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FKBP51 protects 661w cell culture from staurosporine-induced apoptosis.

Daudt DR, Yorio T - Mol. Vis. (2011)

Bottom Line: In this study, we examine the effects of FKBP51 as a neuroprotective agent on a neuronal cell line.FK506 treatment significantly protected 661w neuronal cultures from staurosporine-induced apoptosis.These results suggest that FKBP51 protects 661w cell cultures from apoptosis induced by staurosporine.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

ABSTRACT

Purpose: Neurodegenerative diseases and neurotraumas typically result in apoptosis of specific neurons leading to the pathology observed during the disease state. Existing treatments target the symptoms instead of preventing the death of these neurons. Although neuroprotective drugs should be useful as a treatment to prevent further loss of neurons, efficacious molecules are lacking. FK506 (tacrolimus), a widely used immunosuppressant drug, has significant neuroprotective and neuroregenerative properties throughout the central nervous system, including the eye. FK506 achieves these properties through interaction with FK506 binding proteins (FKBP), including FK506 binding protein 51 (FKBP51). In this study, we examine the effects of FKBP51 as a neuroprotective agent on a neuronal cell line.

Methods: We cultured 661w cell cultures with or without FK506, or stably transfected them with an FKBP51 expression vector. These cells were then exposed to the apoptosis-inducing agent staurosporine. Cell viability was determined using a calcein AM/propidium iodide assay. Protein levels and activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were determined by western immunoblot analysis.

Results: FKBP51 overexpression significantly protected 661w cell cultures from staurosporine-induced apoptosis. FKBP51 overexpression also significantly increased NF-κB p65 protein levels and activated NF-κB p65. FK506 treatment significantly protected 661w neuronal cultures from staurosporine-induced apoptosis. FK506 increased FKBP51, NF-κB p65, and levels of activated NF-κB p65 protein.

Conclusions: These results suggest that FKBP51 protects 661w cell cultures from apoptosis induced by staurosporine. Additionally, FK506 protected 661w cell cultures from apoptosis and displayed a mechanism similar to that of FKBP51 overexpression. Both FK506 and FKBP51 appear to act through activation of NF-κB p65 protein, suggesting a common pathway for neuroprotection. These findings implicate FKBP51 as a protein important to neuronal cell culture survival. FKBP51 may be a potential therapeutic drug target for preventing the neurodegeneration and neurotrauma that occur during neurodegenerative diseases.

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Related in: MedlinePlus

FK506 (tacrolimus) protects 661w cell cultures from staurosporine-induced cell death. A: FK506 (1 μM) significantly protected 661w neuronal cell cultures from staurosporine-induced apoptosis. Cell survival was monitored with the calcein-AM/propidium iodide cell-survival assay (scale bar=100 µm). The 661w neuronal cell cultures were treated with 1 μM FK506, with 1 μM staurosporine, with both 1 μM FK506 and 1 μM staurosporine, or with vehicle for 24 h. B: The 1 μM FK506 treatments significantly protected 661w neuronal cell cultures from 1 μM staurosporine-induced apoptosis (p<0.001, n=3). Error bars represent SEM.
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f6: FK506 (tacrolimus) protects 661w cell cultures from staurosporine-induced cell death. A: FK506 (1 μM) significantly protected 661w neuronal cell cultures from staurosporine-induced apoptosis. Cell survival was monitored with the calcein-AM/propidium iodide cell-survival assay (scale bar=100 µm). The 661w neuronal cell cultures were treated with 1 μM FK506, with 1 μM staurosporine, with both 1 μM FK506 and 1 μM staurosporine, or with vehicle for 24 h. B: The 1 μM FK506 treatments significantly protected 661w neuronal cell cultures from 1 μM staurosporine-induced apoptosis (p<0.001, n=3). Error bars represent SEM.

Mentions: FK506 is neuroprotective against several forms of toxicity as well as in several in vivo [5,6] and in vitro models [7,8]. Although FK506 is significantly neuroprotective outside of the eye, we want to determine whether FK506 protects ocular neuronal cell cultures from staurosporine-induced-apoptosis. The 661w neuronal cell cultures were processed for determination of apoptosis using calcein AM/propidium iodide double-staining [34] following 1 μM staurosporine and 1 μM FK506 treatments. Virtually all 661w neuronal cell cultures were alive when untreated or treated with 1 μM FK506. In contrast, when the 661w neuronal cell cultures were exposed to 1 μM staurosporine for 24 h, only 28±0.05% of the cell cultures survived. The addition of 1 μM FK506 significantly protected the 661w neuronal cell cultures (p≤0.001) from 1 μM staurosporine-induced apoptosis over the 24 h treatment, by increasing the survival rate to 95±0.01 (p=012; Figure 6).


FKBP51 protects 661w cell culture from staurosporine-induced apoptosis.

Daudt DR, Yorio T - Mol. Vis. (2011)

FK506 (tacrolimus) protects 661w cell cultures from staurosporine-induced cell death. A: FK506 (1 μM) significantly protected 661w neuronal cell cultures from staurosporine-induced apoptosis. Cell survival was monitored with the calcein-AM/propidium iodide cell-survival assay (scale bar=100 µm). The 661w neuronal cell cultures were treated with 1 μM FK506, with 1 μM staurosporine, with both 1 μM FK506 and 1 μM staurosporine, or with vehicle for 24 h. B: The 1 μM FK506 treatments significantly protected 661w neuronal cell cultures from 1 μM staurosporine-induced apoptosis (p<0.001, n=3). Error bars represent SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102027&req=5

f6: FK506 (tacrolimus) protects 661w cell cultures from staurosporine-induced cell death. A: FK506 (1 μM) significantly protected 661w neuronal cell cultures from staurosporine-induced apoptosis. Cell survival was monitored with the calcein-AM/propidium iodide cell-survival assay (scale bar=100 µm). The 661w neuronal cell cultures were treated with 1 μM FK506, with 1 μM staurosporine, with both 1 μM FK506 and 1 μM staurosporine, or with vehicle for 24 h. B: The 1 μM FK506 treatments significantly protected 661w neuronal cell cultures from 1 μM staurosporine-induced apoptosis (p<0.001, n=3). Error bars represent SEM.
Mentions: FK506 is neuroprotective against several forms of toxicity as well as in several in vivo [5,6] and in vitro models [7,8]. Although FK506 is significantly neuroprotective outside of the eye, we want to determine whether FK506 protects ocular neuronal cell cultures from staurosporine-induced-apoptosis. The 661w neuronal cell cultures were processed for determination of apoptosis using calcein AM/propidium iodide double-staining [34] following 1 μM staurosporine and 1 μM FK506 treatments. Virtually all 661w neuronal cell cultures were alive when untreated or treated with 1 μM FK506. In contrast, when the 661w neuronal cell cultures were exposed to 1 μM staurosporine for 24 h, only 28±0.05% of the cell cultures survived. The addition of 1 μM FK506 significantly protected the 661w neuronal cell cultures (p≤0.001) from 1 μM staurosporine-induced apoptosis over the 24 h treatment, by increasing the survival rate to 95±0.01 (p=012; Figure 6).

Bottom Line: In this study, we examine the effects of FKBP51 as a neuroprotective agent on a neuronal cell line.FK506 treatment significantly protected 661w neuronal cultures from staurosporine-induced apoptosis.These results suggest that FKBP51 protects 661w cell cultures from apoptosis induced by staurosporine.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

ABSTRACT

Purpose: Neurodegenerative diseases and neurotraumas typically result in apoptosis of specific neurons leading to the pathology observed during the disease state. Existing treatments target the symptoms instead of preventing the death of these neurons. Although neuroprotective drugs should be useful as a treatment to prevent further loss of neurons, efficacious molecules are lacking. FK506 (tacrolimus), a widely used immunosuppressant drug, has significant neuroprotective and neuroregenerative properties throughout the central nervous system, including the eye. FK506 achieves these properties through interaction with FK506 binding proteins (FKBP), including FK506 binding protein 51 (FKBP51). In this study, we examine the effects of FKBP51 as a neuroprotective agent on a neuronal cell line.

Methods: We cultured 661w cell cultures with or without FK506, or stably transfected them with an FKBP51 expression vector. These cells were then exposed to the apoptosis-inducing agent staurosporine. Cell viability was determined using a calcein AM/propidium iodide assay. Protein levels and activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were determined by western immunoblot analysis.

Results: FKBP51 overexpression significantly protected 661w cell cultures from staurosporine-induced apoptosis. FKBP51 overexpression also significantly increased NF-κB p65 protein levels and activated NF-κB p65. FK506 treatment significantly protected 661w neuronal cultures from staurosporine-induced apoptosis. FK506 increased FKBP51, NF-κB p65, and levels of activated NF-κB p65 protein.

Conclusions: These results suggest that FKBP51 protects 661w cell cultures from apoptosis induced by staurosporine. Additionally, FK506 protected 661w cell cultures from apoptosis and displayed a mechanism similar to that of FKBP51 overexpression. Both FK506 and FKBP51 appear to act through activation of NF-κB p65 protein, suggesting a common pathway for neuroprotection. These findings implicate FKBP51 as a protein important to neuronal cell culture survival. FKBP51 may be a potential therapeutic drug target for preventing the neurodegeneration and neurotrauma that occur during neurodegenerative diseases.

Show MeSH
Related in: MedlinePlus