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How to reduce the accumulation of autophagic vacuoles in NPC1-deficient neurons: A comparison of two pharmacological strategies
Neuropharmacology 89 (2015) 282e289
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
How to reduce the accumulation of autophagic vacuoles in NPC1deficient neurons: A comparison of two pharmacological strategies Volker Meske*, Timm Priesnitz, Frank Albert, Thomas Georg Ohm Center of Anatomy, Institute of Integrative Neuroanatomy, Department of Clinical Cell- and Neurobiology, Charit e, Charit e-Platz 1, 10098 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2014 Received in revised form 3 September 2014 Accepted 6 October 2014 Available online 16 October 2014
A disturbed autophagic pathway leads to chronically increased levels of autophagic vacuoles in Niemann Pick Type-C 1 (NPC1) deficient neurons. Since these accumulations potentially contribute to neuronal cell death associated with the disease, we investigated two pharmacological strategies which potentially reduce the number of autophagic structures under following aspects: efficiency, sustainability and effect on neuronal cell viability. The strategies comprised (i) an interruption of the autophagic flux by the class III PI3K inhibitor 3-methyladenine (3-MA) and (ii) an acceleration of the autophagic execution by 2hydroxypropyl-b-cyclodextrin (pCD). Our data show that the inhibition of autophagy with 3-MA only initially reduced the number of autophagic vacuoles in cultured neurons. Prolonged treatments with the PI3K-inhibitor reversed this lowering effect. The re-increase in the number of autophagic vacuoles was combined with a defect in the integrity of lysosomes which endangered further survival of cells. The treatment with pCD evoked a slow but sustained reduction of autophagic structures and had no negative effects on neuronal survival. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Neurons Niemann-Pick type C disease Autophagy Cyclodextrin PI3K-inhibitor
1. Introduction NPC is a lipid storage disease with defects either in the NPC1- or NPC2- protein (Carstea et al., 1997; Naureckiene et al., 2000). Cholesterol and glycosphingolipids accumulate in the late endosomal/lysosomal compartment of NPC1 and/or NPC2-deficient cells as a consequence from a disrupted lipid transport. Several studies reported that the autophagic flux in cells is chronically disturbed leading to an abnormal intracellular accumulation of autophagic vacuoles in NPC1-deficient cells (Liao et al., 2007; Pacheco et al., 2007; Pacheco and Lieberman, 2007; Ishibashi et al., 2009; Elrick et al., 2012; Ordonez et al., 2012; Meske et al., 2014). Some authors suggested that this defect in autophagy may contribute to neurodegeneration in NPC-disease (Ko et al., 2005; Liao et al., 2007). In a recent study with primary neuron cultures we could prove this hypothesis and demonstrated that the reduction of the abnormal lipid load inside the acidic organelles with b-hydroxypropyl cyclodextrin (pCD) is sufficient to correct the abnormal
autophagic flux and prevents NPC1/ neurons from premature death under autophagic stress (Meske et al., 2014). Nevertheless, it has been shown that not only restoration of the defective lysosomal clearance by treatments with cyclodextrin (Ordonez et al., 2012; Meske et al., 2014), but also the inhibition of the autophagosomeformation with PI3K class III inhibitors like 3-methyladenine (3MA) reduces the number of autophagic vacuoles in NPC1deficient cells (Pacheco et al., 2007; Elrick et al., 2012; Ordonez et al., 2012). In the present study we compare both strategies in primary cultures of NPC1-deficient neurons for their efficiency, sustainability and effects on cellular survival. Our results indicate that therapeutic treatments to improve the autophagic state in NPC1-deficient neurons should concentrate on agents which accelerate the execution of autophagy in cells instead on substances which impede autophagy.
2. Material and methods 2.1. Primary neuronal cell culture
Abbreviations: CD, cyclodextrin; pCD, 2-hydroxy-propyl-b-cyclodextrin; LC3, microtubule-associated protein 1 light chain 3; LC3-I, phosphatidylethanolaminedeconjugated LC3; LC3-II, phosphatidylethanolamine-conjugated LC3; (3)-MA, (3)methyladenine; NPC, Niemann-Pick type C. * Corresponding author. Tel.: þ49 (0)30 450 528257; fax: þ49 (0)30 450 528913. E-mail address: [email protected] (V. Meske). http://dx.doi.org/10.1016/j.neuropharm.2014.10.006 0028-3908/© 2014 Elsevier Ltd. All rights reserved.
Neuron cultures were prepared from cortices of embryos (E16) derived from mating of npc1nihBalb/cJ mice (Jackson Laboratories) heterozygous for NPC1 (NPC1þ/ ) as described earlier (Deisz et al., 2005). Breeding and handling of the animals were performed according to the German law of animal care (Tierschutzgesetz) which is in accordance with EU directive 2010/63/EU. Cells with NPC1/ and NPC1þ/þ (wildtype ¼ wt) genotypes were cultured as described earlier (Meske et al., 2008). For experiments 10e14 days old cultures were used.
V. Meske et al. / Neuropharmacology 89 (2015) 282e289 2.2. Treatments of cell cultures 3-methyladenine (SigmaeAldrich): was dissolved in culture medium to a working-concentration of 3 mM. 2-hydroxypropyl-b-cyclodextrin (SigmaeAldrich): a stock solution of 10% (w/v) in culture-medium was further diluted with medium to 0.1% (w/v). 2.3. Neuronal viability We assessed neuronal survival with MTT (methyl-thiazol-tetrazolium [SigmaeAldrich])-assay as described by Mosmann (1983). The colour intensities were measured with an ELISA reader (Kontron) at 580 nm. Data were analysed with EXCEL (Microsoft). 2.4. Sample preparation for protein analysis Samples were prepared as described previously (Meske et al., 2008). Extraction buffer: PBS (phosphate buffered saline) pH 7.4, protease-inhibitor cocktail (Roche), 1 mM activated sodium ortho-vanadate (SigmaeAldrich), 20 mM sodium fluoride (SigmaeAldrich) and 100 nM okadaic acid (SigmaeAldrich) from a 100 mM stock solution in DMSO (dimethylsulfoxide), 2% (v/v) Triton-X100. 2.5. SDS-PAGE/western blot SDS-PAGE (10 and 15% PAGE), blotting-procedure and densitometric analysis of the blots followed a protocol described earlier (Meske et al., 2008). 2.6. Antibodies used for analysis LC3B-antibody was purchased from Cell-Signalling-Technology, LC3B (#2775). As secondary we used horseradish peroxidase e conjugated anti-rabbit IgG (Vector). 2.7. Stainings 2.7.1. General cell staining For staining viable cells we used cells grown on glass cover slips and applied 1 mM Calcein-AM (Molecular probes) to the culture medium for 30 min. Afterwards cells were transferred into a measuring chamber filled with Hanks-balanced-saltsolution, ([with Ca2þ/Mg2,Gibco], supplemented with Hepes 10 mM, pH 7.4). For visualization we used a fluorescence-microscope (IX70, Olympus) attached to an imaging device (Till photonics).
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processed in Adobe Photoshop®. The analysis of areas (filipin-positive vacuoles and acidic compartment) was performed with the software Image J. The results were saved as EXCEL-sheets for further statistical analysis. 2.8. Statistics We used program SPSS for statistical data analysis. Significance was assessed by parametric Student's t-test or non-parametric Whitman-test, when two means were compared. For the comparison of more than two means ANOVA with a subsequent post hoc test (NewmaneKeuls) was performed.
3. Results 3.1. Effect of 3-MA and pCD on LC3BII-contents measured by westernblots In order to reduce the abnormal high number of autophagic vacuoles in cells we tested two strategies. First, we used the PI3K inhibitor 3-methyladenine (3-MA) to interrupt the formation of autophagosomes and second, we used 2-OH-propyl-b-cyclodextrin (pCD) to accelerate the lysosomal clearance of autophagosomes. Westernblot analysis showed that both treatments reduced the LC3B-II contents in NPC1-deficient neurons (Fig. 1A, B) over time. As control we used actin contents, or calculated the ratio of LC3B-I/ LC3BII (see Supplement Figs. S1 and S2). Considerable differences between both treatments occurred when the incubation was extended beyond the time required for a significant reduction. In detail: the treatment with 3-MA required a continuous application of the agent at a relatively high concentration (3 mM) and reduced
2.7.2. Co-staining of the acidic compartment and LC3B Staining of the acidic compartment with LysoTracker (Molecular Probes) and immunofluorescent co-staining of LC3B (antibody #2775, Cell-Signalling) was performed as described earlier (Meske et al., 2014). For analysis of the staining, we used a laser-scan microscope LSM5 (exciter: helium/neon- and argon-laser) attached to an AX10 microscope equipped with a 63 oil immersion objective and control software Zen 2008 (Zeiss). Pictures of the acidic compartment: red channel (ex: 546 nm; em: longpass filter >580 nm), of the LC3B positive vesicles: green-channel (ex: 488 nm; em: pass filter 505e530 nm) and of transmission-light were analysed in Image J (NIH) and Adobe Photoshop®. Using the software Adobe Photoshop, pictures from the same field of view were combined in one picture with three image planes, single cells were delineated, cut out and analysed separately. Areas of colocalization were displayed with the submenu channel-calculation/multiply signals (redegreen channels). Using the software Image J we measured the area of signals in the three related pictures (acidic compartment, LC3B-positive vesicles, colocalized areas). Since all pictures analysed in this study had the same magnification scale (1200), areas were expressed in square pixels. Subtraction of the areas of colocalized punctae from areas of total LC3B punctae/neuron resulted in the number of cytosolic free autophagosomes/neuron. The results were saved as EXCEL-sheets for further statistical analysis. 2.7.3. Co-staining of the acidic compartment and lamp2 The co-staining of neurons with LysoTracker and lamp2 followed a protocol described earlier (Meske et al., 2014), with one exception we permeabilized cells with 0.1% (w/v) saponin (Sigma) after fixation and omitted Triton-X in the washing/ incubation buffers. We used anti-lamp2 antibody (Sressgen) diluted 1: 250 in TBS and as secondary antibody Alexa-488 goat anti-rat IgG (Molecular Probes) diluted 1:500 in TBS. For documentation of the staining, we used the same equipment as described in the preceding chapter. 2.7.4. Co-staining of acidic compartments and un-esterified cholesterol Cells grown on glass cover slips were stained for the acidic compartment with LysoTracker® as described above. After fixation and washings, un-esterified cholesterol was stained with filipin-complex (SigmaeAldrich) at a concentration of 50 mg/ml in PBS for 120 min at 4 C. After washings (PBS) the cover slips were mounted with anti-fade reagent (Vectorshield). Cells were analysed using a fluorescence-microscope (IX70, Olympus) attached to a Fura-imaging device (Till photonics). Pictures of single cells were taken with filter-setups: for LysoTracker® Red DND-99 excitation 545 nm, emission 595 nm and for filipin excitation 360 nm, emission 515 nm. Grey scale pictures of each channel were saved and further
Fig. 1. A: Effect of 3-MA on LC3B-II contents in NPC1/ neurons. Each data point represents the mean value in % relative to the corresponding NPC1/ control (MA ¼ 100%), ±SD, n ¼ 6 for treated and control cells and each time point. Note that treatments for 12 and 24 h lower the LC3B-II content in NPC1/ neurons significantly (*P < 0.05). The lowering effect is transient, after 36 h the treatment leads to a reincrease of LC3B-II in NPC1/ neurons which partially exceeds the level found in untreated control cells. B: Effect of pCD on LC3B-II contents in NPC1/ neurons. Each data point represents the mean value in % relative to the corresponding NPC1/ control (pCD ¼ 100%), ±SD, n ¼ 6 for treated and control cells and each time point. Note that after treatments for 24 h the LC3B-II content in NPC1/ neurons decreases significantly. Prolonged exposures to cyclodextrin do not attenuate this effect (24, 48 h: *P < 0.05).
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LC3B-II contents significantly after 8 h. The reduction lasted for further 16 h. A prolonged incubation reversed the lowering effect of the drug and LC3B-II contents increased over the levels detected in untreated NPC1/ cells (Fig. 1A). The treatment with pCD was performed analogous to 3-MA in a continuous mode. Concentrations of 0.1% (w/v) were sufficient to reduce LC3B-II contents of cells after an exposure time of 24 h. In contrast to 3-MA the reduction under pCD was robust and persisted for several days (data for 48 h are shown in Fig. 1B). In a previous study we could show that a transient (4e6 h) application of pCD at
a higher dose (0.25% w/v), also lowered the contents of autophagic structures in NPC1-deficient neurons in a sustainable manner (for at least 48 h) (Meske et al., 2014). 3.2. Effect of 3-MA and pCD on the content and the distribution of autophagic vacuoles Using lysotracker-staining in combination with indirect immunofluorescence staining of LC3B we analysed the total areas and the distribution of autophagosomes in single NPC1/ neurons under
Fig. 2. Co-staining of the acidic compartment and LC3B-positive structures and quantitative fluorometric analysis of the sub-cellular distribution of LC3B-punctae in NPC1/ neurons (un-)treated with MA. A: Each sequence of micrographs show a representative cell of the respective group with pictures from its acidic compartment (red channel), its autophagic structures (green channel) and a merged picture indicating co-localisation of the former signals (yellow). The charts below the micrographs show the corresponding areas of signals which were used for further statistical analysis. B: The diagram shows the calculated mean area/neuron (square pixels) of total LC3B-positive punctae in NPC1deficient neurons. Bars represent mean-values, ±SEM, n ¼ 85 (MA), n ¼ 47 (þMA: 18 h), n ¼ 42 (þMA: 36 h). Note that the amount of LC3B-positive vesicles/cell is significantly reduced after 18 h (**P < 0.01). After 36 h the number of autophagic vacuoles/neuron re-increases to a level detected in control cells. C: The diagrams show the mean area of LC3B-positive vesicles (square pixels)/neuron within the cytosolic and the acidic compartment of NPC1/ treated for 18 and 36 h þ/MA. Bars represent mean-values, ±SEM, n ¼ 52 (MA: 18 h), n ¼ 47 (þMA: 18 h), n ¼ 33 (MA: 36 h) n ¼ 42 (þMA: 36 h). Note that after 18 h the number of LC3B-vesicles is significantly reduced in both compartments (**P < 0.01, *P < 0.05). After 36 h the amount of LC3B-positive vesicles in the cytosol exceeds significantly the number found in untreated cells (**P < 0.01), whereas the number of vesicles co-localized with the acidic compartment is strongly reduced (**P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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the influence of 3-MA and pCD. We could distinguish between two groups of LC3B-punctae considered to be autophagic vacuoles. One was located in the cytosolic compartment, presumably newly formed autophagosomes, and the other one was co-localized with the acidic compartment, most likely autolysosomes. Under the influence of 3-MA the area of total LC3B-punctae/ neuron initially (18 h) dropped to 50 percent of control (Fig. 2A, B). Prolonged treatments led to a re-increase, so that after 36 h the measured areas of LC3B-punctae were similar in treated and untreated cells (Fig. 2A, B). So far the observed effects confirm the results obtained by the westernblots. When the intracellular distribution of autophagic structures was specified, we detected that after 18 h the area of LC3B-positive vacuoles were significantly
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reduced in both the cytosolic and the lysosomal compartment (Fig. 2C). After 36 h the distribution was completely different. In most cells (>80%), we measured high numbers of free cytosolic autophagosomes, which significantly exceeded the values obtained from untreated NPC1/ neurons (approx 2.5 fold). Furthermore we detected that the acidic compartment was massively reduced in these neurons, so that only few (<20% of control, P < 0.01) LC3Bpunctea were found to be associated with acidic structures (Fig. 2C). Under the influence of pCD the areas of total LC3B-punctae/ neuron were significantly reduced after 24 h and 48 h (Fig. 3A, B) which is in accordance with our western blot-data. The quantitative analysis of the intracellular distribution revealed that at both time points only the part of autophagosomes which co-localized with
Fig. 3. Co-staining of the acidic compartment and LC3B-positive structures and quantitative fluorometric analysis of the sub-cellular distribution of LC3B-punctae in NPC1/ neurons (un-)treated with pCD. A: Each sequence of micrographs show a representative cell of the respective group with pictures from its acidic compartment (red channel), its autophagic structures (green channel) and a merged picture indicating co-localisation of the former signals (yellow). The charts below the micrographs show the corresponding areas of signals which were used for further statistical analysis. B: The diagram shows the calculated mean area/neuron of total LC3B-positive punctae in NPC1-deficient neurons. Bars represent mean-values, ±SEM, n ¼ 79 (pCD), n ¼ 53 (þpCD: 24 h), n ¼ 57 (þpCD: 48 h). Note that the amount of LC3B-positive vesicles/cell is significantly reduced after 24 and 48 h (**P < 0.01). C: The diagram shows the contents of LC3B-positive vesicles (square pixels) within the cytosolic and the acidic compartment of NPC1/ neurons and NPC1/ neurons treated þ/CD for 24 and 48 h. Bars represent mean-values, ±SEM, n ¼ 38 (pCD: 24 h), n ¼ 53 (þpCD: 24 h), n ¼ 41 (pCD: 48 h), n ¼ 57 (þpCD: 48 h). The treatment of NPC1/ neurons with pCD reduces the number of LC3B-vesicles which are co-localized with late endosomes/lysosomes significantly (***P < 0.005) at both time points. The amount of cytosolic LC3B-vesicles remains largely unchanged. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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the acidic compartment was significantly reduced, whereas the area/number of LC3B-positive vacuoles located in the cytosol remained largely unchanged (Fig. 3C).
3.3. Effect of treatments on the acidic compartment
the loss of accumulated lysotracker/cell was significantly more pronounced than the loss of lamp2-positive organelles/cell in MAtreated neurons (Fig. 4B). This observation indicates that the MAinduced damages on lytic structures may begin with a collapse of the inherent proton gradient. In contrast to MA, the treatment with pCD had minor effects on the lytic compartment of neurons (Fig. 4A, B).
In order to examine the effects of 3-MA and pCD on the lytic compartment in detail, we loaded differentially treated neurons with lysotracker, followed by a fixation and co-staining of lamp2 in respective cells. The analysis of the stained area/neuron proved that treatments with the PI3K-inhibitor for 36 h and longer strongly reduced the accumulation of lysotracker (80% reduction) (Fig 4A). Also the lamp2-staining of acidic structures in cells was significantly lowered by 3-MA treatment (30% reduction) (Fig 4A). Comparing the absolute area of the stains per neuron showed that
Fig. 4. Effect of 3-MA and pCD on the acidic compartment of NPC1/ neurons costained with lysotracker and lamp2. A: Each data-point represents the mean area stained per cell in % relative to the corresponding NPC1/ control (MA, pCD ¼ 100%) ± SEM, n ¼ 35 (MA/pCD: 36 h), n ¼ 39 (þMA: 36 h); n ¼ 40 (þpCD: 36 h). Note that the treatment with MA for 36 h strongly reduces (**P < 0.01) the accumulation of lysotracker inside organelles. The reduction of lamp2-staining is less pronounced but also significant (*P < 0.05). pCD (36 h) causes no significant effects. B: Data of Fig. 4A depicted in mean values of the absolute area per cell for each stain. Note that only MA-treatment (36 h) leads to a discrepancy between the two stains. The mean value of the lysotracker-stained area is significant (*P < 0.05) lower than the value of the corresponding lamp2-positive area.
Fig. 5. Effect of 3-MA and pCD on filipin-stained cholesterol in the acidic compartment in NPC1/ neurons. Micrographs represent false-colour encoded signal intensities of filipin and lysotracker co-staining in a representative group of cells. The first row: cells MA; pCD, the second row: cells þMA for 36 h; the third row: cells þpCD for 36 h. The diagram shows the calculated relative fluorescence (intensity area)/cell expressed in % of the corresponding control cells (neurons: pCD, MA ¼ 100%), ±SD, n ¼ 86 (MA, pCD: 36 h), n ¼ 49 (þpCD: 36 h); n ¼ 64 (þMA: 36 h). MA-treatment reduced both the lysotracker- and the cholesterol-stain significantly in lysosomes of most cells (closed arrowheads) (*P < 0.05). Note that some cells (<20%) still contained lysotracker-positive lysosomes (open arrowhead). These lysosomes always contained high cholesterol loads (strong staining with filipin). pCD-treatment reduced only the cholesterol content in lysosomes (**P < 0.01) but not their ability to accumulate lysotracker. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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3.4. Effect of treatments on lysosomal cholesterol accumulations We examined the lipid-lowering potentials of the compounds. As expected all lysosomes/late endosomes in untreated NPC1deficient neurons were strongly filled with cholesterol. Treatments with 3-MA for 36 h significantly reduced both the cholesterol- and the lysotracker-staining in most of the cells. A minor part of the neurons remained unaffected (<20%) showing staining patterns and intensities like untreated NPC1-deficient control cells. Treatments with pCD (36 h) reduced lysosomal cholesterol in NPC1-deficient neurons without impairing the accumulation of the lysotracker within these organelles (Fig. 5). 3.5. Effect of treatments on neuronal survival Since long-term treatments with 3-MA impair the integrity of the lytic compartment, we wanted to clarify whether survival of
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cells was also affected. We observed that treatments with 3-MA significantly reduced neuronal survival after 48 h. Viability stains revealed typical changes in the cell morphological preceding the cell death, like shrunken somata and neuritic swellings. Long term treatments with pCD had no effects, neither on survival nor on cell morphology (Fig. 6). 4. Discussion Inhibiting the formation or forcing the clearance of autophagosomes, both methods lower the number of autophagic vacuoles in NPC1-deficient neurons. In order to inhibit the formation of autophagosomes which ceases the autophagic flux we used 3methyladenin (3-MA), a commonly used compound in this context. 3-MA belongs to a group of PI kinase inhibitors like wortmannin, LY 294002 and exerts its specific effects on autophagy by the inhibition of class III PI kinase activity (PI3K) (Seglen and
Fig. 6. Effect of 3-MA and pCD on survival of NPC1/ neurons. Micrographs show cells incubated with calcein-AM (live stain) after treatments with the compounds (pCD, MA) for the indicated periods of time. The diagram depicts survival of neurons relative to the untreated control cells (¼100%). Survival was determined with MTT-assays, n ¼ 5 for each time point and treatment (MA, pCD; þMA; þpCD). Note that MA treatment reduces survival of neurons significantly after 48 h (***P < 0.005), whereas pCD has no negative effects on survival.
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Gordon, 1982; Petiot et al., 2000; Huang and Sinicrope, 2010). The PI3K/hVps34/Beclin1 complex phosphorylates PI to generate phosphatidylinositol 3-phosphate (PI3P) which is essential for the initiation of autophagy via recruitment of other ATG proteins at the isolation membrane (Yang and Klionsky, 2010). The disadvantage of PI-kinase inhibitors is that they are not absolutely specific for one class of PI kinases. In the concentrationranges used, they also inhibit the class I PI kinase which leads to an inactivation of mTor raptor (Meske et al., 2008) that in turn induces autophagy mediated by ULK1, a core complex essential for the formation of the phagophore. We observed that the suppressing effect of 3-MA on autophagy was only transient in neurons. This result is in accordance with data coming from a study on mouse fibroblasts (Wu et al., 2010). The authors also noticed that a prolonged incubation with the inhibitor reversed its suppressing effect on autophagy (after 9 h). The authors attributed the subsequent promoting activity to differential temporal effects of 3-MA on class I and class III PI3K. We observed that 3-MA influenced the integrity of the lysosomes. Usually lysotracker rapidly accumulates in the acidic compartment by ion-trap mechanism. A long-lasting treatment of neurons with 3-MA impeded this mechanism. This indicates that the lysosomal pH-gradient was destroyed. A similar effect of 3-MA on lysosomal pH in cultured hepatocytes has been described by Caro et al. (1988). In this context we found that disease-specific lysosomal cholesterol accumulations also vanished after prolonged MA-treatments. A similar effect was observed in NPC1-deficient fibroblasts (Elrick et al., 2012). The authors proposed that autophagy is a major source for the entrapped cholesterol in lysosomes and that the inhibition of autophagy with 3-MA reduces the lipid-ballast inside the lytic compartment. We observed that reduced cholesterol-staining of lysosomes was strictly coupled with the loss of lysotracker accumulations. We think that this indicates that the integrity of lysosomal membrane is compromised by long-lasting 3-MA treatments, leading to both: the release of lysosomal contents and to the destruction of the pHgradient. In this context Madge et al. (2003) showed that the inhibition of PI3K in human vascular endothelial cells induces the release of cathepsin B to the cytosol arguing for a direct role of PI3K in preserving lysosomal membrane integrity. The acidic pH inside the lysosomes is the basis for maturation and clearance of autophagosomes (Yamamoto et al., 1998; Klionsky et al., 2008). We conclude that the loss of the acidic environment inside the lysosomes prevents maturation/clearance of autophagosomes and contributes considerably to their accumulation in the cytosol observed after prolonged incubations with the PI3Kinhibitor. Although we can not exclude that an attenuated inhibition of the class III PI3K also contributes to this process as proposed by Wu et al. (2010). Furthermore, there is indication that PI3K-inhibitors sensitize cells to mitochondrion-independent lysosomal death pathways €a €ttela €, 2009). This may (Madge et al., 2003; Kirkegaard and Ja explain the observed chronological order of lysosomal damage and cell death elucidated by 3-MA in our study. Side-effects of the chosen inhibitor may not be the only problem arising with this kind of pharmacological intervention because neuronal cytoarchitecture and survival depend on a functional autophagic flux. A prolonged disturbance at any site of this complex pathway causes axonal dystrophy and eventually cell death (Bi et al., 1999; Hara et al., 2008; Komatsu et al., 2006; Lee et al., 2011). b-cyclodextrins reduce the number of autophagosomes and autolysosomes by accelerating the delayed execution of autophagy in NPC1-deficient cells (Ordonez et al., 2012; Meske et al., 2014). These compounds mobilize endosomal/lysosomal entrapped cholesterol (Abi-Mosleh et al., 2009; Rosenbaum et al., 2010; Peake and Vance, 2012; Meske et al., 2014) which improves the clearance
rate of autophagosomes (Elrick et al., 2012) and fully reconstitutes the autophagic flux in NPC1-deficient neurons (Meske et al., 2014). However, the concentration of b-CD used for extraction of lipids from NPC1-deficient cells seems to be a critical point which influences both autophagy and cell survival differently. Low concentrations (0.1e1 mM) restrict the depletion primarily to the lytic compartment and restore the autophagic flux to “normal” levels (Ordonez et al., 2012; Meske et al., 2014). High doses of b-CD (>5 mM), deplete cholesterol from the plasma membrane and induce autophagy in cells, probably via a class I PI3K-dependent inactivation of Akt and downstream mTor raptor (Cheng et al., 2006). High doses of b-CD, in contrast to low doses, were found to be potentially cytotoxic to a variety of brain cells (Peake and Vance, 2012). In the search for alternative methods to lower the cholesterolload in NPC1-deficient cells it was found that statins worsen the autophagic defect. The inhibition of endogenous isoprenoid synthesis rather activates the autophagy machinery in NPC1-deficient cells, leading to a further increase of the autophagosomal ballast (Cheng et al., 2006; Ishibashi et al., 2009). 5. Conclusions Our study clearly demonstrates that pharmacological treatments to reduce/prevent the accumulation of autophagic vacuoles in NPC1-deficient neurons should concentrate on compounds that restore the disturbed flux in mutated cells. Although an interruption of autophagy also leads to a (transient) decrease in the number of autophagic structures, such a treatment runs the risk of irreversible cell damages caused either by side effects of the inhibitor or by the long-lasting suppression of autophagy. The use of cyclic polysaccharides to repair the defect in the execution of autophagy is reasonable all the more because cyclodextrin derivates were already shown to alleviate clinical symptoms of the NPC-disease in animal studies (Liu et al., 2009; Ramirez et al., 2010; Aqul et al., 2011) and increased the life-span of afflicted animals (Liu et al., 2008, 2010). Acknowledgements This work contains parts of the doctoral thesis of Timm Priesnitz. This work has been supported in part by the Deutsche Forschungsgemeinschaft (ME 1494/2-1). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuropharm.2014.10.006. References Abi-Mosleh, L., Infante, R.E., Radhakrishnan, A., Goldstein, J.L., Brown, M.S., 2009. Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells. Proc. Natl. Acad. Sci. U. S. A. 106, 19316e19321. Aqul, A., Liu, B., Ramirez, C.M., Pieper, A.A., Estill, S.J., Burns, D.K., Repa, J.J., Turley, S.D., Dietschy, J.M., 2011. Unesterified cholesterol accumulation in late endosomes/lysosomes causes neurodegeneration and is prevented by driving cholesterol export from this compartment. J. Neurosci. 31, 9404e9413. Bi, X., Zhou, J., Lynch, G., 1999. Lysosomal protease inhibitors induce meganeurites and tangle-like structures in entorhinohippocampal regions vulnerable to Alzheimer's disease. Exp. Neurol. 158, 312e327. Caro, L.H.P., Plomp, P.J.A.M., Wolvetang, E.J., Kerkhof, C., Meijer, A.J., 1988. 3Methyladenine, an inhibitor of autophagy, has multiple effects on metabolism. Eur. J. Biochem. 175, 325e329. Carstea, E.D., Morris, J.A., Coleman, K.G., Loftus, S.K., Zhang, D., Cummings, C., et al., 1997. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277, 228e231.
V. Meske et al. / Neuropharmacology 89 (2015) 282e289 Cheng, J., Ohsaki, Y., Tauchi-Sato, K., Fujita, A., Fujimoto, T., 2006. Cholesterol depletion induces autophagy. Biochem. Biophys. Res. Commun. 351, 246e252. Deisz, R.A., Meske, V., Treiber-Held, S., Albert, F., Ohm, T.G., 2005. Pathological cholesterol metabolism fails to modify electrophysiological properties of afflicted neurones in Niemann-Pick disease type C. Neurscience 130, 867e873. Elrick, M.J., Yu, T., Chung, C., Lieberman, A.P., 2012. Impaired proteolysis underlies autophagic dysfunction in Niemann-Pick type C disease. Hum. Mol. Genet. 21, 4876e4887. Hara, T., Takamura, A., Kishi, C., Iemura, S., Natsume, T., Guan, J.L., et al., 2008. FIP200, a ULK interacting protein, is required for autophagosome formation in mammalian cells. J. Cell Biol. 181, 497e510. Huang, S., Sinicrope, F.A., 2010. Celecoxib-induced apoptosis is enhanced by ABT737 and by inhibition of autophagy in human colorectal cancer cells. Autophagy 6, 256e269. Ishibashi, S., Yamazaki, T., Okamoto, K., 2009. Association of autophagy with cholesterol-accumulated compartments in Niemann-Pick disease type C cells. J. Clin. Neurosci. 16, 954e959. €ttela €, M., 2009. Lysosomal involvement in cell death and cancer. Kirkegaard, T., J€ aa Biochim. Biophys. Acta 1793, 746e754. Klionsky, D.J., Zvulun Elazar, Z., Seglen, P.O., Rubinsztein, D.C., 2008. Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy 4, 849e850. Ko, D.C., Milenkovic, L., Beier, S.M., Manuel, H., Buchanan, J., Scott, M.P., 2005. Cellautonomous death of cerebellar purkinje neurons with autophagy in NiemannPick type C disease. PLoS Genet. 1, 81e95. Komatsu, M., Waguri, S., Chiba, T., Murata, S., Iwata, J., Tanida, I., Ueno, T., Koike, M., Uchiyama, Y., Kominami, E., Tanaka, K., 2006. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 441, 880e884. Lee, S., Sato, Y., Nixon, R.A., 2011. Primary lysosomal dysfunction causes cargospecific deficits of axonal transport leading to Alzheimer-like neuritic dystrophy. Autophagy 7, 1562e1563. Liao, G., Yao, Y., Liu, J., Yu, Z., Cheung, S., Xie, A., et al., 2007. Cholesterol accumulation is associated with lysosomal dysfunction and autophagic stress in Npc1/ mouse brain. Am. J. Pathol. 171, 962e975. Liu, B., Li, H., Repa, J.J., Turley, S.D., Dietschy, J.M., 2008. Genetic variations and treatments that affect the lifespan of the NPC1 mouse. J Lipid Res. 49, 663e669. Liu, B., Turley, S.D., Burns, D.K., Miller, A.M., Repa, J.J., Dietschy, J.M., 2009. Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1/ mouse. Proc. Natl. Acad. Sci. U. S. A. 106, 2377e2382. Liu, B., Ramirez, C.M., Miller, A.M., Repa, J.J., Turley, S.D., Dietschy, J.M., 2010. Cyclodextrin overcomes the transport defect in nearly every organ of NPC1 mice leading to excretion of sequestered cholesterol as bile acid. J Lipid Res. 51, 933e944. Madge, L.A., Li, J.H., Choi, J., Pober, J.S., 2003. Inhibition of phosphatidylinositol 3kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsindependent apoptosis. J. Biol. Chem. 278, 21295e21306.
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Meske, V., Albert, F., Ohm, T.G., 2008. Coupling of mammalian target of rapamycin with phosphoinositide 3-kinase signaling pathway regulates protein phosphatase 2A- and glycogen synthase kinase-3 -dependent phosphorylation of Tau. J. Biol. Chem. 283, 100e109. Meske, V., Erz, J., Priesnitz, T., Ohm, T.G., 2014. The autophagic defect in NiemannPick disease type C neurons differs from somatic cells and reduces neuronal viability. Neurobiol. Dis. 64, 88e97. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55e63. Naureckiene, S., Sleat, D.E., Lackland, H., Fensom, A., Vanier, M.T., Wattiaux, R., et al., 2000. Identification of HE1 as the second gene of Niemann-Pick C disease. Science 290, 2298e2301. Ordonez, M.P., Roberts, E.A., Kidwell, C.U., Yuan, S.H., Plaisted, W.C., Goldstein, L.S.B., 2012. Disruption and therapeutic rescue of autophagy in a human neuronal model of Niemann Pick type C1. Hum. Mol. Genet. 21, 2651e2662. Pacheco, C.D., Kunkel, R., Lieberman, A.P., 2007. Autophagy in Niemann-Pick C disease is dependent upon Beclin-1 and responsive to lipid trafficking defects. Hum. Mol. Genet. 16, 1495e1503. Pacheco, C.D., Lieberman, A.P., 2007. Lipid trafficking defects increase Beclin-1 and activate autophagy in Niemann-Pick Type C disease. Autophagy 3, 487e489. Peake, K.B., Vance, J.E., 2012. Normalization of cholesterol homeostasis by 2hydroxypropyl-beta-cyclodextrin in neurons and glia from Niemann-Pick C1 (NPC1)-deficient mice. J. Biol. Chem. 287, 9290e9298. Petiot, A., Ogier-Denis, E., Blommaart, E.F., Meijer, A.J., Codogno, P., 2000. Distinct classes of phosphatidylinositol 30 -kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J. Biol. Chem. 275, 992e998. Ramirez, C.M., Liu, B., Taylor, A.M., Repa, J.J., Burns, D.K., Weinberg, A.G., Turley, S.D., Dietschy, J.M., 2010. Weekly cyclodextrin administration normalizes cholesterol metabolism in nearly every organ of the Niemann-pick type C1 mouse and markedly prolongs life. Pediatr. Res. 68, 309e315. Rosenbaum, A.I., Zhang, G., Warren, J.D., Maxfield, F.R., 2010. Endocytosis of betacyclodextrins is responsible for cholesterol reduction in Niemann-Pick type C mutant cells. Proc. Natl. Acad. Sci. U. S. A. 107, 5477e5482. Seglen, P.O., Gordon, P.B., 1982. 3-Methyladenine: specific inhibitor of autophagic/ lysosomal protein degradation in isolated rat hepatocytes. Proc. Natl. Acad. Sci. U. S. A. 79, 1889e1892. Wu, Y.T., Tan, H.L., Shui, G., Bauvy, C., Huang, Q., Wenk, M.R., Ong, C.N., Codogno, P., Shen, H.M., 2010. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3kinase. J. Biol. Chem. 285, 10850e10861. Yamamoto, A., Tagawa, Y., Yoshimori, T., Moriyama, Y., Masaki, R., Tashiro, Y., 1998. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct. Funct. 23, 33e42. Yang, Z., Klionsky, D.J., 2010. Mammalian autophagy: core molecular machinery and signaling regulation. Curr. Opin. Cell Biol. 22, 124e131.
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
How to reduce the accumulation of autophagic vacuoles in NPC1deficient neurons: A comparison of two pharmacological strategies Volker Meske*, Timm Priesnitz, Frank Albert, Thomas Georg Ohm Center of Anatomy, Institute of Integrative Neuroanatomy, Department of Clinical Cell- and Neurobiology, Charit e, Charit e-Platz 1, 10098 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2014 Received in revised form 3 September 2014 Accepted 6 October 2014 Available online 16 October 2014
A disturbed autophagic pathway leads to chronically increased levels of autophagic vacuoles in Niemann Pick Type-C 1 (NPC1) deficient neurons. Since these accumulations potentially contribute to neuronal cell death associated with the disease, we investigated two pharmacological strategies which potentially reduce the number of autophagic structures under following aspects: efficiency, sustainability and effect on neuronal cell viability. The strategies comprised (i) an interruption of the autophagic flux by the class III PI3K inhibitor 3-methyladenine (3-MA) and (ii) an acceleration of the autophagic execution by 2hydroxypropyl-b-cyclodextrin (pCD). Our data show that the inhibition of autophagy with 3-MA only initially reduced the number of autophagic vacuoles in cultured neurons. Prolonged treatments with the PI3K-inhibitor reversed this lowering effect. The re-increase in the number of autophagic vacuoles was combined with a defect in the integrity of lysosomes which endangered further survival of cells. The treatment with pCD evoked a slow but sustained reduction of autophagic structures and had no negative effects on neuronal survival. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Neurons Niemann-Pick type C disease Autophagy Cyclodextrin PI3K-inhibitor
1. Introduction NPC is a lipid storage disease with defects either in the NPC1- or NPC2- protein (Carstea et al., 1997; Naureckiene et al., 2000). Cholesterol and glycosphingolipids accumulate in the late endosomal/lysosomal compartment of NPC1 and/or NPC2-deficient cells as a consequence from a disrupted lipid transport. Several studies reported that the autophagic flux in cells is chronically disturbed leading to an abnormal intracellular accumulation of autophagic vacuoles in NPC1-deficient cells (Liao et al., 2007; Pacheco et al., 2007; Pacheco and Lieberman, 2007; Ishibashi et al., 2009; Elrick et al., 2012; Ordonez et al., 2012; Meske et al., 2014). Some authors suggested that this defect in autophagy may contribute to neurodegeneration in NPC-disease (Ko et al., 2005; Liao et al., 2007). In a recent study with primary neuron cultures we could prove this hypothesis and demonstrated that the reduction of the abnormal lipid load inside the acidic organelles with b-hydroxypropyl cyclodextrin (pCD) is sufficient to correct the abnormal
autophagic flux and prevents NPC1/ neurons from premature death under autophagic stress (Meske et al., 2014). Nevertheless, it has been shown that not only restoration of the defective lysosomal clearance by treatments with cyclodextrin (Ordonez et al., 2012; Meske et al., 2014), but also the inhibition of the autophagosomeformation with PI3K class III inhibitors like 3-methyladenine (3MA) reduces the number of autophagic vacuoles in NPC1deficient cells (Pacheco et al., 2007; Elrick et al., 2012; Ordonez et al., 2012). In the present study we compare both strategies in primary cultures of NPC1-deficient neurons for their efficiency, sustainability and effects on cellular survival. Our results indicate that therapeutic treatments to improve the autophagic state in NPC1-deficient neurons should concentrate on agents which accelerate the execution of autophagy in cells instead on substances which impede autophagy.
2. Material and methods 2.1. Primary neuronal cell culture
Abbreviations: CD, cyclodextrin; pCD, 2-hydroxy-propyl-b-cyclodextrin; LC3, microtubule-associated protein 1 light chain 3; LC3-I, phosphatidylethanolaminedeconjugated LC3; LC3-II, phosphatidylethanolamine-conjugated LC3; (3)-MA, (3)methyladenine; NPC, Niemann-Pick type C. * Corresponding author. Tel.: þ49 (0)30 450 528257; fax: þ49 (0)30 450 528913. E-mail address: [email protected] (V. Meske). http://dx.doi.org/10.1016/j.neuropharm.2014.10.006 0028-3908/© 2014 Elsevier Ltd. All rights reserved.
Neuron cultures were prepared from cortices of embryos (E16) derived from mating of npc1nihBalb/cJ mice (Jackson Laboratories) heterozygous for NPC1 (NPC1þ/ ) as described earlier (Deisz et al., 2005). Breeding and handling of the animals were performed according to the German law of animal care (Tierschutzgesetz) which is in accordance with EU directive 2010/63/EU. Cells with NPC1/ and NPC1þ/þ (wildtype ¼ wt) genotypes were cultured as described earlier (Meske et al., 2008). For experiments 10e14 days old cultures were used.
V. Meske et al. / Neuropharmacology 89 (2015) 282e289 2.2. Treatments of cell cultures 3-methyladenine (SigmaeAldrich): was dissolved in culture medium to a working-concentration of 3 mM. 2-hydroxypropyl-b-cyclodextrin (SigmaeAldrich): a stock solution of 10% (w/v) in culture-medium was further diluted with medium to 0.1% (w/v). 2.3. Neuronal viability We assessed neuronal survival with MTT (methyl-thiazol-tetrazolium [SigmaeAldrich])-assay as described by Mosmann (1983). The colour intensities were measured with an ELISA reader (Kontron) at 580 nm. Data were analysed with EXCEL (Microsoft). 2.4. Sample preparation for protein analysis Samples were prepared as described previously (Meske et al., 2008). Extraction buffer: PBS (phosphate buffered saline) pH 7.4, protease-inhibitor cocktail (Roche), 1 mM activated sodium ortho-vanadate (SigmaeAldrich), 20 mM sodium fluoride (SigmaeAldrich) and 100 nM okadaic acid (SigmaeAldrich) from a 100 mM stock solution in DMSO (dimethylsulfoxide), 2% (v/v) Triton-X100. 2.5. SDS-PAGE/western blot SDS-PAGE (10 and 15% PAGE), blotting-procedure and densitometric analysis of the blots followed a protocol described earlier (Meske et al., 2008). 2.6. Antibodies used for analysis LC3B-antibody was purchased from Cell-Signalling-Technology, LC3B (#2775). As secondary we used horseradish peroxidase e conjugated anti-rabbit IgG (Vector). 2.7. Stainings 2.7.1. General cell staining For staining viable cells we used cells grown on glass cover slips and applied 1 mM Calcein-AM (Molecular probes) to the culture medium for 30 min. Afterwards cells were transferred into a measuring chamber filled with Hanks-balanced-saltsolution, ([with Ca2þ/Mg2,Gibco], supplemented with Hepes 10 mM, pH 7.4). For visualization we used a fluorescence-microscope (IX70, Olympus) attached to an imaging device (Till photonics).
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processed in Adobe Photoshop®. The analysis of areas (filipin-positive vacuoles and acidic compartment) was performed with the software Image J. The results were saved as EXCEL-sheets for further statistical analysis. 2.8. Statistics We used program SPSS for statistical data analysis. Significance was assessed by parametric Student's t-test or non-parametric Whitman-test, when two means were compared. For the comparison of more than two means ANOVA with a subsequent post hoc test (NewmaneKeuls) was performed.
3. Results 3.1. Effect of 3-MA and pCD on LC3BII-contents measured by westernblots In order to reduce the abnormal high number of autophagic vacuoles in cells we tested two strategies. First, we used the PI3K inhibitor 3-methyladenine (3-MA) to interrupt the formation of autophagosomes and second, we used 2-OH-propyl-b-cyclodextrin (pCD) to accelerate the lysosomal clearance of autophagosomes. Westernblot analysis showed that both treatments reduced the LC3B-II contents in NPC1-deficient neurons (Fig. 1A, B) over time. As control we used actin contents, or calculated the ratio of LC3B-I/ LC3BII (see Supplement Figs. S1 and S2). Considerable differences between both treatments occurred when the incubation was extended beyond the time required for a significant reduction. In detail: the treatment with 3-MA required a continuous application of the agent at a relatively high concentration (3 mM) and reduced
2.7.2. Co-staining of the acidic compartment and LC3B Staining of the acidic compartment with LysoTracker (Molecular Probes) and immunofluorescent co-staining of LC3B (antibody #2775, Cell-Signalling) was performed as described earlier (Meske et al., 2014). For analysis of the staining, we used a laser-scan microscope LSM5 (exciter: helium/neon- and argon-laser) attached to an AX10 microscope equipped with a 63 oil immersion objective and control software Zen 2008 (Zeiss). Pictures of the acidic compartment: red channel (ex: 546 nm; em: longpass filter >580 nm), of the LC3B positive vesicles: green-channel (ex: 488 nm; em: pass filter 505e530 nm) and of transmission-light were analysed in Image J (NIH) and Adobe Photoshop®. Using the software Adobe Photoshop, pictures from the same field of view were combined in one picture with three image planes, single cells were delineated, cut out and analysed separately. Areas of colocalization were displayed with the submenu channel-calculation/multiply signals (redegreen channels). Using the software Image J we measured the area of signals in the three related pictures (acidic compartment, LC3B-positive vesicles, colocalized areas). Since all pictures analysed in this study had the same magnification scale (1200), areas were expressed in square pixels. Subtraction of the areas of colocalized punctae from areas of total LC3B punctae/neuron resulted in the number of cytosolic free autophagosomes/neuron. The results were saved as EXCEL-sheets for further statistical analysis. 2.7.3. Co-staining of the acidic compartment and lamp2 The co-staining of neurons with LysoTracker and lamp2 followed a protocol described earlier (Meske et al., 2014), with one exception we permeabilized cells with 0.1% (w/v) saponin (Sigma) after fixation and omitted Triton-X in the washing/ incubation buffers. We used anti-lamp2 antibody (Sressgen) diluted 1: 250 in TBS and as secondary antibody Alexa-488 goat anti-rat IgG (Molecular Probes) diluted 1:500 in TBS. For documentation of the staining, we used the same equipment as described in the preceding chapter. 2.7.4. Co-staining of acidic compartments and un-esterified cholesterol Cells grown on glass cover slips were stained for the acidic compartment with LysoTracker® as described above. After fixation and washings, un-esterified cholesterol was stained with filipin-complex (SigmaeAldrich) at a concentration of 50 mg/ml in PBS for 120 min at 4 C. After washings (PBS) the cover slips were mounted with anti-fade reagent (Vectorshield). Cells were analysed using a fluorescence-microscope (IX70, Olympus) attached to a Fura-imaging device (Till photonics). Pictures of single cells were taken with filter-setups: for LysoTracker® Red DND-99 excitation 545 nm, emission 595 nm and for filipin excitation 360 nm, emission 515 nm. Grey scale pictures of each channel were saved and further
Fig. 1. A: Effect of 3-MA on LC3B-II contents in NPC1/ neurons. Each data point represents the mean value in % relative to the corresponding NPC1/ control (MA ¼ 100%), ±SD, n ¼ 6 for treated and control cells and each time point. Note that treatments for 12 and 24 h lower the LC3B-II content in NPC1/ neurons significantly (*P < 0.05). The lowering effect is transient, after 36 h the treatment leads to a reincrease of LC3B-II in NPC1/ neurons which partially exceeds the level found in untreated control cells. B: Effect of pCD on LC3B-II contents in NPC1/ neurons. Each data point represents the mean value in % relative to the corresponding NPC1/ control (pCD ¼ 100%), ±SD, n ¼ 6 for treated and control cells and each time point. Note that after treatments for 24 h the LC3B-II content in NPC1/ neurons decreases significantly. Prolonged exposures to cyclodextrin do not attenuate this effect (24, 48 h: *P < 0.05).
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LC3B-II contents significantly after 8 h. The reduction lasted for further 16 h. A prolonged incubation reversed the lowering effect of the drug and LC3B-II contents increased over the levels detected in untreated NPC1/ cells (Fig. 1A). The treatment with pCD was performed analogous to 3-MA in a continuous mode. Concentrations of 0.1% (w/v) were sufficient to reduce LC3B-II contents of cells after an exposure time of 24 h. In contrast to 3-MA the reduction under pCD was robust and persisted for several days (data for 48 h are shown in Fig. 1B). In a previous study we could show that a transient (4e6 h) application of pCD at
a higher dose (0.25% w/v), also lowered the contents of autophagic structures in NPC1-deficient neurons in a sustainable manner (for at least 48 h) (Meske et al., 2014). 3.2. Effect of 3-MA and pCD on the content and the distribution of autophagic vacuoles Using lysotracker-staining in combination with indirect immunofluorescence staining of LC3B we analysed the total areas and the distribution of autophagosomes in single NPC1/ neurons under
Fig. 2. Co-staining of the acidic compartment and LC3B-positive structures and quantitative fluorometric analysis of the sub-cellular distribution of LC3B-punctae in NPC1/ neurons (un-)treated with MA. A: Each sequence of micrographs show a representative cell of the respective group with pictures from its acidic compartment (red channel), its autophagic structures (green channel) and a merged picture indicating co-localisation of the former signals (yellow). The charts below the micrographs show the corresponding areas of signals which were used for further statistical analysis. B: The diagram shows the calculated mean area/neuron (square pixels) of total LC3B-positive punctae in NPC1deficient neurons. Bars represent mean-values, ±SEM, n ¼ 85 (MA), n ¼ 47 (þMA: 18 h), n ¼ 42 (þMA: 36 h). Note that the amount of LC3B-positive vesicles/cell is significantly reduced after 18 h (**P < 0.01). After 36 h the number of autophagic vacuoles/neuron re-increases to a level detected in control cells. C: The diagrams show the mean area of LC3B-positive vesicles (square pixels)/neuron within the cytosolic and the acidic compartment of NPC1/ treated for 18 and 36 h þ/MA. Bars represent mean-values, ±SEM, n ¼ 52 (MA: 18 h), n ¼ 47 (þMA: 18 h), n ¼ 33 (MA: 36 h) n ¼ 42 (þMA: 36 h). Note that after 18 h the number of LC3B-vesicles is significantly reduced in both compartments (**P < 0.01, *P < 0.05). After 36 h the amount of LC3B-positive vesicles in the cytosol exceeds significantly the number found in untreated cells (**P < 0.01), whereas the number of vesicles co-localized with the acidic compartment is strongly reduced (**P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
V. Meske et al. / Neuropharmacology 89 (2015) 282e289
the influence of 3-MA and pCD. We could distinguish between two groups of LC3B-punctae considered to be autophagic vacuoles. One was located in the cytosolic compartment, presumably newly formed autophagosomes, and the other one was co-localized with the acidic compartment, most likely autolysosomes. Under the influence of 3-MA the area of total LC3B-punctae/ neuron initially (18 h) dropped to 50 percent of control (Fig. 2A, B). Prolonged treatments led to a re-increase, so that after 36 h the measured areas of LC3B-punctae were similar in treated and untreated cells (Fig. 2A, B). So far the observed effects confirm the results obtained by the westernblots. When the intracellular distribution of autophagic structures was specified, we detected that after 18 h the area of LC3B-positive vacuoles were significantly
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reduced in both the cytosolic and the lysosomal compartment (Fig. 2C). After 36 h the distribution was completely different. In most cells (>80%), we measured high numbers of free cytosolic autophagosomes, which significantly exceeded the values obtained from untreated NPC1/ neurons (approx 2.5 fold). Furthermore we detected that the acidic compartment was massively reduced in these neurons, so that only few (<20% of control, P < 0.01) LC3Bpunctea were found to be associated with acidic structures (Fig. 2C). Under the influence of pCD the areas of total LC3B-punctae/ neuron were significantly reduced after 24 h and 48 h (Fig. 3A, B) which is in accordance with our western blot-data. The quantitative analysis of the intracellular distribution revealed that at both time points only the part of autophagosomes which co-localized with
Fig. 3. Co-staining of the acidic compartment and LC3B-positive structures and quantitative fluorometric analysis of the sub-cellular distribution of LC3B-punctae in NPC1/ neurons (un-)treated with pCD. A: Each sequence of micrographs show a representative cell of the respective group with pictures from its acidic compartment (red channel), its autophagic structures (green channel) and a merged picture indicating co-localisation of the former signals (yellow). The charts below the micrographs show the corresponding areas of signals which were used for further statistical analysis. B: The diagram shows the calculated mean area/neuron of total LC3B-positive punctae in NPC1-deficient neurons. Bars represent mean-values, ±SEM, n ¼ 79 (pCD), n ¼ 53 (þpCD: 24 h), n ¼ 57 (þpCD: 48 h). Note that the amount of LC3B-positive vesicles/cell is significantly reduced after 24 and 48 h (**P < 0.01). C: The diagram shows the contents of LC3B-positive vesicles (square pixels) within the cytosolic and the acidic compartment of NPC1/ neurons and NPC1/ neurons treated þ/CD for 24 and 48 h. Bars represent mean-values, ±SEM, n ¼ 38 (pCD: 24 h), n ¼ 53 (þpCD: 24 h), n ¼ 41 (pCD: 48 h), n ¼ 57 (þpCD: 48 h). The treatment of NPC1/ neurons with pCD reduces the number of LC3B-vesicles which are co-localized with late endosomes/lysosomes significantly (***P < 0.005) at both time points. The amount of cytosolic LC3B-vesicles remains largely unchanged. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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the acidic compartment was significantly reduced, whereas the area/number of LC3B-positive vacuoles located in the cytosol remained largely unchanged (Fig. 3C).
3.3. Effect of treatments on the acidic compartment
the loss of accumulated lysotracker/cell was significantly more pronounced than the loss of lamp2-positive organelles/cell in MAtreated neurons (Fig. 4B). This observation indicates that the MAinduced damages on lytic structures may begin with a collapse of the inherent proton gradient. In contrast to MA, the treatment with pCD had minor effects on the lytic compartment of neurons (Fig. 4A, B).
In order to examine the effects of 3-MA and pCD on the lytic compartment in detail, we loaded differentially treated neurons with lysotracker, followed by a fixation and co-staining of lamp2 in respective cells. The analysis of the stained area/neuron proved that treatments with the PI3K-inhibitor for 36 h and longer strongly reduced the accumulation of lysotracker (80% reduction) (Fig 4A). Also the lamp2-staining of acidic structures in cells was significantly lowered by 3-MA treatment (30% reduction) (Fig 4A). Comparing the absolute area of the stains per neuron showed that
Fig. 4. Effect of 3-MA and pCD on the acidic compartment of NPC1/ neurons costained with lysotracker and lamp2. A: Each data-point represents the mean area stained per cell in % relative to the corresponding NPC1/ control (MA, pCD ¼ 100%) ± SEM, n ¼ 35 (MA/pCD: 36 h), n ¼ 39 (þMA: 36 h); n ¼ 40 (þpCD: 36 h). Note that the treatment with MA for 36 h strongly reduces (**P < 0.01) the accumulation of lysotracker inside organelles. The reduction of lamp2-staining is less pronounced but also significant (*P < 0.05). pCD (36 h) causes no significant effects. B: Data of Fig. 4A depicted in mean values of the absolute area per cell for each stain. Note that only MA-treatment (36 h) leads to a discrepancy between the two stains. The mean value of the lysotracker-stained area is significant (*P < 0.05) lower than the value of the corresponding lamp2-positive area.
Fig. 5. Effect of 3-MA and pCD on filipin-stained cholesterol in the acidic compartment in NPC1/ neurons. Micrographs represent false-colour encoded signal intensities of filipin and lysotracker co-staining in a representative group of cells. The first row: cells MA; pCD, the second row: cells þMA for 36 h; the third row: cells þpCD for 36 h. The diagram shows the calculated relative fluorescence (intensity area)/cell expressed in % of the corresponding control cells (neurons: pCD, MA ¼ 100%), ±SD, n ¼ 86 (MA, pCD: 36 h), n ¼ 49 (þpCD: 36 h); n ¼ 64 (þMA: 36 h). MA-treatment reduced both the lysotracker- and the cholesterol-stain significantly in lysosomes of most cells (closed arrowheads) (*P < 0.05). Note that some cells (<20%) still contained lysotracker-positive lysosomes (open arrowhead). These lysosomes always contained high cholesterol loads (strong staining with filipin). pCD-treatment reduced only the cholesterol content in lysosomes (**P < 0.01) but not their ability to accumulate lysotracker. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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3.4. Effect of treatments on lysosomal cholesterol accumulations We examined the lipid-lowering potentials of the compounds. As expected all lysosomes/late endosomes in untreated NPC1deficient neurons were strongly filled with cholesterol. Treatments with 3-MA for 36 h significantly reduced both the cholesterol- and the lysotracker-staining in most of the cells. A minor part of the neurons remained unaffected (<20%) showing staining patterns and intensities like untreated NPC1-deficient control cells. Treatments with pCD (36 h) reduced lysosomal cholesterol in NPC1-deficient neurons without impairing the accumulation of the lysotracker within these organelles (Fig. 5). 3.5. Effect of treatments on neuronal survival Since long-term treatments with 3-MA impair the integrity of the lytic compartment, we wanted to clarify whether survival of
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cells was also affected. We observed that treatments with 3-MA significantly reduced neuronal survival after 48 h. Viability stains revealed typical changes in the cell morphological preceding the cell death, like shrunken somata and neuritic swellings. Long term treatments with pCD had no effects, neither on survival nor on cell morphology (Fig. 6). 4. Discussion Inhibiting the formation or forcing the clearance of autophagosomes, both methods lower the number of autophagic vacuoles in NPC1-deficient neurons. In order to inhibit the formation of autophagosomes which ceases the autophagic flux we used 3methyladenin (3-MA), a commonly used compound in this context. 3-MA belongs to a group of PI kinase inhibitors like wortmannin, LY 294002 and exerts its specific effects on autophagy by the inhibition of class III PI kinase activity (PI3K) (Seglen and
Fig. 6. Effect of 3-MA and pCD on survival of NPC1/ neurons. Micrographs show cells incubated with calcein-AM (live stain) after treatments with the compounds (pCD, MA) for the indicated periods of time. The diagram depicts survival of neurons relative to the untreated control cells (¼100%). Survival was determined with MTT-assays, n ¼ 5 for each time point and treatment (MA, pCD; þMA; þpCD). Note that MA treatment reduces survival of neurons significantly after 48 h (***P < 0.005), whereas pCD has no negative effects on survival.
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Gordon, 1982; Petiot et al., 2000; Huang and Sinicrope, 2010). The PI3K/hVps34/Beclin1 complex phosphorylates PI to generate phosphatidylinositol 3-phosphate (PI3P) which is essential for the initiation of autophagy via recruitment of other ATG proteins at the isolation membrane (Yang and Klionsky, 2010). The disadvantage of PI-kinase inhibitors is that they are not absolutely specific for one class of PI kinases. In the concentrationranges used, they also inhibit the class I PI kinase which leads to an inactivation of mTor raptor (Meske et al., 2008) that in turn induces autophagy mediated by ULK1, a core complex essential for the formation of the phagophore. We observed that the suppressing effect of 3-MA on autophagy was only transient in neurons. This result is in accordance with data coming from a study on mouse fibroblasts (Wu et al., 2010). The authors also noticed that a prolonged incubation with the inhibitor reversed its suppressing effect on autophagy (after 9 h). The authors attributed the subsequent promoting activity to differential temporal effects of 3-MA on class I and class III PI3K. We observed that 3-MA influenced the integrity of the lysosomes. Usually lysotracker rapidly accumulates in the acidic compartment by ion-trap mechanism. A long-lasting treatment of neurons with 3-MA impeded this mechanism. This indicates that the lysosomal pH-gradient was destroyed. A similar effect of 3-MA on lysosomal pH in cultured hepatocytes has been described by Caro et al. (1988). In this context we found that disease-specific lysosomal cholesterol accumulations also vanished after prolonged MA-treatments. A similar effect was observed in NPC1-deficient fibroblasts (Elrick et al., 2012). The authors proposed that autophagy is a major source for the entrapped cholesterol in lysosomes and that the inhibition of autophagy with 3-MA reduces the lipid-ballast inside the lytic compartment. We observed that reduced cholesterol-staining of lysosomes was strictly coupled with the loss of lysotracker accumulations. We think that this indicates that the integrity of lysosomal membrane is compromised by long-lasting 3-MA treatments, leading to both: the release of lysosomal contents and to the destruction of the pHgradient. In this context Madge et al. (2003) showed that the inhibition of PI3K in human vascular endothelial cells induces the release of cathepsin B to the cytosol arguing for a direct role of PI3K in preserving lysosomal membrane integrity. The acidic pH inside the lysosomes is the basis for maturation and clearance of autophagosomes (Yamamoto et al., 1998; Klionsky et al., 2008). We conclude that the loss of the acidic environment inside the lysosomes prevents maturation/clearance of autophagosomes and contributes considerably to their accumulation in the cytosol observed after prolonged incubations with the PI3Kinhibitor. Although we can not exclude that an attenuated inhibition of the class III PI3K also contributes to this process as proposed by Wu et al. (2010). Furthermore, there is indication that PI3K-inhibitors sensitize cells to mitochondrion-independent lysosomal death pathways €a €ttela €, 2009). This may (Madge et al., 2003; Kirkegaard and Ja explain the observed chronological order of lysosomal damage and cell death elucidated by 3-MA in our study. Side-effects of the chosen inhibitor may not be the only problem arising with this kind of pharmacological intervention because neuronal cytoarchitecture and survival depend on a functional autophagic flux. A prolonged disturbance at any site of this complex pathway causes axonal dystrophy and eventually cell death (Bi et al., 1999; Hara et al., 2008; Komatsu et al., 2006; Lee et al., 2011). b-cyclodextrins reduce the number of autophagosomes and autolysosomes by accelerating the delayed execution of autophagy in NPC1-deficient cells (Ordonez et al., 2012; Meske et al., 2014). These compounds mobilize endosomal/lysosomal entrapped cholesterol (Abi-Mosleh et al., 2009; Rosenbaum et al., 2010; Peake and Vance, 2012; Meske et al., 2014) which improves the clearance
rate of autophagosomes (Elrick et al., 2012) and fully reconstitutes the autophagic flux in NPC1-deficient neurons (Meske et al., 2014). However, the concentration of b-CD used for extraction of lipids from NPC1-deficient cells seems to be a critical point which influences both autophagy and cell survival differently. Low concentrations (0.1e1 mM) restrict the depletion primarily to the lytic compartment and restore the autophagic flux to “normal” levels (Ordonez et al., 2012; Meske et al., 2014). High doses of b-CD (>5 mM), deplete cholesterol from the plasma membrane and induce autophagy in cells, probably via a class I PI3K-dependent inactivation of Akt and downstream mTor raptor (Cheng et al., 2006). High doses of b-CD, in contrast to low doses, were found to be potentially cytotoxic to a variety of brain cells (Peake and Vance, 2012). In the search for alternative methods to lower the cholesterolload in NPC1-deficient cells it was found that statins worsen the autophagic defect. The inhibition of endogenous isoprenoid synthesis rather activates the autophagy machinery in NPC1-deficient cells, leading to a further increase of the autophagosomal ballast (Cheng et al., 2006; Ishibashi et al., 2009). 5. Conclusions Our study clearly demonstrates that pharmacological treatments to reduce/prevent the accumulation of autophagic vacuoles in NPC1-deficient neurons should concentrate on compounds that restore the disturbed flux in mutated cells. Although an interruption of autophagy also leads to a (transient) decrease in the number of autophagic structures, such a treatment runs the risk of irreversible cell damages caused either by side effects of the inhibitor or by the long-lasting suppression of autophagy. The use of cyclic polysaccharides to repair the defect in the execution of autophagy is reasonable all the more because cyclodextrin derivates were already shown to alleviate clinical symptoms of the NPC-disease in animal studies (Liu et al., 2009; Ramirez et al., 2010; Aqul et al., 2011) and increased the life-span of afflicted animals (Liu et al., 2008, 2010). Acknowledgements This work contains parts of the doctoral thesis of Timm Priesnitz. This work has been supported in part by the Deutsche Forschungsgemeinschaft (ME 1494/2-1). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuropharm.2014.10.006. References Abi-Mosleh, L., Infante, R.E., Radhakrishnan, A., Goldstein, J.L., Brown, M.S., 2009. Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells. Proc. Natl. Acad. Sci. U. S. A. 106, 19316e19321. Aqul, A., Liu, B., Ramirez, C.M., Pieper, A.A., Estill, S.J., Burns, D.K., Repa, J.J., Turley, S.D., Dietschy, J.M., 2011. Unesterified cholesterol accumulation in late endosomes/lysosomes causes neurodegeneration and is prevented by driving cholesterol export from this compartment. J. Neurosci. 31, 9404e9413. Bi, X., Zhou, J., Lynch, G., 1999. Lysosomal protease inhibitors induce meganeurites and tangle-like structures in entorhinohippocampal regions vulnerable to Alzheimer's disease. Exp. Neurol. 158, 312e327. Caro, L.H.P., Plomp, P.J.A.M., Wolvetang, E.J., Kerkhof, C., Meijer, A.J., 1988. 3Methyladenine, an inhibitor of autophagy, has multiple effects on metabolism. Eur. J. Biochem. 175, 325e329. Carstea, E.D., Morris, J.A., Coleman, K.G., Loftus, S.K., Zhang, D., Cummings, C., et al., 1997. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277, 228e231.
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