Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant β-Glucocerebrosidase Activity and Mitochondrial Function in Neuronopathic Forms of Gaucher Disease

Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes lysosomal β-glucocerebrosidase. Homozygosity for the L444P mutation in GBA1 is associated with high risk of neurological manifestations which are not improved by enzyme replacement therapy. Alternatively, pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the mutant enzyme represent promising alternative therapies.Here, we report on how the L444P mutation affects mitochondrial function in primary fibroblast derived from GD patients. Mitochondrial dysfunction was associated with reduced mitochondrial membrane potential, increased reactive oxygen species (ROS), mitophagy activation and impaired autophagic flux.Both abnormalities, mitochondrial dysfunction and deficient β-glucocerebrosidase activity, were partially restored by supplementation with coenzyme Q10 (CoQ) or a L-idonojirimycin derivative, N-[N’-(4-adamantan-1-ylcarboxamidobutyl)thiocarbamoyl]-1,6-anhydro-L-idonojirimycin (NAdBT-AIJ), and more markedly by the combination of both treatments. These data suggest that targeting both mitochondria function by CoQ and protein misfolding by PCs can be promising therapies in neurological forms of GD.

20% in Gaucher A, Gaucher B and Gaucher C fibroblasts respect to controls (Fig. 1a). Given the critical role of CoQ in mitochondria function, transferring reducing equivalents from complexes I and II to complex III, it has been suggested that CoQ levels could be a useful biological marker of mitochondrial function 26 . Therefore, we next assessed whether fibroblasts derived from the patients with GD were deficient in CoQ. The average CoQ content of the three Gaucher fibroblasts cell lines was reduced by 24% relative to the average value of three control fibroblasts (Fig. 1b).
To assess the functional consequences of reduced respiratory chain enzyme activities and CoQ levels in Gaucher fibroblasts, we determined the mitochondrial membrane potential (Δ Ψ m) in both control and Gaucher fibroblasts by MitoTracker staining coupled with flow cytometry analysis. As shown in Fig 1c, Gaucher fibroblasts showed a significant reduction in Δ Ψ m, suggesting that the capacity to produce ATP may be diminished in Gaucher fibroblasts. Mitochondrial depolarization was also confirmed by measuring the ratio of TMRM (tetramethylrhodamine methyl ester) to MTG (MitoTracker Green FM) signal by flow cytometry (Supplementary Figure S1a).
Indeed, ATP levels were reduced to 30% of the control value in Gaucher fibroblasts (Fig. 1d). MitoTracker staining and imaging analysis of individual mitochondria confirmed the presence of normal tubular mitochondria (Fig. 1e,f,g, red arrow) along with a population of small rounded mitochondria with a striking depolarization (Fig. 1e,f,g; yellow arrow).
To elucidate whether bioenergetic impairment in Gaucher fibroblasts could be attenuated by improving mitochondrial function by CoQ and restoring GCase folding by PC administration, we quantified Δ Ψ m and ATP levels after CoQ (25 μ M), PC NAdBT-AIJ (25 μ M) or CoQ + PC (25 μ M + 25 μ M) supplementation. Both CoQ or PC treatments and specially the combined treatment of CoQ + PC resulted in a significant increase in Δ Ψ m and cellular ATP levels in Gaucher fibroblasts, but it had no effect in control cells (Fig. 2b). CoQ + PC effectiveness in improving Δ Ψ m and ATP levels was confirmed in the three Gaucher cell lines (Supplementary Figure S1b and S1c).
Next, we determined the effect of the L444P/L444P GCase mutation on cellular proliferation, as an indicator of correct or altered cell homeostasis, after CoQ and/or PC treatments. Proliferation rate was increased in Gaucher fibroblasts after both CoQ or PC treatments, and more significantly under the combined treatment CoQ + PC (Fig. 2c).
Staining with MitoTracker Red and cytochrome c immunostaining revealed discrete staining of mitochondria in control cells, with mitochondria organized in a tubular network, whereas small, rounded depolarized mitochondria were observed in Gaucher fibroblasts (Supplementary Figure S2a and S2b). The fact that Mitotracker staining colocalized with cytochrome c, a mitochondrial marker, (Supplentary Figure S2a) confirmed the specificity of MitoTracker staining and verified that mitochondrial depolarization in Gaucher fibroblasts was not a result of cytochrome c release, as occurs in apoptosis 27

Effect of CoQ and PC NAdBT-AIJ on Reactive Oxygen Species (ROS) Production in Gaucher
Fibroblasts. It is well established that mitochondrial dysfunction is associated with increased ROS production. Therefore, we examined mitochondrial ROS levels using the mitochondrial superoxide indicator MitoSOX Red in control and L444P Gaucher fibroblasts. At the same time, we estimated mitochondrial mass with 10-N-nonyl acridine orange (NAO) and determined the ratio of MitoSOX signal to NAO fluorescence. H 2 O 2 levels were also measured using CMH 2 -DCFDA and flow cytometry analysis. Mitochondrial superoxide production and H 2 O 2 levels were increased approximately by 2.5-fold (Fig. 3a,b) and by 2-fold respectively, suggesting increased oxidative stress in Gaucher fibroblasts. Supplementation with CoQ (25 μ M), PC NAdBT-AIJ (25 μ M) and specially the combined treatment of both CoQ + PC induced a considerable reduction in mitochondrial superoxide and H 2 O 2 levels in Gaucher cultures, but had no effect in control cultures (Fig. 3c,d). CoQ + PC effectiveness in reducing ROS levels was confirmed in the three Gaucher cell lines (Supplementary Figure S4). Autophagy in Gaucher Fibroblasts. Recent evidence suggests the involvement of ROS in autophagy activation 28 . To determine if autophagy was increased in Gaucher L444P fibroblasts, we first measured the amount of acidic vacuoles by LysoTracker staining and fluorescence microscopy and flow cytometry analysis. Using this technique, a 1.4 fold increase in LysoTracker staining was observed in Gaucher as compared to control fibroblasts (Fig. 4a,b). To elucidate whether autophagy in Gaucher fibroblasts could be attenuated by improving mitochondrial function by CoQ and mutant GCase folding by NAdBT-AIJ chaperone supplementation, we quantified the number of lysosomes in the various cultures following CoQ (25 μ M), PC NAdBT-AIJ (25μ M) or CoQ + PC supplementation. Treatment was associated with a reduction in the intensity of LysoTracker staining in Gaucher fibroblasts (Fig. 4c), indicating that    lysosomal activity was reduced following CoQ or PC supplementation and more significantly with the combined treatment CoQ + PC. CoQ + PC effectiveness in reducing the amount of acidic vacuoles was confirmed in the three Gaucher cell lines (Supplementary Figure S5a and S5b).
To further verify that autophagy was activated in Gaucher fibroblasts, we examined the expression levels of autophagic and lysosomal proteins by Western blotting. First, we investigated the conversion of LC3-I (microtubule-associated light chain3) to LC3-II, which is closely correlated with the number of autophagosomes. The ratio of LC3-II to LC3-I was significantly increased in Gaucher fibroblasts ( Fig. 4d), indicating enhanced autophagosome formation in Gaucher fibroblasts. As autophagosome formation involves an ubiquitin-like conjugation system in which Atg12 is covalently bound to Atg5, we also determined the expression levels of ATG12-ATG5 conjugates. ATG12-ATG5 expression levels were significantly increased in Gaucher fibroblasts. Likewise, the expression levels of BECLIN1, another essential protein required for the initiation of the formation of the autophagosome, was slightly increased in Gaucher fibroblasts when compared to control cells (Fig. 4d). We also studied autophagy activation in Gaucher fibroblasts by examining the expression levels of cathepsin D, a lysosomal enzyme. Compared to control cultures, there was a significant increase in cathepsin D expression levels in Gaucher fibroblasts. Supplementation with CoQ (25 μ M) or PC NAdBT-AIJ (25 μ M) partially reduced the expression levels of autophagic and lysosomal proteins (Fig. 4d). However, the combined treatment of both CoQ + PC was highly effective and restored the autophagic and lysosomal protein expression levels to near control levels (Fig. 4d). The densitometric analysis of Western blottings is showed in Supplementary Figure S6.

Mitophagy in Gaucher Fibroblasts.
To determine whether selective mitochondrial degradation or mitophagy was increased in Gaucher fibroblasts, we performed immunofluorescence double staining with antibodies against LC3 (autophagosome marker) and cytochrome c (mitochondrial marker) ( Fig. 5a,b,c). LC3 staining was hardly detectable in control cells (Fig. 6a), whereas normal tubular mitochondria, negative for LC3, along with many small, fragmented mitochondria, positive for LC3, were observed in Gaucher fibroblasts ( Fig. 5a,b,c). The population of small, rounded mitochondria showed a high colocalization of cytochrome c with LC3 staining, (r = 0.8929), whereas the tubular mitochondria did not (r = 0.0526) (Fig. 5a,b,c).
Supplementation with CoQ (25 μ M) or PC NAdBT-AIJ (25 μ M) partially reduced the number of LC3/cytochrome c punctata (Fig. 5c). However, the combined treatment of both CoQ+ PC, was highly effective and drastically reduced the number of LC3/cytochrome c punctata (Fig. 5c).
Mitophagy activation in Gaucher fibroblasts was also confirmed by checking that the expression levels of mitochondrial proteins such as complex I (30 kDa subunit), complex III (core 1 subunit), and porin were reduced compared to controls fibroblasts and, on the contrary, the expression levels of Golgi (Golgi marker), endoplasmic reticulum (PDI, protein disulfide isomerase), and peroxisome (catalase) proteins markers were not affected (Fig. 6a). Supplementation with CoQ (25 μ M) or PC NAdBT-AIJ (25 μ M) partially restored mitochondrial protein expression levels (Fig. 6a). However, the combined treatment of both CoQ + PC was highly effective and mitochondrial protein expression levels were increased near to control values (Fig. 6a). The densitometric analysis of Western blottings is showed in Supplementary Figure S7.
Autophagic Flux is Impaired in Gaucher Fibroblasts. To examine autophagosome/autophagolysosomal maturation and to ascertain whether or not the autophagic flux is impaired in Gaucher fibroblasts, we also checked the levels of LC3-II in the presence of bafilomycin A1 (Baf), a specific inhibitor of vacuolar H + -ATPases and a blocker of autophagosome-lysosome fusion. As expected, bafilomycin A1 treatment in control fibroblast cells led to a significant increase in the amount of LC3-II, suggesting that autophagic flux was normal (Fig. 6b,c). However, bafilomycin A1 treatment in Gaucher fibroblasts had no effect on LC3-II expression levels (Fig. 6b,c), indicating that autophagic flux was impaired in this case. We also examined whether mitochondrial proteins levels were affected by inhibition of autophagic flux. Inhibition of autophagosome-lysosome fusion by bafilomycin A1 induced an increase of porin and cytochrome c levels in Gaucher fibroblasts but not in control fibroblasts (Fig. 6a,b), suggesting increased mitochondrial delivery and degradation within autophagolysosomes in mutant cells.
Altogether, these results support the hypothesis that both mitophagy activation and impaired autophagic flux coexist in Gaucher fibroblasts.

Autophagy is a Protective Mechanism in Gaucher Fibroblasts.
To verify the role of autophagy in survival of GCase deficient cells, apoptosis was examined in GCase deficient wild-type and Atg5-/-mouse embryonic fibroblasts (MEFs). GCase deficiency in MEFs was produced by treatment with the specific GCase inhibitor CBE. We verified that CBE treatment indeed induced GCase deficiency in wild-type and Atg5-/-knockout fibroblasts (data not shown). We then analyzed cell viability and apoptosis in both GCase-deficient wild-type and knockout fibroblasts. GCase deficiency induced a high viability and low level of apoptosis in wild-type MEFs as determined by detecting caspase activation, cytochrome c release, and nuclear DNA condensation. In contrast, Atg5-/-MEFs were significantly more sensitive to GCase deficiency induction with a significant decrease in cell viability and a 1.5 fold increase in apoptosis  Determination of LC3-II, porin and cytochrome c levels in the presence and absence of bafilomycin A1 in control and Gaucher-B fibroblasts. Control and Gaucher fibroblasts were incubated with bafilomycin A1 (100 nM for 12 h). Total cellular extracts were analyzed by immunoblotting with antibodies against LC3, porin and cytochrome c. α -Tubulin was used as a loading control. (c) Densitometry of Western blotting was performed using the ImageJ software. Data represent the mean ± SD of three separate experiments. *p < 0.01 between control and Gaucher fibroblasts. a p < 0.05 between the presence and the absence of bafilomycin A1. (d) Apoptosis is increased in GCase-deficient Atg5-/-cells. Apoptosis is increased in GCasedeficient Atg5-/-cells. GCase deficiency in wild-type and Atg5-/-MEFs was induced by treatment with CBE 2,5 mM for 48 hours. Apoptosis was assessed in both autophagy proficient (wild-type), and autophagydeficient cells (Atg5-/-) treated with CBE as described in Material and Methods. CBE-treated MEFs were also supplemented with, CoQ+ PC NAdBT-AIJ for 96 hours to verify the specificity of apoptosis induction by GCase deficiency. Results are expressed as mean ± SD of three independent experiments *p < 0.01 between Atg5-/-and wild-type MEFs. a p < 0.01 between the presence and the absence of CBE . b p < 0.01 between Atg5-/-in the presence or absence of CoQ+ PC.

Treatment with CoQ+PC NAdBT-AIJ Increases GCase Activity, Improves Enzyme Traffic and
Reduces Intracellular GlcCer Levels. GCase activities in the three Gaucher fibroblasts cell lines were reduced to 10%, 8% and 6% respectively, compared to control values (Supplementary Figure S8a). To elucidate whether GCase activities could be restored by improving mitochondrial function by CoQ and protein folding of the mutant GCase by PC NAdBT-AIJ supplementation, we determined GCase activities after treatment with CoQ (25 μ M), PC NAdBT-AIJ (25 μ M) or CoQ + PC. Supplementation with CoQ, PC and specially the combination of CoQ + PC induced a significant increase of 2-fold, 2.4-fold and 3.4-fold, respectively, of GCase activities (Fig. 7a). CoQ + PC treatment effectiveness in improving GCase activity was also confirmed in the three Gaucher cell lines (Supplementary Figure S8b).
Furthermore, deficient GCase activities were associated with reduced levels of GCase in Gaucher fibroblasts (Fig. 7b,c). As expected, both CoQ or PC and more significantly CoQ + PC treatment induced a marked increase in GCase expression levels (Fig. 7b,c).
Accordingly, CoQ + PC treatment was also able to facilitate the efficient translocation of the mutant GCase to the lysosome compartment in Gaucher fibroblasts. The intracellular localization of mutant GCase before and after treatment was determined by using indirect fluorescent immunostaining. Cells were co-stained with IgGs against GCase and either a marker for lysosomes (LAMP-1) or an ER marker (PDI) (Fig. 8a,b). In untreated cells, GCase staining was diffuse and distinct from the punctate staining pattern of LAMP-1 (Fig. 8b). Instead, GCase staining colocalized (indicated by yellow colour) with the ER marker PDI (Fig. 8a). However, when Gaucher fibroblasts were treated with CoQ co-administered with PC NAdBT-AIJ, their GCase staining pattern increased in fluorescence intensity, became more punctate and exhibited a greater co-localization with LAMP-1, as indicated by the increased yellow colour in the Merge column (Fig. 8b). Furthermore, there was a notable decrease in the overlap between GCase and PDI staining of cells treated with CoQ + PC (Fig. 8a). In the case of CoQ + PC-treated cells, there was also an observable decrease in the overall intensity level of PDI staining, suggesting a decrease in ER stress, for which PDI is also a marker, as compared to untreated cells (Fig. 8a).
To verify increased mutant GCase folding and trafficking from the ER to the lysosomes, we performed endoglycosidase H (Endo-H) digestion assay on cell lysates from control and Gaucher fibroblasts, as it has been previously reported 15 . ER retained GCase proteins carrying N-linked glycans which are sensitive to Endo-H cleavage, producing a low molecular weight band of GCase on Western blotting (Endo-H-S).
In control fibroblasts most of GCase was Endo-H resistant (Endo-H-R), indicating that the protein passed already the mid-Golgi (probably mature lysosomal) (Fig. 8c,d). On the other, a lesser amount of the enzyme were Endo-H-R in Gaucher fibroblasts, suggesting that a significant fraction of GCase in these cells did not reach the mid-Golgi and therefore was not lysosomal. The increased levels of mutant GCase protein Endo-H-R in response to treatments, particularly CoQ + PC, support the hypothesis of a partial restoration of mutant GCase folding and post-ER trafficking.
Furthermore, to confirm the effectiveness of PC and/or CoQ treatment in improving Gcase activity, we quantified GlcCer levels by Dot-blot assays in control and Gaucher fibroblasts after CoQ (25 μ M), PC NAdBT-AIJ (25 μ M) or CoQ + PC (25 μ M + 25 μ M) supplementation. Both CoQ and PC and specially the combined treatment of CoQ+ PC resulted in a significant reduction in GlcCer levels in Gaucher fibroblasts (Supplementary Figure S9a and S9b). Fluorescence microscopy examination also confirmed that CoQ + PC treatment reduced GlcCer accumulation both in the lysosomal and mitochondrial compartment (Supplementary Figure S10).

Discussion
In this work, we studied the pathophysiology of the L444P/L444P mutation in primary cultured fibroblasts derived from GD patients with neurological involvement. We assessed both GCase deficiency and mitochondrial function in cultured fibroblasts. We found mitochondrial dysfunction associated with reduced activity and expression levels of GCase in Gaucher fibroblasts. In addition, mitophagy activation as a mechanism to eliminate dysfunctional mitochondria and impaired autophagic flux were observed in Gaucher fibroblasts. Next, we evaluated the effects of treatments targeting mitochondrial dysfunction and GCase misfolding on the improvement of the pathophysiological alterations of GD. Coenzyme Q 10 (CoQ), a commercially available 1,4-benzoquinone derivative with cellular energy production and antioxidant functions occurring naturally in most eukaryotic cells, and N-[N'-(4-adamantan-1-ylcarboxamidobutyl) thiocarbamoyl]-1,6-anhydro-L-idonojirimycin (NAdBT-AIJ), a synthetic sp 2 -iminosugar-type PC recently shown to be active in restoring L444P GCase folding and trafficking in GD fibroblasts 20 , respectively, were used for those purposes. Our results showed that although single treatments are partially effective, the combined treatment of both CoQ and PC markedly increased GCase activity and enzyme trafficking to the lysosomes, and also improved mitochondrial function and the associated pathophysiological alterations.
Interestingly, the amount of CoQ, an essential electron carrier of the mitochondrial respiratory chain, was reduced in Gaucher fibroblasts. Concomitantly, a large proportion of the mitochondrial population was depolarized. Both alterations may further compromise normal mitochondrial function and cellular   energy metabolism, which can aggravate the lysosomal functional defect. CoQ deficiency has been identified in a wide range of diseases, including mitochondrial diseases, lysosomal diseases, neurodegenerative diseases, such as Parkinson's disease, cancer, fibromyalgia, and in patients undergoing statin treatment 29 .
In addition to CoQ deficiency and decreased mitochondrial membrane potential, we identified increased ROS production and H 2 O 2 content, as well as increased expression of autophagic proteins. All these pathological conditions could be significantly ameliorated by CoQ + PC treatment in fibroblasts harboring the L444P/L444P mutation.
An alteration of autophagy in GD has been previously postulated 30 . GCase deficiency leads to both an induction of autophagy and an impairment of autophagic flux leading to accumulation of autophagosomes and autophagic substrates. The impairment in degradation of autophagic substrates may contribute to several aspects of Gaucher neuropathology, including the accumulation of ubiquitinated proteins and ROS production. Thus, oxidative stress may play a prominent role in the induction of the mitochondrial damage and autophagy activation, which we observed in Gaucher fibroblasts. In addition, the elimination of dysfunctional mitochondria could play a crucial role in protecting cells from the damage caused by perturbed mitochondrial function. Our finding that the autophagic protein LC3 colocalized with structurally abnormal mitochondria, but not with normal tubular mitochondria in Gaucher fibroblasts, suggests that autophagy specifically targets dysfunctional mitochondria in these cells. Formerly reported evidences on several LSDs showed increased ROS, dysfunctional mitochondria, blocked autophagy, aberrant inflammatory and apoptotic signaling and perturbed calcium homeostasis, among other biochemical alterations 1 .
Besides mitophagy activation, we found impaired autophagic flux in Gaucher fibroblasts. In GD, a reduction of autophagic flux may have a major impact on mitochondrial function and on cytoplasmic proteostasis. Constitutive macroautophagy maintains mitochondrial quality by selectively degrading dysfunctional mitochondria by mitophagy 31 . Mitochondrial proteins are consistently found in the proteomes of highly purified autolysosomes, especially subunits of the mitochondrial ATPase 32 . Reduced autophagic flux in GD may lead to the persistence of dysfunctional mitochondria, which is highly pronounced in other LSDs cells such as Batten's disease neurons 33 . Several LSDs (mucolipidosis types IV, IIIA [pseudo-Hurler polydystrophy], and II [I-cell disease], late infantile neuronal ceroid lipifuscinosis [CLN2], mucopolysaccharidosis VI, and GM1 gangliosidosis) also display mitochondrial abnormalities, including replacement of the extended tubular mitochondrial network with high numbers of relatively rounded depolarized mitochondria 1 . Studies into aging and autophagosome formation have shown that mitochondria are involved in signaling pathways regulating apoptosis and innate immunity, and that reduced autophagic flux and subsequent accumulation of dysfunctional, reactive oxygen species-generating mitochondria renders cells more sensitive to apoptotic and inflammatory stimuli 34 . Therefore, the aberrant functioning of mitochondria may be responsible for apoptosis and inflammation in the CNS of multiple LSDs, including GD.
Defective activity of GCase in GD results in intracellular accumulation of the glycosphingolipids, glucosylceramide (GlcCer) and psychosine. Recently, it has been demonstrated in a mouse model of neuronopathic GD that glucosylceramide (GlcCer) and psychosine accumulation in GD brains precede the appearance of neuroinflammation, although only GlcCer accumulation correlated with neuroinflammation and neuron loss 35 . Some authors have hypothesized that variations in lipids involved in ceramide metabolism, including both GlcCer and ceramide, might play an important role in the associated mitochondrial dysfunction in GD. The possible deleterious effect of glycosphingolipids in the mitochondrial membrane and cellular bioenergetics was already discussed by Strasberg et al. 36 and it has been suggested that the accumulation of the GlcCer and and GlcSph is the basis for the extensive neuronal cell loss in the neuronopathic forms GD type II/III 35,37 . Furthermore, it has been reported that the sphingolipid ceramides provoke oxidative stress by disrupting mitochondria and induce lethal mitophagy 38 . Interestingly, sub-cellular fractionation studies of Gaucher fibroblasts showed that GlcCer was not confined to the lysosomes but its concentration increases throughout the cell 39 . In agreement with these results, we found that GlcCer is accumulated mainly in the lysosomal and mitochondrial compartments in Gaucher fibroblasts. We propose that both accumulation of GlcCer and impairment of autophagic flux induce mitochondrial dysfunction in Gaucher fibroblast. Dysfunctional mitochondria are involved in the pathogenesis of several neurodegenerative diseases, but the experimental evidence for a role in altered mitochondria dynamics is especially strong in PD 6 . The proper elimination of damaged mitochondria is needed in post-mitotic neurons because progressive accumulation of damaged mitochondria might lead to eventual cell death.
Mitochondrial dysfunction with reduced respiratory chain complex activities, increased ROS production and decreased potential in neurons and astrocytes has recently been reported in a mouse model of type II neuronopathic Gaucher disease 37 . Thus, a primary lysosomal defect may cause accumulation of dysfunctional mitochondria. The neurological and pathological symptoms observed in Gaucher disease patients are similar to those displayed in Parkinson's disease, including loss of dopaminergic neurons in the substantia nigra, alpha-synuclein accumulation, tremors at rest, bradykinesia, and rigidity 6 . Indeed, parkinsonism has been reported in many Gaucher disease patients, suggesting a mechanistic link between the two disease states. Further supporting this, large, multicenter genome-wide association studies have reported a high incidence of GBA mutations in patients with sporadic Parkinson's disease and an increased risk of developing Parkinson's disease in GBA carriers 40 .
An emerging strategy for the treatment of LSDs is pharmacological chaperone therapy (PCT), based on the use of chaperone molecules that assist the folding of mutated enzymes and improve their stability and lysosomal trafficking 41 . These PC can cross the BBB and therefore have a potential for the treatment of diseases with CNS involvement 42 . Our findings confirmed previous results showing the effectiveness of bicyclic derivatives of L-idonojirimycin as pharmacological chaperones for neuronopathic forms of Gaucher disease 20 . The PC compound NAdBT-AIJ increased GCase activity and also facilitated translocation of the enzyme to the lysosome in Gaucher fibroblasts harboring the L444P mutation.
Likewise, given that defects in energy metabolism and oxidative stress have been demonstrated to play a role in the pathogenesis of GD, we envisioned that the treatment with coenzyme CoQ could also exert beneficial therapeutic effects. The fundamental role of CoQ in mitochondrial bioenergetics and its well-acknowledged antioxidant properties constitute the basis for its clinical applications, although some of its effects may be related to a gene induction mechanism 29 . Interestingly, CoQ is also able to cross the BBB 43 . Therefore, the combined treatment of CoQ with PC NAdBT-AIJ may represent a therapeutic strategy, supplementary or alternative to ERT in Gaucher patients with CNS involvement. Indeed, our results suggest that the combined treatment of CoQ with PC NAdBT-AIJ is high effective in improving the biochemical phenotype in cellular models of GD harboring the L444P/L444P mutation.
In summary, our studies provide experimental evidence that targeting mitochondrial dysfunction and GCase misfolding by the combined supplementation of CoQ and the L444P-active pharmacological chaperone NAdBT-AIJ could be beneficial in the treatment of patients with neuronopathic GD. However, additional experiments in neuronal or animal models are essential for the confirmation of these results.
Our findings support the hypothesis that secondary mitochondrial dysfunction by GlcCer accumulation aggravates the effects of the L444P/L444P mutation and therefore, the improvement of mitochondrial function by CoQ treatment also improves the activity of the mutant GCase. The fact that both CoQ and PC NAdBT-AIJ can cross the BBB makes them promising candidates for the treatment of neuronopathic forms of GD that are not responsive to ERT.

Fibroblast Cultures. Cultured fibroblasts from 3 Gaucher patients (Gaucher-A, Gaucher-B and
Gaucher-C) homozygous for L444P GCase were obtained from Coriell Cell Repositories (Camden, NJ, USA). Control primary fibroblasts were obtained from healthy volunteers; any control value represent the mean value for at least two control fibroblasts cell lines. Samples from patients and controls were obtained according to the Helsinki Declarations of 1964, as revised in 2001. This study has been approved by the institutional ethical committee of Pablo de Olavide University. Informed consent was obtained from all subjects biopsied. Control and Gaucher fibroblasts were cultured at 37 °C in Dulbecco's Modified Eagle Medium (DMEM) (4.5 g/L glucose), plus pyruvate supplemented with 20% fetal bovine serum (FBS), and an antibiotic/anti-mycotic solution.
Mouse Embryonic Fibroblasts (MEFs) derived from wild-type and Atg5-/-mouse embryos were a kind gift of Noboru Mizushima, Tokyo Medical and Dental University, Japan 44 .

Synthesis of the Pharmacological Chaperone NAdBT-AIJ. The preparation of the PC
NAdBT-AIJ was accomplished in two steps and 95% overall yield from 5-amino-5-deoxy-1,2-O-isopropylidene-β -L-idofuranose, which at its turn is readily accessible from commercial D-glucuronolactone, by nucleophilic addition of 4-(1-adamantanylcarboxamido)butyl isothiocyanate 45 followed by acid-catalyzed acetonide hydrolysis of the resulting thiourea adduct, following the procedure previously reported 20 . Mitochondrial Respiratory Chain and Citrate Synthase Enzyme Activities. Activities of Activities of NADH:coenzyme Q1 oxidoreductase (complex I), succinate dehydrogenase (complex II), ubiquinol:cytochrome c oxidoreductase (complex III), succinate:cytochrome c reductase (complex II+ III) and citrate synthase (CS) were determined in sonicated permeabilized fibroblasts using previously described spectrophotometric methods 46 . Results are expressed as % of activity respect to control values. Proteins of fibroblasts homogenates were analyzed by the Lowry procedure 47 .

Measurement of CoQ Levels.
CoQ levels in cultured fibroblasts were performed using a method previously described by our group 48 .
Adenosine-5'-Triphosphate Levels. Adenosine-5'-triphosphate (ATP) levels were determined using a bioluminescence assay (ATP determination kit, Invitrogen-Molecular Probes, Eugene, OR). Proliferation Rate. Two hundred thousand cells were cultured with or without 25 μ M CoQ and/or 25 μ M PC for 96 h. Cell counting was performed from 10 high power fields using an inverted microscope and a 40× objective.
GCase Activity Assay. GCase activities in cell lysates were determined by using 4-methylumbelliferone-conjugated β -D-glucopyranoside as a substrate. The lysates (10 μ L) were incubated at 37 °C with the substrate solution (20 μ L) in 0.1 M citrate buffer, pH 5.2, supplemented with sodium taurocholate (0.8% w/v) for 1 h. The reactions were terminated by adding 0.2 μ L of 0.2 M glycine sodium hydroxide buffer (pH 10.7). The liberated 4-methylumbelliferone was measured spectrophotometrically using a fluorescence Microplate Reader (excitation wave length: 340 nm; emission: 460 nm). One unit of enzyme activity was defined as 1 nmol of 4-methylumbelliferone released per hour and normalized for the amount of protein contained in the lysates.
Immunofluorescence Microscopy. Immunofluorescence microscopy was performed using standard methods as previously described 49 . Cover slips were analyzed using a fluorescence microscope (Leica DMRE, Leica Microsystems GmbH, Wetzlar, Germany). Deconvolution studies and 3-dimensional projections were performed using a DeltaVision system (Applied Precision, Issaquah, WA) with an Olympus IX-71microscope. The deconvolved images were derived from optical sections taken at 30-nm intervals using a 60× PLAPON objective with a 1.42 numerical aperture.

Measurement of Mitochondrial Reactive Oxygen Species (ROS) Generation. Mitochondrial
ROS generation was assessed using the mitochondrial superoxide indicator MitoSOX Red, according to the manufacturer's instructions. ROS levels were expressed relative to mitochondrial mass (ROS signal/ NAO signal), determined by flow cytometry and fluorescence microscopy of cells stained with 10 μ M NAO for 10 minutes at 37 °C in the dark.

Determination of Mitochondrial Membrane Potential (ΔΨm).
Cells were cultured in six-well 35 mm plates and, at confluence, were incubated for 30 minutes with 100 nM Mitotracker Red CMXRos. Cells washed with fresh medium were subsequently harvested and analyzed by flow cytometry. Fibroblasts were grown on 1-mm width (Goldseal No. 1, Thermo Fisher Scientific Inc, Waltham, MA) glass coverslips for 24 to 48 hours, incubated with 100 nM MitoTracker Red for 30 minutes, fixed, and immunostained with anti-cytochrome c (a mitochondrial marker), and examined by fluorescence microscopy. Colocalization of both markers and quantification of MitoTracker fluorescence in individual mitochondria was assessed by the DeltaVision software (Applied Precision).
Mitochondria were classified as small-depolarized and tubular-polarized mitochondria (tubular, mitochondrial tubule of 5 μ m in length is present in a cell). When this network was disrupted and mitochondria appeared predominantly spherical and small they were classified as depolarized. Quantification of fluorescence signal was performed in 100 cells using the ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
Δ Ψ m was also measured by double staining with 20 nM TMRM (a potentiometric fluorescent indicator) and 100 nM MTG (a non-potentiometric dyes that relies on mitochondrial mass independent of Δ Ψ m) and flow cytometry analysis. Relative Δ Ψ m values were calculated by determining the ratio of TMRM signal to MTG fluorescence in arbitrary units.
Immunoblotting Analysis. Western blotting was performed using a standard protocol 48  LysoTracker Red assay. LysoTracker Red (100 nM) was added to cultured fibroblasts for 30 minutes each, well was washed twice with fresh DMEM, and then the cells were fixed with 2% paraformaldehyde in PBS for 10 minutes at 4 °C. LysoTracker fluorescence was quantified by flow cytometry.

Analysis of apoptotic and viable cells. Apoptosis was assessed by observing nuclei fragmentation
by Hoechst staining, cytochrome c release, and caspase 3 activation. Viable cells were determined from their normal nuclear morphology and exclusion of propidium iodide. In each case 10 random fields and more of 500 cells were counted. Dot blot assay for glucosylceramide (GlcCer). Intracellular GlcCer levels was performed by a dot blot assays as described previously 50 .
Endoglycosidase-H treatment. Samples of cell lysates, containing 70 μ g of protein, were denatured with glycoprotein denaturing buffer (New England Biolabs 5% SDS, 0.4 M dithiothreitol) at 100 °C for 15 minutes. The lysate was subjected to an overnight incubation with endoglycosidase-H and reaction buffer (New England Biolabs 0.5 M sodium citrate, pH 5.5) according to the manufacturer's instructions. The lysates were then analyzed by Western blot with the anti GCase antibody.
Statistical Analysis. All results are expressed as mean± SD of 3 independent experiments. The measurements were statistically analyzed using the Student's t test for comparing 2 groups and analysis of variance for more than 2 groups. The level of significance was set at p < 0.05.