Synapsin III deficiency hampers α-synuclein aggregation, striatal synaptic damage and nigral cell loss in an AAV-based mouse model of Parkinson's disease.

Parkinson's disease (PD), the most common neurodegenerative movement disorder, is characterized by the progressive loss of nigral dopamine neurons. The deposition of fibrillary aggregated α-synuclein in Lewy bodies (LB), that is considered to play a causative role in the disease, constitutes another key neuropathological hallmark of PD. We have recently described that synapsin III (Syn III), a synaptic phosphoprotein that regulates dopamine release in cooperation with α-synuclein, is present in the α-synuclein insoluble fibrils composing the LB of patients affected by PD. Moreover, we observed that silencing of Syn III gene could prevent α-synuclein fibrillary aggregation in vitro. This evidence suggests that Syn III might be crucially involved in α-synuclein pathological deposition. To test this hypothesis, we studied whether mice knock-out (ko) for Syn III might be protected from α-synuclein aggregation and nigrostriatal neuron degeneration resulting from the unilateral injection of adeno-associated viral vectors (AAV)-mediating human wild-type (wt) α-synuclein overexpression (AAV-hαsyn). We found that Syn III ko mice injected with AAV-hαsyn did not develop fibrillary insoluble α-synuclein aggregates, showed reduced amount of α-synuclein oligomers detected by in situ proximity ligation assay (PLA) and lower levels of Ser129-phosphorylated α-synuclein. Moreover, the nigrostriatal neurons of Syn III ko mice were protected from both synaptic damage and degeneration triggered by the AAV-hαsyn injection. Our observations indicate that Syn III constitutes a crucial mediator of α-synuclein aggregation and toxicity and identify Syn III as a novel therapeutic target for PD.

Synergistic Association of Valproate and Resveratrol Reduces Brain Injury in Ischemic Stroke.

Histone deacetylation, together with altered acetylation of NF-κB/RelA, encompassing the K310 residue acetylation, occur during brain ischemia. By restoring the normal acetylation condition, we previously reported that sub-threshold doses of resveratrol and entinostat (MS-275), respectively, an activator of the AMP-activated kinase (AMPK)-sirtuin 1 pathway and an inhibitor of class I histone deacetylases (HDACs), synergistically elicited neuroprotection in a mouse model of ischemic stroke. To improve the translational power of this approach, we investigated the efficacy of MS-275 replacement with valproate, the antiepileptic drug also reported to be a class I HDAC blocker. In cortical neurons previously exposed to oxygen glucose deprivation (OGD), valproate elicited neuroprotection at 100 nmol/mL concentration when used alone and at 1 nmol/mL concentration when associated with resveratrol (3 nmol/mL). Resveratrol and valproate restored the acetylation of histone H3 (K9/18), and they reduced the RelA(K310) acetylation and the Bim level in neurons exposed to OGD. Chromatin immunoprecipitation analysis showed that the synergistic drug association impaired the RelA binding to the Bim promoter, as well as the promoter-specific H3 (K9/18) acetylation. In mice subjected to 60 min of middle cerebral artery occlusion (MCAO), the association of resveratrol 680 µg/kg and valproate 200 µg/kg significantly reduced the infarct volume as well as the neurological deficits. The present study suggests that valproate and resveratrol may represent a promising ready-to-use strategy to treat post-ischemic brain damage.

Neuroprotective and Anti-Apoptotic Effects of CSP-1103 in Primary Cortical Neurons Exposed to Oxygen and Glucose Deprivation.

CSP-1103 (formerly CHF5074) has been shown to reverse memory impairment and reduce amyloid plaque as well as inflammatory microglia activation in preclinical models of Alzheimer's disease. Moreover, it was found to improve cognition and reduce brain inflammation in patients with mild cognitive impairment. Recent evidence suggests that CSP-1103 acts through a single molecular target, the amyloid precursor protein intracellular domain (AICD), a transcriptional regulator implicated in inflammation and apoptosis. We here tested the possible anti-apoptotic and neuroprotective activity of CSP-1103 in a cell-based model of post-ischemic injury, wherein the primary mouse cortical neurons were exposed to oxygen-glucose deprivation (OGD). When added after OGD, CSP-1103 prevented the apoptosis cascade by reducing cytochrome c release and caspase-3 activation and the secondary necrosis. Additionally, CSP-1103 limited earlier activation of p38 and nuclear factor κB (NF-κB) pathways. These results demonstrate that CSP-1103 is neuroprotective in a model of post-ischemic brain injury and provide further mechanistic insights as regards its ability to reduce apoptosis and potential production of pro-inflammatory cytokines. In conclusion, these findings suggest a potential use of CSP-1103 for the treatment of brain ischemia.

An Integrated Approach for a Structural and Functional Evaluation of Biosimilars: Implications for Erythropoietin.

BACKGROUND: Authorization to market a biosimilar product by the appropriate institutions is expected based on biosimilarity with its originator product. The analogy between the originator and its biosimilar(s) is assessed through safety, purity, and potency analyses. OBJECTIVE: In this study, we proposed a useful quality control system for rapid and economic primary screening of potential biosimilar drugs. For this purpose, chemical and functional characterization of the originator rhEPO alfa and two of its biosimilars was discussed. METHODS: Qualitative and quantitative analyses of the originator rhEPO alfa and its biosimilars were performed using reversed-phase high-performance liquid chromatography (RP-HPLC). The identification of proteins and the separation of isoforms were studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and two-dimensional gel electrophoresis (2D-PAGE), respectively. Furthermore, the biological activity of these drugs was measured both in vitro, evaluating the TF-1 cell proliferation rate, and in vivo, using the innovative experimental animal model of the zebrafish embryos. RESULTS: Chemical analyses showed that the quantitative concentrations of rhEPO alfa were in agreement with the labeled claims by the corresponding manufacturers. The qualitative analyses performed demonstrated that the three drugs were pure and that they had the same amino acid sequence. Chemical differences were found only at the level of isoforms containing N-glycosylation; however, functional in vitro and in vivo studies did not show any significant differences from a biosimilar point of view. CONCLUSION: These rapid and economic structural and functional analyses were effective in the evaluation of the biosimilarity between the originator rhEPO alfa and the biosimilars analyzed.

Alpha-synuclein modulates NR2B-containing NMDA receptors and decreases their levels after rotenone exposure.

Alpha-synuclein (α-syn) is the main protein component of Lewy bodies (LBs), that together with nigrostriatal dopamine neuron loss constitute typical pathological hallmarks of Parkinson's disease (PD). Glutamate N-methyl-d-aspartate receptor (NMDAR) abnormalities, peculiarly involving NR2B-containing NMDAR, have been observed in the brain of PD patients and in several experimental models of the disease. Recent findings, indicating that α-syn can modulate NMDAR trafficking and function, suggest that this protein may be a pivotal regulator of NMDAR activity. Prompted by these evidences, we used fluorescence immunocytochemistry, western blotting and ratiometric Ca(2+) measurements to investigate whether wild type (wt) or C-terminally truncated α-syn can specifically modulate NR2B-containing NMDAR levels, subcellular trafficking and function. In addition, we evaluated whether the exposure of primary cortical neurons to increasing concentrations of rotenone could differentially regulate NR2B levels and cell viability in the presence or in the absence of α-syn. Our results indicate that both wt and C-terminally truncated α-syn negatively modulate NR2B-containing NMDAR levels, membrane translocation and function. Moreover, we found that absence of α-syn abolishes the rotenone-dependent decrease of NR2B levels and reduces neuronal vulnerability in primary cortical neurons. These findings suggest that α-syn can modulate neuronal resilience by regulating NR2B-containing NMDAR, whose specific alterations could connect α-syn pathology to neuronal degeneration in PD.

GPNMB/OA protein increases the invasiveness of human metastatic prostate cancer cell lines DU145 and PC3 through MMP-2 and MMP-9 activity.

Non-metastatic glycoprotein melanoma protein B (GPNMB), also known as osteoactivin (OA) is expressed in a wide array of tumors and represents an emerging target for drug development. In this study, we investigated the role of GPNMB/OA in the progression of human metastatic DU145 and PC3 prostate cancer cells. GPNMB/OA contribution in PCa malignant phenotype has been analyzed by small interfering RNA-induced GPNMB/OA silencing. We found that following GPNMB/OA silencing the migration capability of both DU145 and PC3 cells, evaluated by using in vitro invasivity assay, as well as the metalloproteinases MMP-2 and MMP-9 activity were equally strongly inhibited. By contrast knocking down GPNMB/OA weakly attenuated cell proliferation rate of DU145, an effect that paralleled with an increase number of apoptotic cells. However, PC3 cell growth seems to be not affected by GPNMB/OA. Together, these data reveal that GPNMB/OA acts as a critical molecular mediator promoting the acquisition of the more aggressive, pro-metastatic phenotype distinctive of human DU145 and PC3 cell lines.

Different muscarinic receptor subtypes modulate proliferation of primary human detrusor smooth muscle cells via Akt/PI3K and map kinases.

While acetylcholine (ACh) and muscarinic receptors in the bladder are mainly known for their role in the regulation of smooth muscle contractility, in other tissues they are involved in tissue remodelling and promote cell growth and proliferation. In the present study we have used primary cultures of human detrusor smooth muscle cells (HDSMCs), in order to investigate the role of muscarinic receptors in HDSMC proliferation. Samples were obtained as discarded tissue from men >65 years undergoing radical cystectomy for bladder cancer and cut in pieces that were either immediately frozen or placed in culture medium for the cell culture establishment. HDSMCs were isolated from samples, propagated and maintained in culture. [(3)H]-QNB radioligand binding on biopsies revealed the presence of muscarinic receptors, with a Kd of 0.10±0.02nM and a Bmax of 72.8±0.1fmol/mg protein. The relative expression of muscarinic receptor subtypes, based on Q-RT-PCR, was similar in biopsies and HDSMC with a rank order of M2≥M3>M1>M4>M5. The cholinergic agonist carbachol (CCh, 1-100µM) concentration-dependently increased [(3)H]-thymidine incorporation (up to 46±4%). This was concentration-dependently inhibited by the general muscarinic receptor antagonist atropine and by subtype-preferring antagonists with an order of potency of darifenacin >4-DAMP>AF-DX 116. The CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation. This work shows that M2 and M3 receptors can mediate not only HDSM contraction but also proliferation; they may also contribute bladder remodelling including detrusor hypertrophy.

Nicotine-induced structural plasticity in mesencephalic dopaminergic neurons is mediated by dopamine D3 receptors and Akt-mTORC1 signaling.

Although long-term exposure to nicotine is highly addictive, one beneficial consequence of chronic tobacco use is a reduced risk for Parkinson's disease. Of interest, these effects both reflect structural and functional plasticity of brain circuits controlling reward and motor behavior and, specifically, recruitment of nicotinic acetylcholine receptors (nAChR) in mesencephalic dopaminergic neurons. Because the underlying cellular mechanisms are poorly understood, we addressed this issue with use of primary cultures of mouse mesencephalic dopaminergic neurons. Exposure to nicotine (1-10 µM) for 72 hours in vitro increased dendritic arborization and soma size in primary cultures. These effects were blocked by mecamylamine and dihydro-ß-erythroidine, but not methyllycaconitine. The involvement of α4ß2 nAChR was supported by the lack of nicotine-induced structural remodeling in neurons from α4 null mutant mice (KO). Challenge with nicotine triggered phosphorylation of the extracellular signal-regulated kinase (ERK) and the thymoma viral proto-oncogene (Akt), followed by activation of the mammalian target of rapamycin complex 1 (mTORC1)-dependent p70 ribosomal S6 protein kinase. Upstream pathway blockade using the phosphatidylinositol 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride] resulted in suppression of nicotine-induced phosphorylations and structural plasticity. These effects were dependent on functional DA D3 receptor (D3R), because nicotine was inactive both in cultures from D3R KO mice and after pharmacologic blockade with D3R antagonist trans-N-4-2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethylcyclohexyl-4-quinolinecarboxamide (SB-277011-A) (50 nM). Finally, exposure to nicotine in utero (5 mg/kg/day for 5 days) resulted in increased soma area of DAergic neurons of newborn mice, effects not observed in D3 receptor null mutant mice mice. These findings indicate that nicotine-induced structural plasticity at mesencephalic dopaminergic neurons involves α4ß2 nAChRs together with dopamine D3R-mediated recruitment of ERK/Akt-mTORC1 signaling.

Targeted acetylation of NF-kappaB/RelA and histones by epigenetic drugs reduces post-ischemic brain injury in mice with an extended therapeutic window.

UNLABELLED: Nuclear factor-kappaB (NF-κB) p50/RelA is a key molecule with a dual effect in the progression of ischemic stroke. In harmful ischemia, but not in preconditioning insult, neurotoxic activation of p50/RelA is characterized by RelA-specific acetylation at Lys310 (K310) and deacetylation at other Lys residues. The derangement of RelA acetylation is associated with activation of Bim promoter. OBJECTIVE: With the aim of producing neuroprotection by correcting altered acetylation of RelA in brain ischemia, we combined the pharmacological inhibition of histone deacetylase (HDAC) 1-3, the enzymes known to reduce global RelA acetylation, and the activation of sirtuin 1, endowed with a specific deacetylase activity on the K310 residue of RelA. To afford this aim, we tested the clinically used HDAC 1-3 inhibitor entinostat (MS-275) and the sirtuin 1 activator resveratrol. METHODS: We used the mouse model of transient middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to oxygen glucose deprivation (OGD). RESULTS: The combined use of MS-275 and resveratrol, by restoring normal RelA acetylation, elicited a synergistic neuroprotection in neurons exposed to OGD. This effect correlated with MS-275 capability to increase total RelA acetylation and resveratrol capability to reduce RelA K310 acetylation through the activation of an AMP-activated protein kinase-sirtuin 1 pathway. The synergistic treatment reproduced the acetylation state of RelA peculiar of preconditioning ischemia. Neurons exposed to the combined drugs totally recovered the optimal histone H3 acetylation. Neuroprotection was reproduced in mice subjected to MCAO and treated with MS-275 (20µg/kg and 200µg/kg) or resveratrol (6800µg/kg) individually. However, the administration of lowest doses of MS-275 (2µg/kg) and resveratrol (68µg/kg) synergistically reduced infarct volume and neurological deficits. Importantly, the treatment was effective even when administered 7h after the stroke onset. Chromatin immunoprecipitation analysis of cortices harvested from treated mice showed that the RelA binding and histone acetylation increased at the Bcl-xL promoter and decreased at the Bim promoter. CONCLUSION: Our study reveals that epigenetic therapy shaping acetylation of both RelA and histones may be a promising strategy to limit post-ischemic injury with an extended therapeutic window.

Involvement of target gene polymorphisms in 5-Fluorouracil toxicity: a case report.

Personalized medicine is becoming an important tool in oncology, both in preventing disease and in optimizing the treatment of existing cancers. Here we describe the cases of 2 patients with relevant systemic toxicity following 5-fluorouracil (5-FU) therapy and we study the more frequent polymorphisms in the target genes, in particular: (1) the variability in the number of 28-base repetitions present in the 5'-untranslated sequence of the thymidine synthase gene; (2) the presence of single-nucleotide polymorphisms in the methylene tetrahydrofolate reductase gene, and (3) the presence of mRNA splicing in intron 14 of the hepatic enzyme dihydropyrimidine dehydrogenase. The 5-FU gene profile of our patients strongly suggested that the polymorphisms expressed may contribute to the adverse effects seen during the therapy. To what extent these polymorphisms induced adverse effects cannot be established at present; however, our results strengthen the relevance of the 5-FU-related pharmacogenomic profile to predict the response outcome and the chemotherapy toxicity.

Clinical outcome after extended endovascular recanalization in Buerger's disease in 20 consecutive cases.

BACKGROUND: To present our experience of extended endovascular management for thromboangiitis obliterans (Buerger's disease) patients with critical limb ischemia (CLI). METHODS: Between January 2005 and July 2010, a consecutive series of 17 Buerger's disease patients with CLI in 20 limbs were admitted and the diagnosis confirmed. The mean age of the patients was 41.5 years (standard error: ±1.7). All patients presented with history of smoking, one patient presented with hypertension, and eight patients presented with dyslipidemia. According to Rutherford classification, all patients were found to be between grades 3 and 5. Ultrasonography first, and angiography examination later, confirmed a severe arterial disease involving almost exclusively below-the-knee and foot arteries in all cases. A new approach for revascularization, defined as extended angioplasty of each tibial and foot artery obstruction, was performed to achieve direct perfusion of at least one foot artery. RESULTS: An extensive endovascular treatment was intended in all patients with success in 19 of 20 limbs, achieving a technical success in 95%. No mortality or complication related to the procedure was observed. During a mean follow-up of 23 months (standard error: ±4.05), amputation-free survival with no need of major amputation in any case and sustained clinical improvement was achieved in 16 of the 19 limbs (84.2%) successfully treated, resulting in a 100% limb salvage rate (19/19). CONCLUSION: In this first experience, in patients with thromboangiitis obliterans, extended endovascular intervention was a feasible and effective revascularization procedure in case of CLI. High technical success, amputation-free survival, and sustained clinical improvement rates were achieved at midterm follow-up was achieved.

Induction of the unfolded protein response by α-synuclein in experimental models of Parkinson's disease.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) is the main event leading to the induction of the ER stress-related unfolded protein response (UPR). Recent postmortem evaluation, showing that the UPR pathway is activated in nigral dopaminergic neurons bearing α-synuclein inclusions in the brain of Parkinson's disease (PD) patients, suggests that the activation of the UPR may be induced by the accumulation of α-synuclein. In this study, we show that the misfolded protein-sensor/UPR activator glucose-regulated protein 78/immunoglobulin heavy chain-binding protein was bound to α-synuclein and was increased in 'in vitro' and 'in vivo' models showing aggregated α-synuclein accumulation. Moreover, α-synuclein accumulation induced the expression of the UPR-related activating transcription factor 4/cAMP-responsive element-2. These findings indicate that activation of the UPR pathway in the PD brain is associated with α-synuclein accumulation occurring in part within the ER.

Nerve growth factor signaling in prostate health and disease.

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.

Dimerization of dopamine D1 and D3 receptors in the regulation of striatal function.

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.

NF-kappaB p50/RelA and c-Rel-containing dimers: opposite regulators of neuron vulnerability to ischaemia.

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.

Molecular and pharmacological detection of dopaminergic receptors in the human male urinary tract.

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.

Effects of olanzapine on glucose transport, proliferation and survival in C2C12 myoblasts.

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].

Identification and characterization of two nuclear factor-kappaB sites in the regulatory region of the dopamine D2 receptor.

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.

Blockade of the tumor necrosis factor-related apoptosis inducing ligand death receptor DR5 prevents beta-amyloid neurotoxicity.

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.

Presenilin 2 mutations alter cystatin C trafficking in mouse primary neurons.

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.