Blood calcium concentration is maintained within a narrow range despite large variations in dietary input and body demand. The Transient Receptor Potential ion channel TRPV5 has been implicated in this process. We report here that TRPV5 is stimulated by the mammalian hormone klotho. Klotho, a beta-glucuronidase, hydrolyzes extracellular sugar residues on TRPV5, entrapping the channel in the plasma membrane. This maintains durable calcium channel activity and membrane calcium permeability in kidney. Thus, klotho activates a cell surface channel by hydrolysis of its extracellular N-linked oligosaccharides.

DNA methylation is an epigenetic mechanism for gene silencing engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to cytosine residues in gene regulatory regions. It is unknown if aberrant DNA methylation can cause neurodegeneration. We tested the hypothesis that Dnmts can mediate neuronal cell death. Enforced expression of Dnmt3a induced degeneration of cultured NSC34 cells. During apoptosis of NSC34 cells induced by camptothecin, levels of Dnmt1 and Dnmt3a increased five-fold and two-fold, respectively, and 5-methylcytosine accumulated in nuclei. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocked apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with RG108 and procainamide protected cultured neurons from excessive DNA methylation and apoptosis. In vivo, Dnmt1 and Dnmt3a are expressed differentially during mouse brain and spinal cord maturation and in adulthood when Dnmt3a is abundant in synapses and mitochondria. Dnmt1 and Dnmt3a are expressed in motor neurons of adult mouse spinal cord, and, during their apoptosis induced by sciatic nerve avulsion, nuclear and cytoplasmic 5-methylcytosine immunoreactivity, Dnmt3a protein levels, and Dnmt enzyme activity increased preapoptotically. Inhibition of Dnmts with RG108 blocked completely the increase in 5-methycytosine and the apoptosis of motor neurons in mice. In human amyotrophic lateral sclerosis (ALS), motor neurons showed changes in Dnmt1, Dnmt3a, and 5-methylcytosine similar to experimental models. Thus, motor neurons can engage epigenetic mechanisms to drive apoptosis, involving Dnmt upregulation and increased DNA methylation. These cellular mechanisms could be relevant to human ALS pathobiology and disease treatment.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes paralysis. Some forms of ALS are inherited, caused by mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human mutant SOD1 (mSOD1) toxicity to MNs are unresolved. Mitochondria in MNs might be key sites for ALS pathogenesis, but cause-effect relationships between mSOD1 and mitochrondiopathy need further study. We used transgenic mSOD1 mice to test the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the MN degeneration of ALS. Components of the multi-protein mPTP are expressed highly in mouse MNs, including the voltage-dependent anion channel, adenine nucleotide translocator (ANT), and cyclophilin D (CyPD), and are present in mitochondria marked by manganese SOD. MNs in pre-symptomatic mSOD1-G93A mice form swollen megamitochondria with CyPD immunoreactivity. Early disease is associated with mitochondrial cristae remodeling and matrix vesiculation in ventral horn neuron dendrites. MN cell bodies accumulate mitochondria derived from the distal axons projecting to skeletal muscle. Incipient disease in spinal cord is associated with increased oxidative and nitrative stress, indicated by protein carbonyls and nitration of CyPD and ANT. Reducing the levels of CyPD by genetic ablation significantly delays disease onset and extends the lifespan of G93A-mSOD1 mice expressing high and low levels of mutant protein in a gender-dependent pattern. These results demonstrate that mitochondria have causal roles in the disease mechanisms in MNs in ALS mice. This work defines a new mitochondrial mechanism for MN degeneration in ALS.

Altered motoneuron excitability is involved in amyotrophic lateral sclerosis pathobiology. To test the hypothesis that inhibitory interneuron innervation of spinal motoneurons is abnormal in an amyotrophic lateral sclerosis mouse model, we measured GABAergic, glycinergic, and cholinergic immunoreactive terminals on spinal motoneurons in mice expressing a mutant form of human superoxide dismutase-1 with a Gly933 Ala substitution (G93A-SOD1) and in controls at different ages. Glutamic acid decarboxylase, glycine transporter-2, and choline acetyltransferase were used as markers for GABAergic, glycinergic, and cholinergic terminals, respectively. Triple immunofluorescent labeling of boutons contacting motoneurons was visualized by confocal microscopy and analyzed quantitatively. Glycine transporter-2-bouton density on lateral motoneurons was decreased significantly in G93A-SOD1 mice compared with controls. This reduction was absent at 6 weeks of age but present in asymptomatic 8-week-old mice and worsened with disease progression from 12 to 14 weeks of age. Motoneurons lost most glycinergic innervation by 16 weeks of age (end-stage) when there was a significant decrease in the numbers of motoneurons and choline acetyltransferase-positive boutons. No significant differences in glutamic acid decarboxylase-bouton densities were found in G93A-SOD1 mice. Reduction of glycinergic innervation preceded mitochondrial swelling and vacuolization. Calbindin-positive Renshaw cell number was decreased significantly at 12 weeks of age in G93A-SOD1 mice. Thus, either the selective loss of inhibitory glycinergic regulation of motoneuron function or glycinergic interneuron degeneration contributes to motoneuron degeneration in amyotrophic lateral sclerosis. (Am J

Gitelman syndrome (GS) is a recessive salt-losing tubulopathy that is caused by mutations in the SLC12A3 gene that encodes the sodium-chloride co-transporter (NCC). GS is characterized by significant inter-and intrafamilial phenotype variability, with early onset and/or severe clinical manifestations in some patients. No correlations between the disease variability and the position/nature of SLC12A3 mutations have been investigated thus far. In this study, extensive mutational analyses of SLC12A3 were performed in 27 patients with GS, including genomic DNA sequencing, multiplex ligation-dependent probe amplification, cDNA analysis, and quantification of allele-specific transcripts, in parallel with functional analyses in Xenopus laevis oocytes and detailed phenotyping. Twenty-six SLC12A3 mutations were identified in 25 patients with GS, including eight novel (detection rate 80%). Transcript analysis demonstrated that splicing mutations of SLC12A3 lead to frameshifted mRNA subject to degradation by nonsense-mediated decay. Heterologous expression documented a novel class of NCC mutants with defective intrinsic transport activity. A subgroup of patients presented with early onset, growth retardation, and/or detrimental manifestations, confirming the potential severity of GS. The mutations that were associated with a severe presentation were the combination at least for one allele of a missplicing resulting in a truncated transcript that was downregulated by nonsense-mediated decay or a nonfunctional, cell surface-absent mutant. The most recurrent mutation on the second allele was a newly described NCC mutant that affected the functional properties of the co-transporter. These data suggest that the nature/position of SLC12A3 mutation, combined with male gender, is a determinant factor in the severity of GS and provide new insights in the underlying pathogenic mechanisms of the disease.

This study was conducted to investigate the effect of fish protein hydrolysate on growth performance, insulin-like growth factor I (IGF-I) levels and the expression levels of liver IGF-I mRNA in juvenile Japanese flounder (Paralichthys olivaceus). Fish hydrolysate was produced by enzymatic (alcalase and flavourzyme) treatment and size-fractionated by ultrafiltration. The permeate after ultrafiltration (UF) and the non-ultrafiltered fish hydrolysate were tested as feed ingredients using high plant protein diets. Fish meal was used in the control diet (FM). The feeding trial lasted for 60 days, and fish fed with 37 g kg )1 UF showed the best growth, feed efficiency, digestibility and protein utilization. Plasma IGF-I level was examined with radioimmunoassay, and the expression levels of liver IGF-I mRNA were evaluated using real-time PCR normalized against the 18S rRNA gene. Plasma IGF-I levels were significantly increased by inclusion of fish protein hydrolysate. Liver IGF-I mRNA expression was significantly higher in fish fed with 37 g kg )1 UF diet than fish fed with control diet. The results indicated that small molecular weight compounds from fish protein hydrolysate showed a positive effect on growth and feed utilization in juvenile Japanese flounder. Dietary fish protein hydrolysate could improve plasma IGF-I levels and liver IGF-I mRNA expression in Japanese flounder.

Amyotrophic lateral sclerosis (ALS) is a rapidly evolving and fatal adult-onset neurological disease characterized by progressive degeneration of motoneurons. Our previous study showed that glycinergic innervation of spinal motoneurons is deficient in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly933 Ala substitution (G93A-SOD1). In this study, we have examined, using whole-cell patch-clamp recordings, glycine receptor (GlyR)-mediated currents in spinal motoneurons from these transgenic mice. We developed a dissociated spinal cord culture model using embryonic transgenic mice expressing enhanced green fluorescent protein (eGFP) driven by the Hb9 promoter. Motoneurons were identified as Hb9 -eGFP-expressing (Hb9 -eGFP ϩ ) neurons with a characteristic morphology. To examine GlyRs in ALS motoneurons, we bred G93A-SOD1 mice to Hb9 -eGFP mice and compared glycine-evoked currents in cultured Hb9 -eGFP ϩ motoneurons prepared from G93A-SOD1 embryos and from their nontransgenic littermates. Glycine-evoked current density was significantly smaller in the G93A-SOD1 motoneurons compared with control. Furthermore, the averaged current densities of spontaneous glycinergic miniature IPSCs (mIPSCs) were significantly smaller in the G93A-SOD1 motoneurons than in control motoneurons. No significant differences in GABA-induced currents and GABAergic mIPSCs were observed between G93A-SOD1 and control motoneurons. Quantitative single-cell reverse transcription-PCR found lower GlyR␣1 subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the reduction of GlyR current may result from the downregulation of GlyR mRNA expression in motoneurons. Immunocytochemistry demonstrated a decrease of surface postsynaptic GlyR on G93A-SOD1 motoneurons. Our study suggests that selective alterations in GlyR function contribute to inhibitory insufficiency in motoneurons early in the disease process of ALS.

. Oocytes coexpressing wild-type TRPV5 and TRPV5⌬N or TRPV5⌬C showed virtually no wild-type TRPV5 expression on the plasma membrane, whereas co-expression of wild-type TRPV5 and TRPV5⌬N⌬C displayed normal channel surface expression. This indicates that TRPV5 trafficking toward the plasma membrane was disturbed by assembly with TRPV5⌬N or TRPV5⌬C but not with TRPV5⌬N⌬C. TRPV5 channel assembly signals were refined between amino acid positions 64 -77 and 596 -601 in the N-tail and C-tail, respectively. Pull-down assays and co-immunoprecipitations demonstrated that N-or C-tail mutants lacking these critical assembly domains were unable to interact with tails of TRPV5. In conclusion, two domains in the N-tail (residues 64 -77) and C-tail (residues 596 -601) of TRPV5 are important for channel subunit assembly, subsequent trafficking of the TRPV5 channel complex to the plasma membrane, and channel activity.TRPV5 and TRPV6 constitute the Ca 2ϩ influx pathway in 1,25-dihydroxyvitamin D 3 -responsive epithelia, including small intestine, kidney, and placenta, and play a vital role in the process of Ca 2ϩ (re)absorption (1-4). Both channels belong to a distinct subfamily (TRPV) within the superfamily of transient receptor potential channels (TRP). 1 The TRP family consists of a diverse group of non-voltage-gated cation channels, including TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies, which varies significantly in their selectivity and mode of activation (5). The understanding of the function, gating, regulation, and structure assembly of the TRP family is developing rapidly. Initially, it was demonstrated that the Drosophila TRP and TRPL members form heteromultimeric channels associated in a supramolecular signaling complex with specific receptors and regulators (6). Moreover, it has been identified that there are many channel compositions within the TRPC family, e.g. TRPC1/3, TRPC1/5, TRPC4/5, and TRPC3/ 6/7 (7-9). Within the TRPV family, the oligomeric structure of TRPV1 was studied by biochemical cross-linking, and the predominant existence of tetramers was suggested (10). More recently, it has been reported that TRPV5 and TRPV6 form homo-or heterotetramers in order to generate a pleiotropic set of functional channels with different Ca 2ϩ transport (11-13). TRPV5 and TRPV6 share 75% sequence homology at the amino acid level (11-13) and display several similar functional properties, including the permeation profile for monovalent and divalent cations (14) and regulation by calciotropic hormones (15-21). However, detailed sequence comparison of the N-and C-tails of the TRPV5 and TRPV6 channels reveals significant differences, which may account for the unique electrophysiological properties including differences of inactivation, kinetic properties, and affinity for the blocker ruthenium red between these two homologous channels (22).A considerable amount of information in channel subunit assembly has been accumulated by studies on voltage-gated K ϩ (K v ) channels that are structurally related to t...