The mTOR kinase inhibitor Everolimus decreases S6 kinase phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of Huntington's disease
© Fox et al; licensee BioMed Central Ltd. 2010
Received: 2 May 2010
Accepted: 22 June 2010
Published: 22 June 2010
Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion within the huntingtin gene. Mutant huntingtin protein misfolds and accumulates within neurons where it mediates its toxic effects. Promoting mutant huntingtin clearance by activating macroautophagy is one approach for treating Huntington's disease (HD). In this study, we evaluated the mTOR kinase inhibitor and macroautophagy promoting drug everolimus in the R6/2 mouse model of HD.
Everolimus decreased phosphorylation of the mTOR target protein S6 kinase indicating brain penetration. However, everolimus did not activate brain macroautophagy as measured by LC3B Western blot analysis. Everolimus protected against early declines in motor performance; however, we found no evidence for neuroprotection as determined by brain pathology. In muscle but not brain, everolimus significantly decreased soluble mutant huntingtin levels.
Our data suggests that beneficial behavioral effects of everolimus in R6/2 mice result primarily from effects on muscle. Even though everolimus significantly modulated its target brain S6 kinase, this did not decrease mutant huntingtin levels or provide neuroprotection.
Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a glutamine-encoding CAG repeat expansion within the huntingtin gene . Neurodegeneration is most prominent within striatum and neocortex and results in abnormal movements, cognitive decline and psychiatric symptoms. Mutant huntingtin misfolds and accumulates as soluble and insoluble aggregated species primarily in neurons.
Macroautophagy is a lysosomal-dependent process that mediates the turnover of organelles and misfolded proteins that are too large to be degraded by the ubiquitin proteosomal system [2, 3]. Steps involve biochemical induction, the sequestering of cytoplasmic fragments into double-membrane bound autophagic vacuoles, subsequent fusion with lysosomes and degradation within autolysosomes . The process involves the coordinated expression and regulation of many core and autophagy-related  as well as lysosomal proteins . There is activation of macroautophagy in HD models [3, 7]. Macroautophagy is also involved in the pathogenesis of Parkinson's, Alzheimer's and prion diseases [8–10].
Promoting clearance of mutant huntingtin (mhtt) by induction of macroautophagy is one approach for treating human HD [7, 11]. Everolimus (formerly called RAD001) is an inhibitor of mammalian target of rapamycin (mTOR), a protein that is part of an intra-cellular signaling pathway regulating cell metabolism. Everolimus, like rapamycin, inhibits the kinase activity of the raptor-mTOR complex (mTORC1) by binding to the protein FKBP-12, which forms an inhibitory complex with mTOR [12, 13]. mTOR kinase is a cytosolic protein that receives inputs from nutrient signaling pathways and is an inhibitor of macroautophagy [14, 15]. Everolimus inhibition of mTOR kinase promotes macroautophagy in a number of model systems [16, 17]. mTOR-kinase-independent macrophagy inducers have also been identified [9, 11] and these may offer an alternative pathway to modulate autophagy. However, the class of mTOR-kinase-inhibiting drugs is well characterized and in clinical use for their anti-neoplastic and anti-solid organ graft rejection effects [18, 19]. These compounds would offer advantages of availability and rapid progression into clinical trials if found to have significant beneficial effects in HD models.
The goal of this study was to evaluate the effect of everolimus in the R6/2 transgenic mouse model of HD. These mice express the exon-1 encoded fragment of mutant huntingtin under the control of the huntingtin promoter which results in protein expression in brain and skeletal muscle . We found that everolimus retarded declines in motor improvements. In brain, everolimus inhibited phosphorylation of the mTOR kinase target protein S6 kinase, but did not decrease mutant huntingtin levels, or decrease brain and neuronal atrophy. However, in skeletal muscle everolimus significantly decreased levels of soluble mutant huntingtin protein. While our data demonstrates a beneficial effect of everolimus in R6/2 HD mice, we could not demonstrate neuroprotection.
Pharmacokinetic analysis of everolimus in R6/2 mice
Everolimus decreases brain S6 kinase phosphorylation
Effect of everolimus on mouse performance and brain pathology
Everolimus normalizes increased LAMP1 in muscle but not in brain
Everolimus decreases soluble mutant huntingtin levels in muscle but not brain
R6/2 HD mice have been used extensively in pre-clinical drug trials and numerous compounds, including one that promotes macroautophagy, have shown neuroprotective effects [9, 27]. We studied the effect of everolimus, a O-(2-hydroxyethyl) chain substitution of rapamycin  in these mice. We hypothesized that everolimus would decrease mutant huntingtin levels in brain and have neuroprotective effects as measured by decreased brain atrophy. To verify that everolimus could interact with and inhibit its target, brain mTOR kinase, we performed a pharmacokinetic study and also evaluated the phosphorylation state of the mTOR target, S6 kinase. Our results demonstrate that everolimus slowly penetrates brain at doses of 10 and 30 μmol/kg (Figure 1). Brain: plasma ratios 4 hours after a final dose were 2-3% which is consistent with plasma contamination (Figure 1C). However, at 24 hours, brain: plasma ratios were 12-14% which is significantly greater than mouse fore-brain vascular volume of ~6% indicating significant brain penetration . Brain everolimus concentrations were in the range 50-180 nM at 24 hours. The IC50 of everolimus in cell proliferation assays is in the sub-nano molar range  which is consistent with the concentrations we found in brain being sufficient to strongly inhibit mTOR kinase. To address this, we used S6 kinase phosphoepitope Western blot analysis. We reduced our high dose from 30 to 20 μmol/kg at this point because of body weight loss following prolonged treatment at the 30 μmol/kg dose. Even though we did not repeat the pharmacokinetic analysis using the 20 μmol/kg dose our Western blot analysis clearly shows decreased S6 kinase phosphorylation at the 20 but not the 10 μmol/kg dose, confirming brain penetration at the higher dose used (Figure 2). Decreased S6 kinase phosphorylation in R6/2 HD cortex at the S235-236 epitope, as compared to wild-type mice, is consistent with findings in the N171-82Q HD mouse .
Despite demonstrating penetration of everolimus into brain and modulation of its target, we were unable to demonstrate any protective effects in R6/2 HD mouse brain as determined by a detailed quantitative neuropathology study (Figure 3D-G) and using three independent methods to quantify mutant huntingtin levels (Figures 3G, 6, see also Additional file 1: Figure S1) all at 12 weeks of age. Our body weight data (Figure 3B) demonstrate that the high (20 μmol/kg) dose we used in our efficacy study was the maximum tolerated dose using our working definition of a 10% loss of body weight over the study period. As brain weights were slightly but significantly lower in the 20 μmol/kg versus placebo group (Figure 3D), this further suggests that doses higher than that used would not have shown beneficial effects. The three times a week dosing paradigm that we used has been reported previously for the closely related rapamycin analog CCI-779 in a study in N171-82Q HD mice . The suitability of this dosing frequency is also supported by our own data in which mice sacrificed four hours after the last dose of an eight week dosing study had decreased brain S6 kinase phosphorylation (Figure 2) even though everolimus did not enter brain 4 hours after a final dose in the pharmokinetic study (Figure 1). This suggests that with repeated, three times a week, dosing everolimus has a prolonged effect on S6 kinase phosphorylation. Taken together, our results suggest that failure to find a neuroprotective effect of everolimus in R6/2 HD mice was not due to insufficient inhibition of brain mTOR kinase activity.
Muscle is a target of mutant huntingtin in mouse and human HD [31, 32]. We found that everolimus demonstrated an early protective effect on Rota-rod performance that was stronger for the low (10 μmol/kg) dose group (Figure 3A). As there was no effect of this low dose on brain S6 kinase phosphorylation (Figure 2) we reasoned that a beneficial effect of everolimus in muscle could explain why the 10 μmol/kg group performed better than the 20 μmol/kg group on Rota-Rod analysis. We did not measure S6 kinase phosphorylation in muscle. However, both mTOR and S6 kinase are expressed in muscle . Given the sub-nanomolar IC50 of everolimus for mTOR kinase and expected high penetration of everolimus into muscle, we would expect strong suppression of S6 kinase phosphorylation. Instead, we measured mutant huntingtin levels in everolimus treated R6/2 mice at 12 weeks by FRET. We found significantly decreased soluble mutant huntingtin at the high dose and a trend towards decreased levels at the low dose (Figure 6). There was no effect on aggregated huntingtin levels (see Additional file 1: Figure S1 C-D). Better Rota-rod performance in the low dose group (Figure 3A) could be related to the high dose having both beneficial and toxic effects (Figure 3B). Therefore, our data are consistent with Rota-Rod effects of everolimus being, at least in part, due to beneficial effects in skeletal muscle. We cannot rule out the possibility of a transient suppression of mutant huntingtin levels in brain occurring in the 6-8 week period contributing to behavioral benefits. However, as several compounds demonstrate prolonged protective activity in R6/2 brain , including one that promotes autophagy [9, 27], this would suggest that even if everolimus decreased brain mutant huntingtin levels transiently that mTOR kinase inhibition is not as effective as modulation of other therapeutic targets in R6/2 mice.
LAMP1 is a type I transmembrane glycoprotein and a marker of lysosomes and autolysosomes , organelles critical for downstream steps of the autophagy cascade. While not a core macroautophagy protein, LAMP1 is a lysosome marker and therefore should reflect activity within the terminal clearance steps of the autophagic cascade. We evaluated LAMP1 in our study to determine if levels are increased in R6/2 HD mice and to determine the effect of everolimus. As expected, we found that LAMP1 protein levels were increased in muscle and brain (Figure 5) of R6/2 as compared to wild-type mice. Everolimus had a significant effect on LAMP1 in muscle, but not brain. In muscle, high dose everolimus decreased LAMP1 towards wild-type levels and there was a trend towards a decrease with the low dose (Figure 5E-F). This result was unexpected and the reasons are not clear. We speculate that everolimus may promote clearance of autolysosomes. Further studies are clearly needed to better understand the mechanism(s) by which everolimus decreases mutant huntingtin and LAMP1 levels in muscle.
CCI-779 is a rapamycin dihydoxymethyl propionic acid ester  that has been shown to demonstrate behavioral benefits and decrease aggregate density in N171-82Q HD mice . Our results using everolimus, a related rapamycin derivative in the R6/2 HD model demonstrate a different effect. While our data confirms entry of everolimus into brain, as measured by direct chemical analysis and S6 kinase phosphorylation levels, we could not demonstrate decreased neurodegeneration or brain mutant huntingtin levels. The difference between our findings and that of Ravikumar et al (2004) could be for a number of reasons. R6/2 HD mice have a more aggressive phenotype than the N171-82Q HD mice used in the CCI-779 study [21, 35]. Factors such as longer polyglutamine expansion in R6/2 compared to N171-82Q mice could also be important.
Our findings suggest that mTOR kinase inhibition in R6/2 HD mice using everolimus has, at most, a minimal effect on disease progression in brain. Everolimus effectively modulated brain S6 kinase, which is upstream of macroautophagy induction, but had no effect on mutant huntingtin levels. Everolimus did decrease soluble mutant huntingtin levels in muscle. While the exact mechanisms by which this occurs were not fully established, our data indicates that a beneficial effect of everolimus in muscle is a likely mechanism for the protective behavioral effects observed in our HD mice. Given the discrepancy between our findings using everolimus in R6/2 mice and Ravikumar et al  using CCI-779 in N171-82Q mice, side-by-side testing of these molecules in both R6/2 and N171-82Q mice, and perhaps as well in a full-length huntingtin mouse model, would provide additional insight into the value of this class of molecule as a treatment for HD.
Materials and methods
R6/2 mice were maintained by crossing R6/2 males (available from The Jackson Laboratory, Bar Harbor, ME) with C57BL/6 X CBA F1 females. Tail tips were obtained at 14 days. Tail DNA CAG expansion sizes of male breeder mice were in the range 120-130, as determined by Laragen Inc. Mice were weaned at 22 days, and assigned to treatment groups 1-2 days later. Systematic assignment to treatment groups was used to minimize the effects of body weight variability, litter and paternal effects. Everolimus was prepared as described . Mice were female and were dosed by gavage on Mondays, Wednesdays and Fridays (see discussion). Body weights were measured weekly and used to adjust doses.
One-hundred μl plasma was extracted three times in 500 μl ethyl acetate. Extracts were dried under a stream of nitrogen before re-dissolving in 100 μl acetonitrile. Brains were weighed then homogenized in water (1:5 w/v) using an Ultra-Turrax® Mod T8 for 30 seconds. Two 100 μl aliquots of each homogenate were extracted thrice with 500 μl ethyl acetate, then processed as for plasma. Calibration standards were prepared by supplementing 100 μl of mouse plasma (from untreated animals) with everolimus and the analysis quality monitored by routine use of an external standard. For HPLC separation, a Nucleosil CC-125/2 C4 reversed phase column (Macherey & Nagel, Oensingen, Switzerland) under isocratic conditions using 60% acetonitrile and 0.05% formic acid in H2O (v/v) with a column temperature of 40°C was used. The flow rate was 0.35 ml/minute and sample injection volume was 10 μl. Retention times were 1.6 and 2.2 minutes for everolimus and NVP-BDF461, respectively. Column efflux was introduced directly into the ion source of a Micromass Platform II LC detector (single quadrupole). The MS conditions were as follows: ionization APCI negative polarity, corona voltage set to 3.2 kV, fragmentor voltage (cone) 50 V, source temperature 350°C. Quantitative analysis was performed by selected ion recording over the de-protonated molecular ion [M+H-] of everolimus (m/z 956.8 ± 0.5). Peaks were integrated using MassLynx (Micromass). Two independent extractions were analyzed per animal. Standard curves were prepared by spiking plasma and brain homogenates originating from untreated animals with five concentrations of everolimus as external standard. A second set of standards in acetonitrile was directly analyzed to estimate extraction yield. A linear calibration was calculated for each analytical batch from the ratio between calibrant and internal standard and the calibrant in spiked plasma or brain samples. Regression was performed using Origin® software. Unknown concentrations were calculated from the calibration parameters obtained with extracted samples containing internal standard.
Western blot analysis
Antibodies used were: mutant huntingtin (MAB5492-Chemicon), actin (AC40-Sigma), LC3B (Novus Biologicals) and LAMP1 (BioLegend). Total S6 (54D2) and phosphor-S6 protein (serines 235/236 and 240/244) antibodies (Cell Signaling). Primary antibodies were used at 1:2000, except for AC40 (1:4000). For mutant huntingtin analysis, dissected brain regions were homogenized in 20 volumes of 20 mM TRIS (pH 7.2), 150 mM sodium chloride, 1 mM EDTA, 1 mM DTT and HALT protease inhibitor cocktail (Pierce) using a Pellet pestle® (Kontes). Samples were cleared at 18000 g for 15 minutes at 4°C. Thirty μg protein was resolved by SDS-PAGE and transferred to PVDF. Membranes were blocked in 5% milk powder in TRIS-buffered saline containing 1% Tween-20 (TBST) and then probed with primary antibody overnight in blocking buffer at 4°C. After washing and incubation in HRP-conjugated secondary antibody membranes were developed using Western Lightening™ chemiluminescent reagent plus (Perkin-Elmer). For analyses of all other proteins, the procedure was identical to that described above except for the homogenization buffer; this comprised 25 mM HEPES (pH 7.4), 75 mM sodium chloride, 12.5 mM β-glycerophosphate, 12.5 mM sodium fluoride, 2.5 mM EGTA, 0.5 mM EDTA, 7.5 mM sodium pyrophosphate, 2 mM sodium vanadate, 0.1% Nonidet-P40 and HALT protease inhibitor. For muscle analysis, the same procedures were used except that tissues were homogenized using a Tissuemiser® (Fisher). For brain, actin was used to normalize Western blots. For muscle, we used parallel run coomassie gels because we do not have a validated housekeeping gene for R6/2 muscle and because actin mRNA is down regulated in this tissue .
Rota-Rod endurance was assessed using an accelerating Rota-rod (Stoelting). The rod speed accelerated from 4.5-45 rpm at a constant rate. Measurements were first taken prior to dosing at 3.5 weeks, then at every 2 weeks of age. For each time point, mice were evaluated on four consecutive days. Day 1 was a training day. On days 2-4 accelerating Rota-rod endurance was evaluated once/day up to a maximum of 15 minutes and endurance times recorded. The average of three trials was used for statistical analysis.
At 12 weeks of age, mice were deeply anesthetized with a tribromoethanol-based anesthetic. They were then perfused with freshly prepared room temperature 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 minutes at a flow rate of 12 mls/minute. Perfused mice were stored at 4°C for 2 hours prior to brain removal. Brains were post-fixed in the same fixative overnight at 4°C prior to cryoprotection for 3 days in 10% glycerol, 2% DMSO and 0.1 M phosphate buffer. The entire striatum was sectioned coronally at 50 μm and every eighth section was mounted and stained for Nissl substance using the thionin method.
Fifty-μm sections at the level of the anterior commissure were used for immunohistochemical (IHC) staining for mutant huntingtin aggregates and LAMP1 immunofluorescence (IF). For IHC, we used a 1:2000 dilution of EM48 antibody incubated with floating sections in PBS containing 0.5% Tween-20 for 48 hours to aid penetrance. After washing, sections were incubated in a biotinylated anti-rabbit antibody overnight. Reactivity was developed using the Vectastain ABC kit (Vector Laboratories). Sections were mounted in aqueous medium (Fluoromount G, Southern Biotech) to prevent z-axis shrinkage. For IF, sections were incubated in 1:100 anti-LAMP1 (BioLegend) in PBS containing 0.1% Tween-20 and 10% normal goat serum for 48 hours. Sections were washed three times in PBS, then incubated in AlexaFluor-488 labeled anti-rat antibody (Invitrogen) for 4-hours at 25°C. After washing in PBS, sections were stained with the nucleic acid stain ethidium dimer at 5 μM for 2 hours in the dark. Sections were washed in PBS before mounting. Three-layered z-stack images were collected using a Leica TCS SL confocal microscope. Neurons were identified within the central stack using ethidium dimer signal that delineates nuclei and cytosol. LAMP1 signal was quantified by outlining neuronal cell body outlines and quantifying fluorescence using Metamorph software (Molecular Devices).
The methodologies used were exactly the same as we have fully described previously .
Biochemical quantification of mutant huntingtin levels
Soluble mutant huntingtin was quantified by time-resolved FRET as described . In brief, muscle samples were homogenized in 20 volumes of PBS containing 0.4% (v/v) Triton-X100 and protease inhibitor using a Precellys®24 (Bertin technologies) for 2 × 30 seconds at 5000 rpm. Homogenates were cleared at 3000 rpm for 10 minutes at 4°C. The supernatant was transferred into a fresh tube and total protein was measured using the BCA-Protein detection kit (Perbio, Cramlington, UK). Brain samples were homogenized in 25 mM HEPES (pH 7.4), 75 mM sodium chloride, 12.5 mM beta-glycerophosphate, 12.5 mM sodium fluoride, 2.5 mM EGTA, 0.5 mM EDTA, 7.5 mM sodium pyrophosphate, 2 mM sodium vanadate, 1 mM dithiothreitol, 0.1% nonidet p40, and 0.1% HALT protease inhibitor. The amount of homogenate needed to reach levels in the linear range of time-resolved FRET detection was determined and resulted in 2 μl of homogenate for brain samples (~12 μg protein/well), and 5 μl of homogenate for muscle samples (~17 μg protein/well). Brain or muscle homogenates were mixed with an antibody solution (5 μl) composed of 2B7-Europium-Cryptate (1 ng) and MW1-d2 (10 ng) dissolved in NaH2PO4 (50 mM, pH 7.4), NaF (400 mM), BSA (0.1% w/v), and Tween 20, (0.05% v/v) in a low-volume 384-well plate and incubated at 4 degrees centigrade overnight. The final volume was 15 μl. Time-resolved FRET with excitation at 320 nm and emission at 620 and 665 nm was measured using an Envision fluorimeter (Perkin-Elmer). Time-resolved FRET signals are given as the 665/620 nm ratio. Background levels (wild-type) were deducted and values normalized for protein concentration. Results are expressed as a percentage of vehicle treated animals. Aggregated huntingtin was quantified using the recently described agarose gel electrophoresis for resolving aggregates (AGERA) method . Briefly, mouse brain samples were homogenized in 10 volumes (w/v) tris-saline (100 mM Tris, pH 7.4, 150 mM NaCl) and Complete Protease Inhibitor (Roche Diagnostics) by 10 ultrasound pulses with a Branson sonifier and stored at −80°C. For 1.7% agarose gels, 1.7 g agarose (Biorad, #161-3101) was dissolved in 100 mL 375 mmol/L Tris-HCl, pH 8.8 and brought to boiling in a microwave oven. After melting, SDS was added to a final concentration of 0.1% (w/v). Gels were poured on short Biorad DNA Sub Cell™ trays. Samples were diluted 1: 1 into non-reducing Laemmli sample buffer (150 mmol/L Tris-HCl pH 6.8, 33% glycerol, 1.2% SDS and bromophenol blue) and incubated for 20 minutes at 95°C. Two-hundred μg of protein was loaded per AGERA lane. After loading, gels were run in Laemmli running buffer (192 mmol/L glycine, 25 mmol/L Tris-base, 0.1% (w/v) SDS) at 100 V, 2 A until the bromophenol blue running front reached the bottom of the gel. Semi-dry electroblotter model B (Ancos, Højby, Denmark) was used to blot the gels on PDVF membranes (Millipore) at 200 mA for 1 hour. Membranes were then developed using MW8 mouse monoclonal antibodies (3 μg/ml), and aggregate quantification performed by densitometry analysis.
All data was analyzed using SAS version 9.1 software (Cary, NC). Rota-rod and body weight data was analyzed using a mixed-model method that included age by treatment interaction effects. Slice functions and t-tests were used to determine significant differences. All other data was analyzed by one-way analysis of variance (ANOVA) using a generalized-linear model procedure followed by pair-wise comparisons. All p-values < 0.05 were considered significant.
This work was supported by the BeatHD Collaborative grant from the Novartis Institutes for BioMedical Research (NIBR).
- The Huntington's Disease Collaborative Research Group: A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell. 1993, 72: 971-983. 10.1016/0092-8674(93)90585-E.View ArticleGoogle Scholar
- Rideout HJ, Lang-Rollin I, Stefanis L: Involvement of macroautophagy in the dissolution of neuronal inclusions. Int J Biochem Cell Biol. 2004, 36: 2551-2562. 10.1016/j.biocel.2004.05.008.PubMedView ArticleGoogle Scholar
- Kegel KB, Kim M, Sapp E, McIntyre C, Castano JG, Aronin N, DiFiglia M: Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy. J Neurosci. 2000, 20: 7268-7278.PubMedGoogle Scholar
- Klionsky DJ, Emr SD: Autophagy as a regulated pathway of cellular degradation. Science. 2000, 290: 1717-1721. 10.1126/science.290.5497.1717.PubMedPubMed CentralView ArticleGoogle Scholar
- Klionsky DJ, Cregg JM, Dunn WA, Emr SD, Sakai Y, Sandoval IV, Sibirny A, Subramani S, Thumm M, Veenhuis M, Ohsumi Y: A unified nomenclature for yeast autophagy-related genes. Dev Cell. 2003, 5: 539-545. 10.1016/S1534-5807(03)00296-X.PubMedView ArticleGoogle Scholar
- Schroder B, Wrocklage C, Pan C, Jager R, Kosters B, Schafer H, Elsasser HP, Mann M, Hasilik A: Integral and associated lysosomal membrane proteins. Traffic. 2007, 8: 1676-1686. 10.1111/j.1600-0854.2007.00643.x.PubMedView ArticleGoogle Scholar
- Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O'Kane CJ, Rubinsztein DC: 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. 10.1038/ng1362.PubMedView ArticleGoogle Scholar
- Aguib Y, Heiseke A, Gilch S, Riemer C, Baier M, Schatzl HM, Ertmer A: Autophagy induction by trehalose counteracts cellular prion infection. Autophagy. 2009, 5: 361-369. 10.4161/auto.5.3.7662.PubMedView ArticleGoogle Scholar
- Sarkar S, Davies JE, Huang Z, Tunnacliffe A, Rubinsztein DC: Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein. J Biol Chem. 2007, 282: 5641-5652. 10.1074/jbc.M609532200.PubMedView ArticleGoogle Scholar
- Boland B, Kumar A, Lee S, Platt FM, Wegiel J, Yu WH, Nixon RA: Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease. J Neurosci. 2008, 28: 6926-6937. 10.1523/JNEUROSCI.0800-08.2008.PubMedPubMed CentralView ArticleGoogle Scholar
- Sarkar S, Perlstein EO, Imarisio S, Pineau S, Cordenier A, Maglathlin RL, Webster JA, Lewis TA, O'Kane CJ, Schreiber SL, Rubinsztein DC: Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nat Chem Biol. 2007, 3: 331-338. 10.1038/nchembio883.PubMedPubMed CentralView ArticleGoogle Scholar
- Guertin DA, Sabatini DM: The pharmacology of mTOR inhibition. Sci Signal. 2009, 2: pe24-10.1126/scisignal.267pe24.PubMedView ArticleGoogle Scholar
- Dowling RJ, Topisirovic I, Fonseca BD, Sonenberg N: Dissecting the role of mTOR: lessons from mTOR inhibitors. Biochim Biophys Acta. 2010, 1804: 433-439.PubMedView ArticleGoogle Scholar
- Inoki K, Corradetti MN, Guan KL: Dysregulation of the TSC-mTOR pathway in human disease. Nat Genet. 2005, 37: 19-24. 10.1038/ng1494.PubMedView ArticleGoogle Scholar
- Sarkar S, Ravikumar B, Floto RA, Rubinsztein DC: Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell Death Differ. 2009, 16: 46-56. 10.1038/cdd.2008.110.PubMedView ArticleGoogle Scholar
- Alonso MM, Jiang H, Yokoyama T, Xu J, Bekele NB, Lang FF, Kondo S, Gomez-Manzano C, Fueyo J: Delta-24-RGD in combination with RAD001 induces enhanced anti-glioma effect via autophagic cell death. Mol Ther. 2008, 16: 487-493. 10.1038/sj.mt.6300400.PubMedView ArticleGoogle Scholar
- Crazzolara R, Bradstock KF, Bendall LJ: RAD001 (everolimus) induces autophagy in acute lymphoblastic leukemia. Autophagy. 2009, 5: 10.4161/auto.5.5.8507.Google Scholar
- Eisen HJ, Tuzcu EM, Dorent R, Kobashigawa J, Mancini D, Valantine-von Kaeppler HA, Starling RC, Sorensen K, Hummel M, Lind JM, et al: Everolimus for the prevention of allograft rejection and vasculopathy in cardiac-transplant recipients. N Engl J Med. 2003, 349: 847-858. 10.1056/NEJMoa022171.PubMedView ArticleGoogle Scholar
- Motzer RJ, Escudier B, Oudard S, Hutson TE, Porta C, Bracarda S, Grunwald V, Thompson JA, Figlin RA, Hollaender N, et al: Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet. 2008, 372: 449-456. 10.1016/S0140-6736(08)61039-9.PubMedView ArticleGoogle Scholar
- Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, Hetherington C, Lawton M, Trottier Y, Lehrach H, Davies SW, Bates GP: Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell. 1996, 87: 493-506. 10.1016/S0092-8674(00)81369-0.PubMedView ArticleGoogle Scholar
- Stack EC, Kubilus JK, Smith K, Cormier K, Del Signore SJ, Guelin E, Ryu H, Hersch SM, Ferrante RJ: Chronology of behavioral symptoms and neuropathological sequela in R6/2 Huntington's disease transgenic mice. J Comp Neurol. 2005, 490: 354-370. 10.1002/cne.20680.PubMedView ArticleGoogle Scholar
- Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J. 2000, 19: 5720-5728. 10.1093/emboj/19.21.5720.PubMedPubMed CentralView ArticleGoogle Scholar
- Devon RS, Orban PC, Gerrow K, Barbieri MA, Schwab C, Cao LP, Helm JR, Bissada N, Cruz-Aguado R, Davidson TL, et al: Als2-deficient mice exhibit disturbances in endosome trafficking associated with motor behavioral abnormalities. Proc Natl Acad Sci USA. 2006, 103: 9595-9600. 10.1073/pnas.0510197103.PubMedPubMed CentralView ArticleGoogle Scholar
- Andrejewski N, Punnonen EL, Guhde G, Tanaka Y, Lullmann-Rauch R, Hartmann D, von Figura K, Saftig P: Normal lysosomal morphology and function in LAMP-1-deficient mice. J Biol Chem. 1999, 274: 12692-12701. 10.1074/jbc.274.18.12692.PubMedView ArticleGoogle Scholar
- Weiss A, Abramowski D, Bibel M, Bodner R, Chopra V, DiFiglia M, Fox J, Kegel K, Klein C, Grueninger S, et al: Single-Step Detection of Mutant Huntingtin in Animal and Human Tissues: a BioAssay for Huntington's Disease. Anal Biochem. 2009, 395: 8-15. 10.1016/j.ab.2009.08.001.PubMedView ArticleGoogle Scholar
- Weiss A, Klein C, Woodman B, Sathasivam K, Bibel M, Regulier E, Bates GP, Paganetti P: Sensitive biochemical aggregate detection reveals aggregation onset before symptom development in cellular and murine models of Huntington's disease. J Neurochem. 2008, 104: 846-858.PubMedGoogle Scholar
- Tanaka M, Machida Y, Niu S, Ikeda T, Jana NR, Doi H, Kurosawa M, Nekooki M, Nukina N: Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. Nat Med. 2004, 10: 148-154. 10.1038/nm985.PubMedView ArticleGoogle Scholar
- Ballou LM, Lin RZ: Rapamycin and mTOR kinase inhibitors. J Chem Biol. 2008, 1: 27-36. 10.1007/s12154-008-0003-5.PubMedPubMed CentralView ArticleGoogle Scholar
- Chugh BP, Lerch JP, Yu LX, Pienkowski M, Harrison RV, Henkelman RM, Sled JG: Measurement of cerebral blood volume in mouse brain regions using micro-computed tomography. Neuroimage. 2009, 47: 1312-1318. 10.1016/j.neuroimage.2009.03.083.PubMedView ArticleGoogle Scholar
- Schuler W, Sedrani R, Cottens S, Haberlin B, Schulz M, Schuurman HJ, Zenke G, Zerwes HG, Schreier MH: SDZ RAD, a new rapamycin derivative: pharmacological properties in vitro and in vivo. Transplantation. 1997, 64: 36-42. 10.1097/00007890-199707150-00008.PubMedView ArticleGoogle Scholar
- Kosinski CM, Schlangen C, Gellerich FN, Gizatullina Z, Deschauer M, Schiefer J, Young AB, Landwehrmeyer GB, Toyka KV, Sellhaus B, Lindenberg KS: Myopathy as a first symptom of Huntington's disease in a Marathon runner. Mov Disord. 2007, 22: 1637-1640. 10.1002/mds.21550.PubMedView ArticleGoogle Scholar
- Luthi-Carter R, Hanson SA, Strand AD, Bergstrom DA, Chun W, Peters NL, Woods AM, Chan EY, Kooperberg C, Krainc D, et al: Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain. Hum Mol Genet. 2002, 11: 1911-1926. 10.1093/hmg/11.17.1911.PubMedView ArticleGoogle Scholar
- Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulos GD: Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol. 2001, 3: 1014-1019. 10.1038/ncb1101-1014.PubMedView ArticleGoogle Scholar
- Ferrante RJ, Kubilus JK, Lee J, Ryu H, Beesen A, Zucker B, Smith K, Kowall NW, Ratan RR, Luthi-Carter R, Hersch SM: Histone deacetylase inhibition by sodium butyrate chemotherapy ameliorates the neurodegenerative phenotype in Huntington's disease mice. J Neurosci. 2003, 23: 9418-9427.PubMedGoogle Scholar
- Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, Slunt HH, Ratovitski T, Cooper JK, Jenkins NA, et al: Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet. 1999, 8: 397-407. 10.1093/hmg/8.3.397.PubMedView ArticleGoogle Scholar
- Crazzolara R, Cisterne A, Thien M, Hewson J, Baraz R, Bradstock KF, Bendall LJ: Potentiating effects of RAD001 (Everolimus) on vincristine therapy in childhood acute lymphoblastic leukemia. Blood. 2009, 113: 3297-3306. 10.1182/blood-2008-02-137752.PubMedView ArticleGoogle Scholar
- Chopra V, Fox JH, Lieberman G, Dorsey K, Matson W, Waldmeier P, Housman DE, Kazantsev A, Young AB, Hersch S: A small-molecule therapeutic lead for Huntington's disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse. Proc Natl Acad Sci USA. 2007, 104: 16685-16689. 10.1073/pnas.0707842104.PubMedPubMed CentralView ArticleGoogle Scholar
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