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7S nerve growth factor has different biological activity from 2.5S nerve growth factor in vitro
338
Brahz Research, 609 (1993) 338-340 Elsevier Science Publishers B.V.
BRES 25629
7S Nerve growth factor has different biological activity from 2.5S nerve growth factor in vitro Ningsheng Shao, Huixin Wang, Tingchong Z h o u and C h u a n Liu Beijing Institute of Basic Medical Sciences, Beijing (People's Republic of China) (Accepted 19 January 1993)
Key words: Nerve growth factor; Neuron; Neurotrophic effect; Astrocyte; Mitogen; Central nervous system
It is considered that two molecular forms of nerve growth factor (NGF), that is 7S NGF and 2.5S NGF have the identical neurotrophic effect on neurons. We now report that 7S NGF has different biological activities from 2.5S NGF in vitro. 7S NGF could promote the survival and neurite outgrowth of neurons from newborn rat hippocampus, cortex and cerebellum and stimulate the proliferation of astrocytes in vitro, but 2.5S NGF had no such effect.
Nerve growth factor (NGF) from the mouse submaxillary gland is a complex of three subunits known as a, /3, y with a composition of a2/3y 2. The intact complex has a sedimentation coefficient of 7S (hence the name 7S) with a molecular weight of about 130,000. The /3-subunit, so-called/3 NGF, is the active subunit of 7S NGF, and exhibits all the biological activities classically ascribed to NGF (i.e. support of neuronal survival, promotion of neurite outgrowth)./3 NGF is active only when dissociated from the 7S complex./3 NGF usually loses several amino acid residues of the C- or N-terminal during isolation, to form the 2.5S NGF which has the identical activities to /3 NGF. Until now it is considered that 7S NGF is biological active because it dissociates easily in solution, releasing/3 NGF and has the same neurotrophic effect to 2.5S NGF 4'6. Our study showed that 7S NGF has different roles from 2.5S NGF in vitro. Primary cultures of neurons were obtained from newborn Wistar rats (1-2 days old). The brain regions tested in the present study were the cortex, hippocampus and cerebellum. The desired brain tissues were dissected carefully free from meninges and collected in Ca2+-Mg2+-free Hank's balance salt solution (CMF). After incubation in 0.25% trypsin/CMF for 10 min at 37°C, tissues were washed twice in Dulbecco's modified Eagle's medium (DMEM) supplemented to 10%
horse serum and mechanically dissociated into single cells by pipetting. The cell suspensions were plated in poly-L-lysine- (Sigma, mol. wt. 20,000-30,000; coated plastic 96-well plates (6 mm diameter) at a density of 2 × 104 cells/well with DMEM supplemented with 10% horse serum. Twenty h after plating, the medium was changed to serum-free medium (SFM, that is, DMEM supplemented with 33.3 mmol glucose, 15 mmol HEPES buffer, 5 ~ g / m l insulin, 5/xg/ml transferrin and 0.02% bovine serum albumin) and the growth factors (7S NGF and 2.5S NGF are both from mouse submaxillary gland; 7S NGF is a Sigma product, 2.5S NGF is purified by Dr. Liu 1. The biological activity of these two factors has been tested using 8-day chick embryo dorsal root ganglia. Both of these two factors could induce neurite outgrowth of ganglia.). After 3 days, the cultures were fixed with 4% paraformaldehyde and the number of surviving neurons in each well were counted under a phase-contrast microscope. Only the neurons with process longer than the cell body diameter were counted. The neuronal cells were distinguished from non-neuronal cells by immunostaining with neuron-specific enolase antibodys. In the present cultures, more than 90% of the cells were neurons. Primary rat astrocyte cultures were prepared by the method of McCathy and de Vellis 5. Cultures used in this study were firstly plated in Falcon plastic tissue
Correspondence: N. Shao, Beijing Institute of Basic Medical Sciences, PO Box 130, Beijing 100850, P.R. China.
339 I control []50ng/ml 1~250ng/ml I~lO00ng/ml
go
I control ~ 5 0 ng/ml t~1200ng/ml r~qlOOOng/ml
10 O0 m
800 0
* \I
0 600
C
¢-
.0
I 0
,it
, •
0 Q) JO
\ \ \ \ \ \
0
\ \
I {/) 0
o EL
\i
0
\! NGF(7S)
0
NGF(2.5S)
Fig. 1. Effect of 7S N G F and 2.5S N G F on the survival of neurons from newborn rat hippocampus. Neurons were cultured in serum-free medium (SFM) for 3 days. Only the neurons with the process longer than one cell body diameter were counted under a phase contrast microscope in each well. The data are expressed asthe mean + S.E.M. (n = 3). * represents P < 0.01 compared with the control group.
culture flasks (75 cm 2) in DMEM supplemented with 10% baby calf serum (BCS). After 10 days, the astrocytes were suspended using 0.25% trypsin for 10 min and washed with the complete medium by centrifugation. Then the cells were planted into plastic 24-well plates with a density of 2 × 105 cells/well. After 2 days, cells were washed twice with SFM, and then added fresh SFM. After 12 h incubation, growth factors and [3H]TdR (1 ~Ci/ml) were added. The radioactivity was measured by liquid scintillation counting 24 h later. NGF (2.5S) has well been known to have little neurotrophic effect on neurons in the central nervous system (CNS) except septal and forebrain cholinergic 500
I control ~ 5 0 ng/ml ~200ng/ml [] lO00ng/ml
400
NGF(2.5S)
neurons 2'3. In present study, we also conformed that NGF (2.5S) has no significant neurotrophic effect on the neurons derived from cortex, hippocampus and cerebellum. Interestingly, we found that NGF (7S) had strong effect to promote the survival and the neurite outgrowth of neurons from above regions. Figs. 1-3 show that NGF (7S) could significantly promote the survival of hippocampal and cerebellum neurons at the doses of 50-1,000 ng/ml and cortical neurons at the concentration of 200-1,000 ng/ml. The neurotrophic effect was dose-dependent. More interestingly, we discovered that 7S NGF not only was a neurotrophic growth factor to neurons, but also a mitogen to astrocytes. Fig. 4 shows that 7S NGF could stimulate the [3H]TdR incorporation into DNA r~~ "
9O
I control IE~ 100 ng/mt IZ~ 500 ng/ml
Io X
Er7 60
0 I:~ C
NGF(7S)
Fig. 3. Effect of 7S N G F and 2.5S N G F on the survival of neurons from newborn rat cerebellum. Neurons were cultured in serum-free medium (SFM) for 3 days. Only the neurons with the process longer than one cell body diameter were counted under a phase contrast microscope in each well. The data are expressed as the mean + S.E.M. (n = 3). * represents P < 0.01 compared with the control group.
3OO
C 0 /// /// /// ///
0 0
.a
0 el.
NOF(7S)
iPln NGF(2.SS)
Fig. 2. Effect of 7S N G F and 2.5S N G F on the survival of neurons from newborn rat cortex. Neurons were cultured in serum-free medium (SFM) for 3 days. Only the neurons with the process longer than one cell body diameter were counted under a phase contrast microscope in each well. The data are expressed as the mean 5: S.E.M. (n = 3). * represents P < 0.01 compared with the control group.
o
50
0:2 "0
I I
/// Ff¢
7S NGF
2.5S
NGF
Fig. 4. Effect of 7S N G F and 2.5S N G F on the [3H]TdR incorporation into D N A synthesis of astrocytes derived from newborn rat brain. Astrocytes were treated with growth factors for 24 h, then the radioactivity was measured by liquid scintillation counting. The data are expressed as the m e a n + S . E . M . (n = 3). * represents P < 0.05, • * represents P < 0.01. (compared with the control group)
340 synthesis of astrocytes from newborn rat brain at the doses of more than 100 n g / m l . The mitogenic effect of 7S N G F was also dose-dependent. 2.5S N G F had no mitogenic effect on astrocytes. This result further supported our proposal that 7S N G F and 2.5S N G F had different biological activity in vitro. In summary, we have shown for the first time that 7S N G F had different biological roles on neurons and astrocytes in vitro. Considering the structural difference between the two molecules, we propose that the biological activities of 7S N G F on neurons and astrocytes may be related to the presence of c~, y beside/3 subunits in 7S NGF.
1 Chuan, L., Bull. Acad. Mill. Med. Sci., 11 (1987)28. 2 Gage, F.H. et al., Nerve growth factor function in the central nervous system. In M. Bothwell (Ed.), Current Topics m Microhiol~ ogy and Immunology, Vol. 165, Springer, Berlin, 1991, pp. 71 93. 3 Korsching, S., The role of nerve growth factor in the CNS, Trend.s Neurosci., 9 (1986) 570-573. 4 Longo, F.M. et al., Purification of nerve growth factor. In R.A. Rush (Ed.), Nerce Growth Factor, Wiley, New York, 1989, pp. 3-30. 5 McCarthy, K.D. and Vellis, J.D., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue, J. Cell Biol., 85 (1980) 890-902. 6 Moore, J.B. et al., Proteolytic modification of the /3-nerve growth factor protein, Biochemistry, 13 (1974) 833-840.
Brahz Research, 609 (1993) 338-340 Elsevier Science Publishers B.V.
BRES 25629
7S Nerve growth factor has different biological activity from 2.5S nerve growth factor in vitro Ningsheng Shao, Huixin Wang, Tingchong Z h o u and C h u a n Liu Beijing Institute of Basic Medical Sciences, Beijing (People's Republic of China) (Accepted 19 January 1993)
Key words: Nerve growth factor; Neuron; Neurotrophic effect; Astrocyte; Mitogen; Central nervous system
It is considered that two molecular forms of nerve growth factor (NGF), that is 7S NGF and 2.5S NGF have the identical neurotrophic effect on neurons. We now report that 7S NGF has different biological activities from 2.5S NGF in vitro. 7S NGF could promote the survival and neurite outgrowth of neurons from newborn rat hippocampus, cortex and cerebellum and stimulate the proliferation of astrocytes in vitro, but 2.5S NGF had no such effect.
Nerve growth factor (NGF) from the mouse submaxillary gland is a complex of three subunits known as a, /3, y with a composition of a2/3y 2. The intact complex has a sedimentation coefficient of 7S (hence the name 7S) with a molecular weight of about 130,000. The /3-subunit, so-called/3 NGF, is the active subunit of 7S NGF, and exhibits all the biological activities classically ascribed to NGF (i.e. support of neuronal survival, promotion of neurite outgrowth)./3 NGF is active only when dissociated from the 7S complex./3 NGF usually loses several amino acid residues of the C- or N-terminal during isolation, to form the 2.5S NGF which has the identical activities to /3 NGF. Until now it is considered that 7S NGF is biological active because it dissociates easily in solution, releasing/3 NGF and has the same neurotrophic effect to 2.5S NGF 4'6. Our study showed that 7S NGF has different roles from 2.5S NGF in vitro. Primary cultures of neurons were obtained from newborn Wistar rats (1-2 days old). The brain regions tested in the present study were the cortex, hippocampus and cerebellum. The desired brain tissues were dissected carefully free from meninges and collected in Ca2+-Mg2+-free Hank's balance salt solution (CMF). After incubation in 0.25% trypsin/CMF for 10 min at 37°C, tissues were washed twice in Dulbecco's modified Eagle's medium (DMEM) supplemented to 10%
horse serum and mechanically dissociated into single cells by pipetting. The cell suspensions were plated in poly-L-lysine- (Sigma, mol. wt. 20,000-30,000; coated plastic 96-well plates (6 mm diameter) at a density of 2 × 104 cells/well with DMEM supplemented with 10% horse serum. Twenty h after plating, the medium was changed to serum-free medium (SFM, that is, DMEM supplemented with 33.3 mmol glucose, 15 mmol HEPES buffer, 5 ~ g / m l insulin, 5/xg/ml transferrin and 0.02% bovine serum albumin) and the growth factors (7S NGF and 2.5S NGF are both from mouse submaxillary gland; 7S NGF is a Sigma product, 2.5S NGF is purified by Dr. Liu 1. The biological activity of these two factors has been tested using 8-day chick embryo dorsal root ganglia. Both of these two factors could induce neurite outgrowth of ganglia.). After 3 days, the cultures were fixed with 4% paraformaldehyde and the number of surviving neurons in each well were counted under a phase-contrast microscope. Only the neurons with process longer than the cell body diameter were counted. The neuronal cells were distinguished from non-neuronal cells by immunostaining with neuron-specific enolase antibodys. In the present cultures, more than 90% of the cells were neurons. Primary rat astrocyte cultures were prepared by the method of McCathy and de Vellis 5. Cultures used in this study were firstly plated in Falcon plastic tissue
Correspondence: N. Shao, Beijing Institute of Basic Medical Sciences, PO Box 130, Beijing 100850, P.R. China.
339 I control []50ng/ml 1~250ng/ml I~lO00ng/ml
go
I control ~ 5 0 ng/ml t~1200ng/ml r~qlOOOng/ml
10 O0 m
800 0
* \I
0 600
C
¢-
.0
I 0
,it
, •
0 Q) JO
\ \ \ \ \ \
0
\ \
I {/) 0
o EL
\i
0
\! NGF(7S)
0
NGF(2.5S)
Fig. 1. Effect of 7S N G F and 2.5S N G F on the survival of neurons from newborn rat hippocampus. Neurons were cultured in serum-free medium (SFM) for 3 days. Only the neurons with the process longer than one cell body diameter were counted under a phase contrast microscope in each well. The data are expressed asthe mean + S.E.M. (n = 3). * represents P < 0.01 compared with the control group.
culture flasks (75 cm 2) in DMEM supplemented with 10% baby calf serum (BCS). After 10 days, the astrocytes were suspended using 0.25% trypsin for 10 min and washed with the complete medium by centrifugation. Then the cells were planted into plastic 24-well plates with a density of 2 × 105 cells/well. After 2 days, cells were washed twice with SFM, and then added fresh SFM. After 12 h incubation, growth factors and [3H]TdR (1 ~Ci/ml) were added. The radioactivity was measured by liquid scintillation counting 24 h later. NGF (2.5S) has well been known to have little neurotrophic effect on neurons in the central nervous system (CNS) except septal and forebrain cholinergic 500
I control ~ 5 0 ng/ml ~200ng/ml [] lO00ng/ml
400
NGF(2.5S)
neurons 2'3. In present study, we also conformed that NGF (2.5S) has no significant neurotrophic effect on the neurons derived from cortex, hippocampus and cerebellum. Interestingly, we found that NGF (7S) had strong effect to promote the survival and the neurite outgrowth of neurons from above regions. Figs. 1-3 show that NGF (7S) could significantly promote the survival of hippocampal and cerebellum neurons at the doses of 50-1,000 ng/ml and cortical neurons at the concentration of 200-1,000 ng/ml. The neurotrophic effect was dose-dependent. More interestingly, we discovered that 7S NGF not only was a neurotrophic growth factor to neurons, but also a mitogen to astrocytes. Fig. 4 shows that 7S NGF could stimulate the [3H]TdR incorporation into DNA r~~ "
9O
I control IE~ 100 ng/mt IZ~ 500 ng/ml
Io X
Er7 60
0 I:~ C
NGF(7S)
Fig. 3. Effect of 7S N G F and 2.5S N G F on the survival of neurons from newborn rat cerebellum. Neurons were cultured in serum-free medium (SFM) for 3 days. Only the neurons with the process longer than one cell body diameter were counted under a phase contrast microscope in each well. The data are expressed as the mean + S.E.M. (n = 3). * represents P < 0.01 compared with the control group.
3OO
C 0 /// /// /// ///
0 0
.a
0 el.
NOF(7S)
iPln NGF(2.SS)
Fig. 2. Effect of 7S N G F and 2.5S N G F on the survival of neurons from newborn rat cortex. Neurons were cultured in serum-free medium (SFM) for 3 days. Only the neurons with the process longer than one cell body diameter were counted under a phase contrast microscope in each well. The data are expressed as the mean 5: S.E.M. (n = 3). * represents P < 0.01 compared with the control group.
o
50
0:2 "0
I I
/// Ff¢
7S NGF
2.5S
NGF
Fig. 4. Effect of 7S N G F and 2.5S N G F on the [3H]TdR incorporation into D N A synthesis of astrocytes derived from newborn rat brain. Astrocytes were treated with growth factors for 24 h, then the radioactivity was measured by liquid scintillation counting. The data are expressed as the m e a n + S . E . M . (n = 3). * represents P < 0.05, • * represents P < 0.01. (compared with the control group)
340 synthesis of astrocytes from newborn rat brain at the doses of more than 100 n g / m l . The mitogenic effect of 7S N G F was also dose-dependent. 2.5S N G F had no mitogenic effect on astrocytes. This result further supported our proposal that 7S N G F and 2.5S N G F had different biological activity in vitro. In summary, we have shown for the first time that 7S N G F had different biological roles on neurons and astrocytes in vitro. Considering the structural difference between the two molecules, we propose that the biological activities of 7S N G F on neurons and astrocytes may be related to the presence of c~, y beside/3 subunits in 7S NGF.
1 Chuan, L., Bull. Acad. Mill. Med. Sci., 11 (1987)28. 2 Gage, F.H. et al., Nerve growth factor function in the central nervous system. In M. Bothwell (Ed.), Current Topics m Microhiol~ ogy and Immunology, Vol. 165, Springer, Berlin, 1991, pp. 71 93. 3 Korsching, S., The role of nerve growth factor in the CNS, Trend.s Neurosci., 9 (1986) 570-573. 4 Longo, F.M. et al., Purification of nerve growth factor. In R.A. Rush (Ed.), Nerce Growth Factor, Wiley, New York, 1989, pp. 3-30. 5 McCarthy, K.D. and Vellis, J.D., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue, J. Cell Biol., 85 (1980) 890-902. 6 Moore, J.B. et al., Proteolytic modification of the /3-nerve growth factor protein, Biochemistry, 13 (1974) 833-840.