Curcumin Treatment Improves Motor Behavior in α-Synuclein Transgenic Mice

<div><p>The curry spice curcumin plays a protective role in mouse models of neurodegenerative diseases, and can also directly modulate aggregation of α-synuclein protein <i>in vitro</i>, yet no studies have described the interaction of curcumin and α-synuclein in genetic synucleinopathy mouse models. Here we examined the effect of chronic and acute curcumin treatment in the Syn-GFP mouse line, which overexpresses wild-type human α-synuclein protein. We discovered that curcumin diet intervention significantly improved gait impairments and resulted in an increase in phosphorylated forms of α-synuclein at cortical presynaptic terminals. Acute curcumin treatment also caused an increase in phosphorylated α-synuclein in terminals, but had no direct effect on α-synuclein aggregation, as measured by <i>in vivo</i> multiphoton imaging and Proteinase-K digestion. Using LC-MS/MS, we detected ~5 ng/mL and ~12 ng/mL free curcumin in the plasma of chronic or acutely treated mice, with a glucuronidation rate of 94% and 97%, respectively. Despite the low plasma levels and extensive metabolism of curcumin, these results show that dietary curcumin intervention correlates with significant behavioral and molecular changes in a genetic synucleinopathy mouse model that mimics human disease.</p></div

No change in in vivo FRAP kinetics in acutely treated curcumin mice.

<p>(A) Photobleaching and recovery imaging of presynaptic Syn-GFP protein in cortical brain tissue, through a cranial window in the skull. (B) FRAP recovery curves from DMSO and curcumin (C) treated animals, at pre-treatment, 1 week, and 2 week time points. (D) Immobile Fraction and recovery time constant tau values (E) do not change over time or with treatment (n = 3 animals curcumin, n = 2 animals DMSO, 2–5 regions per animal per time point; yellow box indicates bleach ROI; Scale bar = 10 μm).</p

LC-MS/MS detection of curcumin in plasma from mice on a curcumin diet.

<p>Food consumption (A) and body weight (B) for mice on a 500 ppm curcumin diet or control diet, recorded weekly for 6 months. Extracted-ion chromatogram for plasma matrix spiked with 100 ng/mL curcumin (C), showing distinct peaks for curcumin and honokiol internal standards.</p

Curcumin diet mice show improved gait.

<p>Syn-GFP mice on a 6 month curcumin diet intervention had significant changes in gait compare to control diet mice, including increased fore paw swing duration (A), decreased fore paw ataxia coefficient (B), decreased for paw stride length variability (C), and decreased fore paw stance width variability (D). The ataxia coefficient, and variability in stride length and stance width, are all associated with a PD-like phenotype in both humans and mice (flat platform run, n = 4 animals per group, *p<0.05, **p<0.01).</p

Increased S129-P-α-synuclein in acutely treated curcumin mice.

<p>Immunohistochemical detection of S129-P-α-synuclein in cortical tissue from mice treated for 2 weeks with DMSO (A) or 15mg/kg/day curcumin (B). (C) Quantification of GFP and S129-P fluorescence at individual presynaptic terminals shows significant changes in S129-P puncta, but no change in GFP. (D) Analysis of GFP/S129-P colocalization (CoLoc channel), and quantification of the Pearson’s Colocalization Coefficient (E), shows a decrease in S129-P at GFP-positive terminals. (F) No correlation between plasma curcumin levels and terminal S129-P fluorescence in individual mice. (n = 2 mice for DMSO treatment, n = 3 mice for curcumin treatment; each data point represents the average value for a 3μm z-stack, with 4–12 z-stacks analyzed per mouse; pink arrows indicate colocalized synapses, white arrows indicate lack of colocalization; *p<0.05,**p<0.01,***p<0.001, Scale bar = 5 μm).</p

Increased S129-P-α-synuclein in curcumin diet mice.

<p>Immunohistochemical detection of S129-P-α-synuclein in cortical tissue from control (A) and curcumin (B) diet mice. (C) Quantification of GFP and S129-P fluorescence at individual presynaptic terminals shows significant changes in S129-P puncta, but no change in GFP. (D) Analysis of GFP/S129-P colocalization (CoLoc channel), and quantification of the Pearson’s Colocalization Coefficient (E), demonstrates an increase in S129-P at GFP-positive terminals. (F) Strong correlation between plasma curcumin levels and terminal S129-P fluorescence in individual mice. (n = 4 mice for control diet, n = 4 mice for curcumin diet; each data point represents the average value for a 3μm z-stack, with 7–15 z-stacks analyzed per mouse; pink arrows indicate colocalized synapses, white arrows indicate lack of colocalization; **p<0.01, ***p<0.0005, ****p<0.0001, Scale bar = 5 μm).</p

Curcumin stains Lewy Bodies.

<p>(A) Individual fluorescence spectra of curcumin, Alexa-647 secondary antibody, and two autofluorescent components, defined by spectral imaging and linear unmixing. Autofluorescence components (B, C) were clearly separated from adjacent curcumin-positive (D) structures, which were defined as Lewy Bodies because they co-labeled with S129-P-α-synuclein-A647 antibodies (E, G). Minimal unassigned residual light (F) demonstrates a high efficiency of the unmixing technique for the defined spectra. (Scale bar = 10 μm)</p

LC-MS/MS parameters.

<p>LC-MS/MS parameters.</p

No change in Proteinase-K-resistant aggregates in curcumin diet mice.

<p>(A) Western blot detection of Syn-GFP microaggregates, following Proteinase-K digestion of synaptosome and cytosolic protein fractions from control and curcumin diet mice. (B) Quantification of Syn-GFP band intensity shows no change in the resistant fraction between control and curcumin diet mice.</p

<i>par-2</i> mutants.

<p>A. DIC time-lapse images of wild-type <i>par-2(or373 </i>ts<i>)</i>, <i>par-2(or539 </i>ts<i>)</i>, and <i>par-2(or640 </i>ts<i>)</i> embryos. The blastomeres in the <i>par-2</i> mutants were of similar size at the two cell stage and initiated mitosis simultaneously, in contrast to the wild type. The <i>par-2(or373 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 5 hours prior to imaging. The <i>par-2(or539 </i>ts<i>)</i> and par-<i>2(or540 </i>ts<i>)</i> embryos were obtained from hermaphrodites shifted to the restrictive temperature for 30 minutes prior to imaging. Arrows indicate mitotic spindles at the two cell stage. Times in min:sec are given relative to AB NEBD. Scale bar, 10 µm. B. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; Normal one cell embryo; 2; assymetric two cell embyro, 3; asynchronous two cell divisions. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites transferred to the restrictive temperature for 30 minutes. C. Amino acid alteration in the <i>par-2(or373 </i>ts<i>)</i> mutant. Asterisk indicates the changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein.</p