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, 10 (1), 1817

Felodipine Induces Autophagy in Mouse Brains With Pharmacokinetics Amenable to Repurposing

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Felodipine Induces Autophagy in Mouse Brains With Pharmacokinetics Amenable to Repurposing

Farah H Siddiqi et al. Nat Commun.

Erratum in

Abstract

Neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Huntington's disease manifest with the neuronal accumulation of toxic proteins. Since autophagy upregulation enhances the clearance of such proteins and ameliorates their toxicities in animal models, we and others have sought to re-position/re-profile existing compounds used in humans to identify those that may induce autophagy in the brain. A key challenge with this approach is to assess if any hits identified can induce neuronal autophagy at concentrations that would be seen in humans taking the drug for its conventional indication. Here we report that felodipine, an L-type calcium channel blocker and anti-hypertensive drug, induces autophagy and clears diverse aggregate-prone, neurodegenerative disease-associated proteins. Felodipine can clear mutant α-synuclein in mouse brains at plasma concentrations similar to those that would be seen in humans taking the drug. This is associated with neuroprotection in mice, suggesting the promise of this compound for use in neurodegeneration.

Conflict of interest statement

F.M.M. is employed by Eli Lilly and E.K. is employed by and holds stock in Abbvie. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Screening of L-type calcium channel blockers in primary neurons and zebrafish. a Quantification of autophagosomes and autolysosomes in primary neurons from mRFP-GFP-LC3 mice treated with L-type calcium channel blockers (at 1 μM). Data represent mean number of vesicles/neuron +/− SEM, 4 independent experiments, two–tailed unpaired t-test. P values shown for autolysosomes and total vesicle number. bc Wild-type primary neurons were treated with 1 and 5 μM felodipine for 5 h with and without 400 nM bafilomycin (last 4 h). Graph shows mean densitometry +/− SEM (LC3II vs. actin; n = 4 independent experiments; one-tailed, unpaired t-test). Data were normalised to the control (set to 100). c Representative western blots for LC3II in wild-type primary cortical neurons. de Verapamil and felodipine were tested in Rho::EGFP-Taucu7 fish—either in autophagy-null (atg7/) or wild-type-autophagy siblings (atg7+/+). d Representative fluorescence images of the GFP-positive rod photoreceptors in sections of Rho::EGFP-Taucu7 fish treated with DMSO, verapamil or felodipine, scale bar = 50 μm. e Quantification of rod photoreceptors from images of sections through the central retina after verapamil or felodipine treatment (n = 40 eyes/group); one-way ANOVA with post hoc Tukey’s multiple comparison test. Exact p values are provided for control (DMSO) compared to drugs for the same genotype. f Verapamil and felodipine ameliorated morphological defects in Dendra-tau-A152T fish (see Supplementary Fig. 2a for details of phenotype scoring). Data represent means +/− SEM; n ≥ 20 fish/ treatment group from ≥9 clutches. Two-tailed unpaired t-test. g Verapamil and felodipine increased the levels of sarkosyl-soluble tau and reduced the levels of insoluble tau in fractions from Dendra-tauA152T fish. h Graph shows densitometry of the mean ratios (±SEM) of soluble and insoluble tau vs. tubulin from fractions of 3 independent clutches (50 fish/group) normalised to mean DMSO value (set to 100); two-tailed unpaired t-test
Fig. 2
Fig. 2
Felodipine in vivo dose-response experiments. ac mRFP-GFP-LC3 transgenic mice (males and females, 6–7 weeks old) were injected i.p. with various doses of felodipine (1, 2, 5, 10 mg/kg body weight). ab Treatment with 5 and 10 mg felodipine resulted in a statistically significant increase in autolysosomes and total vesicle number in the cerebral cortex with a similar trend observed in Purkinje cells. Autophagosome and autolysosome numbers are shown as mean +/− SEM; mean values of each vesicle type (autophagosome, autolysosome or total vesicles) for each dose of felodipine were compared with the mean value of same vesicle type to those in vehicle controls using one-sample, one-tailed unpaired t-test. Data shown was from 7 litters collected at different time-points (i.e., independent experiments), all values were normalised to mean autolysosome levels in the vehicle controls. c Representative images. Scale bar represents 10 µm in both cerebral cortex and Purkinje cells. de A six-week study was carried out on B6HD (N171–82Q) female mice. Mice were injected i.p. three times a week with 1, 2 or 5 mg/kg body weight of felodipine or vehicle control. Felodipine treatment resulted in a significant decrease in aggregates in the motor cortex (at 5 mg) and piriform cortex (2 and 5 mg). d Representative images of neuronal inclusions in piriform cortex and motor cortex of B6HD mice. Arrows indicate the neurons with huntingtin aggregates or inclusions, scale bar represents 10 µm. e Aggregate number was blind-counted in the motor cortex and piriform cortex. Data are presented as percentage of neurons with aggregates (mean +/− SEM) (n = 5 mice per group); analysed using one-way ANOVA with post hoc Dunnett’s multiple comparison test; exact p values for felodipine dose vs. control are shown
Fig. 3
Fig. 3
Efficacy study of felodipine in N171-82Q (B6HD) mice. B6HD male mice were given i.p. injections of 5 mg/kg felodipine or carrier substance (vehicle control) three times a week from 6 weeks of age. Wild-type mice littermates were also given felodipine or vehicle control injections with the same frequency. n = 22 mice for B6HD mice for both felodipine and vehicle control groups, while n = 11 for wild-type felodipine-treated littermates and n = 12 for wild-type vehicle-treated littermates. a Felodipine improved grip strength in B6HD mice. All B6HD mice showed some age dependent decline in grip strength compared to wild-type littermates regardless of treatment. However, felodipine significantly improved the decline in grip strength in B6HD mice relative to vehicle-treated B6HD mice at the time-points indicated. Data are shown as mean values +/− SEM; one-way ANOVA with post hoc Fisher’s LSD test; exact p values for B6HD-felodipine vs. B6HD-vehicle control are shown. Felodipine treatment had no effect on grip strength in wild-type mice. b The severity of tremors in B6HD mice was significantly improved by felodipine treatment at 17 and 19 weeks. Mann-Whitney two-tailed test was performed for ranked data; exact p values are shown. c Felodipine treatment improved performance at the wire manoeuvre task at 17 and 19 weeks in B6HD mice. Mice were scored on their ability to perform the wire manoeuvre task, data for the active grip with hind limbs are shown and analysed using one-way ANOVA for non-parametric data (Kruskal-Wallis with Dunn’s multiple comparison test); exact p values for felodipine vs. vehicle control in B6HD mice are shown. d Felodipine treatment improved rotarod performance in B6HD mice at 18 weeks. Data are presented as mean values +/− SEM; one-way ANOVA with post hoc Fisher’s LSD test; exact p values for felodipine vs. vehicle treated B6HD mice are shown. HD-vehicle control mice had a significantly reduced performance on the rotarod at week 10 and 14, compared to wild-type littermates (both felodipine-treated and vehicle-treated), as would be expected based on their known disease phenotypes
Fig. 4
Fig. 4
Felodipine pharmacokinetics and bioavailability. a Plasma and brain felodipine concentrations at 0, 5, 10, 60, 120, 240, and 480 min after single i.p. injection of 5 mg/kg body weight of felodipine in C57Bl6 wild-type mice, n = 3 mice per time-point. Means +/− SEM. b Felodipine (50 nM, 100 nM and 1 μM) in primary cortical neurons from mRFP-GFP-LC3 mice. Mean numbers of vesicles +/− SEM per neuron normalised to number of autolysosomes in DMSO (control); one sample, one-tailed unpaired t-test; exact p values for comparison of autolysosome numbers are shown. c Percentages of EGFP-positive neurons with aggregates (means +/− SEM (n = 3 independent experiments)). Data normalised to DMSO control in each experiment, set at 90. One-tailed unpaired t-test. df Double transgenic mRFP-GFP-LC3/B6HD male mice were implanted with felodipine-loaded minipumps (5 mg/kg/day) with 0.25 μl/h flow rate for 28 days. d Felodipine treatment increased the number of autolysosomes. Mean values +/− SEM; mean values for each vesicle type (autophagosomes, autolysosomes and total vesicles) for felodipine were compared with the same vesicle type of vehicle control, one-tailed unpaired t-test; exact p values are shown for autolysosomes. Only mice expressing detectable fluorescence of the mRFP-GFP-LC3 transgene were included in the final analysis. Representative images of cerebral cortex—scale bar = 10 µm. e Representative images of huntingtin inclusions in motor and piriform cortices. Scale bar = 10 µm. f Felodipine significantly reduced the percentage of cells with aggregates in both regions. Data are mean percentage of cells with aggregates per field with +/− SEM, using one-tailed unpaired t-test; exact p values for felodipine vs. vehicle control for each region are shown
Fig. 5
Fig. 5
Felodipine clears mutant A53T a-synuclein in iPSC-derived human neurons and in vivo. a Representative western blots for α-synuclein in iPSC-derived human neurons homozygous for A53T α-synuclein mutation treated with 100 nM felodpine for 5 days. Densitometry data mean +/− SEM (α-synuclein vs. actin loading control; n = 3 independent experiments in triplicate; one-tailed, one sample t-test). Data normalised to control, set at 100. All nine data points are shown for the control and felodipine-treated samples. The data were also significant (p = 0.008) when each of the nine data points for control and treated samples were compared by t test. be Male and female SNCA mice (6.5 months-old; n = 20 felodipine; n = 19 vehicle) were implanted with subcutaneous felodipine-loaded osmotic minipumps (ALZET Model 2002) for 28 days, which were replaced once at day 14. At day 27, 17 felodipine and 18 vehicle control mice were available for analysis—some mice were culled for health reasons other than drug side effects. Behaviour was tested once before implanting and 14 days and 27 days after treatment (7 and 7.5 months-old, respectively). At the end of the study, 11 mice per group were used for biochemistry (b, c) and the remainder (9 felodipine; 8 vehicle) were used for histology. b Soluble and insoluble α-synuclein fractions of brain lysates from cerebral cortex and brain stem of SNCA mice. c Quantification of densitometry insoluble α-synuclein/GAPDH. Soluble α-synuclein was not significantly different between drug and vehicle (not shown). Data are mean α-synuclein vs. GAPDH ratio +/− SEM; two-tailed unpaired t-test. d Grip strength data are means +/− SEM; two-tailed, unpaired t-test. e Unbiased estimates of the number of TH-positive neurons in SN region of felodipine-treated and vehicle-treated SNCA mice. Data are means +/− SEM; two-tailed unpaired t-test. If the data are restricted to mice that survived until the end of the study, then the p values are: c (cerebral cortex): 0.057; (Brain stem): 0.025; e: 0.015

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References

    1. Berger Z, et al. Rapamycin alleviates toxicity of different aggregate-prone proteins. Hum. Mol. Genet. 2006;15:433–442. doi: 10.1093/hmg/ddi458. - DOI - PubMed
    1. Ravikumar B, Duden R, Rubinsztein DC. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. Mol. Genet. 2002;11:1107–1117. doi: 10.1093/hmg/11.9.1107. - DOI - PubMed
    1. Ravikumar B, et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 2004;36:585–595. doi: 10.1038/ng1362. - DOI - PubMed
    1. Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC. Alpha-Synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 2003;278:25009–25013. doi: 10.1074/jbc.M300227200. - DOI - PubMed
    1. Menzies FM, et al. Autophagy and neurodegeneration: pathogenic mechanisms and therapeutic opportunities. Neuron. 2017;93:1015–1034. doi: 10.1016/j.neuron.2017.01.022. - DOI - PubMed

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