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Rapamycin improves motor function, reduces 4-hydroxynonenal adducted protein in brain, and attenuates synaptic injury in a mouse model of synucleinopathy.

Bai X, Wey MC, Fernandez E, Hart MJ, Gelfond J, Bokov AF, Rani S, Strong R - Pathobiol Aging Age Relat Dis (2015)

Bottom Line: Rapamycin did not alter A53T α-synuclein content.Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice.Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

ABSTRACT

Background: Synucleinopathy is any of a group of age-related neurodegenerative disorders including Parkinson's disease, multiple system atrophy, and dementia with Lewy Bodies, which is characterized by α-synuclein inclusions and parkinsonian motor deficits affecting millions of patients worldwide. But there is no cure at present for synucleinopathy. Rapamycin has been shown to be neuroprotective in several in vitro and in vivo synucleinopathy models. However, there are no reports on the long-term effects of RAPA on motor function or measures of neurodegeneration in models of synucleinopathy.

Methods: We determined whether long-term feeding a rapamycin diet (14 ppm in diet; 2.25 mg/kg body weight/day) improves motor function in neuronal A53T α-synuclein transgenic mice (TG) and explored underlying mechanisms using a variety of behavioral and biochemical approaches.

Results: After 24 weeks of treatment, rapamycin improved performance on the forepaw stepping adjustment test, accelerating rotarod and pole test. Rapamycin did not alter A53T α-synuclein content. There was no effect of rapamycin treatment on midbrain or striatal monoamines or their metabolites. Proteins adducted to the lipid peroxidation product 4-hydroxynonenal were decreased in brain regions of both wild-type and TG mice treated with rapamycin. Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice. Rapamycin attenuated the loss of synaptophysin protein in the affected brain regions. Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal.

Conclusion: Taken together, these data suggest that rapamycin, an FDA approved drug, may prove useful in the treatment of synucleinopathy.

No MeSH data available.


Related in: MedlinePlus

Effect of RAPA on 4-HNE-induced loss of neurites in SH-SY5Y cells. (a) experiment design. SH-SY5Y cells were plated in normal culture medium which was replaced the next day with differentiation medium which contained reduced serum (2.5% FBS) and 10 mM retinoic acid. Two days later, different doses of RAPA and corresponding volume of vehicle in normal culture medium was added to cells, followed by 4-HNE (15 µM) treatment 19 h later for another 5 h followed by protein extraction. Blank controls had no reagent, including vehicle, added to cells. (b) representative images captured using PerkinElmer High Content Screen Imaging system (20X). Orange staining is for cell membrane (neurite), blue staining is for nuclei. (c,d) effect of RAPA on 4-HNE-induced decrease in maximum neurite length (c) and total neurite length (d) in SH-SY5Y cells. Cells (n=1119±94.6, mean±SEM) in 3–4 images per experiment were analyzed using Columbus Image Data Storage and Analysis System. Data were analyzed by two-way ANOVA with followed by Tukey post hoc tests to compare individual means. ****p=0.0000, **p<0.01. Experiments were repeated three times.
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Figure 0005: Effect of RAPA on 4-HNE-induced loss of neurites in SH-SY5Y cells. (a) experiment design. SH-SY5Y cells were plated in normal culture medium which was replaced the next day with differentiation medium which contained reduced serum (2.5% FBS) and 10 mM retinoic acid. Two days later, different doses of RAPA and corresponding volume of vehicle in normal culture medium was added to cells, followed by 4-HNE (15 µM) treatment 19 h later for another 5 h followed by protein extraction. Blank controls had no reagent, including vehicle, added to cells. (b) representative images captured using PerkinElmer High Content Screen Imaging system (20X). Orange staining is for cell membrane (neurite), blue staining is for nuclei. (c,d) effect of RAPA on 4-HNE-induced decrease in maximum neurite length (c) and total neurite length (d) in SH-SY5Y cells. Cells (n=1119±94.6, mean±SEM) in 3–4 images per experiment were analyzed using Columbus Image Data Storage and Analysis System. Data were analyzed by two-way ANOVA with followed by Tukey post hoc tests to compare individual means. ****p=0.0000, **p<0.01. Experiments were repeated three times.

Mentions: SH-SY5Y cells (1.4×105/well) were seeded on 12-well plates. Culture medium was replaced the next day with differentiation medium containing 2.5% FBS serum and 10 mM retinoic acid. The differentiation medium was replaced with normal culture medium 2 days later. Different doses of RAPA and corresponding volumes of vehicle were added to each well. Cells then were treated with 4-HNE (15 µM) 19 h later for 5 h in the same medium as shown in Fig. 5a. A non-treated control consisted of cells without any added reagent.


Rapamycin improves motor function, reduces 4-hydroxynonenal adducted protein in brain, and attenuates synaptic injury in a mouse model of synucleinopathy.

Bai X, Wey MC, Fernandez E, Hart MJ, Gelfond J, Bokov AF, Rani S, Strong R - Pathobiol Aging Age Relat Dis (2015)

Effect of RAPA on 4-HNE-induced loss of neurites in SH-SY5Y cells. (a) experiment design. SH-SY5Y cells were plated in normal culture medium which was replaced the next day with differentiation medium which contained reduced serum (2.5% FBS) and 10 mM retinoic acid. Two days later, different doses of RAPA and corresponding volume of vehicle in normal culture medium was added to cells, followed by 4-HNE (15 µM) treatment 19 h later for another 5 h followed by protein extraction. Blank controls had no reagent, including vehicle, added to cells. (b) representative images captured using PerkinElmer High Content Screen Imaging system (20X). Orange staining is for cell membrane (neurite), blue staining is for nuclei. (c,d) effect of RAPA on 4-HNE-induced decrease in maximum neurite length (c) and total neurite length (d) in SH-SY5Y cells. Cells (n=1119±94.6, mean±SEM) in 3–4 images per experiment were analyzed using Columbus Image Data Storage and Analysis System. Data were analyzed by two-way ANOVA with followed by Tukey post hoc tests to compare individual means. ****p=0.0000, **p<0.01. Experiments were repeated three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549373&req=5

Figure 0005: Effect of RAPA on 4-HNE-induced loss of neurites in SH-SY5Y cells. (a) experiment design. SH-SY5Y cells were plated in normal culture medium which was replaced the next day with differentiation medium which contained reduced serum (2.5% FBS) and 10 mM retinoic acid. Two days later, different doses of RAPA and corresponding volume of vehicle in normal culture medium was added to cells, followed by 4-HNE (15 µM) treatment 19 h later for another 5 h followed by protein extraction. Blank controls had no reagent, including vehicle, added to cells. (b) representative images captured using PerkinElmer High Content Screen Imaging system (20X). Orange staining is for cell membrane (neurite), blue staining is for nuclei. (c,d) effect of RAPA on 4-HNE-induced decrease in maximum neurite length (c) and total neurite length (d) in SH-SY5Y cells. Cells (n=1119±94.6, mean±SEM) in 3–4 images per experiment were analyzed using Columbus Image Data Storage and Analysis System. Data were analyzed by two-way ANOVA with followed by Tukey post hoc tests to compare individual means. ****p=0.0000, **p<0.01. Experiments were repeated three times.
Mentions: SH-SY5Y cells (1.4×105/well) were seeded on 12-well plates. Culture medium was replaced the next day with differentiation medium containing 2.5% FBS serum and 10 mM retinoic acid. The differentiation medium was replaced with normal culture medium 2 days later. Different doses of RAPA and corresponding volumes of vehicle were added to each well. Cells then were treated with 4-HNE (15 µM) 19 h later for 5 h in the same medium as shown in Fig. 5a. A non-treated control consisted of cells without any added reagent.

Bottom Line: Rapamycin did not alter A53T α-synuclein content.Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice.Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

ABSTRACT

Background: Synucleinopathy is any of a group of age-related neurodegenerative disorders including Parkinson's disease, multiple system atrophy, and dementia with Lewy Bodies, which is characterized by α-synuclein inclusions and parkinsonian motor deficits affecting millions of patients worldwide. But there is no cure at present for synucleinopathy. Rapamycin has been shown to be neuroprotective in several in vitro and in vivo synucleinopathy models. However, there are no reports on the long-term effects of RAPA on motor function or measures of neurodegeneration in models of synucleinopathy.

Methods: We determined whether long-term feeding a rapamycin diet (14 ppm in diet; 2.25 mg/kg body weight/day) improves motor function in neuronal A53T α-synuclein transgenic mice (TG) and explored underlying mechanisms using a variety of behavioral and biochemical approaches.

Results: After 24 weeks of treatment, rapamycin improved performance on the forepaw stepping adjustment test, accelerating rotarod and pole test. Rapamycin did not alter A53T α-synuclein content. There was no effect of rapamycin treatment on midbrain or striatal monoamines or their metabolites. Proteins adducted to the lipid peroxidation product 4-hydroxynonenal were decreased in brain regions of both wild-type and TG mice treated with rapamycin. Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice. Rapamycin attenuated the loss of synaptophysin protein in the affected brain regions. Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal.

Conclusion: Taken together, these data suggest that rapamycin, an FDA approved drug, may prove useful in the treatment of synucleinopathy.

No MeSH data available.


Related in: MedlinePlus