Role of abnormal neutral endopeptidase-like activities in Hyp mouse bone cells in renal phosphate transport

We investigated whether the absence of Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) in the Hyp mouse affects the expression and activity of neprilysin (NEP) and of endothelin-converting enzyme-like endopeptidase (ECEL1/DINE) in bone marrow stromal cells (BMSC) and osteoblasts (Ob). Total NEP-like activity was higher in Ob than in BMSC regardless of genotype, and Hyp cells showed higher activities than normal. Conditioned media (CM) from Hyp BMSC and Ob inhibited inorganic phosphate (P(i)) uptake by mouse proximal tubule cells, and incubating Hyp Ob with phosphoramidon prevented the production of the inhibitor of renal P(i) uptake. A linear relationship was observed between the NEP-like activity of Hyp and normal cells and the inhibition of P(i) uptake. NEP and ECEL1/DINE mRNA levels were higher in Hyp cells than in normal cells, and in situ hybridization of ECEL1/DINE confirmed higher levels of expression in the Hyp mouse than in normal cells. In conclusion, we observed a correlation between the inhibition of P(i) uptake by CM from Hyp cells and elevated NEP-like activities.     

Endothelin-converting enzyme-like 1 (ECEL1; 'XCE'): a putative metallopeptidase crucially involved in the nervous control of respiration

Endothelin-converting enzyme-like 1 (ECEL1) is a putative zinc metalloprotease that was recently identified on the basis of its strong similarity to endothelin-converting enzyme 1. The physiological function of ECEL1 remains unknown so far; the failure to identify a substrate for ECEL1 could be related to the endoplasmic reticulum subcellular localization found by immunofluorescence in recombinant systems. However, clues to the function of ECEL1 were provided by the inactivation of its gene in mice, which resulted in neonatal lethality. The phenotype of homozygous ECEL1(-/-) mice, together with the very specific expression profile of its mRNA in the central nervous system, suggests that ECEL1 is crucially involved in the nervous control of respiration.     

Neprilysin degrades both amyloid beta peptides 1-40 and 1-42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases

To identify the amyloid beta peptide (Abeta) 1-42-degrading enzyme whose activity is inhibited by thiorphan and phosphoramidon in vivo, we searched for neprilysin (NEP) homologues and cloned neprilysin-like peptidase (NEPLP) alpha, NEPLP beta, and NEPLP gamma cDNAs. We expressed NEP, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PEX), NEPLPs, and damage-induced neuronal endopeptidase (DINE) in 293 cells as 95- to 125-kDa proteins and found that the enzymatic activities of PEX, NEPLP alpha, and NEPLP beta, as well as those of NEP and DINE, were sensitive to thiorphan and phosphoramidon. Among the peptidases tested, NEP degraded both synthetic and cell-secreted Abeta1-40 and Abeta1-42 most rapidly and efficiently. PEX degraded cold Abeta1-40 and NEPLP alpha degraded both cold Abeta1-40 and Abeta1-42, although the rates and the extents of the digestion were slower and less efficient than those exhibited by NEP. These data suggest that, among the endopeptidases whose activities are sensitive to thiorphan and phosphoramidon, NEP is the most potent Abeta-degrading enzyme in vivo. Therefore, manipulating the activity of NEP would be a useful approach in regulating Abeta levels in the brain.     

Complex arrangement of genes within a 220-kb region of double-duplicated DNA on human 2q37.1

Gene duplication events are followed by divergence of initially identical gene copies, due to the subsequent accumulation of mutations. These mutations tend to be degenerative and may lead to either nonfunctionalization or subfunctionalization of the gene copies. Here we report the molecular characterization of a 220-kb genomic DNA fragment from human 2q37.1, in which a double duplication and a partial triplication event has taken place. As a result, this region contains four copies of alkaline phosphatase (P), four copies of the ECEL1 gene (X), two copies of a newly identified gene (N), and two copies of a cholinergic receptor subunit (R), in the order N-P-X-P-X-P-X-N-P-X-R-R. While three of the four ECEL1 copies, one copy of the phosphatase gene and one copy of the newly identified gene have lost their function, three phosphatase gene copies and the two receptor subunits are still functionally active and thus may provide an example for subfunctionalization of duplicated genes.     

Identification of spin diffusion pathways in proteins by isotope-assisted NMR cross- relaxation network editing

A new isotope-assisted cross-relaxation editing experiment, [1H-13C]DINE-NOESY[1H-15N]HSQC (DINE = Double INEPT Edited), is proposed. It is based on the selectiveinversion of CH/CH3 or CH2 protons in the middle of the mixing time. The experiment sortsout the spin diffusion paths according to the principal mediators, either the CH/CH3 or theCH2 protons. This is useful in the structure refinement process, as it enables proper alignmentof the aliphatic protons in the vicinity of NH protons.     

Postnatal development- and age-related changes in DNA-methylation patterns in the human genome

Alterations in DNA methylation have been reported to occur during development and aging; however, much remains to be learned regarding post-natal and age-associated epigenome dynamics, and few if any investigations have compared human methylome patterns on a whole genome basis in cells from newborns and adults. The aim of this study was to reveal genomic regions with distinct structure and sequence characteristics that render them subject to dynamic post-natal developmental remodeling or age-related dysregulation of epigenome structure. DNA samples derived from peripheral blood monocytes and in vitro differentiated dendritic cells were analyzed by methylated DNA Immunoprecipitation (MeDIP) or, for selected loci, bisulfite modification, followed by next generation sequencing. Regions of interest that emerged from the analysis included tandem or interspersed-tandem gene sequence repeats (PCDHG, FAM90A, HRNR, ECEL1P2), and genes with strong homology to other family members elsewhere in the genome (FZD1, FZD7 and FGF17). Our results raise the possibility that selected gene sequences with highly homologous copies may serve to facilitate, perhaps even provide a clock-like function for, developmental and age-related epigenome remodeling. If so, this would represent a fundamental feature of genome architecture in higher eukaryotic organisms.     

Neuronal pentraxin 1 induction in hypoxic-ischemic neuronal death is regulated via a glycogen synthase kinase-3α/β dependent mechanism

Intracellular signaling pathways that regulate the production of lethal proteins in central neurons are not fully characterized. Previously, we reported induction of a novel neuronal protein neuronal pentraxin 1 (NP1) in neonatal brain injury following hypoxia-ischemia (HI); however, how NP1 is induced in hypoxic-ischemic neuronal death remains elusive. Here, we have elucidated the intracellular signaling regulation of NP1 induction in neuronal death. Primary cortical neurons showed a hypoxic-ischemia time-dependent increase in cell death and that NP1 induction preceded the actual neuronal death. NP1 gene silencing by NP1-specific siRNA significantly reduced neuronal death. The specificity of NP1 induction in neuronal death was further confirmed by using NP1 (−/−) null primary cortical neurons. Declines in phospho-Akt (i.e. deactivation) were observed concurrent with decreased phosphorylation of its downstream substrate GSK-3α/β (at Ser21/Ser9) (i.e. activation) and increased GSK-3α and GSK-3β kinase activities, which occurred prior to NP1 induction. Expression of a dominant-negative inhibitor of Akt (Akt-kd) blocked phosphorylation of GSK-3α/β and subsequently enhanced NP1 induction. Whereas, overexpression of constitutively activated Akt (Akt-myr) or wild-type Akt (wtAkt) increased GSK-α/β phosphorylation and attenuated NP1 induction. Transfection of neurons with GSK-3α siRNA completely blocked NP1 induction and cell death. Similarly, overexpression of the GSK-3β inhibitor Frat1 or the kinase mutant GSK-3βKM, but not the wild-type GSK-3βWT, blocked NP1 induction and rescued neurons from death. Our findings clearly implicate both GSK-3α- and GSK-3β-dependent mechanism of NP1 induction and point to a novel mechanism in the regulation of hypoxic-ischemic neuronal death.     

Expression of CD47/integrin-associated protein induces death of cultured cerebral cortical neurons

The death and survival of neuronal cells are regulated by various signaling pathways during development of the brain and in neuronal diseases. Previously, we demonstrated that the neuronal adhesion molecule brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1 (BIT/SHPS-1) is involved in brain-derived neurotrophic factor (BDNF)-promoted neuronal cell survival. Here, we report the apoptosis-inducing effect of CD47/integrin-associated protein (IAP), the heterophilic binding partner of BIT/SHPS-1, on neuronal cells. We generated a recombinant adenovirus vector expressing a neuronal form of CD47/IAP, and found that the expression of CD47/IAP by infection with CD47/IAP adenovirus induced the death of cultured cerebral cortical neurons. The numbers of TdT-mediated biotin-dUTP nick-end labelling (TUNEL)-positive neurons and of cells displaying apoptotic nuclei increased by expression of CD47/IAP. Neuronal cell death was prevented by the addition of the broad-spectrum caspase inhibitor Z-VAD-fmk. Furthermore, we observed that co-expression of CD47/IAP with BIT/SHPS-1 enhanced neuronal cell death, and that BDNF prevented it. These results suggest that CD47/IAP is involved in a novel pathway which regulates caspase-dependent apoptosis of cultured cerebral cortical neurons. CD47/IAP-induced death of cultured cortical neurons may be regulated by the interaction of CD47/IAP with BIT/SHPS-1 and by BDNF.     

LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission

Although the aberrant activation of cell cycle proteins has a critical role in neuronal death, effectors or mediators of cyclin D1/cyclin-dependent kinase 4 (CDK4)-mediated death signal are still unknown. Here, we describe a previously unsuspected role of LIM kinase 2 (LIMK2) in programmed necrotic neuronal death. Downregulation of p27^Kip1 expression by Rho kinase (ROCK) activation induced cyclin D1/CDK4 expression levels in neurons vulnerable to status epilepticus (SE). Cyclin D1/CDK4 complex subsequently increased LIMK2 expression independent of caspase-3 and receptor interacting protein kinase 1 activity. In turn, upregulated LIMK2 impaired dynamic-related protein-1 (DRP1)-mediated mitochondrial fission without alterations in cofilin phosphorylation/expression and finally resulted in necrotic neuronal death. Inhibition of LIMK2 expression and rescue of DRP1 function attenuated this programmed necrotic neuronal death induced by SE. Therefore, we suggest that the ROCK-p27^Kip1-cyclin D1/CDK4-LIMK2-DRP1-mediated programmed necrosis may be new therapeutic targets for neuronal death.     

Co-localization of hypocretin-1 and hypocretin-2 in the cat hypothalamus and brainstem

Hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2) are two recently discovered hypothalamic neuropeptides. In the present study, using double immunofluorescent techniques, the co-localization of hcrt-1 and hcrt-2 was examined in neuronal soma and fibers/terminals located, respectively, in the cat hypothalamus and brainstem. In the hypothalamus, all hcrt-1 positive neuronal soma also displayed hcrt-2 immunoreactivity. In the brainstem, both hcrt-1 and hcrt-2 antibodies labeled the same fibers/terminals, indicating that hcrt-1 and hcrt-2 co-localize not only in the neuronal soma (hypothalamus) but also in their fibers/terminals (brainstem). If both peptides are released following neuronal activity, then the distinct effects of these peptides in the brain are likely to depend on the types of postsynaptic receptors that are activated.     

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.     

Methylcap-seq reveals novel DNA methylation markers for the diagnosis and recurrence prediction of bladder cancer in a Chinese population

Purpose

There is a need to supplement or supplant the conventional diagnostic tools, namely, cystoscopy and B-type ultrasound, for bladder cancer (BC). We aimed to identify novel DNA methylation markers for BC through genome-wide profiling of BC cell lines and subsequent methylation-specific PCR (MSP) screening of clinical urine samples.

Experimental Design

The methyl-DNA binding domain (MBD) capture technique, methylCap/seq, was performed to screen for specific hypermethylated CpG islands in two BC cell lines (5637 and T24). The top one hundred hypermethylated targets were sequentially screened by MSP in urine samples to gradually narrow the target number and optimize the composition of the diagnostic panel. The diagnostic performance of the obtained panel was evaluated in different clinical scenarios.

Results

A total of 1,627 hypermethylated promoter targets in the BC cell lines was identified by Illumina sequencing. The top 104 hypermethylated targets were reduced to eight genes (VAX1, KCNV1, ECEL1, TMEM26, TAL1, PROX1, SLC6A20, and LMX1A) after the urine DNA screening in a small sample size of 8 normal control and 18 BC subjects. Validation in an independent sample of 212 BC patients enabled the optimization of five methylation targets, including VAX1, KCNV1, TAL1, PPOX1, and CFTR, which was obtained in our previous study, for BC diagnosis with a sensitivity and specificity of 88.68% and 87.25%, respectively. In addition, the methylation of VAX1 and LMX1A was found to be associated with BC recurrence.

Conclusions

We identified a promising diagnostic marker panel for early non-invasive detection and subsequent BC surveillance.     

Cellular prion protein coupling to TACE-dependent TNF-α shedding controls neurotransmitter catabolism in neuronal cells

Despite considerable efforts to unravel the role of cellular prion protein (PrP^C) in neuronal functions, the mechanisms by which PrP^C takes part in the homeostasis of a defined neuronal phenotype remain poorly characterized. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C11^5-HT) or noradrenergic (1C11^NE) neurons, we assessed the contribution of PrP^C to bioaminergic cell functions. We established that in 1C11-derived neuronal cells antibody-mediated PrP^C ligation triggered tumor necrosis factor (TNF)-α release, through recruitement of the metalloproteinase TNF-α converting enzyme (TACE). TNF-α shed in response to PrP^C acts as a second message signal, eliciting serotonin (5-HT) or norepinephrine (NE) degradation in 1C11^5-HT or 1C11^NE cells, respectively. Our data thus introduced TNF-α as a PrP^C-dependent modulator of neuronal metabolism. Of note, we previously reported on a control of neurotransmitter catabolism by 5-HT_2B or α_1D autoreceptors in 1C11 bioaminergic neurons, via the same TACE/TNF-α pathway (Ann. N Y Acad. Sci. 1091, 123). Here, we show that combined stimulation of PrP^C and these two bioaminergic receptors add their effects on neurotransmitter degradation. Overall, these observations unveil a novel contribution of PrP^C to the control of neuronal functions and may have implications regarding dysfunction of the bioaminergic systems in prion diseases.