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1.
PLoS One ; 11(6): e0157052, 2016.
Article in English | MEDLINE | ID: mdl-27272053

ABSTRACT

LAMP5 is member of the LAMP family of membrane proteins. In contrast to the canonical members of this protein family, LAMP1 and LAMP2, which show widespread expression in many tissues, LAMP 5 is brain specific in mice. In C. elegans, the LAMP5 ortholog UNC-46 has been suggested to act a trafficking chaperone, essential for the correct targeting of the nematode vesicular GABA-transporter UNC-47. We show here that in the mouse brain LAMP5 is expressed in subpopulations of GABAergic forebrain neurons in the striato-nigral system and the olfactory bulb. The protein was present at synaptic terminals, overlapping with the mammalian vesicular GABA-transporter VGAT. In LAMP5-deficient mice localization of the transporter was unaffected arguing against a conserved role in VGAT trafficking. Electrophysiological analyses in mutants showed alterations in short term synaptic plasticity suggesting that LAMP5 is involved in controlling the dynamics of evoked GABAergic transmission. At the behavioral level, LAMP5 mutant mice showed decreased anxiety and deficits in olfactory discrimination. Altogether, this work implicates LAMP5 function in GABAergic neurotransmission in defined neuronal subpopulations.


Subject(s)
GABAergic Neurons/metabolism , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Presynaptic Terminals/metabolism , Vesicular Inhibitory Amino Acid Transport Proteins/metabolism , Animals , Corpus Striatum/metabolism , Male , Mice , Olfactory Bulb/metabolism , Substantia Nigra/metabolism , Synaptic Transmission
2.
Handb Exp Pharmacol ; 220: 193-221, 2014.
Article in English | MEDLINE | ID: mdl-24668474

ABSTRACT

The neurotrophins play crucial roles regulating survival and apoptosis in the developing and injured nervous system. The four neurotrophins exert profound and crucial survival effects on developing peripheral neurons, and their expression and action is intimately tied to successful innervation of peripheral targets. In the central nervous system, they are dispensable for neuronal survival during development but support neuronal survival after lesion or other forms of injury. Neurotrophins also regulate apoptosis of both peripheral and central neurons, and we now recognize that there are regulatory advantages to having the same molecules regulate life and death decisions. This chapter examines the biological contexts in which these events take place and highlights the specific ligands, receptors, and signaling mechanisms that allow them to occur.


Subject(s)
Apoptosis , Cell Survival , Nerve Growth Factors/physiology , Animals , Humans , Nerve Growth Factor/physiology , Protein Precursors/physiology , Receptor, Nerve Growth Factor/physiology , Receptor, trkA/physiology
3.
FASEB J ; 25(6): 2061-70, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21411748

ABSTRACT

Signaling by TrkA and TrkB receptor tyrosine kinase is required for peripheral neuron survival. TrkA and TrkB signaling is facilitated by the p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, through mechanisms that remain obscure. Here, we demonstrate that TrkA and TrkB induces MEK-dependent phosphorylation of the transmembrane cysteine protease ADAM17 (a disintegrin and metalloprotease 17) at the intracellular residue threonine 735. Phosphorylation at this site activates ADAM17 and causes cleavage of p75NTR and production of the receptors' intracellular domain (p75NTR(ICD)) in PC12 cells and in primary cerebellar granule neurons. We show that Trk-induced ADAM17 phosphorylation and generation of the p75NTR(ICD) is required for neurotrophin-induced Erk and Akt activation and for neurotrophin-dependent survival signaling. Survival of PC12 cells maintained in 10 ng/ml nerve growth factor drops by 47% in cells depleted of ADAM17; this survival deficit is resolved if the p75NTR(ICD) is overexpressed in the ADAM17 depleted cells. These studies identify a novel signaling circuit in which Trk activates ADAM17-dependent p75NTR(ICD) production to feedback to sustain Trk signaling and Trk-dependent survival.


Subject(s)
ADAM Proteins/metabolism , Nerve Growth Factors/metabolism , Receptor, Nerve Growth Factor/metabolism , Receptor, trkA/metabolism , Signal Transduction/physiology , ADAM Proteins/genetics , ADAM17 Protein , Animals , Cerebellum/cytology , Gene Silencing , Nerve Growth Factors/genetics , Neurons/metabolism , PC12 Cells , Rats , Rats, Sprague-Dawley , Receptor, Nerve Growth Factor/genetics , Receptor, trkA/genetics
4.
J Cell Sci ; 123(Pt 13): 2299-307, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20530577

ABSTRACT

The p75 neurotrophin receptor (p75NTR) potentiates Trk signaling, but the underlying mechanisms remain uncertain. Here, we examine the relationship between p75NTR cleavage and Trk signaling. We found that, in PC12 cells, nerve growth factor (NGF) induces rapid and robust alpha-secretase- and gamma-secretase-dependent cleavage of p75NTR, releasing the resulting intracellular domain into the cytosol. Brain-derived neurotrophic factor similarly induces p75NTR cleavage in primary cerebellar granule neurons. p75NTR cleavage occurs by means of Trk-dependent activation of MEK-Erk signaling and induction of alpha-secretase activity, and is independent of ligand binding to p75NTR. Neurons and PC12 cells lacking p75NTR display defects in neurotrophin-dependent Akt activation. Normal Akt activation is rescued using full-length p75NTR or the p75 intracellular domain, but not cleavage-resistant p75NTR. We then demonstrate that NGF-dependent growth arrest of PC12 cells requires p75NTR cleavage and generation of the intracellular domain. We conclude that generation of the soluble p75NTR intracellular domain by Trk-induced cleavage plays a fundamental role in Trk-dependent signaling events.


Subject(s)
Nerve Growth Factors/metabolism , Receptor, Nerve Growth Factor/metabolism , Receptor, trkA/metabolism , Signal Transduction/physiology , Amyloid Precursor Protein Secretases/metabolism , Animals , Brain-Derived Neurotrophic Factor/metabolism , Cerebellum/cytology , Enzyme Activation , Humans , MAP Kinase Kinase Kinases/metabolism , Mice , Mice, Inbred C57BL , Nerve Growth Factor/metabolism , PC12 Cells , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Nerve Growth Factor/genetics , Receptor, trkA/genetics , Receptor, trkB/metabolism
5.
J Biol Chem ; 283(19): 12709-16, 2008 May 09.
Article in English | MEDLINE | ID: mdl-18299325

ABSTRACT

The uncleaved, pro-form of nerve growth factor (proNGF) functions as a pro-apoptotic ligand for the p75 neurotrophin receptor (p75NTR). However, some reports have indicated that proneurotrophins bind and activate Trk receptors. In this study, we have examined proneurotrophin receptor binding and activation properties in an attempt to reconcile these findings. We show that proNGF readily binds p75NTR expressed in HEK293T cells but does not interact with TrkA expressed under similar circumstances. Importantly, proNGF activates TrkA tyrosine phosphorylation, induces Erk and Akt activation, and causes PC12 cell differentiation. We show that inhibiting endocytosis or furin activity reduced TrkA activation induced by proNGF but not that induced by mature NGF and that proNGF123, a mutant form of NGF lacking dibasic cleavage sites in the prodomain, does not induce TrkA phosphorylation in PC12 cells. Therefore, endocytosis and cleavage appear to be prerequisites for proNGF-induced TrkA activity. We also found that proBDNF induces activation of TrkB in cerebellar granule neurons and that proBDNF cleavage by furin and metalloproteases facilitates this effect. Taken together, these data indicate that under physiological conditions, proneurotrophins do not directly bind or activate Trk receptors. However, endocytosis and cleavage of proneurotrophins produce processed forms of neurotrophins that are capable of inducing Trk activation.


Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Endocytosis , Nerve Growth Factor/metabolism , Peptide Hydrolases/metabolism , Protein Precursors/metabolism , Receptor, trkA/metabolism , Animals , Brain-Derived Neurotrophic Factor/genetics , Cell Line , Enzyme Activation , Gene Expression Regulation , Humans , Nerve Growth Factor/genetics , Protein Binding , Protein Precursors/genetics , Rats , Signal Transduction
6.
Biochem J ; 395(2): 417-26, 2006 Apr 15.
Article in English | MEDLINE | ID: mdl-16411897

ABSTRACT

cADPR (cADP-ribose), a metabolite of NAD+, is known to modulate intracellular calcium levels and to be involved in calcium-dependent processes, including synaptic transmission, plasticity and neuronal excitability. However, the enzyme that is responsible for producing cADPR in the cytoplasm of neural cells, and particularly at the synaptic terminals of neurons, remains unknown. In the present study, we show that endogenous concentrations of cADPR are much higher in embryonic and neonate mouse brain compared with the adult tissue. We also demonstrate, by comparing wild-type and Cd38-/- tissues, that brain cADPR content is independent of the presence of CD38 (the best characterized mammalian ADP-ribosyl cyclase) not only in adult but also in developing tissues. We show that Cd38-/- synaptosome preparations contain high ADP-ribosyl cyclase activities, which are more important in neonates than in adults, in line with the levels of endogenous cyclic nucleotide. By using an HPLC method and adapting the cycling assay developed initially to study endogenous cADPR, we accurately examined the properties of the synaptosomal ADP-ribosyl cyclase. This intracellular enzyme has an estimated K(m) for NAD+ of 21 microM, a broad optimal pH at 6.0-7.0, and the concentration of free calcium has no major effect on its cADPR production. It binds NGD+ (nicotinamide-guanine dinucleotide), which inhibits its NAD+-metabolizing activities (K(i)=24 microM), despite its incapacity to cyclize this analogue. Interestingly, it is fully inhibited by low (micromolar) concentrations of zinc. We propose that this novel mammalian ADP-ribosyl cyclase regulates the production of cADPR and therefore calcium levels within brain synaptic terminals. In addition, this enzyme might be a potential target of neurotoxic Zn2+.


Subject(s)
ADP-ribosyl Cyclase 1/metabolism , ADP-ribosyl Cyclase/metabolism , Aging/physiology , Brain/enzymology , Synaptosomes/enzymology , ADP-ribosyl Cyclase/antagonists & inhibitors , ADP-ribosyl Cyclase 1/deficiency , Animals , Animals, Newborn , Brain/drug effects , Brain/growth & development , Cyclic ADP-Ribose/metabolism , Guanine Nucleotides/pharmacology , Hydrogen-Ion Concentration , Kinetics , Mice , Mice, Knockout , NAD/analogs & derivatives , NAD/pharmacology , Synaptosomes/drug effects , Zinc/pharmacology
7.
EMBO Rep ; 6(10): 936-41, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16113645

ABSTRACT

Target-derived neurotrophins regulate neuronal survival and growth by interacting with cell-surface tyrosine kinase receptors. The p75 neurotrophin receptor (p75 NTR) is coexpressed with Trk receptors in long-range projection neurons, in which it facilitates neurotrophin binding to Trk and enhances Trk activity. Here, we show that TrkA and TrkB receptors undergo robust ligand-dependent ubiquitination that is dependent on activation of the endogenous Trk activity of the receptors. Coexpression of p75 NTR attenuated ubiquitination of TrkA and TrkB and delayed nerve growth factor-induced TrkA receptor internalization and receptor degradation. These results indicate that p75 NTR may prolong cell-surface Trk-dependent signalling events by negatively regulating receptor ubiquitination.


Subject(s)
Receptor, Nerve Growth Factor/metabolism , Receptor, trkA/metabolism , Receptor, trkB/metabolism , Ubiquitin/metabolism , Animals , Brain-Derived Neurotrophic Factor/pharmacology , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Humans , Immunoblotting , Immunoprecipitation , Nerve Growth Factor/pharmacology , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , PC12 Cells , Rats , Signal Transduction/physiology , Transfection
8.
Neuron ; 46(6): 839-40, 2005 Jun 16.
Article in English | MEDLINE | ID: mdl-15953410

ABSTRACT

The p75 neurotrophin receptor (p75NTR) collaborates with the Nogo receptor (NgR) and LINGO-1 to activate RhoA in response to myelin-based growth inhibitors such as myelin-associated glycoprotein (MAG). In this issue of Neuron, Domeniconi et al., in a surprising turn, show that MAG induces intramembrane proteolysis (RIP) of p75NTR and find that p75NTR cleavage is required for MAG-induced RhoA activation and growth inhibition.


Subject(s)
Myelin-Associated Glycoprotein/pharmacology , Neurites/drug effects , Neurons/cytology , Animals , Models, Biological , Neurites/metabolism , Protein Structure, Tertiary/physiology , rhoA GTP-Binding Protein/metabolism
9.
J Biol Chem ; 278(42): 40670-8, 2003 Oct 17.
Article in English | MEDLINE | ID: mdl-12909645

ABSTRACT

Cyclic ADP-ribose, a metabolite of NAD+, is known to modulate intracellular calcium levels and signaling in various cell types, including neural cells. The enzymes responsible for producing cyclic ADP-ribose in the cytoplasm of mammalian cells remain unknown; however, two mammalian enzymes that are capable of producing cyclic ADP-ribose extracellularly have been identified, CD38 and CD157. The present study investigated whether an ADP-ribosyl cyclase/NAD+-glycohydrolase independent of CD38 is present in brain tissue. To address this question, NAD+ metabolizing activities were accurately examined in developing and adult Cd38-/- mouse brain protein extracts and cells. Low ADP-ribosyl cyclase and NAD+-glycohydrolase activities (in the range of pmol of product formed/mg of protein/min) were detected in Cd38-/- brain at all developmental stages studied. Both activities were found to be associated with cell membranes. The activities were significantly higher in Triton X-100-treated neural cells compared with intact cells, suggesting an intracellular location of the novel cyclase. The cyclase and glycohydrolase activities were optimal at pH 6.0 and were inhibited by zinc, properties which are distinct from those of CD157. Both activities were enhanced by guanosine 5'-O-(3-thiotriphosphate), a result suggesting that the novel enzyme may be regulated by a G protein-dependent mechanism. Altogether our results indicate the presence of an intracellular membrane-bound ADP-ribosyl cyclase/NAD+-glycohydrolase distinct from CD38 and from CD157 in mouse brain. This novel enzyme, which is more active in the developing brain than in the adult tissue, may play an important role in cyclic ADP-ribose-mediated calcium signaling during brain development as well as in adult tissue.


Subject(s)
ADP-ribosyl Cyclase/genetics , ADP-ribosyl Cyclase/metabolism , Antigens, CD/genetics , Brain/metabolism , NAD+ Nucleosidase/chemistry , ADP-ribosyl Cyclase 1 , Animals , Calcium/metabolism , Cell Membrane/metabolism , Chlorides/pharmacology , Chromatography, High Pressure Liquid , Detergents/pharmacology , Dose-Response Relationship, Drug , Hydrogen-Ion Concentration , Membrane Glycoproteins , Mice , Neurons/metabolism , Octoxynol/pharmacology , Signal Transduction , Time Factors , Zinc/metabolism , Zinc Compounds/pharmacology
10.
Biochem J ; 370(Pt 1): 175-83, 2003 Feb 15.
Article in English | MEDLINE | ID: mdl-12403647

ABSTRACT

CD38 is a transmembrane glycoprotein that is expressed in many tissues throughout the body. In addition to its major NAD+-glycohydrolase activity, CD38 is also able to synthesize cyclic ADP-ribose, an endogenous calcium-regulating molecule, from NAD+. In the present study, we have compared ADP-ribosyl cyclase and NAD+-glycohydrolase activities in protein extracts of brains from developing and adult wild-type and Cd38 -/- mice. In extracts from wild-type brain, cyclase activity was detected spectrofluorimetrically, using nicotinamide-guanine dinucleotide as a substrate (GDP-ribosyl cyclase activity), as early as embryonic day 15. The level of cyclase activity was similar in the neonate brain (postnatal day 1) and then increased greatly in the adult brain. Using [14C]NAD+ as a substrate and HPLC analysis, we found that ADP-ribose is the major product formed in the brain at all developmental stages. Under the same experimental conditions, neither NAD+-glycohydrolase nor GDP-ribosyl cyclase activity could be detected in extracts of brains from developing or adult Cd38 -/- mice, demonstrating that CD38 is the predominant constitutive enzyme endowed with these activities in brain at all developmental stages. The activity measurements correlated with the level of CD38 transcripts present in the brains of developing and adult wild-type mice. Using confocal microscopy we showed, in primary cultures of hippocampal cells, that CD38 is expressed by both neurons and glial cells, and is enriched in neuronal perikarya. Intracellular NAD+-glycohydrolase activity was measured in hippocampal cell cultures, and CD38-dependent cyclase activity was higher in brain fractions enriched in intracellular membranes. Taken together, these results lead us to speculate that CD38 might have an intracellular location in neural cells in addition to its plasma membrane location, and may play an important role in intracellular cyclic ADP-ribose-mediated calcium signalling in brain tissue.


Subject(s)
ADP-ribosyl Cyclase/metabolism , Antigens, CD/metabolism , Hippocampus/enzymology , ADP-ribosyl Cyclase/genetics , ADP-ribosyl Cyclase 1 , Animals , Antigens, CD/genetics , Base Sequence , Blotting, Western , Calcium Signaling , Cells, Cultured , Centrifugation , DNA Primers , Membrane Glycoproteins , Mice , Microscopy, Confocal , Microscopy, Fluorescence , NAD/metabolism , RNA, Messenger/genetics
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