and c-Rel subunits of NF-kappaB, while glutamate activated only p50 and p65 proteins. Cell stimulation by glutamate, correlated with expression of the pro-apoptotic genes Caspase-3, Caspase-2L and Bax. Conversely, IL-1beta induced the expression of the short anti-apoptotic isoform of Caspase-2. Finally, we analysed the effect of the inhibition of IkappaBalpha degradation on glutamate-induced toxicity by using BAY 11-7082, a selective inhibitor of IkappaBalpha phosphorylation. Our results suggest that BAY 11-7082 preserves neuron viability from the glutamate-mediated injury.
Abstract: Nerve growth factor (NGF) is known to play a role as a circulating neurokine, integrating signals from the neuro-immuno-endocrine system. The ability of NGF to activate the pituitary-adrenocortical axis, together with the increase of its serum concentration in pregnancy and lactation, supports the hypothesis that NGF is secreted by the pituitary gland and plays a role as modulator of endocrine functions. Evidence obtained both in vitro and in vivo in experimental animal models suggests that lactotroph cells secrete both prolactin (PRL) and NGF. Furthermore, in previous studies we demonstrate that cell lines derived from dopamine (DA)-sensitive human prolactinomas express high levels of NGF messenger RNA and protein. On these basis, we studied serum NGF concentrations in female patients with microprolactinoma (n = 4) and in control women (n = 5). PRL and NGF were measured at the diagnosis, during the thyrotropin releasing hormone (TRH) test and after the therapy with DA D2 receptor agonist cabergoline (0.25 mg, twice a week). Results obtained suggested that hyperprolactinemia (70.3+/-8.4 ng/ml) paralleled markedly higher NGF levels (58.4+/-18.7 pg/ml) compared to controls (PRL 8.7+/-3.2 ng/ml, NGF 8.4+/-1.3 pg/ml). Serum concentrations of NGF and PRL during the TRH test were closely associated (r = 0.943, p < 0.01). Cabergoline therapy normalized PRL (7.9+/-3.6 ng/ml) and induced a significant decrease of NGF levels (12.5+/-4.9 pg/ml). In conclusions, data reported here indicated that, in human microprolactinomas, NGF is released in the bloodstream paralleling PRL-secretion and it is modulated by a neurotransmitter-regulated mechanism, since the normalization of PRL elicited by the DA D2 receptor agonist cabergoline induced a significant decrease of serum NGF as well.
Abstract: We investigated the effects of IL-6 and a chimeric derivative of IL-6 and soluble IL-6 receptor (IL6RIL6 chimera) on excitotoxic injury in rat organotypic hippocampal slices. Brief application of N-methyl-d-aspartate (NMDA) induced astrocyte reactivity, neuron cell death, and oligodendrocyte degeneration, the latter caused by secondary activation of AMPA/kainate receptors. Both these cytokines rescued neurons and oligodendrocytes, albeit the chimeric compound was much more potent and efficient than IL-6. No change was produced on reactive astrocytosis. The cytokines preserved myelin basic protein (MBP) production in slices exposed to excitotoxic insult, and when applied singularly for a week, they also enhanced both MBP and proteolipid protein expression. These effects occurred through activating the signal transducer gp130 and were associated with stimulation of transcription factors STAT1 and STAT3. Our results suggest that IL-6 and IL6RIL6 may prove to be valuable in treating neurodegenerative and demyelinating diseases.
Abstract: A detailed study of the presence of alpha1 AR binding sites and alpha1 AR subtype mRNA expression in human urinary bladder areas involved in the micturition (i.e. detrusor, trigone and neck) is reported here, investigating whether or not there are differences between sexes. Results obtained indicated that alpha1 AR proteins were detectable in each bladder area. In both sexes, the detrusor and the neck expressed similar levels of alpha1 ARs: respectively, detrusor: 14.6 +/- 1.2 in men and 13.1 +/- 1.1 fmol/mg prot in women; neck: 16.9 +/- 3.2 in men and 17.5 +/- 4.1 fmol/mg prot in women. In the trigone, significantly higher alpha1ARs were found in women compared to men (20.6 +/- 1.1 vs 11.7 +/- 0.7 fmol/mg prot). Subtype analysis indicated that in women, each area was endowed with mRNA encoding for each alpha1 AR subtype. The men detrusor expressed alpha1a and alpha1d ARs, while in the trigone and the neck, each subtype was present. Since the detrusor muscle hypertrophy is a marker of bladder obstructive outlet, the selective alpha1 AR subtype targeting arouses much interest, as evidence indicates that there are differences in signalling pathways among the subtypes. Furthermore, the significance of the alpha1 ARs coexpression is still unknown; interestingly, recent papers demonstrate that alpha1 AR subtypes could dimerize. Thus, in the human urinary bladder it may be suggested a potential level of alpha1 AR complexity that could have an impact on drug development.
Abstract: Differentiated human NT2-N neurons were shown to express CCR5 and CXCR4 chemokine receptor mRNA and protein, and to be responsive to the chemokines CCL5 and CXCL12. Using cDNA microarray technology, CCL5 was found to induce a distinct transcriptional program, with reproducible induction of 46 and 9 genes after 2 and 8 hr of treatment, respectively. Conversely, downregulation of 20 and 7 genes was observed after 2 and 8 hr of treatment, respectively. Modulation of a selected panel of CCL5-responsive genes was also confirmed by quantitative RT-PCR and Western blot and compared to gene expression changes induced by CXCL12 treatment. Gene clustering identified distinct functional subsets of CCL5-responsive molecules, and a significant number of expressed sequence tags encoding unknown genes. CCL5-responsive genes comprise a significant number of enzymes, transcription factors, and miscellaneous molecules involved in neuronal survival and differentiation, including neurite outgrowth and synaptogenesis. Our results suggest that CCL5 biological functions might go beyond its recognized chemotactic activity in the central nervous system, in particular with regard to the control of neural plasticity events both during development and in postnatal life.
