34 research outputs found
Erythropoietin Receptor and Hemoglobin, two commonly blood – associated proteins, in mesencephalic dopaminergic neurons
No full textDopaminergic neurons (DA) are an anatomically and functionally heterogeneous group of cells involved in a wide range of neuronal network activities and behaviour. Among them, mesencephalic dopaminergic neurons (mDA) are the major source of dopamine in the brain. They present two main groups of projecting cells: the A9 neurons of the Substantia Nigra (SN) and the A10 cells of the Ventral Tegmental Area (VTA). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson’s disease (PD) and the loss of DA synapses in the striatum is believed to be primary cause for the disruption of the ability to control movements. A10 cells constitute the mesocorticolimbic pathway playing a fundamental role in reward and attention. Their abnormal function has been linked to schizophrenia, attention deficit and addiction while they are relatively spared in PD (Meyer-Lindenberg et al., 2002). The description of the repertory of genes of mDA neurons may provide crucial information on their physiology as well as on the mechanisms of cell-type specific dysfunction. Interestingly, in previous gene expression profiling experiments, mDA cells groups presented a limited number of differentially expressed genes (Chung et al., 2005; Greene et al., 2005). By a combination of different gene expression platforms with Laser Capture Microdissection (LCM), it has been unveiled the existence of an alternative splice variant of Erythropoietin Receptor (EpoR) in A9 neurons. Moreover, the transcripts of hemoglobin alpha, adult chain 1 (Hba-a1) and hemoglobin beta, adult chain 1 (Hbb-b1) have been identified. The main goal of this study is the understanding of the role of Erythropoietin receptor (EpoR) and of alpha and β-globin in dopaminergic neurons as well as in Parkinson’s disease (PD).
In blood Epo regulates erythrocyte differentiation and Hb production. Since Epo has been recently shown to protect dopaminergic neurons in neurochemical PD models and it is a very important pharmacological target, it has been analyzed by the RACE technique the expression of EpoR in spleen (as control) and in A9 mouse mesencephalic cells collected by Laser Capture Microdissection.
5’RACE analysis of DA neurons indicate the existence of an alternative transcription start site for the EpoR (referred to as DA-EpoR). EpoR expression was validated by PCR experiments and was restricted to A9 DA cells, which selectively degenerate in PD. The truncated cDNA corresponding to DA EpoR has been cloned from the A9 mesencephalic neurons, while the full-length WT EpoR has been cloned from the spleen. Both cDNAs have been expressed in HEK-T cells in order to unveil their function. DA EpoR may act as a decoy toward the WT EpoR.
To unveil the role of α and β globin in the brain it has been investigated an animal model of β-Thalassemia. I took advantage of a mouse model of β-Thalassemia, in which β-globin gene is deleted in heterozigosity. The goal is to study the status of global gene expression in A9 and A10 dopaminergic neurons in a thalassemic carrier genotype. This condition is present in 3 million individuals in Italy. No brain studies have been carried out so far both in mouse models and in post-mortem human brains. I bred β-Thalassemic mice with TH-GFP mice where dopaminergic neurons are labelled. Then I took advantage of Laser Capture Microdissection to harvest A9 and A10 dopaminergic neurons from 12 months old wild type and heterozygous mice for β-chain of Hb for four replicas. Liver and cerebellum cells were used as controls, since liver presents iron accumulation in old thalassemic carriers. Sample cells have been analyzed at the Affymetrix core facility. I identified among the genes differentially expressed Atg4, a major regulator of autophagy, and Hepcidin, the hormone controlling iron metabolism. Relevant target genes have been verified by qPCR
Comments on ''Geranylgeraniol--a new potential therapeutic approach to bisphosphonate associated osteonecrosis of the jaw" by Ziebart T et al. (2011).
No full texttherapeutic approach to bisphosphonate associated osteonecrosis of the jaw’’ in Oral Oncology, by Ziebart T et al., and would like to compare and discuss these results with our recently published data.1 The design of the two studies is very similar: natural isoprenoids have been used to revert the mevalonate pathway inhibition induced by amino-bisphosphonates (N-BPs). Isoprenoid compounds are hypothesized to enter the mevalonate pathway after the N-BPs block, being metabolized as farnesyl-pyrophosphate and bypass the biochemical inhibition mediate by N-BPs on farnesyl- pyrophosphate synthase, restoring the metabolites flux along the pathway.2,3 Ziebart T. et al. empathized that the isoprenoid geranylgeraniol (GGOH) can antagonize the effects of N-BPs in the processes of osteoclast formation, apoptosis, bone resorption and in tumor cells (i.e. prostate cancer cells, human myeloma cells) as previously described in several studies,4,5 suggesting a therapeutic use of GGOH in bisphosphonate associated osteonecrosis of the jaw (BP-ONJ). Recently, our group demonstrated that GGOH and several other isoprenoids (farnesol, geranygeraiol, mentol, limonene) are able to revert the pro-inflammatory effect induced by the combination of N-BPs and bacterial lipoplyshaccaride (LPS) or muramyldipeptide (MDP) both in Balb/c mice and in human and murine monocytes. 2,3,6 We also proposed isoprenoid compounds as eligible treatment for the rare and still orphan disease mevalonate kinase deficiency (MKD, OMIM: 251170), characterized by genetic defect in the second enzyme of the mevalonate pathway. All this considered we would like to discuss the following issues: – We do agree with Ziebart T. et al.1 about the anti-N-BPs effect of geraniol, emphasizing that this effect is not dependent on the N-BP used on the isoprenoids. Ziebart’ group used ibandronate, pamidronate and zoledronate in their model, whereas we treated Balb/c mice and monocytes with alendronate or pamidronate obtaining comparable and reproducible findings. 2,7 Moreover geranylgeraniol, farnesol, menthol and limonene showed a comparable effect in contrasting N-BP action in our models,3 suggesting a common mechanism of action for these compounds in the context of NBP inhibition. – It is interesting to note that GGOH is able to contrast N-BP effect independently of the cellular model implied (osteoblast or monocytes) and the different outcome of N-BP inhibition. Since the block of the mevalonate pathway affects the prenylation of several signalling molecules involved in cell cycle, differentiation and cell response to extracellular stimulus, probably it results in different defects depending on the cell types. – We suggest Ziebart T. et al.1, to test other isoprenoids in addition to GGOH, such as geraniol, farnesol, menthol or limonene, in order to identify the most effective isoprenoids to treat in combination within aminobisphosphonate associated osteonecrosis of the jaw. While the dose/effect of the isoprenoid is comparable within Ziebart T. et al. and our models, additionally we would like to emphasize the importance of the timing in isoprenoid administration. – In our animal model the isoprenoid timing necessary to revert the pro-inflammatory action of alendronate or pamidronate was the critical point, because the isoprenoid must be injected the day before and/or after the N-BP.2,7 – Recently we showed that farnesyltransferase inhibitor (FTI) such as manumycin A, Tipifarnib or Lonafarnib, currently used in clinical trials as anticancer drugs, were able to contrast N-BP effect leading to a redistribution of mevalonate intermediates along its pathway.6 We propose Ziebart T. et al. to evaluate the effects of these pharmacological agents which could be an alternative therapeutic approach in the case of N-BP-induced osteonecrosis of the jaw. In summary, we do agree with the study by Ziebart T. et al., and emphasize the pivotal role of isoprenoid to rescue the phenotype inflammation induced by aminobisphosphonate treatment
Letter to the editor: acute effects of intravenous administration of pamidronate in patients with osteoporosis.
No full textWe read the interesting article “Acute Effects of Intravenous Administration of Pamidronate in Patients with Osteoporosis” in the Journal of Korean Medical Science by Lim et al. (1). We would like to comment and compare these data to a study recently published by our research group (2). The two studies had different initial aims, but still they share the same results in determining the modulatory effect of inflammation of aminobisphosphonates, such as pamidronate. The pamidronate belongs to the family of aminobisphosphonates (N-BPs), currently the major class of drugs used for the treatment of osteoporosis and other diseases characterized by increased bone resorption. The immune modulation exerted by pamidronate has not yet fully been understood (3). In vitro experiments have shown an anti-inflammatory effect of this N-BP; (4, 5) as well as a pro-inflammatory one (6, 7). Moreover contrasting results were obtained when pamidronate was used for the treatment of different inflammatory or immunologic diseases, such as rheumatoid arthritis (8,9) or systemic sclerosis. The aminobiphosphonates act on farnesylpyrophosphate synthase (FPPS) and inhibit the mevalonate pathway, the latter being responsible for production of cholesterol and isoprenoid lipids. In particular we can hypothesize that the inflammatory phenotype is due to lack of enzymes downstream the FPPS, and in particular the lack of geranylgeranyl-pyrophosphate (GGPP) could be associated to the activation of caspase-1 and the high IL-1β release. Lim et al. (1) emphasized that in vivo infusion of pamidronate at a therapeutic dose of 30 mg increased production of two inflammatory cytokines, IL-6 and TNF-α in serum. The increase is an acute effect after intravenous injection (1). Recently, our group demonstrated that pamidronate is able to increase the sensitivity to bacterial compounds both in the murine macrophagic cell line (Raw 264.7) and in Balb/c mice, by an incremental release of IL1β. These findings are in agreement with published data concerning inflammatory modulation in alendronate treated-mice (2). Moreover the effect of pamidronate does not depend on its concentration, whereas it may be involved in the increase of susceptibility to pro-inflammatory compounds such as muramildipeptide or lipopolysaccaride (2). In summary, we agree with the study by Lim et al. (1) and we emphasize the pivotal role of pamidronate in the modulation of inflammatory response
TRAF6 promotes atypical ubiquitination of mutant DJ-1 and alpha-synuclein and is localized to Lewy bodies in sporadic Parkinson's disease brains
No full textParkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the Substantia Nigra and the formation of ubiquitin- and alpha-synuclein (aSYN)-positive cytoplasmic inclusions called Lewy bodies (LBs). Although most PD cases are sporadic, families with genetic mutations have been found. Mutations in PARK7/DJ-1 have been associated with autosomal recessive early-onset PD, while missense mutations or duplications of aSYN (PARK1, PARK4) have been linked to dominant forms of the disease. In this study, we identify the E3 ubiquitin ligase tumor necrosis factor-receptor associated factor 6 (TRAF6) as a common player in genetic and sporadic cases. TRAF6 binds misfolded mutant DJ-1 and aSYN. Both proteins are substrates of TRAF6 ligase activity in vivo. Interestingly, rather than conventional K63 assembly, TRAF6 promotes atypical ubiquitin linkage formation to both PD targets that share K6-, K27- and K29- mediated ubiquitination. Importantly, TRAF6 stimulates the accumulation of insoluble and polyubiquitinated mutant DJ-1 into cytoplasmic aggregates. In human post-mortem brains of PD patients, TRAF6 protein colocalizes with aSYN in LBs. These results reveal a novel role for TRAF6 and for atypical ubiquitination in PD pathogenesis
Hemoglobin is present as a canonical α2β2 tetramer in dopaminergic neurons
No full textAbstractHemoglobin is the oxygen carrier in blood erythrocytes. Oxygen coordination is mediated by α2β2 tetrameric structure via binding of the ligand to the heme iron atom. This structure is essential for hemoglobin function in the blood. In the last few years, expression of hemoglobin has been found in atypical sites, including the brain. Transcripts for α and β chains of hemoglobin as well as hemoglobin immunoreactivity have been shown in mesencephalic A9 dopaminergic neurons, whose selective degeneration leads to Parkinson's disease. To gain further insights into the roles of hemoglobin in the brain, we examined its quaternary structure in dopaminergic neurons in vitro and in vivo. Our results indicate that (i) in mouse dopaminergic cell line stably over-expressing α and β chains, hemoglobin exists as an α2β2 tetramer; (ii) similarly to the over-expressed protein, endogenous hemoglobin forms a tetramer of 64kDa; (iii) hemoglobin also forms high molecular weight insoluble aggregates; and (iv) endogenous hemoglobin retains its tetrameric structure in mouse mesencephalon in vivo. In conclusion, these results suggest that neuronal hemoglobin may be endowed with some of the biochemical activities and biological function associated to its role in erythroid cells. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins
Cytokine profiles of women with vulvodynia: Identification of a panel of pro-inflammatory molecular targets
No full textObjective: The vulvar pain syndrome (VPS) is a multifactorial disease severely influencing the lifestyle of affected women. Among possible etiological factors, local injury, peripheral and/or central sensitization of the nervous system, and a chronic inflammatory status have been positively associated with the development of VPS. The identification of a constitutive altered local inflammatory profile in VPS women may represent an important point in the characterization of patients’ phenotype as a useful marker influencing the vulvar micro-environment. The aim of this study was to investigative the possible role of the local cytokines production in women with VPS in comparison to healthy women.
Study design: In this study were collected vaginal swabs from 57 healthy women (HC) who never suffered from VPS and from 30 patients diagnosed with vulvodynia (VPS) by at least 3 years and currently symptomatic. All patients included in this study showed the absence of Sexually Transmitted (STD) diseases and Reproductive Tract Infection. Real-time PCR was performed to assess the genomic sequences of ST pathogens. The Luminex Bio-Plex platform was used for the analysis of a panel of 48 immune factors.
Results: Eleven molecules, specifically involved in the pro-inflammatory pathway were significantly modulated in VPS patients in comparison to healthy women, suggesting a persistent inflammatory process.
Conclusions: Therefore, these inflammatory factors could be possible biological markers involved in this disease. Nevertheless, other studies are needed to consider this specific immune profile as avalid marker of
the vulvodynia
Further studies on Caribbean tenebrionid beetles
No full textThe study of some newly collected material from the West Indies may justify a fourth paper on Caribbean Tenebrionidae in these “Studies”. Thanks to dr. P. WAGENAAR HUMMELINCK’S collecting work, the Tenebrionid fauna of the Antilles and the adjacent South American mainland shores may be considered to be pretty well known — at least as far as the Melasomes are concerned. Thus zoogeographical conclusions — though not differing essentially from those published in 1962 — appear to have a rather solid basis. Unfortunately much less is known about planticolous Tenebrionids, which anyhow are relatively less interesting for zoogeographical purposes, than the geophilous ones.
We also had the privilege of consulting the collections of the I.N.R.A. at Guadeloupe (see MARCUZZI & D’AGUILAR 1971) which considerably increased our knowledge of the Tenebrionid fauna of that and neighbouring islands. Several specimens on hand at the Institute of Marine Biology, Mayagüez, proved extremely useful for obtaining a better knowledge of the Tenebrionid fauna of the old, sedimentary island of Puerto Rico. In a few single cases material from other sources (British Museum, Museum G. Frey and the private collection of the author) has been used
Isoforms of the Erythropoietin receptor in dopaminergic neurons of the Substantia Nigra
No full textErythropoietin receptor (EpoR) regulates erythrocytes differentiation in blood. In the brain, EpoR has been shown to protect several neuronal cell types from cell death, including the A9 dopaminergic neurons (DA) of the Substantia Nigra (SN). These cells form the nigrostriatal pathway and are devoted to the control of postural reflexes and voluntary movements. Selective degeneration of A9 DA neurons leads to Parkinson's disease. By the use of nanoCAGE, a technology that allows the identification of Transcription Start Sites (TSSs) at a genome-wide level, we have described the promoter-level expression atlas of mouse A9 DA neurons purified with Laser Capture Microdissection (LCM). Here, we identify mRNA variants of the Erythropoietin Receptor (DA-EpoR) transcribed from alternative TSSs. Experimental validation and full-length cDNA cloning is integrated with gene expression analysis in the FANTOM5 database. In DA neurons, the EpoR gene encodes for a N-terminal truncated receptor. Based on STAT5 phosphorylation assays, we show that the new variant of N-terminally truncated EpoR acts as decoy when co-expressed with the full-length form. A similar isoform is also found in human. This work highlights new complexities in the regulation of Erythropoietin (EPO) signaling in the brain. (Figure presented.
Cell behaviour and cell-matrix interactions of human palmar aponeurotic cells in vitro
No full textThe present investigation has been performed to better characterize, in vitro, normal aponeurotic cells in comparison with dermal fibroblasts and with cells derived from Dupuytren ́s affected aponeuroses. Cells were cultured in monolayer and/or into three-dimensional collagen gels. Cell structure, adhesion, and spreading capability on different substrates, as well as integrin expression were investigated by light and electron microscopy and by flow cytometry. Cell-matrix interactions were also analyzed by gel retraction experiments in the presence, or absence, of RGD peptides and anti-integrin antibodies. Normal aponeurotic cells, compared with dermal fibroblasts, exhibited in vitro peculiar structural features, which were substantially maintained in Dupuytren ́s aponeurotic cells, irrespective of the substrate they were grown on. By contrast, the aponeurotic cell behavior was different in normal and diseased cells, these latter approaching that of dermal fibroblasts. Normal aponeurotic cells, in fact, were characterized by low efficiency in retracting the collagen gel, low alpha(2), alpha(1) and alpha(5) integrin subunit expression and low adhesion properties onto collagen and fibronectin, whereas cells isolated from the aponeuroses of Dupuytren ́s patients exhibited higher capability of retracting the collagen gel, increased adhesion properties toward collagen and fibronectin, and higher levels of integrin expression. No differences were observed between dermal fibroblasts from Dupuytren ́s patients or from normal subjects. These in vitro results are consistent with those previously obtained in situ, suggesting that palmar aponeurotic cells have a peculiar phenotype and that changes in cell-matrix interactions occur in Dupuytren ́s contracture. Moreover, by comparing data obtained from the retracted fibrotic cords and the still clinically unaffected aponeuroses of the same patients, it may be noted that Dupuytren ́s disease is not only confined to the clinically involved branches, but includes the whole aponeurosis of the affected hand
