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Original article

Human neuroblastoma cells transfected with two Chinese presenilin 1 mutations are sensitized to trophic factor withdrawal and protected by insulin-like growth factor-1

FANG, Bo-yan; JIA, Jian-ping

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Abstract

Mutations in the presenilin 1 (PS1) gene are the main causes of most of the early-onset familial Alzheimer's disease (FAD).1,2 Since neuronal loss is one of the main pathological changes in AD, the connection between mutant PS1 and neuron death sensitivity has been explored in many investigations.3–7 Some of the results suggested that PS1 gene mutations predispose cells to apoptosis by mechanisms involving perturbed calcium homeostasis,8 oxidative stress and caspase activation.9,10 PS1 gene mutations also sensitize cells to death by influencing unfolded protein reaction (UPR)6,11 and increasing toxic β-amyloid (Aβ) generation.12,13 Recent data reported that the impairment of glucose uptake, usage and metabolism may contribute to the development of AD since they found the glucose metabolism decreased in the cortex of temporal and parietal lobe of AD patients.14,15 Whether there are any links between PS1 mutation and impaired glucose metabolism remain unclear. Here, we employed two novel PS1 mutations (PS1 V97L and PS1 A136G) we found in previous studies16,17 to explore the effect of the mutations on apoptosis, glucose uptake and insulin-like growth factor-1 (IGF-1) protection in cell culture and to further elucidate the pathogenesis of Chinese FAD.

METHODS

Cells culture and transfection

The construct strategy used to generate PS1 mutant cells models is detailed elsewhere.18,19 The plasmids containing mutant human PS1 (V97L) cDNA and PS1 (A136G) cDNA were generated from pcDNA 3.1/Zeo(+) containing wild type PS1 cDNA (PS1wt) by a site-directed mutagenesis method. Four kinds of plasmids containing PS1wt, mtPS1(V97L), mtPS1(A136G) and pcDNA 3.1 empty vector (mock) were introduced into SH-SY5Y cells using the LipofectamineTM method. The stably transfected cell lines were cultured with minimum essential medium (MEM) and F12 (nutrient mixture Ham's F-12) (1:1 mixture, Gibco, Invitrogen Co., NY, USA), supplemented with 10% fetal bovine serum (Gibco-BRL, CA, USA), 400 mg/ml Zeocin, at pH 7.4 and maintained in a humidified 5% CO2 atmosphere at 37°C.

Treatment of cells

Cells were seeded in 96-well plates (Corning, Sweden) at about 2×103. When the cells grow to 70%-80% confluence they were washed by phosphate-buffered saline (PBS) once, which was replaced with serum free medium, 400 mg/ml Zeocin (with or without 25 ng/ml IGF-1).20 A culture medium containing 10% fetal bovine serum with 400 mg/ml Zeocin was used for cells in control groups and the cells were incubated at 37°C for 24 hours.

MTT assay

Cell viability was measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) tetrazolium salt assay.4,20 The viability of cells was determined by evaluation of the live cell number through the optical density values after addition of MTT. Cells were treated as described above. MTT tetrazolium salt was dissolved in serum-free MEM at a concentration of 5 mg/ml and then 20 μl was added to cultured cells after treatment. Cells were incubated for 1 hour at 37°C then the medium was aspirated and DMSO was added. For each sample absorbance was measured in 5 wells at OD550 nm in a DIAGNOSTICS LP 400 enzyme label meter (BARSTER, France) and the experiment repeated 3 times. Results were expressed as viability percentage of the value obtained from the treated and untreated cells. The formula is as follows: Cell viability (%)=treatment group OD550/control group OD550×100 %

Nuclei staining

Visualization of apoptosis21 was performed by staining with the DNA-binding fluorescent dye, Hoechst 33258. Cells were grown on cover slips to 80% confluence and treated as described above. Then, following the procedure of the apoptosis Hoechst 33258 staining kit (Beyotime Biotechnology, China), the cells were collected after removing the medium, fixed for 10 minutes and then washed twice with PBS before staining with Hoechst 33258 for 5 minutes. The number of apoptotic cells was counted under a fluorescence microscope (Nikon, Tokyo, Japan, excitation 350 nm/emission 460 nm). Cells were counted in five random fields and scored as a percentage of the total cells counted. A total of at least 500 cells were counted for each experiment and experiments were repeated 3 times. Cells were considered as apoptotic when appearing with highly blue fluorescent condensed or markedly fragmentized nuclei.4 Assessments were performed in a blind manner without knowledge of the source of the cells or their treatment.

Flow cytometry measurement

Annexin-V can conjugate to phosphatidylserine (PS) which can turnover to the outer cell membrane in the early stage of apoptosis. Using propidine iodide (PI) and FITC labeled Annexin-V (AnnexinV-FITC) double staining, apoptotic cells can be assessed using flow cytometry measurement. After the treatment described above, 1×106 cells were trypsinized, washed twice with PBS, re-suspended with 100 μl of Binding Buffer (10 mmol/L HEPES-NaOH, pH 7.4, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2) and AnnexinV-FITC (20 μg/ml) 10μl. Cells were kept in the dark at room temperature (RT) for 30 minutes, then PI (50 μg/ml) 5 μl was added and incubated in the dark for about 5 minutes before mixing with 400 μl Binding Buffer and immediately being examined by scanning. The negative control was also performed at the same time by adding PBS instead of adding AnnexinV-FITC and PI.

Glucose uptake

To measure the glucose uptake,22 cells were cultured in 12-well plates until in logarithmic growth phase with or without 25 ng/ml of IGF-1 for 30 minutes. Then 37 kBq/ml [1-3H]2-deoxy-glucose and 1.0 mmol/L un-labeled glucose was added and incubated for another 15 minutes at 37°C. After incubation cells were washed three times with ice-cold PBS containing 10 mmo/L glucose then lysed with 0.2 mol/L NaOH, containing 1% Sodium dodecyl sulfate (SDS) for 45 minutes at room temperature. Incorporated radioactivity was measured in a liquid scintillation counter (Beckman LS6500, USA).

GLUT1 expression

Cells were treated as above for 30 minutes and cell membranes were prepared as follows in order to detect GLUT1 translocation expression.20,23,24 a cell lysate (10 mmo/L Tris/HCl, 2 mmo/L PMSF, 1 IU/ml Aprotinin) was repeatedly aspirated by a tip on ice for 30 minutes, centrifuged for 5 minutes at 500 r/min at 4°C, then continuously at 4°C for 1 hour at 14 000 r/min to collect the supernatant. The pellet contained the membrane component. The pellet was resuspensed in 10 mmo/L Tris-HCl (pH 7.4) and stored in -20°C. Samples were analyzed by Western blotting using the rabbit anti-GLUT1 AB1341 antiserum (1:2000) (Chemicon, CA). Immunoreactivity was detected by horse radish peroxidase (HRP)-conjugated secondary antibody (1:2000) using the chemiluminescence detection kit (Pierce, USA). Images were collected and analyzed by Tanon gel analysis system 4100 (Tanon, Shanghai, China).

Statistical analysis

All values were expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to evaluate the significance of differences in the cells viability, apoptotic rate, and glucose uptake. A Student's post hoc test was conducted if the ANOVA indicated a significant difference. A P value <0.05 was considered as statistically significant.

RESULTS

Effect of mutant PS1 gene on the viability of SH-SY5Y and protection of IGF-1

There were significant viability differences among the four stably transfected cell lines including PS1 V97L (cells over-expressing the FAD PS1 mutant V97L), PS1 A136G (cells over-expressing the FAD PS1 mutant A136G), PS1wt (cells over-expressing wild-type PS1) and mock (cells transfected with an empty vector) after culture for 24 hours (F=13.867, P=0.0001). The V97L mutant showed the most sensitivity to serum deprivation when compared to PS1wt and mock (P<0.01). The viability of the A136G mutant was also lower than PS1wt (P<0.05) and mock (P<0.01). The addition of IGF-1 improved the viability of the two mutant cell lines significantly (P<0.01, Figure 1).

Figure 1.
Figure 1.:
Comparison of cell viability after serum deprivation and IGF-1 addition. MTT assay against serum deprivation with (+) or without IGF-1 addition (—) on these transfectants was performed to elucidate the sensitivity to the trophic withdrawal and protection of IGF-1. # P<0.01,vs mock by same treatment; *;P<0.05, vs PS1wt by the same treatment; * P<0.01, vs PS1wt by the same treatment; * P<0.01, for comparison with same cell type, treated with serum deprivation and serum deprivation + IGF-1 (25 ng/ml).

Effect of mutant PS1 gene on apoptosis of SH-SY5Y and protection of IGF-1

With Hoechst 33258 staining and flow cytometery we found that V97L and A136G mutant cells were sensitized to apoptosis at 24 hours after serum deprivation. The percentage of condensed nuclei among the four transfectants showed significant differences (F=17.0314, P=0.0000). It was also significantly different when V97L or A136G was compared to mock (P<0.01) or PS1wt (P<0.01). Addition of IGF-1 significantly decreased the percentage of cells with condensed nuclei in all four kinds of cells (P<0.01) (Figure 2).

Figure 2.
Figure 2.:
Percentage of cells with condensed nuclei following serum deprivation and IGF-1 protection. (—) represents serum deprivation; (+) represents serum deprivation + IGF-1 (25 ng/ml). # P<0.01, vs mock by same treatment; * P<0.01, vs PS1wt by same treatment; * P<0.01, for comparison with same cell type, treated with serum deprivation and serum deprivation + IGF-1 (25 ng/ml).

The results of flow cytometery revealed that the two mutant cell lines had a higher percentage of apoptotic cells and necrotic cells than the PS1wt and mock. However, the percentage of apoptotic cells and necrotic cells decreased in the four transfectants when IGF-1 was used (Figure 3).

Figure 3.
Figure 3.:
Flow cytometry measurement using Annexin V-FITC and PI double staining. Lower right quadrant represents apoptotic cells percentage; right superior quadrant represents necrotic cells percentage; left inferior quadrant represents normal cells percentage. A: PS1 V97L(−); B: PS1 A136G(−); C: PS1wt(−); D: mock(−); E: PS1 V97L(+); F: PS1 A136G(+); G: PS1wt(+); H: mock(+). The percentage of each kind of cell shown on the quadrant.

Effect of mutant PS1 gene on glucose uptake of SH-SY5Y and protection of IGF-1

There were no significant differences in the glucose uptake among the four transfectants (F=0.1996, P=0.8947). After addition of IGF-1 for 30 minutes the [3H]-2-DG uptake rate of PS1 V97L increased from (7.74±1.24) nmol•mg−1•min−1 to (9.41±1.68) nmol•mg−1•min−1; the [3H]-2-DG uptake rate of PS1 A136G increased from (7.82±1.03) nmol•mg−1•min−1 to (9.32±1.12) nmol•mg−1•min−1; the [3H]-2-DG uptake rate of PS1wt increased from (7.59±1.27) nmol•mg−1•min−1 to (10.66±1.49) nmol•mg−1•min−1 and the mock transfected group increased from (8.26±1.56) nmol•mg−1•min−1 to (9.93±1.73) nmol•mg−1•min−1; the average increasing rate was about 25%.

GLUT1 expression level increased by IGF-1 addition

Cell membrane GLUT1 expression was slightly increased at 30 minutes after IGF-I addition to the four transfectants. The increased expression was about 15%-20% (Figure 4).

Figure 4.
Figure 4.:
IGF-1 can acutely increase translocation of the GLUT1 protein to the cell surface. GLUT1 protein was immuno-detected by anti-GLUT1 AB1341 antiserum (1:2000) (A). Immunoreactive bands were quantified by densitometric analysis (B). Cell membrane GLUT1 can be enhanced for about 15%-20% by IGF-1 addition. (+) represents serum deprivation + IGF-1 (25 ng/ml).

DISCUSSION

AD is characterized by neuronal death in specific brain regions related to learning and memory. Many studies have focused on the relation between a mutant PS1 gene and neuron death.3–7,25,26 Although different types of cells and different stimulating methods were used, the results obtained were similar. PC12 cells and primary hippocampal neurons expressing mutant PS1 exhibit increased sensitivity to DNA damage-induced death.10 Overexpression of mutated PS1 in primary hippocampal neuronal cultures increased spontaneous apoptosis and down-regulated the survival factor Akt/PKB.27 In the primary cultured cortical neurons of mutant PS1 (mutation M146L) transgenic mice the background and induced apoptotic levels were both increased.28 Here, we studied the effect of serum deprivation on the two Chinese PS1 mutations and found the SH-SY5Y cells carrying the mutant PS1 gene had their mitochondria oxidation-reduction enzymatic activity changed significantly, which led to decreased viability and increased apoptosis in cells. The results suggested that the two Chinese PS1 mutations can sensitize neuron cells to death. Apoptosis and necrosis are both involved in the process of neuron loss. Glucose metabolism is crucial for cells to survive and the glucose metabolism impairment in AD brain has also been confirmed in many studies.14,15

However, in our study, the glucose uptake showed no difference between mutant cells and wild type cells, which suggested that these two PS1 mutations did not affect glucose metabolism directly. There may be other pathways of mutant PS1 involved in cell apoptosis besides glucose metabolism impairment.

Many factors may contribute to the priming of AD pathology, including accumulation of oxidative stress and chronic inflammation, changing of hormone expression and nutritional factors, etc. Recent studies indicated that neuron degeneration related to the disturbed trophic support and intracellular trophic signaling may be compromised in several neurodegenerative diseases.29 As one of the neurotrophic factors, IGF-1 showed multiple neuroprotective effects in several human neurodegenerative diseases,29–31 which has generated increasing interest. So in our study, serum deprivation was chosen as the proapoptosis stimulation and IGF-1 was chosen as the protecting factor. The cell's mitochondrial viability increased and apoptotic cell number decreased significantly when using IGF-1 as the protecting factor. The anti-death effect of IGF-1 was apparent in PS1 V97L and PS1 A136G mutant cell lines. The results indicated that IGF-1 can both eliminate environmental effects and compensate hereditary effects to some degree.

There are a number of potential mechanisms by which IGF-1 could protect cells from apoptosis.29 IGF-1 can interact with other anti-apoptotic proteins such as Bcl-2, BclxL, ICE and CPP32 to block the programmed cell death pathway in primary cultured neuron and nerve cell lines.32 IGF-1 can also prohibit mitochondria membrane depolarization and caspase-3 activition.33,34 IGF-1 can regulate the expression level of insulin degrading enzyme (IDE) to influence the Aβ deposition.35 Normal levels of serum IGF-1 are required to maintain a broad range of brain functions, including energy supply, formation of new neurons and vessels, clearance of potentially toxic Aβ, stimulation of neuronal excitability, regulation of synaptic plasticity and even modulation of cognition.36–38 When triggered by hereditary or environmental factors, neuron cells death can be further aggravated by low levels of IGF-1, but depressed by replenishing IGF-1.38 IGF-1 can also promote neuronal glucose utilization during brain development and repair processes.36 In addition to all these mechanisms our study showed IGF-1 can regulate glucose uptake to protect cells from apoptosis; the glucose uptake of each transfected cell line increased to about 25% after IGF-1 treatment. The glucose transport protein GLUT1 expression level in cell membrane was found to be increased by about 15%—20% following IGF-I addition; which was consistent with the enhancement of glucose uptake. Our results showed that IGF-1 can compensate for the negative effects of trophic withdrawal through regulating glucose metabolism.

In conclusion, the expression of these two Chinese FAD mutant PS1 genes increases SH-SY5Y cells vulnerability to trophic withdrawal. Although there were no differences of glucose uptake found between mutant and wild or mock cells, IGF-1 still can enhance cells viability through regulation of glucose metabolism, indicating IGF-1 is protective for neurons as a metabolism activation agent.

REFERENCES

1. Hardy J. Amyloid, the presenilins and Alzheimer's disease. Trends Neurosci 1997; 20: 154–159.
2. Mattson MP, Guo Q, Furukawa K, Pedersen WA. Presenilins, the endoplasmic reticulum, and neuronal apoptosis in Alzheimer's disease. J Neurochem 1998; 70: 1–14.
3. Guo Q, Sopher BL, Furukawa K, Pham DG, Robinson N, Martin GM, et al. Alzheimer's presenilin mutation sensitizes neural cells to apoptosis induced by trophic factor withdrawal and amyloid β-peptide: involvement of calcium and oxyradicals. J Neurosci 1997; 11: 4212–4222.
4. Mattson MP, Zhu H, Yu J, Kindy MS. Presenilin 1 mutation increases neuronal vulnerability to focal ischemia in vivo and to hypoxia and glucose deprivation in cell culture: involvement of perturbed calcium homeostasis. J Neurosci 2000; 20: 1358–1364.
5. Popescu BO, Ankarcrona M. Neurons bearing presenilins: weapons for defense or suicide? J Cell Mol Med 2000; 4: 249–261.
6. Katayama T, Imaizumi K, Manabe T, Hitomi J, Kudo T, Tohyama M. Induction of neuronal death by ER stress in Alzheimer's disease. J Chem Neuroanat 2004; 28: 67–78.
7. Mohmmad Abdul H, Sultana R, Keller JN, St Clair DK, Markesbery WR, Butterfield DA. Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease. J Neurochem 2006; 96: 1322–1335.
8. Keller JN, Guo Q, Holtsberg FW, Bruce-Keller AJ, Mattson MP. Increased sensitivity to mitochondrial toxin-induced apoptosis in neural cells expressing mutant presenilin 1 is linked to perturbed calcium homeostasis and enhanced oxyradical production. J Neurosci 1998; 15: 4439–4450.
9. Mattson MP, Gary DS, Chan SL, Duan W. Perturbed endoplasmic reticulum function, synaptic apoptosis and the pathogenesis of Alzheimer's disease. Biochem Soc Symp 2001; 67: 151–162.
10. Chan SL, Culmsee C, Haughey N, Klapper W, Mattson MP. Presenilin 1 mutations sensitize neurons to DNA damage-induced death by a mechanism involving perturbed calcium homeostasis and activation of calpains and caspase-12. Neurobiol Dis 2002; 11: 2–19.
11. van Laar T, van der Eb AJ, Terleth C. Mif1: a missing link between the unfolded protein response pathway and ER-associated protein degradation? Curr Protein Pept Sci 2001; 2: 169–190.
12. Borchelt DR, Thinakaran G, Eckman CB, Lee MK, Davenport F, Ratovitsky T, et al. Familial Alzheimer's disease-linked presenilin 1 variants elevate Aβ25-35/1-40 ratio in vitro and in vivo. Neuron 1996; 17: 1005–1013.
13. Butterfield DA, Bush AI. Alzheimer's amyloid β-peptide (1—42): involvement of methionine residue 35 in the oxidative stress and neurotoxicity properties of this peptide. Neurobiol Aging 2004; 25: 563–568.
14. Harwood DG, Sultzer DL, Feil D, Monserratt L, Freedman E, Mandelkern MA. Frontal lobe hypometabolism and impaired insight in Alzheimer disease. Am J Geriatr Psychiatry 2005; 13: 934–941.
15. Rasgon NL, Kenna HA. Insulin resistance in depressive disorders and Alzheimer's disease: revisiting the missing link hypothesis. Neurobiol Aging 2005; 26(Suppl 1): 103–107.
16. Xu EH, Jia JP, Sun WJ. Mutation site of presenilin 1 gene in familial Alzheimer's disease. Natl Med J China (Chin) 2002; 82: 1518–1520.
17. Jia J, Xu E, Shao Y, Sun Y, Jia J, Li D. One novel presenilin 1 gene mutation in a Chinese pedigree of familial Alzheimer's disease. J Alz Dis 2005; 7: 119–124.
18. Shao YK, Jia JP, Fang BY, Liu XS, Sun YX, Dong XM. Construction of eukaryotic expression vector containing PS1 and the expression in neuroblasoma cell line SH-SY5Y. Chin J Lab Diagn (Chin) 2005; 9: 1–4.
19. Fang BY, Jia LF, Jia JP. Chinese Presenilin-1 V97L mutation enhanced Aβ42 levels in SH-SY5Y neuroblastoma cells. Neurosci Lett 2006; 406:33–37.
20. Russo VC, Kobayashi K, Najdovska S, Baker NL, Werther GA. Neuronal protection from glucose deprivation via modulation of glucose transport and inhibition of apoptosis: a role for the insulin-like growth factor system. Brain Res 2004; 1009: 40–53.
21. Zhu LX, Liu J, Xie YH, Kong YY, Ye Y, Wang CL, et al. Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells. World J Gastroenterol 2004; 10: 2972–2978.
22. Tafuri SR. Troglitazone enhances differentiation, basal glucose uptake, and Glut1 protein levels in 3T3-L1 adipocytes. Endocrinology 1996; 137: 4706–4712.
23. Russo VC, Bach LA, Fosang AJ, Baker NL, Werther GA. Insulin-like growth factor binding protein-2 binds to cell surface proteoglycans in the rat brain olfactory bulb. Endocrinology 1997; 138: 4858–4867.
24. Guo X, Geng M, Du G. Glucose transporter 1, distribution in the brain and in neural disorders: its relationship with transport of neuroactive drugs through the blood-brain barrier. Biochem Genet 2005; 43: 175–187.
25. Tanii H, Ankarcrona M, Flood F, Nilsberth C, Mehta ND, Perez-Tur J, et al. Alzheimer's disease presenilin 1 exon 9 deletion and L250S mutations sensitize SH-SY5Y neuroblastoma cells to hyperosmotic stress-induced apoptosis. Neuroscience 2000; 95: 593–601.
26. Wang HQ, Nakaya Y, Du Z, Yamane T, Shirane M, Kudo T, et al. Interaction of presenilins with FKBP38 promotes apoptosis by reducing mitochondrial Bcl-2. Hum Mol Genet 2005; 14: 1889–1902.
27. Weihl CC, Ghadge GD, Kennedy SG, Hay N, Miller RJ, Roos RP. Mutant presenilin-1 induces apoptosis and downregulates Akt/PKB. J Neurosci 1999; 19: 5360–5369.
28. Terro F, Czech C, Esclaire F, Elyaman W, Yardin C, Baclet MC, et al. Neurons overexpressing mutant presenilin-1 are more sensitive to apoptosis induced by endoplasmic reticulum-golgi stress. J Neurosci Res 2002; 69: 530–539.
29. Trejo JL, Carro E, Garcia-Galloway E, Torres-Aleman I. Role of insulin-like growth factor I signaling in neurodegenerative diseases. J Mol Med 2004; 82: 156–162.
30. Gasparini L, Xu H. Potential roles of insulin and IGF-1 in Alzheimer's disease. Trends Neurosci 2003; 26: 404–406.
31. Bondy CA, Cheng CM. Signaling by insulin-like growth factor 1 in brain. Eur J Pharmacol 2004; 490: 25–31.
32. Singleton JR, Dixit VM, Feldman EL. Type I insulin-like growth factor receptor activation regulates apoptotic proteins. J Biol Chem 1996; 271: 31791–31794.
33. Peruzzi F, Prisco M, Morrione A, Valentinis B, Baserga R. Antiapoptotic signaling of the insulin-like growth factor-I receptor through mitochondrial translocation of c-Raf and Nedd4. J Biol Chem 2001; 276: 25990–25996.
34. Saeki M, Maeda S, Wada K, Kamisaki Y. Insulin-like growth factor-1 protects peroxynitrite-induced cell death by preventing cytochrome c-induced caspase-3 activation. J Cell Biochem 2002; 84: 708–716.
35. Messier C, Teutenberg K. The role of insulin, insulin growth factor, and insulin-degrading enzyme in brain aging and Alzheimer's disease. Neural Plast 2005; 12: 311–328.
36. Bondy CA, Cheng CM. Insulin-like growth factor-1 promotes neuronal glucose utilization during brain development and repair processes. Int Rev Neurobiol 2002; 51: 189–217.
37. Carro E, Torres-Aleman I. The role of insulin and insulin-like growth factor I in the molecular and cellular mechanisms underlying the pathology of Alzheimer's disease. Eur J Pharmacol 2004; 490: 127–133.
38. Jin K, Peel AL, Mao XO, Xie L, Cottrell BA, Henshall DC, et al. Increased hippocampal neurogenesis in Alzheimer's disease. Proc Natl Acad Sci USA 2004; 101: 343–347.
Keywords:

Alzheimer disease; presenilin 1; mutation; apoptosis; insulin-like growth factor 1

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