117
Neuroscience Letters, 130 (1991) 117-119 Elsevier Scientific Publishers Ireland Ltd. ADONIS 0304394091004884 NSL 08009
NGF restores decrease in catalase and increases glutathione peroxidase activity in the brain of aged rats G. Nistic61, M.R. Ciriolo 2, K. Fiskin 2, M. Iannone 1, A. De Martino 2 and G. Rotilio 2 1Chair of Pharmacology and 2Chair of Biochemistry, Department of Biology, University of Rome 'Tor Vergata', Rome (Italy) (Received 13 February 1991; Revised version received 21 May 1991; Accepted 27 May 1991)
Key words: Nerve growth factor; Catalase; Glutathione peroxidase; Ageing; Brain area The effects of subchronic administration of nerve growth factor (NGF) into the lateral ventricle on catalase and selenium-dependent glutathioneperoxidase (GSH-Px) activity in several areas of the brain in 3-, 12- and 24-month-old rats were studied. NGF given daily (1 pg for 28 consecutive days) produced in all brain areas studied a significant increase in catalase activity in 12- and 24-month-old rats. The most important finding was a complete restoration in 12- and 24-month-old rats of catalase activity to levels similar to those occurring in young (3-month-old) rats. In addition, NGF produced in comparison to 3-month-old rats and to same age vehicle-treated rats a significant increase in selenium-dependent GSH-Px in all the brain areas studied in 12- and 24-month-old animals, whereas selenium-independent GSH-Px was unaffected. In conclusion, the present results show that long-term administration of NGF into the lateral ventricle significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of hydrogen peroxide.
The free radicals theory [1, 8, 22] is one of the more persuasive hypotheses advanced to explain the degenerative changes and neuronal loss which occur in specific brain areas during ageing. Free radicals are highly reactive intermediates formed in many biochemical processes. For example, the superoxide and hydroxyl radicals derived from oxygen stimulate lipid peroxidation, disrupt membrane integrity and can lead to neuronal death (the membrane hypothesis of ageing) [20, 21]. Tissues are normally protected from their toxic effects by enzymatic mechanisms. Thus the enzyme superoxide dismutase scavenges the superoxide radical by converting it to molecular oxygen and hydrogen peroxide (H202), which in turn is inactivated by glutathione-peroxidase (GSH-Px) and catalase [6, 18]. Reduced levels of antioxidant enzymes with ageing could therefore explain agerelated neuro-degenerative processes. However, data present in literature regarding changes in antioxidant enzymes occurring in several areas of the brain are controversial [5, 9, 15] and this could be due to the different methods utilized for determination of the enzymatic activities, the life period considered, the brain regions selected, the strain of rats used, the criterion adopted for subcellular fractionation of brain as well as the prior perfusion of the brain.
Correspondence: G. Nistic6, Department of Biology, University of Rome 'Tor Vergata', Via Orazio Raimondo, 00173 Roma, Italy.
Here we report that intraventricular administration of nerve growth factor (NGF) is able to increase the activity of catalase and selenium-dependent GSH-Px activity in the rat brain. In particular, the activities of catalase and seleniumdependent GSH-Px in several brain regions of rats of different ages were determined. The effects of continuous intracerebroventricular infusion of NGF on the activity of these enzymes were also investigated. NGF stimulates growth of central neurones [12] and the concentration of NGF and of its mRNA decreases markedly during senescence [23]. NGF prevents forebrain neuronal death after fimbria-fornix transection [24] and after ibotenic acid lesions [16] and it also increases forebrain choline acetyltransferase (CHAT) activity in both young [7] and aged rats [4] as well as it prevents the decrease in ChAT and high-affinity choline uptake of the ipsilateral cortical level after lesion of the nucleus basalis in rats [2]. Under general anaesthesia, a cannula was stereotactically implanted into the right lateral ventricle and secured. The cannula was connected with polyethylene tubing to an Alzet minipump (model 2002, Alza, Palo Alto, CA, U.S.A.), inserted subcutaneously between the scapulae. The minipump infused 1.0 pg (in 12 /A) of NGF each day, or a similar volume of vehicle (controls) for 28 consecutive days (the pump was changed after 14 days). NGF was kindly provided by Dr. Alberta Leon (Fidia, Abano Terme, Italy). NGF was purified as the 2.5S subunit from the submaxillary glands of adult male
118 TABLE I CATALASE ACTIVITY IN VARIOUS AREAS OF THE BRAIN IN VEHICLE- OR N G F - T R E A T E D RATS OF D I F F E R E N T AGES Levels are the mean ___ S.E.M. of determinations from 6 rats. Brain area
Prefrontal cortex Parietal cortex Hippocampus Hypothalamus Caudate nucleus Mesencephalon Medulla oblongata Pons
Catalase activity (,umol substrate transformed min ~mg protein t) 3 months 12 months vehicle NGF vehicle NGF
24 months vehicle
NGF
4.5 3.0 2.8 9.7 3.4 5.3 11.3 9.4
2.4 2.2 2.4 5.8 2.1 5.0 5.1 6.0
5,9 3.9 4.3 9.6 3.4 8.5 11.2 9.7
_+ 0.7 ± 0.2 ± 0.1 ± 0.1 ± 0.3 ± 0.1 + 0.6 _+ 0.6
6.2 4.2 4.5 10.6 3.2 9.2 11.8 10.7
± 0.6 + 0.2 _+ 0.2 _+ 0.4 ___ 0.2 _ 0.2 + 0.7 ± 0.8
2.0 2.0 2.3 3.6 2.1 4.1 4.7 5.3
_ 0.1 a ± 0.1,' + 0.2 ± 0.2" + 0.1 a q- 0.2 ± 0.4 a ±_ 0.3"
4.3 4.9 3.7 8.5 3,6 7.0 6.7 8.6
± 0.68 + 0.68 + 0.38 _+ 0.38 ± 0.28 ± 0.38 _+ 0.28 _+ 0.38
_+ 0.2" ± 0,2" ± 0.3 + 0.5 a ± 0.1 ~ q- 0.1 + 0.2 ~ ± 0.5"
± ± + ± + + ± ±
0.48 0.i b 0.28 0.38 0.2 b 0.3 b 0.38 0.38
Student's t-test: ~P < 0.05 vs. 3-month-old rats; 8p < 0.05 between N G F - and vehicle-treated rats.
ferent ages (3-, 12- and 24-month-old) were assayed by a U.V. method [14]. GSH-Px (EC 1.11.1.9.) activity was assayed by a color±metric method with hydrogen peroxide or cumene hydroperoxide as substrate [11]. Catalase activity significantly decreased with ageing in the prefrontal and parietal cortex, hypothalamus, caudate nucleus, pons and medulla oblongata whereas it remained almost unchanged in the mesencephalon and hippocampus. N G F restored catalase activity in all areas of the brain to the levels seen at 3 months (Table I). Selenium-dependent GSH-Px activity was similar in brains of 3-, 12- and 24-month-old rats with the exception for a transient decrease only in some areas (hypothalamus, pons and medulla) in 12-month-old animals, suggesting that changes of some enzymatic activities occurring at the beginning of the ageing process (12-monthold rats) can be compensated at later stages of ageing. Infusion of N G F significantly increased selenium-depen-
Swiss albino mice, and its biological activity, evaluated utilizing embryonic chicken dissociated dorsal root ganglion cells in vitro, was in the range of 0.14).2 ng protein/trophic unit. For experimental purposes, N G F was dissolved in phosphate-buffered artificial cerebrospinal fluid containing 0.01% bovine albumin as carrier protein [24]. Twenty-four hours after the end of the treatment, the rats were anaesthetized and rapidly perfused with ice-cold saline (0.9% NaC1, w/v) via the heart, the brain areas dissected out and stored at -80°C. After homogenization in phosphate-buffered saline (1:5 w/v; pH 7.4, Potter Elvehjem glass homogenizer), the samples were cooled to 0°C, sonicated for 1.5 min (applied in 30 s bursts) and centrifuged at 22,000 g for 20 min. Supernatants were used for all assays of enzymatic activities and proteins. Proteins were determined according to Lowry et al. [13] using serum albumin as standard. Levels of catalase (EC. 1.11.1.6.) activity in various areas of the brain in control or NGF-treated rats of difTABLE II
LEVELS OF S E L E N I U M - D E P E N D E N T G L U T A T H I O N E PEROXIDASE (GSH-Px) ACTIVITY IN VARIOUS AREAS OF THE BRAIN IN VEHICLE- OR N G F - T R E A T E D RATS OF D I F F E R E N T AGE Levels are the mean _+ S.E.M. of determinations from 6 rats. Brain area
Prefrontal cortex Parietal cortex Hippocampus Hypothalamus Caudate nucleus Mesencephalon Medulla oblongata Pons
GSH-Px activity (substrate nmol transformed min ' mg protein -~) 3 months 12 months vehicle NGF vehicle NGF
24 months vehicle
NGF
34.9 36.9 35.0 68.3 76.0 59.4 82.7 75.6
35.3 49.4 39.8 56.7 72.2 60.3 86.5 81.8
63.2 66.8 50,5 81.3 102.4 85.7 106.5 92.6
+ 1.4 _+ 1.9 + 0.4 _ 1.7 + 9.0 _+ 1.4 ± 4.2 ± 4.5
40.1 34.7 42.6 50.6 60.2 66.8 63.6 60.5
± 2.8 ± 3.4 ± 4.0 ± 4.7 _+ 2.5 + 2,8 _+ 4,1 _+ 7.1
29.9 28.9 30.8 45,2 62.4 52.7 59.2 56.5
_+ 2.0 ± 3.7 ± 1.0 ± 2.1 ~' ± 4.9 ± 2.0 _+ 2.8 '~ _+ 4.0*
48.6 68.8 55.5 78.7 92.3 82.7 80.5 93.4
Student's t-test: "P
Neuroscience Letters, 130 (1991) 117-119 Elsevier Scientific Publishers Ireland Ltd. ADONIS 0304394091004884 NSL 08009
NGF restores decrease in catalase and increases glutathione peroxidase activity in the brain of aged rats G. Nistic61, M.R. Ciriolo 2, K. Fiskin 2, M. Iannone 1, A. De Martino 2 and G. Rotilio 2 1Chair of Pharmacology and 2Chair of Biochemistry, Department of Biology, University of Rome 'Tor Vergata', Rome (Italy) (Received 13 February 1991; Revised version received 21 May 1991; Accepted 27 May 1991)
Key words: Nerve growth factor; Catalase; Glutathione peroxidase; Ageing; Brain area The effects of subchronic administration of nerve growth factor (NGF) into the lateral ventricle on catalase and selenium-dependent glutathioneperoxidase (GSH-Px) activity in several areas of the brain in 3-, 12- and 24-month-old rats were studied. NGF given daily (1 pg for 28 consecutive days) produced in all brain areas studied a significant increase in catalase activity in 12- and 24-month-old rats. The most important finding was a complete restoration in 12- and 24-month-old rats of catalase activity to levels similar to those occurring in young (3-month-old) rats. In addition, NGF produced in comparison to 3-month-old rats and to same age vehicle-treated rats a significant increase in selenium-dependent GSH-Px in all the brain areas studied in 12- and 24-month-old animals, whereas selenium-independent GSH-Px was unaffected. In conclusion, the present results show that long-term administration of NGF into the lateral ventricle significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of hydrogen peroxide.
The free radicals theory [1, 8, 22] is one of the more persuasive hypotheses advanced to explain the degenerative changes and neuronal loss which occur in specific brain areas during ageing. Free radicals are highly reactive intermediates formed in many biochemical processes. For example, the superoxide and hydroxyl radicals derived from oxygen stimulate lipid peroxidation, disrupt membrane integrity and can lead to neuronal death (the membrane hypothesis of ageing) [20, 21]. Tissues are normally protected from their toxic effects by enzymatic mechanisms. Thus the enzyme superoxide dismutase scavenges the superoxide radical by converting it to molecular oxygen and hydrogen peroxide (H202), which in turn is inactivated by glutathione-peroxidase (GSH-Px) and catalase [6, 18]. Reduced levels of antioxidant enzymes with ageing could therefore explain agerelated neuro-degenerative processes. However, data present in literature regarding changes in antioxidant enzymes occurring in several areas of the brain are controversial [5, 9, 15] and this could be due to the different methods utilized for determination of the enzymatic activities, the life period considered, the brain regions selected, the strain of rats used, the criterion adopted for subcellular fractionation of brain as well as the prior perfusion of the brain.
Correspondence: G. Nistic6, Department of Biology, University of Rome 'Tor Vergata', Via Orazio Raimondo, 00173 Roma, Italy.
Here we report that intraventricular administration of nerve growth factor (NGF) is able to increase the activity of catalase and selenium-dependent GSH-Px activity in the rat brain. In particular, the activities of catalase and seleniumdependent GSH-Px in several brain regions of rats of different ages were determined. The effects of continuous intracerebroventricular infusion of NGF on the activity of these enzymes were also investigated. NGF stimulates growth of central neurones [12] and the concentration of NGF and of its mRNA decreases markedly during senescence [23]. NGF prevents forebrain neuronal death after fimbria-fornix transection [24] and after ibotenic acid lesions [16] and it also increases forebrain choline acetyltransferase (CHAT) activity in both young [7] and aged rats [4] as well as it prevents the decrease in ChAT and high-affinity choline uptake of the ipsilateral cortical level after lesion of the nucleus basalis in rats [2]. Under general anaesthesia, a cannula was stereotactically implanted into the right lateral ventricle and secured. The cannula was connected with polyethylene tubing to an Alzet minipump (model 2002, Alza, Palo Alto, CA, U.S.A.), inserted subcutaneously between the scapulae. The minipump infused 1.0 pg (in 12 /A) of NGF each day, or a similar volume of vehicle (controls) for 28 consecutive days (the pump was changed after 14 days). NGF was kindly provided by Dr. Alberta Leon (Fidia, Abano Terme, Italy). NGF was purified as the 2.5S subunit from the submaxillary glands of adult male
118 TABLE I CATALASE ACTIVITY IN VARIOUS AREAS OF THE BRAIN IN VEHICLE- OR N G F - T R E A T E D RATS OF D I F F E R E N T AGES Levels are the mean ___ S.E.M. of determinations from 6 rats. Brain area
Prefrontal cortex Parietal cortex Hippocampus Hypothalamus Caudate nucleus Mesencephalon Medulla oblongata Pons
Catalase activity (,umol substrate transformed min ~mg protein t) 3 months 12 months vehicle NGF vehicle NGF
24 months vehicle
NGF
4.5 3.0 2.8 9.7 3.4 5.3 11.3 9.4
2.4 2.2 2.4 5.8 2.1 5.0 5.1 6.0
5,9 3.9 4.3 9.6 3.4 8.5 11.2 9.7
_+ 0.7 ± 0.2 ± 0.1 ± 0.1 ± 0.3 ± 0.1 + 0.6 _+ 0.6
6.2 4.2 4.5 10.6 3.2 9.2 11.8 10.7
± 0.6 + 0.2 _+ 0.2 _+ 0.4 ___ 0.2 _ 0.2 + 0.7 ± 0.8
2.0 2.0 2.3 3.6 2.1 4.1 4.7 5.3
_ 0.1 a ± 0.1,' + 0.2 ± 0.2" + 0.1 a q- 0.2 ± 0.4 a ±_ 0.3"
4.3 4.9 3.7 8.5 3,6 7.0 6.7 8.6
± 0.68 + 0.68 + 0.38 _+ 0.38 ± 0.28 ± 0.38 _+ 0.28 _+ 0.38
_+ 0.2" ± 0,2" ± 0.3 + 0.5 a ± 0.1 ~ q- 0.1 + 0.2 ~ ± 0.5"
± ± + ± + + ± ±
0.48 0.i b 0.28 0.38 0.2 b 0.3 b 0.38 0.38
Student's t-test: ~P < 0.05 vs. 3-month-old rats; 8p < 0.05 between N G F - and vehicle-treated rats.
ferent ages (3-, 12- and 24-month-old) were assayed by a U.V. method [14]. GSH-Px (EC 1.11.1.9.) activity was assayed by a color±metric method with hydrogen peroxide or cumene hydroperoxide as substrate [11]. Catalase activity significantly decreased with ageing in the prefrontal and parietal cortex, hypothalamus, caudate nucleus, pons and medulla oblongata whereas it remained almost unchanged in the mesencephalon and hippocampus. N G F restored catalase activity in all areas of the brain to the levels seen at 3 months (Table I). Selenium-dependent GSH-Px activity was similar in brains of 3-, 12- and 24-month-old rats with the exception for a transient decrease only in some areas (hypothalamus, pons and medulla) in 12-month-old animals, suggesting that changes of some enzymatic activities occurring at the beginning of the ageing process (12-monthold rats) can be compensated at later stages of ageing. Infusion of N G F significantly increased selenium-depen-
Swiss albino mice, and its biological activity, evaluated utilizing embryonic chicken dissociated dorsal root ganglion cells in vitro, was in the range of 0.14).2 ng protein/trophic unit. For experimental purposes, N G F was dissolved in phosphate-buffered artificial cerebrospinal fluid containing 0.01% bovine albumin as carrier protein [24]. Twenty-four hours after the end of the treatment, the rats were anaesthetized and rapidly perfused with ice-cold saline (0.9% NaC1, w/v) via the heart, the brain areas dissected out and stored at -80°C. After homogenization in phosphate-buffered saline (1:5 w/v; pH 7.4, Potter Elvehjem glass homogenizer), the samples were cooled to 0°C, sonicated for 1.5 min (applied in 30 s bursts) and centrifuged at 22,000 g for 20 min. Supernatants were used for all assays of enzymatic activities and proteins. Proteins were determined according to Lowry et al. [13] using serum albumin as standard. Levels of catalase (EC. 1.11.1.6.) activity in various areas of the brain in control or NGF-treated rats of difTABLE II
LEVELS OF S E L E N I U M - D E P E N D E N T G L U T A T H I O N E PEROXIDASE (GSH-Px) ACTIVITY IN VARIOUS AREAS OF THE BRAIN IN VEHICLE- OR N G F - T R E A T E D RATS OF D I F F E R E N T AGE Levels are the mean _+ S.E.M. of determinations from 6 rats. Brain area
Prefrontal cortex Parietal cortex Hippocampus Hypothalamus Caudate nucleus Mesencephalon Medulla oblongata Pons
GSH-Px activity (substrate nmol transformed min ' mg protein -~) 3 months 12 months vehicle NGF vehicle NGF
24 months vehicle
NGF
34.9 36.9 35.0 68.3 76.0 59.4 82.7 75.6
35.3 49.4 39.8 56.7 72.2 60.3 86.5 81.8
63.2 66.8 50,5 81.3 102.4 85.7 106.5 92.6
+ 1.4 _+ 1.9 + 0.4 _ 1.7 + 9.0 _+ 1.4 ± 4.2 ± 4.5
40.1 34.7 42.6 50.6 60.2 66.8 63.6 60.5
± 2.8 ± 3.4 ± 4.0 ± 4.7 _+ 2.5 + 2,8 _+ 4,1 _+ 7.1
29.9 28.9 30.8 45,2 62.4 52.7 59.2 56.5
_+ 2.0 ± 3.7 ± 1.0 ± 2.1 ~' ± 4.9 ± 2.0 _+ 2.8 '~ _+ 4.0*
48.6 68.8 55.5 78.7 92.3 82.7 80.5 93.4
Student's t-test: "P
