J Mol Neurosci DOI 10.1007/s12031-013-0202-6
Axonal Transport of Neprilysin in Rat Sciatic Nerves Genki Ohkushi & Noriko Suzuki & Shigeki Kobayashi & Toshiyuki Chikuma
Received: 13 September 2013 / Accepted: 2 December 2013 # Springer Science+Business Media New York 2013
Abstract Axonal transport of neprilysin, a putative neuropeptide degrading-enzyme, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Neprilysin activity was significantly increased not only in the proximal segment but also in the distal segment 12–120 h after ligation, and the maximal neprilysin activity was found in the proximal and distal segments at 96 and 72 h, respectively. Western blot analysis of neprilysin showed that its immunoreactivities in the proximal and distal segments were 2.8- and 2.4-fold higher than that in the middle segment, indicating that neprilysin is transported by anterograde and retrograde axonal flow. These observations suggest that neprilysin may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts. Keywords Neprilysin . Axonal flow . Rat sciatic nerve . Double ligation technique Abbreviations Aβ CCK CPE DFP N-DNS-AG N-DNS-AGNPG N-DNS-R HRP IAA
Amyloid-β Cholecystokinin Carboxypeptidase E Diisopropylfluorophosphate N-Dansyl-D-alanylglycine N-Dansyl-D-alanylglycyl-pnitrophenylalanylglycine N-Dansyl-arginine Horseradish peroxidase Iodoacetic acid
G. Ohkushi : N. Suzuki : S. Kobayashi : T. Chikuma (*) Department of Analytical Chemistry of Medicines, Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen, Machida, Tokyo 194-8543, Japan e-mail: [email protected]
NEP OD pAb PAM PBS PCMS SP TPBS
Neprilysin Optical density Polyclonal antibody Peptidylglycine α-amidating monooxygenase Phosphate-buffered saline p-Chloromercuriphenylsulfonic acid Substance P Tween-PBS
Introduction Several types of neuropeptide function as neurotransmitters or modulators in the peripheral and central nervous systems. The expression of neuropeptide physiological function requires that newly synthesized neuropeptides in the cell body are transported to nerve terminals and released into the synaptic clefts. Previously, we demonstrated that not only neuropeptides (e.g., substance P [SP] and cholecystokinin [CCK]) (Kato et al. 1987; Tozawa et al. 1990) but also the enzymes required for their biosynthesis (e.g., processing endopeptidase, carboxypeptidase E [CPE] and peptidylglycine α-amidating monooxygenase [PAM]) (Tozawa et al. 1990; Yajima et al. 1994; Imaizumi et al. 2000) are transported to nerve terminals by anterograde axoplasmic flow. The functional level of biologically active neuropeptide depends on the rates of synthesis and degradation. It is generally considered that neuropeptides are degraded by enzymes in the extracellular space or synaptic clefts (Mckelvy and Blumberg 1986), and that these neuropeptide-degrading enzymes are also rapidly transported to nerve terminals. Neuropeptide-degrading enzymes may be removed from synaptic clefts by reuptake into dendrites, from which they are
J Mol Neurosci
retrogradely transported to the cell body. Previously, we reported the axonal transport of some enzymes involved in the degradation of neuropeptides (Kato et al. 1987; Yamamoto et al. 2002, 2003; Chikuma et al. 2007). However, the axonal transport of neprilysin (NEP; EC 3.4.24.11), a putative SPand CCK-inactivating zinc metallopeptidase, has not been investigated. NEP, also known as neutral endopeptidase, is a type II integral membrane zinc metalloprotein that does not have a proenzyme form. It is an ectoenzyme with a short cytoplasmic NH2 terminus, a transmembrane hydrophobic region, and a large extracellular domain containing the active site. Depending on tissue source, the molecular weight of NEP ranges from about 85,000 to 110,000 owing to differences in its glycosylation (Relton et al. 1983). It has broad substrate specificity, with its physiological role dependent upon its tissue localization. In the brain, NEP has been shown to be involved in the degradation of amyloid; specifically, it can degrade amyloid-β [Aβ(1–42)], the peptide component of Alzheimer's amyloid, in vivo and in vitro (Iwata et al. 2000, 2001; Marr et al. 2003; Eckman et al. 2006). Therefore, NEP is considered to be a putative target for the prevention or amelioration of Alzheimer’s disease symptoms (Malito et al. 2008; Nalivaeva et al. 2012). Furthermore, Koo et al. (1990) reported anterograde axonal transport of the amyloid precursor protein. In this study, we examined the axonal transport of NEP in rat sciatic nerves using a double ligation technique.
Ligation of Rat Sciatic Nerves
Materials and Methods
Assay for Neprilysin Activity
Materials
The assay for NEP activity is based on the fluorimetric measurement of N-DNS-AG liberated enzymatically from the substrate, N-DNS-AGNPG, after separation by HPLC. The reaction mixture contained 63 mM PIPES–NaOH buffer (pH 7.0), 25 μMN-DNS-AGNPG, and enzyme plus water, in the presence or absence of 100 μM thiorphan, in a total reaction volume of 200 μl. Incubation was carried out at 37 °C, and the reaction was terminated by heating at 95 °C for 5 min in boiling water. After centrifugation, N-DNS-R was added to clear supernatant as the internal standard, and an aliquot of the mixture obtained was subjected to HPLC analysis. The peak area of N-DNS-AG was measured and the amount was determined from the peak area of N-DNS-R added as an internal standard. Analysis of the product was performed using a Shimadzu (Kyoto, Japan) HPLC system consisting of an LC-10 AD pump, RF-10A fluorescence detector, CTO-10A column oven, DGU-12A degasser, SCL10A system controller and a C-R6A chromatopac. The system was operated at room temperature at a flow-rate of 1.0 ml/min employing a Finepak SIL C18S (particle size, 5 μm) reversed-
3,3′-Diaminobenzidine tetrahydrochloride and 1,10phenanthroline monohydrate were purchased from Wako (Tokyo, Japan). N-Dansyl-D-alanylglycine (N-DNS-AG), NDansyl-D-alanylglycyl-p-nitrophenylalanylglycine (N-DNSA G N P G ) , N- D a n s y l - a r g i n i n e ( N- D N S - R ) , diisopropylfluorophosphate (DFP), iodoacetic acid (IAA), pchloromercuriphenylsulfonic acid (PCMS), and phosphoramidon disodium salt were obtained from Sigma (St. Louis, MO, USA). Biotin-conjugated goat anti-rabbit immunoglobulins and streptavidin-conjugated horseradish peroxidase (streptavidin HRP) were obtained from Dako Cytomation (Denmark). Other materials and their sources were sodium pentobarbital (Dainabot, Tokyo, Japan), thiorphan (Endo Life Sciences, Farmingdale, NY, USA) and anti-rat NEP rabbit polyclonal antibody (pAb) (Millipore, Temecula, CA, USA). All other reagents were purchased from commercial sources and were generally of the highest purity available.
Male Wistar rats, weighing 250–300 g, were obtained from Sankyo Laboratory (Tokyo, Japan) and were housed with free access to food and water. A 12 h:12 h light–dark cycle was maintained over a period of 1 week. On the day of the surgery, the animals were anesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.). The right and left sciatic nerves were ligated at two places with surgical silk thread (4–0 gauge) (Figs. 1a and 2a). The animals were killed by decapitation between 0 and 120 h after ligation. Each segment from the nerves was dissected on ice, and used as a source of NEP activity or Western blot analysis in additional experiments. All animal experiments were carried out in accordance with the 1980 Animal Experiment Guidelines of the Japanese Government, and were approved by the Animal Experiment Committee of our University. Sample Preparation Each 3-mm segment was minced with scissors and homogenized by sonication in 100 μl of ice-cold phosphate-buffered saline (PBS). The homogenate was centrifuged at 100,000×g for 15 min and the supernatant obtained was used for the measurement of enzyme activity in each segment. On the other hand, each 5-mm segment of a sciatic nerve 48 h after ligation was minced with scissors, and homogenized in 31 μl of ice-cold PBS by sonication. After centrifugation at 100,000×g for 60 min, the supernatant obtained was used for Western blot analysis.
J Mol Neurosci
(a)
Ligation
P4
P3
P2
P1
Ligation
M1
M2
M3
D1
D2
Middle
Proximal
D3
Distal
3 mm
(b)
7
NEP activity (nmol/min/3 mm)
Fig. 1 Distribution of NEP activity in each segment of rat sciatic nerves 48 h after ligation. The supernatant of each segment was subjected to the NEP enzyme assay. a Schematic illustration of ligation in rat sciatic nerves, where arrows and "Ligation" indicate sites of ligation. b Distribution of NEP activity in rat sciatic nerves. P1–P4 proximal segments, M1–M3 middle segments, D1–D3 distal segments. Data are means ± SEM (bars) values from five to six animals. Statistical comparisons are indicated by solid lines. ANOVA was performed followed by the Student–Newman–Keuls test: ***p
Axonal Transport of Neprilysin in Rat Sciatic Nerves Genki Ohkushi & Noriko Suzuki & Shigeki Kobayashi & Toshiyuki Chikuma
Received: 13 September 2013 / Accepted: 2 December 2013 # Springer Science+Business Media New York 2013
Abstract Axonal transport of neprilysin, a putative neuropeptide degrading-enzyme, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Neprilysin activity was significantly increased not only in the proximal segment but also in the distal segment 12–120 h after ligation, and the maximal neprilysin activity was found in the proximal and distal segments at 96 and 72 h, respectively. Western blot analysis of neprilysin showed that its immunoreactivities in the proximal and distal segments were 2.8- and 2.4-fold higher than that in the middle segment, indicating that neprilysin is transported by anterograde and retrograde axonal flow. These observations suggest that neprilysin may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts. Keywords Neprilysin . Axonal flow . Rat sciatic nerve . Double ligation technique Abbreviations Aβ CCK CPE DFP N-DNS-AG N-DNS-AGNPG N-DNS-R HRP IAA
Amyloid-β Cholecystokinin Carboxypeptidase E Diisopropylfluorophosphate N-Dansyl-D-alanylglycine N-Dansyl-D-alanylglycyl-pnitrophenylalanylglycine N-Dansyl-arginine Horseradish peroxidase Iodoacetic acid
G. Ohkushi : N. Suzuki : S. Kobayashi : T. Chikuma (*) Department of Analytical Chemistry of Medicines, Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen, Machida, Tokyo 194-8543, Japan e-mail: [email protected]
NEP OD pAb PAM PBS PCMS SP TPBS
Neprilysin Optical density Polyclonal antibody Peptidylglycine α-amidating monooxygenase Phosphate-buffered saline p-Chloromercuriphenylsulfonic acid Substance P Tween-PBS
Introduction Several types of neuropeptide function as neurotransmitters or modulators in the peripheral and central nervous systems. The expression of neuropeptide physiological function requires that newly synthesized neuropeptides in the cell body are transported to nerve terminals and released into the synaptic clefts. Previously, we demonstrated that not only neuropeptides (e.g., substance P [SP] and cholecystokinin [CCK]) (Kato et al. 1987; Tozawa et al. 1990) but also the enzymes required for their biosynthesis (e.g., processing endopeptidase, carboxypeptidase E [CPE] and peptidylglycine α-amidating monooxygenase [PAM]) (Tozawa et al. 1990; Yajima et al. 1994; Imaizumi et al. 2000) are transported to nerve terminals by anterograde axoplasmic flow. The functional level of biologically active neuropeptide depends on the rates of synthesis and degradation. It is generally considered that neuropeptides are degraded by enzymes in the extracellular space or synaptic clefts (Mckelvy and Blumberg 1986), and that these neuropeptide-degrading enzymes are also rapidly transported to nerve terminals. Neuropeptide-degrading enzymes may be removed from synaptic clefts by reuptake into dendrites, from which they are
J Mol Neurosci
retrogradely transported to the cell body. Previously, we reported the axonal transport of some enzymes involved in the degradation of neuropeptides (Kato et al. 1987; Yamamoto et al. 2002, 2003; Chikuma et al. 2007). However, the axonal transport of neprilysin (NEP; EC 3.4.24.11), a putative SPand CCK-inactivating zinc metallopeptidase, has not been investigated. NEP, also known as neutral endopeptidase, is a type II integral membrane zinc metalloprotein that does not have a proenzyme form. It is an ectoenzyme with a short cytoplasmic NH2 terminus, a transmembrane hydrophobic region, and a large extracellular domain containing the active site. Depending on tissue source, the molecular weight of NEP ranges from about 85,000 to 110,000 owing to differences in its glycosylation (Relton et al. 1983). It has broad substrate specificity, with its physiological role dependent upon its tissue localization. In the brain, NEP has been shown to be involved in the degradation of amyloid; specifically, it can degrade amyloid-β [Aβ(1–42)], the peptide component of Alzheimer's amyloid, in vivo and in vitro (Iwata et al. 2000, 2001; Marr et al. 2003; Eckman et al. 2006). Therefore, NEP is considered to be a putative target for the prevention or amelioration of Alzheimer’s disease symptoms (Malito et al. 2008; Nalivaeva et al. 2012). Furthermore, Koo et al. (1990) reported anterograde axonal transport of the amyloid precursor protein. In this study, we examined the axonal transport of NEP in rat sciatic nerves using a double ligation technique.
Ligation of Rat Sciatic Nerves
Materials and Methods
Assay for Neprilysin Activity
Materials
The assay for NEP activity is based on the fluorimetric measurement of N-DNS-AG liberated enzymatically from the substrate, N-DNS-AGNPG, after separation by HPLC. The reaction mixture contained 63 mM PIPES–NaOH buffer (pH 7.0), 25 μMN-DNS-AGNPG, and enzyme plus water, in the presence or absence of 100 μM thiorphan, in a total reaction volume of 200 μl. Incubation was carried out at 37 °C, and the reaction was terminated by heating at 95 °C for 5 min in boiling water. After centrifugation, N-DNS-R was added to clear supernatant as the internal standard, and an aliquot of the mixture obtained was subjected to HPLC analysis. The peak area of N-DNS-AG was measured and the amount was determined from the peak area of N-DNS-R added as an internal standard. Analysis of the product was performed using a Shimadzu (Kyoto, Japan) HPLC system consisting of an LC-10 AD pump, RF-10A fluorescence detector, CTO-10A column oven, DGU-12A degasser, SCL10A system controller and a C-R6A chromatopac. The system was operated at room temperature at a flow-rate of 1.0 ml/min employing a Finepak SIL C18S (particle size, 5 μm) reversed-
3,3′-Diaminobenzidine tetrahydrochloride and 1,10phenanthroline monohydrate were purchased from Wako (Tokyo, Japan). N-Dansyl-D-alanylglycine (N-DNS-AG), NDansyl-D-alanylglycyl-p-nitrophenylalanylglycine (N-DNSA G N P G ) , N- D a n s y l - a r g i n i n e ( N- D N S - R ) , diisopropylfluorophosphate (DFP), iodoacetic acid (IAA), pchloromercuriphenylsulfonic acid (PCMS), and phosphoramidon disodium salt were obtained from Sigma (St. Louis, MO, USA). Biotin-conjugated goat anti-rabbit immunoglobulins and streptavidin-conjugated horseradish peroxidase (streptavidin HRP) were obtained from Dako Cytomation (Denmark). Other materials and their sources were sodium pentobarbital (Dainabot, Tokyo, Japan), thiorphan (Endo Life Sciences, Farmingdale, NY, USA) and anti-rat NEP rabbit polyclonal antibody (pAb) (Millipore, Temecula, CA, USA). All other reagents were purchased from commercial sources and were generally of the highest purity available.
Male Wistar rats, weighing 250–300 g, were obtained from Sankyo Laboratory (Tokyo, Japan) and were housed with free access to food and water. A 12 h:12 h light–dark cycle was maintained over a period of 1 week. On the day of the surgery, the animals were anesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.). The right and left sciatic nerves were ligated at two places with surgical silk thread (4–0 gauge) (Figs. 1a and 2a). The animals were killed by decapitation between 0 and 120 h after ligation. Each segment from the nerves was dissected on ice, and used as a source of NEP activity or Western blot analysis in additional experiments. All animal experiments were carried out in accordance with the 1980 Animal Experiment Guidelines of the Japanese Government, and were approved by the Animal Experiment Committee of our University. Sample Preparation Each 3-mm segment was minced with scissors and homogenized by sonication in 100 μl of ice-cold phosphate-buffered saline (PBS). The homogenate was centrifuged at 100,000×g for 15 min and the supernatant obtained was used for the measurement of enzyme activity in each segment. On the other hand, each 5-mm segment of a sciatic nerve 48 h after ligation was minced with scissors, and homogenized in 31 μl of ice-cold PBS by sonication. After centrifugation at 100,000×g for 60 min, the supernatant obtained was used for Western blot analysis.
J Mol Neurosci
(a)
Ligation
P4
P3
P2
P1
Ligation
M1
M2
M3
D1
D2
Middle
Proximal
D3
Distal
3 mm
(b)
7
NEP activity (nmol/min/3 mm)
Fig. 1 Distribution of NEP activity in each segment of rat sciatic nerves 48 h after ligation. The supernatant of each segment was subjected to the NEP enzyme assay. a Schematic illustration of ligation in rat sciatic nerves, where arrows and "Ligation" indicate sites of ligation. b Distribution of NEP activity in rat sciatic nerves. P1–P4 proximal segments, M1–M3 middle segments, D1–D3 distal segments. Data are means ± SEM (bars) values from five to six animals. Statistical comparisons are indicated by solid lines. ANOVA was performed followed by the Student–Newman–Keuls test: ***p
