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. 2015 Aug 24:5:28743.
doi: 10.3402/pba.v5.28743. eCollection 2015.

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

Xiang Bai  1   2 , Margaret Chia-Ying Wey  1   2 , Elizabeth Fernandez  1   2   3 , Matthew J Hart  2   4   5 , Jonathan Gelfond  6 , Alex F Bokov  6 , Sheela Rani  1   2 , Randy Strong  1   2   7
Affiliations

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

Xiang Bai et al. Pathobiol Aging Age Relat Dis. .

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.

Keywords: 4-hydroxynonenal (4-HNE); motor function; rapamycin; synaptic injury; synaptophysin; synucleinopathy.

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Figures

Fig. 1
Fig. 1
Effect of RAPA on performance in tests of motor function. (a) pole test; (b) forepaw stepping adjustment test; (c) accelerating rotarod test. Age-matched WT and TG mice were fed mouse diet containing microencapsulated RAPA or the microencapsulation material Eudragit S100 (Vehicle Control) from 13 weeks of age for 24 weeks. Data represent mean±SEM. Data were analyzed using a mixed-effect regression model. *p<0.05, WT control versus WT RAPA; **p<0.05, WT control versus TG control; ***p<0.05, TG control versus TG RAPA.
Fig. 2
Fig. 2
Effect of RAPA on 4-HNE protein adducts in midbrain, striatum, brain stem, cerebellum, and spinal cord. Age-matched female WT and TG mice were fed mouse diet incorporated with microencapsulated RAPA or the microencapsulation material Eudragit S100 (Control) from 13 weeks of age for 24 weeks. Each well of a 4–12% Criterion gel was loaded with 40 µg brain tissue lysate. Immunoreactive bands were quantified by Odyssey software. Data represent the mean±SEM. Two-way ANOVAs followed by post hoc Bonferonni tests were used to analyze effects of genotype and RAPA on levels of synaptophysin in either sex. *p<0.05, WT control versus WT RAPA; **p<0.05, WT control versus TG control; ***p<0.05, TG control versus TG RAPA.
Fig. 3
Fig. 3
Effect of RAPA on synaptophysin protein level in midbrain, striatum, brain stem, cerebellum, and spinal cord. (a) quantification of Western blot; (b) representative immunoblots. Age-matched female WT and TG mice were fed mouse diet incorporated with microencapsulated RAPA and the microencapsulation material Eudragit S100 (vehicle control) from 13 weeks of age for 24 weeks. Each well of a 4–12% Criterion gel was loaded with 40 µg brain tissue lysate. Immunoreactive bands were quantified by Odyssey software. Data represent the mean±SEM. Two-way ANOVA with post hoc Bonferonni t-tests were used to analyze the effects of genotype and RAPA on levels of synaptophysin in either sex. *p<0.05, WT control versus WT RAPA; **p<0.05, WT control versus TG control; ***p<0.05, TG control versus TG RAPA.
Fig. 4
Fig. 4
Effect of RAPA on 4-HNE-induced loss of synaptophysin protein in SH-SY5Y cells. (a) experiment design. RAPA at different doses and corresponding volumes of Vehicle was added to SH-SY5Y cells. Cells were then treated with 4-HNE (15 µM) 19 h later for 5 h in the same medium before protein extraction. (b) representative immunoblot. Each well of 4–12% Criterion gel was loaded with 100 µg cell lysate. (c) quantification of immunoblot by Odyssey software. Each group contained nine replicates. Data were analyzed by a two-way ANOVA followed by Bonferonni post hoc n=9, p<0.05, **p<0.01.
Fig. 5
Fig. 5
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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