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Keywords:

  • Akt;
  • BDNF;
  • hippocampal neurons;
  • IGF-1;
  • MAPK

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) are trophic factors required for the viability and normal functions of various neuronal cells. However, the detailed intracellular mechanism(s) involved in these effects in neuronal cells remains to be fully elucidated. In present study, the respective intracellular signaling pathway induced by IGF-1 and BDNF and their possible role in neuronal survival were investigated. Both IGF-1 and BDNF protected hippocampal neurons from serum deprivation-induced death with IGF-1 apparently being more potent. Western blot analyses showed that both IGF-1 and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3)/Akt (protein kinase B) kinase and the mitogen-activated protein kinase (MAPK) pathways. The phosphorylation of Akt and its downstream target, FKHRL1, induced by IGF-1 was rapid and sustained while that of MAPK was transient. The reverse situation was observed for BDNF. Moreover, IGF-1 potently induced the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with PI3 kinase while BDNF was weak in these assays. In contrast, the tyrosine phosphorylation of Shc proteins was dramatically stimulated by BDNF, with IGF-1 having only a minimal effect. Most interestingly, only the inhibitor of the PI3K/Akt pathway, LY294002, was able to block the survival effects of both IGF-1 and BDNF; an inhibitor of the MAPK pathway inhibitor, PD98059, being ineffective. Taken together, these data reveal that the survival properties of both IGF-1 and BDNF against serum deprivation are mediated by the activation of the PI3K/Akt, but not the MAPK, pathway in hippocampal neurons.

Abbreviations used
Akt

protein kinase B

BDNF

brain-derived neurotrophic factor

CREB

cAMP response element-binding protein

DMEM

Dulbecco's modified Eagle's medium

DTT

dithiothreitol

FBS

fetal bovine serum

GSK3

glycogen synthase kinase 3

HBSS

Hank's balanced salt solution

HRP

horseradish peroxidase

IGF-1

insulin-like growth factor-1

IRS-1

insulin receptor substrate-1

MAPK

or ERK, mitogen-activated protein kinase

MEK

MAPK kinase

MTT

3-(4,5-dimethylthiazol-2-yl0–2,5-diphenyl-tetrazolium bromide

OKA

okadaic acid

PAGE

polyacrylamide gel electrophoresis

PDK1

PIP3-dependent kinase 1

PDK2

PIP3-dependent kinase 2

PI3

kinase, phosphatidylinositide 3 kinase

PIP2

PI 3,4-biphosphate

PIP3

PI 3,4,5-triphosphate. PLCγ, phospholipase Cγ

SDS

sodium dodecyl sulfate

Trk

tyrosine kinase

Insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) are polypeptide growth factors playing important roles in the normal development and maintenance of cellular integrity of the organism, including of the CNS (Lindholm et al. 1996; Connor and Dragunow 1998; Zheng et al. 2000a; Jones et al. 2003). IGF-1 and BDNF, as well as their respective receptors, are broadly expressed in various brain regions, with a particular enrichment in the hippocampal formation (Barbacid 1995; Dore et al. 1997; Connor and Dragunow 1998; Zheng et al. 2000a). Recent studies have provided direct evidence for the trophic role of these two growth factors in the hippocampus and their neuronal survival-promoting properties against different insults such as β-amyloid peptides and serum deprivation (Lindholm et al. 1996; Dore et al. 1997; Tagami et al. 1997; Arsenijevic and Weiss 1998; Trejo et al. 2001; Yamaguchi et al. 2001; Zheng et al. 2002a).

The biological actions of IGF-1 and BDNF are mediated by high-affinity receptors, the type I IGF and the tyrosine kinase B (TrkB) receptors, respectively. The binding of IGF-1 to the type I IGF-1 receptor or of BDNF to tyrosine kinase B (TrkB) activates the tyrosine kinase domains of these two receptors which in turn phosphorylates various intracellular substrates such as the insulin receptor substrate-1 (IRS-1), phospholipase Cγ (PLCγ), phospatidylinositol 3-kinase (PI3K) and Shc (Klein et al. 1989; Myers et al. 1993; Sasaoka et al. 1994; Barbacid 1995; LeRoith et al. 1995), leading to the activation of multiple signaling pathways including the mitogen-activated protein kinase [MAPK; also called extracellular signal-regulated kinase (ERK) and the PI3K/Akt; Barbacid 1995; Butler et al. 1998; Zheng et al. 2000a] pathways.

The phosphorylation/activation of MAPK enhances the phosphorylation of its downstream targets such as transcription factors and other kinases that mediate various cellular responses induced by trophic factors (Su and Karin 1996). For example, the activation of MAPK phosphorylates MAPK-activated kinases, the Rsks, which then catalyze the phosphorylation of the pro-apoptotic protein Bad at serine (Ser) residue 112 and the transcription factor CREB (cAMP response element-binding protein) at serine residue 133. The phosphorylation of Bad leads to the inactivation of this component of the cell death machinery, while the phosphorylation of CREB promotes cell survival (Butler et al. 1998; Bonni et al. 1999). By comparison, the activation of the PI3K pathway by a trophic factor stimulates the phosphorylation of the survival kinase Akt at Thr-308 and Ser-473 residues, this event activating this kinase (Alessi et al. 1996; Delcommenne et al. 1998; Kitamura et al. 1998; Balendran et al. 1999). Activated Akt in turn phosphorylates and inhibits several pro-apoptotic proteins such as Bad (del Peso et al. 1997), caspase-9 (Cardone et al. 1998), and the winged-helix family of transcription factors, FKHRL1 (Brunet et al. 1999; Zheng et al. 2000b, 2002b), FKHR (Guo et al. 1999; Zheng et al. 2002b) and AFX (Kops et al. 1999; Zheng et al. 2002b), leading to cell survival (Datta et al. 1999; Vivanco and Sawyers 2002).

Although both IGF-1 and BDNF are effective in promoting survival in many cell lines and neuronal phenotypes, the comparative effect of these two growth factors on the survival of hippocampal neurons as well as the underlined mechanisms involved have not been investigated. In present study, we compared the efficacy of IGF-1 and BDNF in promoting the survival of hippocampal neurons and the respective intracellular pathways involved in these effects. Our results showed that both IGF-1 and BDNF can protect hippocampal neurons from cell death induced by serum deprivation. Moreover, the survival effect of these two growth factors is mainly due to the activation of the PI3k/Akt (but not the MAPK) pathway.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Human recombinant IGF-1 was obtained from Genetech Inc (San Francisco, CA, USA). Human recombinant BDNF was from Pepro Tec. (Rocky Hill, NJ, USA). Wortmannin, leupeptin, aprotinin, and sodium vanadate were from Sigma Chemical (St Louis, MO, USA), while LY294002 and PD98059 were purchased from Calbiochem (Bad Soden, Germany). U0126 was from Promega (Madison, WI, USA). Anti-FKHRL1-Ser-253, anti-FKHRL1-Thr-32, anti-FKHRL1, anti-IRS-1/2, anti-PI3 kinase p85, and antiphosphotyrosine (4G10) were obtained from Upstate Biotechnology Inc. (Waltham, MA, USA); Anti-phospho-Akt, antiphospho-MEK (MAPK kinase), and antiphospho-ERK were from Cell Signaling (Beverly, MA, USA); monoclonal anti-IGF-1 receptor (ab-1) antibody was from Oncogen Research Products (Cambridge, MA, USA); anti-Trk antibody C14, anti-ERKs, anti-IGF-1 receptor β subunit, antiphosphotyrosine (PY99), and all secondary antibodies conjugated with horseradish peroxidase (HRP) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell culture reagents were purchased from Gibco Life Technologies (Grand Island, NY, USA), whereas all other reagents were from Sigma Chemical or Fisher Scientific (Nepean, ON, Canada).

Rat hippocampal neuronal cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Hippocampal cultured neurons were prepared as described before with minor modifications (Zheng et al. 2002a). Briefly, hippocampal cultured neurons are prepared from fetuses (embryonic day 19) of pregnant Sprague–Dawley rats (Charles River Breeding Laboratories, St-Constant, Quebec, Canada) and cultured under serum-free conditions. Animal care was according to protocols and guidelines approved by McGill University Animal Care Committee and the Canadian Council for Animal Care. Hippocampi were dissected in calcium- and magnesium-free Hank's balanced salt solution (HBSS) supplemented with 15 mm HEPES, 10 U/mL penicillin, and 10 µg/mL streptomycin. Tissues were collected and washed four to five times with HBSS and then submitted to an enzymatic digestion at 37°C with 0.25% trypsin (Gibco, Grand Island, NY, USA) in HBSS media for 10 min. The reaction was stopped and the cellular suspension was obtained by repeating aspirations through a Pasteur pipette. Following a centrifugation at 800 g for 10 min, the medium was removed and cells were resuspended in a chemically defined serum-free neurobasal medium supplemented with 2% B27 (Gibco), 20 µm l-glutamine, 15 mm HEPES, 10 U/mL penicillin, and 10 µg/mL streptomycin. Neurons were seeded at density of 5–8 × 105 cells/mL in culture plates pre-coated with 10 µg/mL poly-d-lysine under serum-free conditions in the above medium and grown at 37°C with 5% CO2 humidified atmosphere. On the day following plating, the medium was replaced with fresh culture medium as above. Experimental treatments were performed on day 7 after plating.

Treatments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Medium was replaced with neurobasal without B27 2 h before the desired reagents were added. To study the effects of different stimuli on the phosphorylation of various signaling proteins, cells were treated with or without 100 nm IGF-1 or 100 ng/mL BDNF, the respective optimal concentrations of IGF-1 and BDNF for the survival of hippocampal neurons. Alternatively, cells were pre-treated with LY294002 (12.5–100 µm, 30 min), PD98059 (12.5–50 µm, 40 min) or U0126 (20 µm, 30 min), followed by a stimulation with 100 nm IGF-1 or 100 ng/mL BDNF.

Western blotting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Western blotting was performed as described earlier with some modifications (Zheng et al. 1996, 2000c). Briefly, cells from different experimental conditions were rinsed with ice-cold HBSS and lysed in either RIPA buffer [50 mm Tris–HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Igepal CA-630, 0.1% sodium dodecyl sulfate (SDS), 50 mm NaF, 1 mm NaVO3, 5 mm phenyl-methylsulfonyl fluoride, 10 µg/mL leupeptin, 50 µg/mL aprotinin] or sample buffer [62.5 mm Tris-HCl, pH 6.8, 2% w/v sodium dodecyl sulfate (SDS), 10% glycerol, 50 mm dithiothreitol (DTT) and 0.1% w/v bromphenol blue]. Samples with equal amounts of protein were then separated by 4–20% polyacrylamide gel electrophoresis (PAGE), and the resolved proteins were electrotransferred to Hybond-C Nitrocellulose. Membranes were incubated with 5% non-fat milk in TBST (10 mm Tris–HCl, pH 8.0, 150 mm NaCl, and 0.2% Tween-20) for 2 h at room temperature or overnight at 4°C and incubated with appropriate primary antibody at 4°C overnight. Membranes were then washed twice with TBST and probed with corresponding second antibodies conjugated with HRP (anti/rat/mouse/rabbit/goat-HRP) at room temperature for 1 h. Membranes were finally washed several times with TBST to remove unbound secondary antibodies and visualized using an ECL detection kit (Amersham Co, Toronto, ON, Canada). A part of the SDS gel was stained with Coomassie blue to ensure the use of equal amounts of protein.

To evaluate the activation and inactivation of other signaling proteins, phosphorylation of these proteins was determined by western blot using their phospho-antibodies including antiphospho-Akt, antiphospho-MEK, antiphospho-ERK or a mixture of antiphospho-FKHRL1 Thr-32, and phospho-FKHRL1-Ser-253 antibodies, respectively. Blots were stripped and reprobed with antibodies for above proteins to ensure that equal amounts of these proteins were present. In some experiments, two parallel running gels loaded with identical samples were used. One of them was to evaluate the phosphorylation of the kinase while the other was used to determine the corresponding kinase levels in cell extracts. In experiments when the phosphorylation of multiple kinases was investigated, blots were stripped and reprobed with anti-β-actin as control. The effect of BDNF and IGF-1 was determined by comparing the phosphorylation of these proteins with the unphosphorylated protein level in cell extracts determined as above.

Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Tyrosine phosphorylation of these proteins were determined as described (Zheng et al. 2000b, 2000c). Briefly, the supernatants with equal amounts of protein from various samples were incubated overnight at 4°C with either anti-pan-Trk antibody C14, anti-IGF-1 receptor, anti-IRS-1, or anti-Shc antibodies. Protein–antibody immunocomplexes were isolated by protein A/G PLUS-agarose (Santa Cruz Biotechnology), separated by 4–20% SDS gel and then tyrosine phosphorylation was determined by western blot with a mixture of antiphosphotyrosine antibodies 4G10 and PY99. The blots were reprobed with antibodies for corresponding unphosphorylated proteins to ensure the presence of equal amount of proteins.

Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

IRS-1 were separated from hippocampal cultured neurons treated with IGF-1/BDNF at different time points as described above and the isolated IRS-1 and proteins associated with it were separated in 4–20% SDS gel. The amount of PI3 kinase associated with IRS-1 was determined by western blotting using anti-PI3 kinase antibody.

MTT colorimetric assay to determine the viability of hippocampal neurons

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Hippocampal neurons were plated in 96-well plates pre-coated with 10 µg/mL poly-d-lysine at the density of 50 000 cells/well as described before (Zheng et al. 2002a,b). At day 7, culture media were replaced with neurobasal without B27 to induce cell death and treated with or without different reagents for different times as indicated in individual figure legends. Cells were grown for 4–5 days and the culture medium was replaced with 0.5 mg/mL 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in DMEM. These cells were then returned into the incubator for a 3-h period. Cells and MTT formazan crystals were then solubilized by trituration in a solution of anhydrous isopropanol–HCL (0.1 N) and the survival profile of these cells was quantified by spectrophotometrically measuring the plate at 570 nm. Assays were repeated at least three to six times in quadruplicates.

Statistical analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Data are expressed as mean ± SEM. A one-way anova with Newman–Keul test was used to establish statistical significance set at p < 0.05.

Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

The effect of IGF-1 and BDNF on the survival of cultured hippocampal neurons was evaluated. As reported earlier (Zheng et al. 2002a), hippocampal cells grown in a serum-free defined medium supplemented with B27 were highly enriched with neurons (> 95%). Figure 1 shows that deprivation of B27 for 4–5 days induced 40–60% cell loss as determined using the MTT assay. Incubation in the presence of 100 nm IGF-1 or 100 ng/mL BDNF at the beginning of B27 deprivation significantly rescued these neurons. IGF-1 protected 90–100% of hippocampal neurons from B27 deprivation-induced cell losses while BDNF prevented 40–50% of cell death (Fig. 1). It thus appears that IGF-1 is more effective than BDNF in protecting hippocampal neurons from B27 deprivation-induced cell loss. To rule out the possibility that the relative lack of effect of BDNF is due to its relative instability in culture medium, both IGF-1 and BDNF were added each day and cell survival was determined as described in Materials and methods. Results from these experiments are similar to those in which IGF-1 and BDNF was added once (data not shown).

image

Figure 1. Both IGF-1 (100 nm) and BDNF (100 ng/mL) promote the survival of cultured hippocampal neurons from serum deprivation with IGF-1 apparently being more potent. Serum-deprived hippocampal cultures were treated with or without IGF-1 and BDNF for 4–5 days and the survival of neurons was determined by MTT assay as described in Materials and methods. Data represent assays from at least three independent experiments.

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IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

To investigate whether the different effect of IGF-1 and BDNF was related to the differential phophorylation of their respective receptors, the extent of receptor phosphorylation was studied next. Figure 2 shows that both IGF-1 and BDNF induced to a similar extent the tyrosine phosphorylation of their respective receptors.

image

Figure 2. Comparative phosphorylation of TrkB and IGF-1 receptors in hippocampal neurons. Hippocampal neurons were treated with IGF-1 or BDNF for 5 min and the tyrosine phosphorylation of IGFI-R and TrkB were determined. Both IGF-1 and BDNF stimulated the tyrosine phosphorylation of their respective receptors. Blots represent prototypical examples of experiments replicated at least three times.

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IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

To further establish the intracellular mechanism(s) involved in IGF-1/BDNF-mediated neuroprotective effects, the activation/phosphorylation of the PI3K/Akt and MAPK pathways was studied next. Cultured hippocampal neurons were treated with 100 nm IGF-1 or 100 ng/mL BDNF at different time points and the phosphorylation levels of Akt and its downstream target FKHRL1 (Zheng et al. 2002a, 2000b), as well as that of MAPK and MEK, an upstream kinase of MAPK, were determined. Figure 3 shows that both IGF-1 and BDNF time-dependently induced the phosphorylation of Akt, FKHRL1, MAPK, and MEK in cultured hippocampal neurons. IGF-1 rapidly induced the sustained activation of Akt as detected by its phosphorylation at Ser-473 (Fig. 3, row 1, lanes 2–6 vs. 1), this event leading to the inactivation of FKHRL1 as shown by the phosphorylation of this protein (Fig. 3, row 2, lanes 2–6 vs. 1) in hippocampal cultured neurons. The respective phosphorylation of Akt and FKHRL1 began as early as by 2.5 min, reached a maximum at almost the same time and remained stable for at least 40 min. In contrast, the activation of MAPK and MEK by IGF-1 is either transient (for MAPK, Fig. 3, row 4, lanes 2–6 vs. 1) or weak (for MEK, Fig. 3, row 3, lanes 2–6 vs. 1). The reversed situation is seen for BDNF as this trophic factor primarily and potently activated the MAPK pathway with a much lesser effect on the PI3/Akt pathway (Fig. 3, rows 1–4, lanes 8–12 vs. 7).

image

Figure 3. IGF-1 mainly activates the PI3K/Akt pathway while BDNF primarily acts via the Shc/MAPK pathway. Following treatment with IGF-1 or BDNF, the activation of Akt (Akt, FKHRL1) and MAPK (MEK, ERK) pathways in hippocampal neurons was determined by Western blots using antiphospho-FKHRL1/Akt/MEK/MAPK antibodies as described in Materials and methods. IGF-1 strongly stimulated the phosphorylation of Akt and its downstream target FKHRL1 while having much less effect on the MAPK pathway. The opposite was seen for BDNF. Blots represent prototypical examples of experiments replicated at least three times.

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IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

The respective effects of IGF-1 and BDNF on upstream signaling of PI3k/Akt and MAPK were studied next. Cultured hippocampal neurons were treated with either trophic factor and the tyrosine phosphorylation of IRS-1 and Shc was determined by immunoprecipitation. IGF-1 rapidly induced a three- to sixfold increase in the tyrosine phosphorylation of IRS-1 in cultured hippocampal neurons. The tyrosine phosphorylation of IRS-1 reached a maximum at 5 min, remained stable for up to 20 min to decrease thereafter (Fig. 4, row 1, lanes 1–4 vs. 5). In contrast, BDNF failed to significantly induce the tyrosine phosphorylation of IRS-1 (Fig. 4, row 1, lanes 6–9 vs. 5). Shc proteins are a group of adapter proteins consisting of Shc46, Shc52, and Shc66 (Bonfini et al. 1996). Treatment of hippocampal neurons with 100 ng/mL BDNF induced a five- to 10-fold increase in the tyrosine phosphorylation of Shc52 and Shc66, while no effect was seen on Shc46 (Fig. 4, row 2, lanes 6–9 vs. 5). Maximal tyrosine phosphorylation of Shc52 and Shc66 was seen by 5 min to decrease thereafter (Fig. 4, row 2, lanes 6–9 vs. 5). IGF-1 also induced the tyrosine phosphorylation of all three Shc proteins, but only to a minimal extent compared to BDNF (Fig. 4, row 2, lanes 1–4 vs. 5).

image

Figure 4. IGF-1 dramatically stimulates the tyrosine phosphorylation of IRS-1 while BDNF increases the phosphorylation of Shc proteins. Cultured hippocampal neurons pre-treated with 100 nm IGF-1 or 100 ng/mL BDNF and the tyrosine phosphorylation of IRS-1 and Shc was determined as described in Materials and methods. Tyrosine phosphorylation of IRS-1 is clearly stimulated by IGF-1 but not by BDNF. In contrast, BDNF dramatically increased the tyrosine phosphorylation of Shc and not IRS-1. Blots represent prototypical examples of experiments replicated at least three times.

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IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Knowing that IGF-1 mainly stimulates IRS-1 tyrosine phosphorylation, the respective effect of IGF-1 and BDNF on the association of PI3 kinase with IRS-1 was studied next. Hippocampal neurons were treated with either 100 nm IGF-1 or 100 ng/mL BDNF and the association of PI3 kinase with IRS-1 was determined by immunoprecipitation. Figure 5 shows that IGF-1 time-dependently facilitates the association of the PI3 kinase to IRS-1. The time course of this association is parallel to that of the tyrosine phosphorylation of IRS-1 (Fig. 5, lanes 1–4 vs. 5). In contrast to IGF-1, BDNF only weakly promoted the association between PI3 kinase and IRS-1 (Fig. 5).

image

Figure 5. Differential effect of IGF-1 and BDNF on the association of IRS-1 with PI3kinase in hippocampal neurons. Hippocampal neurons were stimulated with IGF-1 or BDNF and IRS-1 was separated with immunoprecipitation as described in Materials and methods. The association of PI3 Kinase with IRS-1 was determined by western blots using antiphospho-PI3K antibody. Only IGF-1 stimulated the association of IRS-1 with PI3 kinase. Blots represent prototypical examples of experiments replicated at least three times.

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IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

To establish the respective role of the PI3 kinase/Akt and MAPK pathways in the survival-promoting effects of IGF-1 and BDNF, cultured hippocampal neurons were pre-treated with the PI3 kinase inhibitor, LY294002, or the MEK inhibitor, PD98059. Pre-treatments with 25–50 µm LY294002 significantly blocked the protective effects of both IGF-1 and BDNF (Fig. 6a) while the MEK inhibitor, PD98059, had no effect (Fig. 7a). Similar data were obtained with another MAPK pathway inhibitor, U0126 (20 µm, data not shown). Parallel studies on the phosphorylation of Akt and MAPKs revealed that the inhibition of the survival effects of both IGF-1 and BDNF are related to the inhibition of the phosphorylation of Akt (Figs 6b and 7b). The combination of IGF-1 and BDNF did not increase the survival of these neurons above IGF-1 alone (Figs 6a and 7a). A similar observation was made regarding the phosphorylation of Akt (Fig. 6b). Figure 8 shows that the PI3 kinase inhibitor, LY294002, concentration-dependently blocked the survival effects of IGF-1, BDNF or a mixture of IGF-1 and BDNF, as well as the phosphorylation of Akt induced by these growth factors. Because the survival effect of BDNF is minor, to rule out the possibility that the inhibition of BDNF's neuroprotection is partly due to the toxic effect of LY294002, concentrations of LY294002 (5–10 µm) that do not have a significant effect by themselves were used in the survival assay. Figure 8(b) shows that 5–10 µm LY294002 significantly inhibited the effect of BDNF.

image

Figure 6. IGF-1 and BDNF promote cell survival by the activation of the PI3k/Akt pathway. Hippocampal neurons were pre-treated with the PI3 kinase inhibitor LY294002 and then exposed to IGF-1 or BDNF and grown for 4–5 days. Cell viability was determined by MTT as described in Materials and methods. Neuronal survival by both IGF-1 and BDNF was blocked by the PI3 kinase inhibitor LY294002 at a concentration required to inhibit the activation of Akt kinase. (a) MTT assays; (b) western blots. Data represent assays from at least three independent experiments (in quadruplicate for MTT assay).

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image

Figure 7. IGF-1 and BDNF-induced cell survival is not mediated by the MAPK pathway. Hippocampal neurons were pre-treated with the MAPK inhibitor PD98059 and then exposed to IGF-1 or BDNF and grown for 4–5 days. Cell viability was determined by MTT as described in Materials and methods. PD98059 has no effect in the survival properties of IGF-1 or BDNF. (a) MTT assays; (b) western blots. Data represent assays from at least three independent experiments (in quadruplicate for MTT assay).

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image

Figure 8. The PI3 kinase inhibitor LY294002, concentration-dependently blocks the survival effect and the activation of Akt induced by IGF-1 and BDNF in hippocampal neurons. Cultured hippocampal neurons pre-treated with different concentrations of LY294002 were exposed to IGF-1 or BDNF, and the phosphorylation of Akt and cell survival were determined. The effects of IGF-1 and BDNF on cell survival are not additive and are parallel to the activation of Akt. Data represent assays from at least three independent experiments (in quadruplicate for MTT assay).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

In this study, we provided evidence that IGF-1 and BDNF protect hippocampal neurons from death induced by serum deprivation with IGF-1 apparently being more effective. In addition, we demonstrated that the survival-promoting effects of both IGF-1 and BDNF are dependent on the activation of the PI3K/Akt pathway. Hence, in contrast to the original hypothesis, BDNF, like IGF-1, is acting via the PI3K/Akt (but not the MAPK pathway), to promote the survival of hippocampal neurons.

IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

Although IGF-1 and BDNF are growth factors promoting the survival of various cell types including neurons, some recent studies have suggested that IGF-1 is more potent than BDNF in most models (Fernandez-Sanchez et al. 1996; Lindholm et al. 1996; Yamada et al. 2001). For example, IGF-1 fully protects cerebellar granule cells from toxicity induced by a protein phosphatase 1 and 2A inhibitor, okadaic acid (OKA), while BDNF has no effect (Frernandez-Sanchez et al. 1996). In cortical neurons, both IGF-1 and BDNF can prevent neuronal death induced by serum deprivation but IGF-1 is much more potent than BDNF (Yamada et al. 2001). In the present study, we observed that IGF-1 is apparently more potent than BDNF in protecting hippocampal neurons against death induced by B27 deprivation. Taken together, it would hence appear that IGF-1 is a potent, broadly effective survival factor in various neuronal cell types. This is apparently not the case for BDNF (Frernandez-Sanchez et al. 1996; Lindholm et al. 1996; Yamada et al. 2001).

IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

The PI3 kinase/Akt pathway and the MAPK pathway are two major intracellular signaling systems used by growth factors involved in cell survival (Butler et al. 1998; Zheng et al. 2002a). Therefore, the respective effects of IGF-1 and BDNF on the activation of these two pathways was investigated next. Our results show that IGF-1 mainly activated the PI3/Akt pathway while BDNF primarily, but not exclusively, stimulated the MAPK pathway in hippocampal neurons. While both IGF-1 and BDNF promoted the activation/phosphorylation of the PI3K/Akt pathway, IGF-1 was clearly more potent and dramatically increased the phosphorylation of Akt and its downstream target FKHRL1; the effects of BDNF on these two proteins was rather weak. However, the reverse situation was seen in the case of the MAPK pathway, with BDNF being much more potent than IGF-1. Moreover, IGF-1 rapidly stimulated the tyrosine phosphorylation of IRS-1 and its association with PI3 kinase while its effect on the tyrosine phosphorylation of Shc was very weak. Again, the opposite results were obtained with BDNF, which was much more potent to induce the tyrosine phosphorylation of Shc than IRS-1. IRS proteins are the major upstream signaling proteins the IGF-1 receptor PI3/Akt pathway while the predominant activators of the Ras-MAPK pathway are the Shc-GRB2 proteins (Sasaoka et al. 1994, 1996; Ricketts et al. 1996; Bruning et al. 1997). Thus, while IGF-1 and BDNF preferentially activated the PI3/Akt kinase and the MAPK pathway, respectively, our data revealed that this is by no means exclusive. Similar results were recently obtained in cultured cortical neurons (Yamada et al. 2001).

The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

As IGF-1 mainly stimulated the PI3K/Akt pathway while BDNF preferentially activated the MAPK pathway, we investigated next the role of these two pathways in the neuroprotective effects of IGF-1 and BDNF in cultured hippocampal neurons. In accordance with earlier studies (Zheng et al. 2000b, 2002a, 2002b), the present results show that the survival effect of IGF-1 is mainly dependent on the activation of the PI3K/Akt pathway as only the PI3 kinase inhibitor, LY294002, and not the MAPK inhibitor, PD98059, blocked the protective effects of IGF-1 against death induced by B27 deprivation. Most interestingly, our data also demonstrated that the PI3K/Akt pathway is the key one involved in the protective effects of BDNF as only LY294002 blocked the effects of BDNF in our model. The MAPK pathway inhibitor, PD98059, was ineffective against the neuroprotective effects of BDNF even at a concentration sufficient to significantly block the phosphorylation of MAPK and MEK (Fig. 7, and data not shown). These results are in accordance with a recent study suggesting that MAPK was not very much involved in the protective effects of BDNF in primary cultured neurons (Bonni et al. 1999; Dolcet et al. 1999). Data further supporting the key role of the PI3K/Akt pathway included the fact that the survival effects of IGF-1 and BDNF are not additive, and that their combined protective effects are only blocked by a pre-treatment with the PI3 kinase inhibitor, the MAPK inhibitor being ineffective. Taken together, the results demonstrate the preferential role of the PI3K/Akt pathway in the neuroprotective effects of IGF-1 and BDNF in hippocampal neurons.

The role of Akt kinases in cell survival was first suggested by Dudek et al. (1997) in their study showing that IGF-1-induced survival of cultured cerebellar granule cells was mediated by Akt. Subsequently, Akt was found to be the key kinase involved in the survival-promoting effects of a broad range of survival factors in various cell types (Dudek et al. 1997; Crowder and Freeman 1999; Datta et al. 1999; Zheng et al. 2000a). The present findings demonstrate that, in embryonic hippocampal neurons, the PI3K/Akt pathway is the major one involved in the neuroprotective effects not only of IGF-1 and but also of BDNF against death induced by serum deprivation.

The exact mechanism(s) by which Akt mediates the survival effects of IGF-1 and BDNF in cultured hippocampal neurons remains to be fully established. It is known that activated Akt phosphorylates (this event leading to their inactivation) proapoptotic proteins such as Bad (del Peso et al. 1997), caspase-9 (Cardone et al. 1998), GSK3 and, most recently, the Forkhead transcription factors (Zheng et al. 2002a, 2000b; 2002b) and ASK1 (Kim et al. 2001). These results suggest that Akt can directly suppress apoptosis by inhibiting the activities of caspase-9, Forkhead, Bad, ASK1, and GSK3. Akt also been shown to indirectly regulate major death survival pathways such as p53, NF-κB, or CREB (Datta et al. 1999; Vivanco et al. 2002). Accordingly, Akt kinases likely mediate cell survival at different levels depending on cell type, target availability, and the requirement of transcriptional or post-transcriptional events to suppress apoptosis (Kaplan and Miller 2000). Further studies will be required to establish the various PI3k/Akt kinase-modulated downstream events involved in the neuroprotective effects of IGF-1 and BDNF in hippocampal neurons.

In summary, the present study demonstrated that IGF-1 is particularly effective in promoting the survival in cultured hippocampal neurons. Moreover, the activation of the PI3K/Akt pathway by IGF-1 and BDNF is critically involved in the neuroprotective effect of these two growth factors while the MAPK pathway likely only has a minimal role.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References

We sincerely thank Dr S. Kar for his excellent discussion. This research was supported by the Canadian Institute of Health Research (CIHR).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Rat hippocampal neuronal cultures
  6. Treatments
  7. Western blotting
  8. Determination of tyrosine phosphorylation of IGF-receptor, TrkB, IRS-1, and Shc by immunoprecipitation
  9. Determination of the association of PI3 kinase with IRS-1 induced by IGF-1/BDNF by co-precipitation
  10. MTT colorimetric assay to determine the viability of hippocampal neurons
  11. Statistical analysis
  12. Results
  13. Both IGF-1 and BDNF protect cultured hippocampal neurons from death induced by B27 deprivation
  14. IGF-1 and BDNF activate their individual receptors at a comparative level under our assay conditions
  15. IGF-1 mainly activates the PI3/Akt kinase pathway while BDNF primarily stimulates the MAPK pathway in cultured hippocampal neurons
  16. IGF-1 potently stimulates the tyrosine phosphorylation of IRS-1 while BDNF induces the tyrosine phosphorylation of Shc in hippocampal neurons
  17. IGF-1 induces the association of PI3 kinase with IRS-1 in hippocampal neurons while BDNF has a limited effect
  18. IGF-1 and BDNF protect hippocampal neurons against death induced by B27 deprivation via the PI3 kinase pathway
  19. Discussion
  20. IGF-1 potently protects cultured hippocampal neurons from death induced by serum deprivation
  21. IGF-1 primarily stimulates the activation of the Akt pathway while BDNF is particularly potent on the MAPK pathway
  22. The PI3/Akt pathway plays a key role in the survival effects of both IGF-1 and BDNF in hippocampal neurons
  23. Acknowledgements
  24. References
  • Alessi D. R., Andjelkovic M., Caudwell B., Cron P., Morrice N., Cohen P. and Hemmings B. A. (1996) Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15, 65416551.
  • Arsenijevic Y. and Weiss S. (1998) Insulin-like growth factor-I is a differentiation factor for post-mitotic CNS stem cell-derived neuronal precursors: distinct actions from those of brain-derived neurotrophic factor. J. Neurosci. 18, 21182128.
  • Balendran A., Casamayor A., Deak M., Paterson A., Gaffney P., Currie R., Downes C. P. and Alessi D. R. (1999) PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr. Biol. 9, 393404.
  • Barbacid M. (1995) Neurotrophic factors and their receptors. Curr. Opin. Cell Biol. 7, 148155.
  • Bonfini L., Migliaccio E., Pelicci G., Lanfrancone L. and Pelicci P. G. (1996) Not all Shc's roads lead to Ras. Trends Biochem. Sci. 21, 257261.
  • Bonni A., Brunet A., West A. E., Datta S. R., Takasu M. A. and Greenberg M. E. (1999) Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286, 13581362.
  • Brunet A., Bonni A., Zigmond M. J. et al. (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857868.
  • Bruning J. C., Winnay J., Cheatham B. and Kahn C. R. (1997) Differential signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1-deficient cells. Mol. Cell. Biol. 17, 15131521.
  • Butler A. A., Yakar S., Gewolb I. H., Karas M., Okubo Y. and LeRoith D. (1998) Insulin-like growth factor-I receptor signal transduction: at the interface between physiology and cell biology. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 121, 1926.
  • Cardone M. H., Roy N., Stennicke H. R., Salvesen G. S., Franke T. F., Stanbridge E., Frisch S. and Reed J. C. (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 13181321.
  • Connor B. and Dragunow M. (1998) The role of neuronal growth factors in neurodegenerative disorders of the human brain. Brain Res. Brain Res. Rev. 27, 139.
  • Crowder R. and Freeman R. (1999) The survival of sympathetic neurons promoted by potassium depolarization, but not by cyclic AMP, requires phosphatidylinositol 3-kinase and Akt. J. Neurochem. 73, 466475.
  • Datta S. R., Brunet A. and Greenberg M. E. (1999) Cellular survival: a play in three Akts. Genes Dev. 13, 29052927.
  • Delcommenne M., Tan C., Gray V., Rue L., Woodgett J. and Dedhar S. (1998) Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc. Natl Acad. Sci. USA 95, 1121111216.
  • Dolcet X., Egea J., Soler R., Martin-Zanca D. and Comella J. (1999) Activation of phosphatidylinositol 3-kinase, but not extracellular-regulated kinases, is necessary to mediate brain-derived neurotrophic factor-induced motoneuron survival. J. Neurochem. 73, 521531.
  • Dore S., Kar S. and Quirion R. (1997) Insulin-like growth factor I protects and rescues hippocampal neurons against β-amyloid- and human amylin-induced toxicity. Proc. Natl Acad. Sci. USA 94, 47724777.
  • Dudek H., Datta S. R., Franke T. F., Birnbaum M. J., Yao R., Cooper G. M., Segal R. A., Kaplan D. R. and Greenberg M. E. (1997) Regulation of neuronal survival by the serine–threonine protein kinase Akt. Science 275, 661665.
  • Fernandez-Sanchez M. T., Garcia-Rodriguez A., Diaz-Trelles R. and Novelli A. (1996) Inhibition of protein phosphatases induces IGF-1-blocked neurotrophin-insensitive neuronal apoptosis. FEBS Lett. 398, 106112.
  • Guo S., Rena G., Cichy S., He X., Cohen P. and Unterman T. (1999) Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J. Biol. Chem. 274, 1718417192.
  • Jones D., Tucker B. A., Rahimtula M. and Mearow K. M. (2003) The synergistic effects of NGF and IGF-1 on neurite growth in adult sensory neurons: convergence on the PI 3-kinase signaling pathway. J. Neurochem. 86, 11161128.
  • Kaplan D. R. and Miller F. D. (2000) Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol. 10, 381391.
  • Kim A. H., Khursigara G., Sun X., Franke T. F. and Chao M. V. (2001) Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol. Cell Biol. 21, 893901.
  • Kitamura T., Ogawa W., Sakaue H. et al. (1998) Requirement for activation of the serine–threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport. Mol. Cell Biol. 18, 37083717.
  • Klein R., Parada L. F., Coulier F. and Barbacid M. (1989) trkB, a novel tyrosine protein kinase receptor expressed during mouse neural development. EMBO J. 8, 37013709.
  • Kops G. J., De Ruiter N. D., Vries-Smits A. M., Powell D. R., Bos J. L. and Burgering B. M. (1999) Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398, 630634.
  • LeRoith D., Werner H., Beitner-Johnson D. and Roberts C. T. Jr (1995) Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr. Rev. 16, 143163.
  • Lindholm D., Carroll P., Tzimagiogis G. and Thoenen H. (1996) Autocrine-paracrine regulation of hippocampal neuron survival by IGF-1 and the neurotrophins BDNF, NT-3 and NT-4. Eur. J. Neurosci. 8, 14521460.
  • Myers M. G., Sun X. J., Cheatham B., Jachna B. R., Glasheen E. M., Backer J. M. and White M. F. (1993) IRS-1 is a common element in insulin and insulin-like growth factor-I signaling to the phosphatidylinositol 3′-kinase. Endocrinology 132, 14211430.
  • Del Peso L., Gonzalez-Garcia M., Page C., Herrera R. and Nunez G. (1997) Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278, 687689.
  • Ricketts W. A., Rose D. W., Shoelson S. and Olefsky J. M. (1996) Functional roles of the Shc phosphotyrosine binding and Src homology 2 domains in insulin and epidermal growth factor signaling. J. Biol. Chem. 27, 26165261659.
  • Sasaoka T., Rose D. W., Jhun B. H., Saltiel A. R., Draznin B. and Olefsky J. M. (1994) Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. J. Biol. Chem. 269, 1368913694.
  • Sasaoka T., Ishiki M., Sawa T., Ishihara H., Takata Y., Imamura T., Usui I., Olefsky J. M. and Kobayashi M. (1996) Comparison of the insulin and insulin-like growth factor 1 mitogenic intracellular signaling pathways. Endocrinology 137, 44274434.
  • Su B. and Karin M. (1996) Mitogen-activated protein kinase cascades and regulation of gene expression. Curr. Opin. Immunol. 8, 402411.
  • Tagami M., Ikeda K., Nara Y., Fujino H., Kubota A., Numano F. and Yamori Y. (1997) Insulin-like growth factor-1 attenuates apoptosis in hippocampal neurons caused by cerebral ischemia and reperfusion in stroke-prone spontaneously hypertensive rats. Lab. Invest. 76, 613617.
  • Trejo J. L., Carro E. and Torres-Aleman I. (2001) Circulating insulin-like growth factor I mediates exercise induced increases in the number of new neurons in the adult hippocampus. J. Neurosci. 21, 16281634.
  • Vivanco I. and Sawyers C. L. (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat. Rev. Cancer. 2, 489501.
  • Yamada M., Tanabe K., Wada K., Shimoke K., Ishikawa Y., Ikeuchi T., Koizumi S. and Hatanaka H. (2001) Differences in survival-promoting effects and intracellular signaling properties of BDNF and IGF-1 in cultured cerebral cortical neurons. J. Neurochem. 78, 940951.
  • Yamaguchi A., Tamatani M., Matsuzaki H., Namikawa K., Kiyama H., Vitek M. P., Mitsuda N. and Tohyama M. (2001) Akt activation protects hippocampal neurons from apoptosis by inhibiting transcriptional activity of p53. J. Biol. Chem. 276, 52562564.
  • Zheng W. H., Fink D. W. Jr and Guroff G. (1996) Role of protein kinase C alpha in nerve growth factor-induced arachidonic acid release from PC12 cells. J. Neurochem. 66, 18681875.
  • Zheng W. H., Kar S., Dore S. and Quirion R. (2000a) Insulin-like growth factor-1 (IGF-1): a neuroprotective trophic factor acting via the Akt kinase pathway. J. Neural. Transm. Suppl. 60, 261272.
  • Zheng W. H., Kar S. and Quirion R. (2000b) Insulin-like growth factor-1-induced phosphorylation of the forkhead family transcription factor FKHRL1 is mediated by akt kinase in PC12 cells. J. Biol. Chem. 275, 3915239158.
  • Zheng W. H., Kar S. and Quirion R. (2000c) Stimulation of protein kinase C modulates insulin-like growth factor-1-induced akt activation in PC12 cells. J. Biol. Chem. 275, 1337713385.
  • Zheng W. H., Kar S. and Quirion R. (2002a) Insulin-like growth factor-1-induced phosphorylation of transcription factor FKHRL1 is mediated by phosphatidylinositol 3-kinase/Akt kinase and role of this pathway in insulin-like growth factor-1-induced survival of cultured hippocampal neurons. Mol. Pharmacol. 62, 225233.
  • Zheng W. H., Kar S. and Quirion R. (2002b) FKHRL1 and its homologs are new targets of nerve growth factor Trk receptor signaling. J. Neurochem. 80, 10491061.