WO2012094600A1 - Methods for treating lysosomal storage diseases using l-type ca2+ channel blockers with a 1,4 dihydropyridine structure and inhibitors of er-associated degradation - Google Patents

Abstract

Methods are provided for the treatment of lysosomal storage diseases, such as Gaucher's disease. In one embodiment, a method comprising administering to a subject a therapeutically effective amount of a L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure is provided.

Description

METHODS FOR TREATING LYSOSOMAL STORAGE DISEASES USING L-TYPE CA²⁺ CHANNEL BLOCKERS WITH A 1,4 DIHYDROPYRIDINE STRUCTURE AND

INHIBITORS OF ER-ASSOCIATED DEGRADATION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/430,326 filed

January 6, 2011, which is incorporated herein by reference.

BACKGROUND

Lysosomal storage diseases consist of a group of more that 40 distinct diseases exhibiting different symptoms and clinical manifestations but characterized by similar cellular pathogenesis, e.g. mutation in lysosomal enzymes that often destabilize native folding and result in partial or complete loss of a fundamental hydrolytic function. These inherited diseases are also characterized by aberrant storage of metabolites in the lysosomes.

In particular, Gaucher' s disease (GD) is the most common lysosomal storage disorder and it is characterized by deficient lysosomal glucocerebrosidase (GC) activity and accumulation of GC substrate, glucosylceramide (Schueler et al., 2004). Mutations in GC encoding gene (GBA, (Hruska et al., 2008)) result in inactive GC variants, which are typically retrotranslocated from the endoplasmic reticulum (ER) to the cytoplasm for ER-associated degradation (ERAD). Extensive ERAD of mutated, unstable proteins is a common theme in the cellular pathogenesis of LSD. For instance, mutations in the β-hexosaminidase A (HexA) cause storage of (GM2) (N-AcGalpl,4(NeuAca2,3)Galpl,4Glc-ceramide) gangliosides and development of Tay-Sachs disease (Jeyakumar et al., 2002) Similar to many LSD, GD is caused by ERAD of mutated unstable enzymes destined to the lysosome (Grace et al. 1994). Native folding of mutated GC variants is limited by rapid disposal of unstable folding intermediates (Wang et al. 201 1 c).

A number of characterized missense mutations destabilize GC native structure without directly impairing its catalytic activity (Schmitz et al., 2005). The most frequently encountered mutations in GC encoding genes do not directly impair enzyme activity. As a result, these unstable GC variants retain biologic activity if forced to fold into their native 3D structure (Sawkar et al, 2002; Sawkar et al., 2006; Yu et al., 2007). One of the most frequently occurring misfolding mutations is the L444P substitution. (Schmitz et al., 2005). This mutation severely destabilizes the GC native structure and results in a complete loss of activity.

1 Ca²⁺ ions, and particularly the gradient of [Ca²⁺] between the ER (1 mM) and the cytoplasm (100 nM), play a signaling role in a number of fundamental cellular activities, including protein folding in the ER (Berridge et al., 1998; Bygrave and Benedetti, 1996). As a result, impairment of Ca²⁺ homeostasis hampers the folding of unstable GC variants. Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate (IP3) receptors on the ER membrane regulate [Ca²⁺]ER efflux, whereas Ca²⁺-ATPases (SERCA pumps) transfer Ca²⁺ from the cytoplasm into the ER (Baumann and Walz, 2001). In GD affected neurons, glucosylceramide accumulation causes excessive [Ca ]ER efflux via RyRs (Korkotian et al., 1999; Lloyd-Evans et al, 2003; Pelled et al, 2005).

Ca²⁺ homeostasis influences the biogenesis of secretory proteins and the activity of a number of ER chaperones (Michalak et al., 2002). It was previously suggested that impairment of Ca²⁺ homeostasis in GD fibroblasts, by compromising ER folding, hampers rescue of highly unstable, degradation-prone L444P GC folding (Wang et al., 201 lb). The application of RyRs blockers was shown to counteract the effect of glucosylceramide accumulation on [Ca ]ER efflux, re-establish Ca²⁺ homeostasis, and create an environment more conducive to native folding of L444P GC. However, it did not lead to a substantial rescue of the folding of L444P GC (Wang et aL, 2011b).

L-type Ca²⁺ channels (LTCC) blockers bind to high voltage activated channels on the plasma membrane and lower cytosolic free [Ca ] (Hockerman et al., 1997; Toggle, 2006). Phenylalkylamines, benzothiazepines, and 1 ,4-dihydropyridines are the three main classes of LTCC blockers and include molecules that bind to three distinct LTCC receptor sites (Hockerman et al., 1997). Verapamil and diltiazem, prototypes of phenylalkylamines and benzothiazepines, respectively, are FDA-approved for the treatment of hypertension and cardiac arrhythmias (Hockerman et al., 1997), and were previously reported to partially rescue the folding of GC variants, but their mechanism remains elusive (Mu et al, 2008a; Sun et al., 2009).

Many lysosomal storage diseases are considered to be "orphan disease" in that they comprise a small patient population. As a result, limited efforts have been devoted to the development of therapeutic solutions for each disease individually. Accordingly, it would be beneficial to find methods that enhance the activity of general cellular folding pathways involved in the processing and maturation of a number of different enzymes, so as to find application in the treatment of multiple lysosomal storage diseases. SUMMARY

The present disclosure generally relates to methods for treating lysosomal storage diseases. More particularly, the present disclosure relates to methods for treating lysosomal storage diseases, such as Gaucher' s disease, using L-type Ca²⁺ channel blockers with a 1,4 dihydropyridine structure, at least one inhibitor of ER-associated degradation, or a combination thereof. .

In one embodiment, the present disclosure provides a method comprising administering to a subject a therapeutically effective amount of a L-type Ca channel blocker with a 1 ,4 dihydropyridine structure.

In another embodiment, the present disclosure also provides a method for restoring enzymatic activity to a mutated enzyme associated with a lysosomal storage disease that comprises introducing an L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure to a subject and allowing the channel blocker to modulate a cellular folding pathway while preventing apoptosis induction so as to at least partially increase the folding of the mutated enzyme.

In another embodiment, the present disclosure provides a method comprising administering to a subject a therapeutically effective amount of at least one inhibitor of ER- associated degradation.

The features and advantages of the present invention will be apparent to those skilled in the art. While numerous changes may be made by those skilled in the art, such changes are within the spirit of the invention.

DRAWINGS

Some specific example embodiments of the disclosure may be understood by referring, in part, to the following description and the accompanying drawings.

Figure 1 shows treatment of GD patient-derived fibroblasts with LTCC blockers enhances L444P GC activity. (A) Relative L444P GC activities in cells treated with lacidipine (20 μΜ), lercanidipine (20 μΜ) and nicardipine (20 μΜ) for 72 hrs, and diltiazem (10 μΜ) and verapamil (5 μΜ) for 120 hrs. Relative GC activities were evaluated normalizing GC activities measured in treated cells to the activity of untreated cells (left y axis), (p<0.01 if not specified; *p<0.001). The corresponding fraction of WT GC activity is reported (right y axis). Experiments were repeated three times and data points are reported as mean ± SD. Relative L444P GC activities were also evaluated in L444P GC cells treated with a proteostasis regulator (MG-132, 0.6 μΜ; celastrol, 0.6 μΜ) and a LTCC blocker: (B) lacidipine, (C) lercanidipine, and (D) nicardipine for 72 hrs, (E) diltiazem, and (F) verapamil for 120 hrs. GC activities were obtained as described in (A) and are reported as mean ± SD. (G) Relative N370S GC activities in cells treated with lacidipine (20 μΜ) and diltiazem (10 μΜ) for 72 hrs. Relative N370S GC activities in cells treated a proteostasis regulator (MG-132, 0.6 μΜ; celastrol, 0.6 μΜ) and a LTCC blocker: (H) lacidipine, and (I) diltiazem.

Figure 2 shows treatment of GD patient-derived fibroblasts with lacidipine promotes L444P GC folding, glycosylation, and trafficking. (A) Western blot analyses of EndoH treated and untreated total protein content of L444P GC fibroblasts cultured with lacidipine (20 μΜ), MG-132 (0.6 μΜ), and celastrol (0.6 μΜ) for 48 hrs and detected using GC-specific antibody. The solid and dashed arrows indicate, respectively, EndoH resistant and EndoH sensitive bands. PR, proteostasis regulator. Lac, lacidipine; Dil, diltiazem; MG, MG-132; Cel, celastrol. (B) Quantification of GC bands detected by western blot. Lower MW, EndoH-sensitive bands corresponding to ER retained GC were quantified and are reported in the white portion of the bars, and quantification of higher MW, EndoH-resistant bands corresponding to lysosomal GC are reported in the black top portions. Band analyses and quantifications were conducted using NIH Java Image analysis software. Immunofluorescence microscopy of (C) GC and CNX (an ER marker), and (D) GC and LAMP1 (a lysosomal marker) in L444P GC fibroblasts. Cells were treated with lacidipine (20 μΜ) and MG-132 (0.6 μΜ) for 48 hrs. Colocalization of CNX (red, column 1) and GC (grey, column 2) is shown in green (column 3). Colocalization of LAMP 1 (red, column 1) and GC (blue, column 2) is also shown in green (column 3).

Figure 3 shows LTCC blockers reduce cytosolic [Ca²⁺] levels in patient-derived fibroblasts. (A) L444P GD, (B) N370S GD, and (C) WT fibroblasts were cultured with lacidipine (20 μΜ) and diltiazem (20 μΜ) for 5, 10, 20, 40 and 60 min respectively. Cytosolic [Ca²⁺] was evaluated by measuring excitation 340/380 ratio of fura-2 acetoxymethyl ester and normalized to that at time zero. The data is reported as mean ± SD.

Figure 4 shows treatment of patient-derived fibroblasts with lacidipine upregulates BiP expression. Relative mRNA expression levels of (A) BiP (pO.01 ), (B) CNX (p<0.05), and (C) CRT (p<0.05) in L444P GC fibroblasts treated with lacidipine (20 μΜ), diltiazem (10 μΜ), MG-132 (0.6 μΜ), and celastrol (0.6 μΜ) for 24 hrs were obtained by quantitative RT-PCR, corrected by the expression of the housekeeping gene GAPDH, and normalized to those of untreated cells. The data is reported as mean ± SD. (D) Western blot analyses of BiP, CNX, CRT, and GAPDH (used as loading control) accumulation in cells treated with lacidipine (20 μΜ) and MG-132 (0.6 μΜ) for 48 hrs. Lac, lacidipine; Dil, diltiazem. Figure 5 shows treatment of patient-derived L444P GC fibroblasts with lacidipine results in upregulation of the UPR without induction of apoptosis. Cells were treated with lacidipine (20 μΜ), diltiazem (10 μΜ), MG-132 (0.6 μΜ), and celastrol (0.6 μΜ) for 24 hrs. (A) Xbp-1 expression and splicing was determined by RT-PCR and separation on agarose gel and quantification of spliced Xbp-1 band intensities (B) conducted using the NIH Java Image analysis software. PR, proteostasis regulator; Lac, lacidipine; Dil, diltiazem; MG, MG-132; Cel, celastrol. Relative mRNA expression levels of (C) ATF6, (D) CHOP, (E) BAX, (F) BAX, and (G) BCl-2 (p<0.01) were obtained by quantitative RT-PCR and calculated as described in Figure 4. The data is reported as mean ± SD.

Figure 6 shows L444P GC expression is upregulated in fibroblasts treated with lacidipine and diltiazem. (A) Relative GC mRNA expression level was evaluated by quantitative RT-PCR in L444P GC fibroblasts treated with lacidipine (20 μΜ), diltiazem (10 μΜ), MG-132 (0.6 μΜ), and celastrol (0.6 μΜ) for 24 hrs (pO.01). mRNA expression levels were calculated as described in Figure 4. The data is reported as mean ± SD. (B) and (C) Western blot analyses of cells treated with lacidipine (20 μΜ), MG-132 (0.6 μΜ), and celastrol (0.6 μΜ) for 48 hrs using GC specific antibody. GC bands were quantified by NIH Java Image analysis software. GAPDH expression was used as a loading control. PR, proteostasis regulator; MG, MG-132; Cel, celastrol.

Figure 7 shows L444P GC activities in patient-derived fibroblasts treated with LTCC blockers. Relative L444P GC activities were evaluated in cells cultured with (A) lacidipine, (B) lercanidipine and (C) nicardipine for 24, 48 and 72 hrs, and with (D) diltiazem and (E) verapamil for 48, 72 and 120 hrs. Reported L444P GC activities were normalized to the activity of untreated cells (left y axis) and corresponded to the fraction of WT GC activity (right y axis). The data is reported as mean ± SD.

Figure 8 shows L444P GC activities of L444P GC patient-derived fibroblasts treated with an LTCC blockers and a proteostasis regulator. GC enzymatic assays were performed in cells treated with a proteostasis regulator (MG-132 0.6 μΜ or celastrol 0.6 μΜ) and an LTCC blocker (A-C) lacidipine, (D-F) lercanidipine, (G-I) nicardipine for 24, 48 and 72 hrs, and (J-L) diltiazem, (M-O) verapamil for 48, 72 and 120 hrs. Relative GC activities were evaluated as described in Figure 7.

Figure 9 shows lacidipine ameliorates cytotoxicity caused by proteostasis regulators in L444P GC patient-derived fibroblasts. Cells were cultured with (A) medium only, (B) lacidipine (20 μΜ), (C) MG-132 (0.6 μΜ), (D) lacidipine and MG-132, (E) celastrol (0.6 μΜ), and (F) lacidipine and celastrol as described in the text. Following cells incubation with Annexin V and PI, samples were analyzed by flow cytometry. Annexin V binding and PI binding are reported in the left and right side of the figure, respectively.

Figure 10 shows ERAD pathways and mechanisms of ERAD inhibition. As newly synthesized polypeptides are translocated into the ER, they are immediately recognized by BiP, which promotes substrate folding and solubility. They are then marked with an oligosaccharide precursor (GlcNAc2-Man9-Glc3), which is sequentially trimmed to allow substrate interaction with the lectin chaperones CNX and CRT. Specifically, interaction with the lectin chaperones occurs upon cleavage of the two terminal glucoses by glucosidase I and II and is terminated by removal of the last outermost glucose residue (GlcNAc₂-Man₉) by glucosidase II. At this point, natively folded proteins exit the ER, whereas partially folded intermediates are reglucosylated by UDP-glucose: glycoprotein glucosyltransferase (UGGT) and re-enter the lectin folding cycle. In order to prevent excessive accumulation of folding intermediates, unstable misfolding-prone substrates are processed by ER mannosidase I, which cleaves three to four mannose residues from the oligosaccharidic group and promotes substrate binding with the ER degradation- enhancing a-mannosidase-like lectins (EDEM). ERAD substrates are then retrotranslocated to the cytoplasm via the Sec61 retrotranslocon and polyubiquitinated. Substrate retrotranslocation is mediated by the p97 complex, which includes ubiquitin fusion degradation 1 (Ufdl) and nuclear protein localization 4 (Npl4). p97 ATPase provides the driving force for substrate extraction and shuffling to the proteasome. As shown in the schematic, kifunensine and eeyarestatin I, small molecules that function as ERAD inhibitors, block different steps of the ERAD pathway. Kifunensine inhibits ER mannosidase I, and eeyarestatin I inhibits p97 ATPase activity.

Figure 11 shows cell treatment with ERAD inhibitors enhances L444P GC activity in GD patient-derived fibroblasts. Relative L444P GC activities were evaluated in cells treated with a range of concentrations of ERAD inhibitors (Eerl or Kif) and constant doses of proteostasis regulators (MG-132 or celastrol: 0.4, 0.6, or 0.8 μΜ). Shown are relative GC activities of L444P cells treated with Eerl and MG-132 for 48 h (A),EerI and celastrol for 48 h (B), Kif and MG-132 for 120 h (C), and Kif and celastrol for 120 h (D). Relative GC activities were evaluated by normalizing GC activities measured in treated cells to the activity of untreated cells (left y axis)(p < 0.01 if not specified; *, p < 0.001). The corresponding fraction of WT GC activity is also reported (right y axis). (E) shows Western blot analyses of Endo Hi- treated and -untreated total protein content from L444P GC fibroblasts cultured with Eerl (6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) for 48 h and detected using GC-specific antibody. The solid and dashed arrows indicate Endo H-resistant and Endo H-sensitive bands, respectively. (F) shows quantification of GC bands detected by Western blot in Endo H-treated samples. Quantification of lower M_r, Endo H-sensitive bands corresponding to ER-retained GC are reported in the white portion of the bars, and quantification of higher M_r, Endo H-resistant bands corresponding to lysosomal GC are reported in the black top portions. Band analyses and quantifications were conducted using National Institutes of Health ImageJ analysis software. Experiments were repeated three times, and data points are reported as mean ± S.D. (error bars). MG is the abbreviation used for MG-132; Cel is the abbreviation used for celastrol.

Figure 12 shows ERAD inhibitors promote L444P GC folding and trafficking in GD patient-derived fibroblasts. Shown is immunofluorescence microscopy of GC and CNX(an ER marker) (A) and GC and LAMP-1 (alysosomal marker)(B) in L444P GC fibroblasts. Cells were treated with Eerl (2 and 6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) for 48 h. Colocalization of CNX (gray, column 1) and GC (red, column 2) is shown in pink (column 3). Colocalization of LAMP-1 (blue, column 1) and GC (red, column 2) is shown in purple (column 3). Heat maps of co-localization images were obtained with National Institutes of Health ImageJ analysis software (column 4). Hot colors represent positive correlation (co-localization), whereas cold colors represent negative correlation (exclusion). (C) shows GC activity of subcellular homogenate fractions. Untreated cells and cells cultured with Eerl (6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) for 48 h were fractionated. Subcellular fractions were collected, numbered from low to high density (from 1 to 8), and subjected to GC activity assays. HexA activity was also measured in each fraction obtained from untreated cells to identify fractions containing the ER (fractions 1 and 2) and lysosomes (fractions 7 and 8). The total protein concentration of each fraction was determined by Nanodrop. GC enzyme activity (left y axis) and HexA enzyme activity (right y axis) of each fraction were normalized to the corresponding protein concentrations. Experiments were repeated three times, and data points are reported as mean ± S.D. MG is the abbreviation used for MG-132.

Figure 13 shows Eerl facilitates N370S GC folding, lysosomal trafficking, and activity in GD patient-derived fibroblasts. (A) Relative N370S GC activities were measured in cells treated with proteostasis regulators (MG-132 (0.2 μΜ) and celastrol (0.2 μΜ)) and a range of Eerl concentrations for 72 h. Relative GC activities were evaluated as described in the legend to Fig. 1 1 (p < 0.01 if not specified; *, p < 0.001). Experiments were repeated three times, and data points are reported as mean ± S.D. (error bars). MG is the abbreviation for MG-132; Cel is the abbreviation for celastrol. Shown are immunofluorescence microscopy images of GC and CNX (an ER marker) (B) and GC and LAMP-1 (a lysosomal marker) (C) in cells treated with Eerl (2 μΜ) and MG-132 (0.2 μΜ) for 48 h. Colocalization images were analyzed as described in the legend to Fig. 12.

Figure 14 shows Eerl enhances G269S HexA activities in Tay-Sachs patient-derived fibroblasts. Cells were cultured with proteostasis regulators (MG-132 (0.2 μΜ) and celastrol (0.2 μΜ)) and a range of Eerl concentrations for 96 h. Relative aG269S/1278insTATC HexA activities (p O.01) were evaluated by normalizing HexA activity of treated cells to that of untreated cells (left y axis). The corresponding fraction of WT HexA activity is also reported (right y axis). Experiments were repeated three times, and data points are reported as mean ± S.D. (error bars). MG is the abbreviation for MG-132; Cel for celastrol.

Figure 15 shows up-regulation of BiP expression in L444P GC fibroblasts treated with ERAD inhibitors. Relative mRNA expression levels of BiP (p < 0.01) (A), CNX (p < 0.05) (B), and CRT (p < 0.05) (C) in L444P GC fibroblasts treated with Eerl (2 and 6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) for 24 h were obtained by quantitative RT-PCR, corrected by the expression of the housekeeping gene GAPDH, and normalized to those of untreated cells. The data are reported as mean ± S.D. (error bars). (D) Western blot analyses of BiP, CNX, CRT, and GAPDH (used as loading control) in cells treated with Eerl (2 and 6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) for 48 h. (E) quantification of Western blot bands. ER chaperone band intensities were quantified with National Institutes of Health ImageJ analysis software, corrected by GAPDH band intensities, and divided by the values obtained in untreated samples.

Figure 16 shows UPR activation in L444P GC fibroblasts treated with ERAD inhibitors. Cells were treated with Eerl (2 and 6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) for 24 h. (A) Xbp-1 mRNA splicing was determined by RT-PCR followed by gel electrophoresis. (B) spliced Xbp-1 band intensities were quantified with National Institutes of Health ImageJ analysis software. Relative mRNA expression levels of CHOP (p < 0.01) (C), ATF4 (p < 0.05) (D), and GC (p < 0.05) (E) were obtained by quantitative RT-PCR and calculated as described in the legend to Fig. 15. The data are reported as mean ± S.D. (error bars). (F) Western blot analysis of cells treated with Eerl (2 and 6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) for 48 h using GC specific antibody. GAPDH expression was used as a loading control. (G) Western blot band quantification. GC bands were quantified by National Institutes of Health ImageJ analysis software and corrected by GAPDH band intensities.

Figure 17 shows apoptosis induction in L444P GC patient-derived fibroblasts treated with ERAD inhibitors. Flow cytometry histograms of annexin V-FITC fluorescence intensities (x axis, log scale) plotted against cell counts (y axis, linear scale) obtained from the analysis of untreated cells and cells treated with MG-132 (0.6 μΜ), Eerl (2 μΜ), and Eerl (2 μΜ) and MG- 132 (0.6 μΜ) (A) or untreated cells and cells treated with G-132 (0.6 μΜ), Kif (50 nM), and Kif (50 nM) and MG-132 (0.6 μΜ) (B) for 16 h. Three independent experiments were conducted, and results of one representative experiment are reported. (C) shows propidium iodide (PI) binding population change (%) of cells treated with Eerl (2 and 6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) for 16 h compared with untreated cells (p < 0.01). The number of total counted cells was 10,000. The data are reported as mean ± S.D. (error bars).

Figure 18 shows cell treatment with Eerl enhances N370S GC folding, lysosomal trafficking and activity in GD patient-derived fibroblasts. Relative N370S GC activities were measured in cells treated with proteostasis regulators (MG-132 0.2 μΜ; celastrol 0.2 μΜ) and a range of Eerl concentrations for 72 hrs. Relative GC activities were evaluated as described in Figure 11 (p<0.01). Experiments were repeated three times and data points are reported as mean ± SD. MG is the abbreviations for MG-132; Cel is the abbreviation for celastrol.

Figure 19 shows inhibition of ERAD and modulation of Ca²⁺ homeostasis synergize to enhance the ER folding capacity in cells derived from patients with Gaucher' s disease. Lacidipine enhances ER folding by restoring Ca²⁺ homeostasis in Gaucher's disease cells. Particularly, lacidipine inhibits extracellular Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels (LTCC) on the plasma membrane and blocks ER Ca²⁺ efflux through ryanodine receptors (RyRs) on the ER membrane, thus restoring the intracellular gradient of [Ca ]. Eerl treatment enhances retention of unstable proteins in the ER. Specifically, Eerl inhibits p97 ATPase activity, which promotes retrotranslocation of misfolded substrates from the ER to the cytoplasm for proteasomal degradation (ERAD).

Figure 20 shows co-treatment of GD patient-derived fibroblasts with Eerl and lacidipine enhances the folding, lysosomal trafficking and activity of L444P GC. (A) Relative L444P GC activities were evaluated in cells treated with a range of concentrations of Eerl and constant doses of lacidipine (5, 10, or 20 μΜ) for 48 hrs. Relative GC activities were evaluated by normalizing GC activities measured in treated cells to the activity in untreated cells (left y axis), (pO.01 if not specified;*p<0.001). The corresponding fraction of WT GC activity is also reported (right y axis). Experiments were repeated three times and data points are reported as mean ± SD. Lac, lacidipine. B-C. Immunofluorescence microscopy of GC and CNX (an ER marker), and GC and LAMP-1 (a lysosomal marker) in L444P GC fibroblasts. Cells were treated with Eerl (6 μΜ), and lacidipine (10 μΜ) for 48 hrs. B. Colocalization of CNX (grey, column 1) and GC (red, column 2) is shown in pink (column 3). C. Colocalization of LAMP-1 (blue, column 1) and GC (red, column 2) is shown in purple (column 3). Heat maps of co- localization images were obtained with NIH ImageJ analysis software (column 4). Hot colors represent positive correlation (co-localization), whereas cold colors represent negative correlation (exclusion).

Figure 21 shows lacidipine treatment attenuates Eerl-mediated apoptosis induction in L444P GC patient derived fibroblasts. A. Flow cytometry histograms of Annexin V-FITC fluorescence intensities (x-axis, log scale) plotted against cell counts (y-axis, linear scale) obtained from the analysis of untreated cells and cells treated with Eerl (6 μΜ) and lacidipine (10 μΜ). Three independent experiments were conducted and results of one representative experiment are reported. B. PI binding population change (%) of cells treated with Eerl (6 μΜ) for 16 hrs compared to untreated cells (p<0.01). Number of total counted cells: 10,000. The data is reported as mean ± SD.

Figure 22 shows lacidipine treatment remodels Eerl-mediated activation of the UPR pathway in L444P GC patient-derived fibroblasts. Cells were treated with Eerl (6 μΜ) and lacidipine (10 μΜ) for 24 hrs. A. Xbp-1 mRNA splicing was determined by RT-PCR followed by gel electrophoresis. B. Spliced Xbp-1 band intensities were quantified with the NIH ImageJ analysis software. Relative mRNA expression levels of C. ATF4, D. CHOP, E. Bcl-2, and F. GC were obtained by quantitative RT-PCR, corrected by the expression of the housekeeping gene GAPDH, and normalized by that of untreated cells. The data is reported as mean ± SD. G. Western blot analysis of cells treated with Eerl (6 μΜ) and lacidipine (10 μΜ) for 48 hrs using GC specific antibody. GAPDH expression was used as a loading control. H Western blot band quantification. GC bands were quantified by NIH ImageJ analysis software and corrected by GAPDH band intensities.

Figure 23 shows lacidipine treatment attenuates BiP upregulation caused by Eerl- mediated UPR activation. A. Western blot analyses of BiP, CNX, CRT, and GAPDH (used as loading control) in cells treated with Eerl (6 μΜ) and lacidipine (10 μΜ) for 48 hrs. B. Quantification of Western blot bands. ER chaperone band intensities were quantified with NIH ImageJ analysis software, corrected by GAPDH band intensities, and divided by the values obtained in untreated samples.

Figure 24 shows upregulation of Bcl-2 protects Gaucher's disease cells from apoptosis caused by proteostasis modulation. A. Relative mRNA expression levels of Bcl-2 in cells treated with Eerl (2 and 6 μΜ), MG-132 (0.6 μΜ), and fluvastatin (100 nM) for 24 hrs were obtained by quantitative RT-PCR and calculated as described in Fig. 22. B. PI binding population change (%) of cells treated with Eerl (2 and 6 μΜ), MG-132 (0.6 μΜ), and fluvastatin (100 nM) for 16 hrs compared to untreated cells (pO.01 ). Number of total counted cells: 10,000. The data is reported as mean ± SD. C. Relative L444P GC activities of GD fibroblasts treated with Eerl (2 and 6 μΜ), MG-132 (0.6 μΜ), and fluvastatin (100 nM) for 48 hrs. Relative GC activities were evaluated as described in Fig. 20 (pO.01). Experiments were repeated three times and data points are reported as mean ± SD. Flu is the abbreviation for fluvastatin.

Figure 25 shows co-treatment of GD patient-derived fibroblasts with Eerl and lacidipine enhances the folding, lysosomal trafficking and activity of L444P GC. Relative L444P GC activities were evaluated in cells treated with a range of concentrations of Eerl and constant doses of lacidipine (5, 10, or 20 μΜ) for 72 hrs. Relative GC activities were evaluated by normalizing GC activities measured in treated cells to the activity in untreated cells (left y axis). The corresponding fraction of WT GC activity is also reported (right y axis). Experiments were repeated three times and data points are reported as mean ± SD. Lac is the abbreviation used for lacidipine.

Figure 26 shows chemically induced upregulation of Bcl-2 enhances mutated GC activity rescue. Relative L444P GC activities of GD fibroblasts treated with Eerl (2 and 6 μΜ), MG-132 (0.6 μΜ), and fluvastatin (100 nM) for 72 hrs. Relative GC activities were evaluated as described in Fig. 20. Experiments were repeated three times and data points are reported as mean ± SD. MG is the abbreviation for MG-132; Flu is the abbreviation for fluvastatin.

Figure 27 shows BiP cellular localization upon lacidipine treatment. (Related to Figure 4). Immunofluorescence microscopy of (A) CNX (an ER marker) and BiP, and (B) GM130 (a Golgi marker) and BiP in L444P GC fibroblasts. Cells were treated with lacidipine (20 μΜ) for 48 hrs. Colocalization of CNX (blue, column 1) and BiP (red, column 2) is shown in green (column 3). Colocalization of GM130 (blue, column 1) and BiP (red, column 2) is also shown in green (column 3).

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are herein described in more detail. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as illustrated, in part, by the appended claims.

DESCRIPTION The present disclosure generally relates to methods for treating lysosomal storage diseases. More particularly, the present disclosure relates to methods for treating lysosomal storage diseases, such as Gaucher' s disease, using L-type Ca²⁺ channel blockers with a 1,4 dihydropyridine structure, at least one inhibitor of ER-associated degradation, or a combination thereof. The methods of the present disclosure aid in the development of effective therapeutic strategies for the treatment of lysosomal storage diseases based on remodeling the proteostasis network to rescue folding of unstable, degradation-prone enzymes.

The main therapeutic option for GD is currently enzyme replacement therapy. Although its safety and effectiveness has been demonstrated for several other lysosomal storage diseases (LSD), including Fabry and Pompe disease, enzyme replacement therapy fails to provide economically sustainable treatment and efficiently address several aspects of the disease. Specifically, enzyme replacement therapy is limited to the treatment of non- neuronopathic symptoms due to inability of the intravenously injected recombinant enzyme to cross the blood-brain barrier.

Patients with GD who are homozygous for the L444P GC allele typically present severe neuronopathic symptoms. However, currently available treatments are inadequate to treat patients with neuronopathic GD. Rescuing the function of mutated GC variants is an appealing alternative to the currently available therapeutic options, mainly enzyme replacement therapy (Cerezyme®) and substrate reduction therapy, which are not adequate for the treatment of neuropathic forms of GD (Sidransky et al., 2007). Hence, considerable effort has been recently devoted to the design of strategies to rescue cellular folding, trafficking, and activity of mutated GC variants associated with neuropathic GD (Mu et al., 2008a; Mu et al., 2008b; Ong et al, 2010; Wang et al., 201 lb),

A number of highly prevalent alleles associated with LSD development contain non- inactivating, destabilizing mutations. Such protein variants retain function if forced to fold into their native structure. Hence, efforts have been recently devoted to the development of strategies to rescue folding, trafficking, and activity of unstable substrates, with particular attention to those associated with the manifestation of neuronopathic symptoms. Inhibition of ERAD enhances folding, trafficking, and lysosomal activity of mutated enzyme variants that cause two clinically distinct LSD, Gaucher and Tay-Sachs disease. The present disclosure suggests that ERAD limits the folding of secretory proteins containing misfolding, destabilizing mutations and provides proof of principle of ERAD inhibition as a viable strategy to rescue loss-of- function phenotypes in fibroblasts derived from individuals with LSD. The ERAD pathway is part of a complex quality control network that ensures correct folding and processing of active proteins and eliminates non-native, off-pathway products. A simplified schematic is reported in Fig. 10. As newly synthesized proteins are translocated into the ER, they immediately interact with BiP, which facilitates their folding while preventing aggregation. Substrates are marked with oligosaccharide precursors (GlcNAc₂-Man₉-Glc₃) and subsequently trimmed by ER glucosidases to allow recognition by the lectin chaperones (CNX and CRT). Upon removal of the outermost glucose residue (GlcNAc Mang), natively folded proteins exit the ER and proceed through the secretory pathway, whereas misfolded intermediates are reglucosylated by UDP-glucose: glycoprotein glucosyltransferase. This cycle repeats itself until substrates either reach native folding or are recognized as irreversibly misfolded by ER degradation-enhancing a-mannosidase-like lectins. Removal of three to four mannose residues by ER mannosidases, and particularly mannosidase I, marks misfolded substrates for degradation, which proceeds through polyubiquitination and retrotranslocation via the p97 complex. (Fig. 10). Although rescue of mutated GC in patient-derived cells treated with proteasome inhibitors has been previous reported, the role of the ERAD pathway in the processing of mutated GC variants remains elusive.

Modulating the proteostasis network to enhance the innate cellular folding capacity restores the folding and function of different GC variants (Mu et al. 2008b). Proteostasis modulation is achieved by chemically inducing the upregulation of molecular chaperones and inhibiting the degradation of misfolded proteins. Small molecule proteostasis regulators, however, typically function by triggering cellular stress, and particularly the unfolded protein response (UPR), which, if sustained, results in activation of apoptosis. Hence, particular effort has been recently devoted to modulate the cellular folding capacity and rescue the folding of GC mutants without causing activation of the apoptotic cascade. Thus, according to one aspect of the present disclosure, simultaneously inhibiting ERAD and enhancing the ER folding capacity by co-administering an L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure and an inhibitor of ER-associated degradation to a subject with GD results in the synergistic rescue of the folding and activity of certain GC variants, particularly the severely destabilized L444P GC variant.

In one embodiment, the methods of the present disclosure comprise administering to a subject a therapeutically effective amount of a L-type Ca²⁺ channel blocker with a 1 ,4 dihydropyridine structure. As used herein, the term "subject" refers to at least one cell or a mammal. As used herein, the term "therapeutically effective amount" refers to the amount that will elicit the desired biological or medical response. In one embodiment, at least one cell may be a fibroblast derived from an individual with LSD. The correlation between the extent of GC variants residual activity in fibroblasts derived from an individual with LSD and clinical severity of the disease, including the occurrence of neuronopathic symptoms, has been established (Beutler et al., 1984; Meivar-Levy et al., 1994; Michelakakis et al., 1995). Hence, fibroblasts derived from an individual with LSD have been repeatedly used to investigate how Ca²⁺ homeostasis modulation affects the folding of mutated GC variants (Mu et al., 2008a; Ong et al., 2010; Wang et al., 2011b).

While not wishing to be bound to any particular theory, it is currently believed that the methods of the present disclosure allow for the rescue of mutated enzyme activity, such as glucosilcerebrosidase activity, by simultaneously i) lowering the cytoplasmic concentration of Ca²⁺ thereby restoring Ca²⁺ homeostasis in the cell, ii) upregulating the expression of the molecular chaperone BiP/GRP78, iii) inducing the unfolded protein response (UPR) which enhances cellular folding and involves upregulation of glucosilcerebrosidase gene, and iv) ameliorating cytotoxicity and preventing apoptosis induction by modulation of pro- and anti- apoptotic genes. The latter is particularly important because proteostasis regulators discovered to date function by inducing the unfolded protein response, which if sustained, results in activation of apoptosis, and thus are inevitably cytotoxic.

Examples of suitable L-type Ca²⁺ channel blockers with a 1,4 dihydropyridine structure may include, but are not limited to, lacidipine, lercanidipine, nifedipine, nitrendipine, nicardipine, nimodipine, nisoldipine, manidipine, amlodipine, isradipine, felodipine, cilnidipine, and benidipine. Other molecules that recapitulate the mechanism of action of L-type Ca²⁺ channel blockers with a 1,4 dihydropyridine structure, particularly, lacidipine, may also be suitable. In some embodiments, lacidipine may also inhibit [Ca²⁺]eR efflux to enhance folding, trafficking and activity of degradation-prone GC variants. In some embodiments, suitable L-

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type Ca channel blockers with a 1 ,4 dihydropyridine structure induce remodeling of mutated GC proteostasis by simultaneously activating a series of distinct molecular mechanisms, namely modulation of Ca²⁺ homeostasis, upregulation of the ER chaperone BiP, and moderate induction of the unfolded protein response.

The nature of the chemical structure of L-type Ca²⁺ channel blockers with a 1 ,4 dihydropyridine structure, particularly their hydrophobicity, likely influences their cell permeability. The hydrophobicity of these blockers may explain why treatment of GD fibroblasts with L-type Ca²⁺ channel blockers with a 1 ,4 dihydropyridine structure, such as lacidipine, resulted in higher depletion of cytosolic [Ca²⁺] and more effective remodeling of L444P GC proteostasis as compared to other L-type Ca²⁺ channel blockers, such as diltiazem and verapamil, which are charged at physiologic pH.

In particular, the chemical properties of L-type Ca²⁺ channel blockers with a 1,4 dihydropyridine structure are different from the two other classes of L-type Ca channel blockers, benzothiazepines and phenylalkylamines. One example of a benzothiazepine is diltiazem. One example of a phenylalkylamine is verapamil. First, both diltiazem and verapamil are charged species at physiologic pH, and thus exhibit hydrophilic properties. They access the channel through a polar and hydrophilic pathway, particularly open channel gates. L-type Ca²⁺ channel blockers with a 1,4 dihydropyridine structure are predominantly hydrophobic, and they access binding sites through membrane-delimited pathways. (Triggle 2003). In a membrane-delimited pathway, a compound generally traverses a short distance through the membrane to a closely associated ion channel. Verapamil and diltiazem exhibit frequency-dependent interactions with L-type channels in that the interaction is enhanced as the frequency of the depolarizing stimulus increases. Since L-type Ca²⁺ channel blockers with a 1 ,4 dihydropyridine structure are neutral and hydrophobic, they interact with depolarized (open and inactivated) channel states. Moreover, increasing levels of maintained depolarization enhance the interaction potency of L-type Ca²⁺ channel blockers with a 1 ,4 dihydropyridine structure with L-type channels (Triggle 2006). Because L-type Ca channel blockers with a 1 ,4 dihydropyridine structure preferentially target depolarized membranes, they exhibit higher specificity for neurons than diltiazem and verapamil. The antagonism of L-type Ca ⁺ channel blockers with a 1 ,4 dihydropyridine structure, particularly isradipine and nimodipine, was shown to effectively stop dendritic Ca²⁺ oscillations but left autonomous pacemaking unchanged (in neuron cells) (Guzman et al. 2009). At concentrations that effectively antagonize L-type channels, L-type Ca²⁺ channel blockers with a 1,4 dihydropyridine structure attenuate intracellular Ca ⁺ oscillations and do not affect the activity of other channels. As proved in dopaminergic neurons of the substantia nigra pars compacta, autonomous pacemaking in these cells is a multiple-channel involved process.

As opposed to previously reported proteostasis regulators that rescue mutated GC folding by activating the unfolded protein response, L-type Ca²⁺ channel blockers with a 1 ,4 dihydropyridine structure suitable for use in the present disclosure do not cause cytotoxicity, but generally prevents apoptosis induction typically associated with sustained activation of the unfolded protein response. Because Ca blockers influence a number of cellular functions, the direct use Ca²⁺ blockers for the treatment of LSD may raise issues, such as, for example, undesired side effects. Thus, the present disclosure aids in understanding the cellular mechanisms activated by L-type Ca²⁺ channel blockers with a 1,4 dihydropyridine structure that facilitate glucosilcerebrosidase rescue. The present disclosure will open the way to the discovery of new drugs with similar mechanisms and no undesired side effects.

In another embodiment, the methods of the present disclosure comprise administering to a subject a therapeutically effective amount of at least one inhibitor of ER-associated degradation. While not wishing to be bound to any particular theory, treatment with at least one inhibitor of ER-associated degradation at least partially restores folding, trafficking, or activity of mutated enzymes by prolonging ER retention.

In another embodiment, the methods of the present disclosure comprise administering to a subject a therapeutically effective amount of a L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure and a therapeutically effective amount of an inhibitor of ER- associated degradation. While not wishing to be bound to any particular theory, it has been reported that the rescue of folding and trafficking of mutated enzymes occurs upon chemical inhibition of specific steps of the ERAD pathway. Generally, certain inhibitors of ER- associated degradation induce UPR and apoptosis. However, administering an L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure lowers the inhibitor mediated UPR induction and apoptosis. Certain anti-apoptotic genes are upregulated by treatment with an L- type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure, which prevents induction of apoptosis that may occur upon treatment with inhibitors of ER-associated degradation.

Examples of suitable inhibitors of ER-associated degradation may include, but are not limited to, eeyarestatin I (Eerl), kifunensine (Kif), and small molecules that recapitulate the mechanism of action of eeyarestatin and kifunensine. Eerl interferes with retrotranslocation of misfolded substrates to the cytoplasm by inhibiting p97 ATPase activity, whereas Kif interferes with recognition of misfolded substrates by inhibiting ER mannosidase I. In particular, treatment of GD cells with Eerl results in dramatic rescue of folding and lysosomal activity of multiple GC variants. ERAD inhibition via Eerl treatment prolongs ER retention of mutated GC variants, thus enhancing the pool of GC folding intermediates amenable to folding rescue. Moreover, rescue of mutated HexA was observed in Tay-Sachs disease cells upon treatment with Eerl. However, Eerl treatment causes a dramatic induction of unfolded protein response and apoptosis. ,Kif-mediated inhibition of early substrate recognition, however, which is likely to prolong ER retention and substrate folding without causing accumulation of irremediably misfolded proteins, causes minimal activation of UPR and does not result in the induction of apoptosis. Restoring Ca²⁺ homeostasis in GD cells would create a folding environment particularly amenable to rescue of mutated GC folding via ERAD inhibition. Therefore remodeling the proteostasis network by inhibiting retrotranslocation and degradation of unstable GC variants to increase their retention in the ER and by restoring Ca homeostasis to enhance chaperone mediated folding may solve the problems associated with the induction of the unfolded protein response and apoptosis by treatment with ERAD inhibitors (Fig. 19). In one embodiment, simultaneously inhibiting ERAD and enhancing the ER folding capacity by coadministering lacidipine and Eerl results in synergistic rescue of the folding and activity of GC variants. Moreover, lacidipine treatment lowers Eerl-mediated unfolded protein response induction and apoptosis. Upregulation of the anti-apoptotic gene Bcl-2 associated with lacidipine treatment plays a key role in preventing the induction of apoptosis in Eerl-treated cells.

In some embodiments, the methods of the present disclosure may be particularly appealing because they hold promise for the treatment of Gaucher's disease types associated with neuropathic symptoms, which are currently a completely unmet medical need. Current therapeutic options for Gaucher's disease are enzyme replacement therapy (based on injection of recombinantly produced glucosilcerebrosidase, which cannot cross the blood brain barrier and is thus limited to the treatment of symptoms that do not affect the central nervous system) and substrate replacement therapy (associated with numerous side effects and limited efficacy).

To facilitate a better understanding of the present invention, the following examples of certain aspects of some embodiments are given. In no way should the following examples be read to limit, or define, the entire scope of the invention.

EXAMPLES

Experimental Procedures:

Reagents and cell cultures

Celastrol and MG-132 were purchased from Alexis Biochemicals. Conduritol B Epoxide (CBE) and lacidipine were from Toronto Research Chemicals. 4-methylumbelliferyl β- D-glucoside (MUG), Nicardipine and Lercanidipine were from Sigma-Aldrich. Diltiazem and verapamil were from Tocris bioscience. Cell culture media were purchased from Gibco. GD patient-derived fibroblasts homozygous for the L444P (1448T>C) mutation (GM10915), and N370S (1226A>G) mutation (GM00852), and wild type fibroblasts (GM00498), were obtained from Coriell Cell Repositories. Fibroblasts were grown at 37°C in 5% C0₂ in minimal essential medium with Earle's salts supplemented with 10% heat-inactivated fetal bovine serum and 1 % glutamine Pen-Strep. Culturing medium was replaced every 3 or 4 days. Monolayers were passaged upon reaching confluency with TrypLE Express.

Glucocerebrosidase (GC) activity assay

The intact cell GC activity assay was performed as previously described (Mu et al., 2008b). Briefly, 10⁴ cells were plated in each well of a 96-well plate (100 μΐ per well) and incubated overnight to allow cell attachment. Cells were washed with PBS, medium replaced with fresh medium containing small molecules and plates were incubated at 37°C (the small molecule concentration and time of incubation are specified in each experiment). The medium was then removed and monolayers were washed with PBS. The assay reaction was started by adding 50 μΐ of 2.5 mM MUG in 0.2 M acetate buffer (pH 4.0) and stopped by adding 150 μΐ of 0.2 M glycine buffer (pH 10.8) to each well after incubating 7 hrs at 37°C. Liberated 4- methylumbelliferone was measured (excitation 365 nm, emission 445 nm) with a SpectraMax Gemini plate reader (Molecular Device). 1 mM CBE was added to control experiments to measure non-lysosomal GC activity and evaluate the background noise. GC activities were normalized to the activity of untreated cells.

Quantitative RT-PCR

RT-PCR was conducted as previously described (Wang et al., 201 1b). Briefly, cells were incubated with small molecules for 24 hrs before total RNA was extracted using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from total RNA using the Reverse Transcription Kit (Invitrogen). Quantitative PCR reactions were performed using 200 ng/μΐ cDNA, the QuantiTect SYBR Green PCR Kit (Applied Biosystems), and primers (listed in Table 1) using a CFX96™ Real-Time PCR detection system (Bio-Rad). Samples were heated for 15 minutes at 95°C and amplified in 45 cycles of 15 s at 94°C, 30 s at 57°C, and 30 s at 72°C. Analysis was done using CFX manager software (Bio-Rad) and threshold cycle (Cj) was extracted from the PCR amplification plot. ACT were calculated by subtracting the Or of a housekeeping gene (GAPDH) from the C of each target gene and. The relative mRNA expression levels of a target gene in cells cultured in the presence of small molecules were normalized to that of untreated cells: relative mRNA expression level = 2 exp [-(Δ CT (treated cells) - Δ CT (untreated cells))]. Each data point was evaluated in triplicate and measured three times. RT-PCR analysis of Xbp-1 splicing was performed using total cDNA, Taq DNA polymerase, and the Xbp-1 primers listed in Table 1 following reverse transcription. PCR products were separated on a 2.5% agarose gel. The intensity of spliced Xbp-1 was quantified by Java Image processing and analysis software (NIH). TABLE 1

Western blot analysis

Cells were incubated with small molecules for 48 hrs, collected and lysated with the lysis-M buffer containing the protease inhibitor cocktail (Roche). Total protein concentrations were determined by Bradford assay and each sample was diluted to the same protein concentration. EndoH treatment was performed by incubating samples at 95 °C for 10 min, followed by incubation with EndoH (New England Biolabs) for 1 hrs at 37°C. Aliquots of cell lysates were separated by 10% SDS-PAGE gel, and Western blot analyses was performed using primary antibodies (rabbit anti-BiP, and mouse anti-CRT (Stressgen), mouse anti-CNX, and rabbit anti-Glucocerebrosidase (Sigma-Aldrich), or rabbit anti-GAPDH (Santa Cruz Biotechnology)) and appropriate secondary antibodies (HRP-conjugated goat anti-rabbit (Santa Cruz Biotechnology) or goat anti-mouse IgG (Stressgen)). Blots were visualized using SuperSignal West Femto Maximum (Pierce) and quantified by Java Image processing and analysis software (NIH).

Immunofluorescence

L444P patient-derived fibroblasts were seeded on glass coverslips, cultured in the presence of small molecules for 48 hrs, and fixed with 4% paraformaldehyde for 30 min. Cells were permeabilized with 0.1 % Triton-X for 5 min and incubated with 8% BSA for 1 hr. Cells were incubated for 1 hrs with primary antibodies (rabbit anti- Glucocerebrosidase and mouse anti-CNX antibodies, Sigma-Aldrich). Upon washing with 0.1% Tween-20/PBS, cells were incubated with secondary antibodies for 1 hr (Dylight 488 goat anti-mouse IgG and Dylight 549 goat antirabbit IgG from KPL, and FITC anti-LAMPl from Biolegend). Images were obtained using an Olympus 1X81 confocal microscope and analyzed using the Fluoview software. Colocalization images were analyzed using MATLAB and green was designated as the merged color.

Intracellular [Ca ] measurement

Fura-2, AM (Anaspec) was used to measure cytosolic [Ca²⁺] according to company's instructions. Briefly, 10⁴ cells were plated in each well of a 96- well plate and incubated overnight to allow cell attachment. Ca²⁺ blockers were added to the medium, cells were washed twice with PBS and incubated with 5 μΜ Fura-2, AM and 0.05% (w/v) Pluronic F-127 (Invitrogen) at 37 °C for 30 min. Following two washing steps, fluorescence was measured using excitation at 340 nm and 380 nm and emission at 510 nm with a TEC AN Infinite Ml 000 fluorescence plate reader. Fluorescence ratio of excitation 340/380 reflects relative intracellular Ca2+ level. Each data point was the average of at least 6 replicates.

Toxicity assay

Toxicity assays were conducted as described previously (Wang et al., 201 l b). L444P patient-derived fibroblasts were cultured in the presence of small molecules for 16 hrs at 37 °C. Cell toxicity was evaluated with the CytoGLO™ Annexin V-FITC Apoptosis Detection Kit (IMGENEX) according to the manufacturer's instructions and quantified by flow cytometry (FACSCantoTM II, Beckon Dickingson) with a 488-nm Argon laser.

Statistical analysis

All data is presented as mean ± s.d., and statistical significance was calculated using a two-tailed t-test.

Results

Treatment with small molecule LTCC and RyRs blockers enhances folding, trafficking and activity of mutated GC in patient-derived fibroblasts.

A series of LTCC blockers with 1 ,4-dihydropyridine structure were investigated, particularly nicardipine, lacidipine, lercanidipine, nifedipine, and nitrendipine (Triggle, 2003). Patient-derived fibroblasts harboring L444P GC were treated with a range of Ca blocker concentrations for 5 days and GC activities were evaluated every 24 hours with the intact cell GC enzymatic activity assay (Mu et al., 2008b) (Figure 7). Verapamil and Diltiazem, prototypes of the other two classes of LTCC blocker (phenylalkylamines and benzothiazepines, respectively) were included for comparison as they were previously reported to partially rescue mutated GC folding (Mu et al., 2008a). Culturing conditions resulting in maximal rescue of L444P GC activity are reported in Figure 1 A. L444P GC activity was observed to increase up to 2.0-fold in cells treated with lacidipine (20 μΜ, final medium concentration, p<0.001) for 72 hrs compared to untreated cells, which corresponds to about 25% of the WT cellular activity, and is expected to ameliorate GD symptoms (Schueler et al., 2004). A milder increase in L444P GC activity (1.2-fold, p<0.01) was detected in the same cells treated with lercanidipine and nicardipine (20 μΜ) for 72 hrs, compared to untreated cells (Figure 1A). Nifedipine and nitrendipine treatment failed to rescue the activity of L444P GC (data not shown). Lacidipine was observed to enhance the activity of L444P GC to a considerably higher degree than diltiazem and verapamil tested under the same conditions (Figure 7). Maximal L444P GC activity increase was observed upon diltiazem (10 μΜ, 1.4-fold) and verapamil (5 μΜ, 1.1 -fold) treatment for 120 hrs (Figure 1 A).

Similar to diltiazem and verapamil, lacidipine blocks LTCC on the plasma membrane as well and RyRs on the ER membrane (Gunther et al., 2008). Nicardipine and lercanipine are known to block LTCC and were reported to interfere with the release of Ca²⁺ from the ER (Wishart et al., 2008), while nifedipine and nitrendipine are thought to only interact with LTCC (Gunther et al., 2008). These reported binding interactions, together with results from the GC activity assays reported above (Figure 1A), suggest a correlation between the mechanism of Ca²⁺ mobilization and the extent of L444P GC folding rescue. Specifically, a higher increase in L444P GC activity results from treatment with Ca²⁺ blockers that antagonize both LTCC and RyRs.

It was then investigated whether treatment with proteostasis regulators, such as MG- 132 and celastrol, applied in combination with LTCC blockers enhance the rescue of L444P GC folding, as previously demonstrated for RyRs blockers (Wang et al., 2011b). Proteostasis regulation was achieved via cell treatment with either MG-132 (0.6 μΜ) or celastrol (0.6 μΜ), which are known to rescue L444P GC folding through a mechanism distinct from Ca²⁺ homeostasis modulation (Mu et al., 2008b). Patient-derived fibroblasts were cultured in medium supplemented with an LTCC blocker and a proteostasis regulator for up to 5 days and GC activity was measured every 24 hours (Figure 1B-F and Figure 8). Co-administration of lacidipine (20 μΜ) and MG-132 for 72 hrs resulted in a dramatic increase in L444P GC activity compared to untreated cells (5.1 -fold, pO.001 ; Figure IB), which corresponds to 64% of wild type GC activity and is significantly higher than what was observed treating the cells only with lacidipine (2.0-fold, Figure 1A) or MG-132 (2.7-fold, Figure IB). Addition of lacidipine was observed to also enhance celastrol mediated increase in L444P GC activity (2.6-fold, Figure IB). Interestingly, lercanidipine (20 μΜ) and nicardipine (5 μΜ) enhanced MG-132 mediated L444P GC activity rescue (4.3-fold and 3.2-fold (pO.001), respectively), but failed to improve celastrol activity (Figure 2C-D). Diltiazem and verapamil were observed to synergize with proteostasis regulators with lower efficiency than 1 ,4-dihydropyridines. Particularly, treatment with diltiazem (10 μΜ) and verapamil (5 μΜ) moderately enhanced celastrol mediated L444P GC folding rescue (2.1- and 1.9-fold, respectively), and failed to alter MG-132 mediated rescue (Figure 2E-F).

N370S GC is the most common GC variant exhibiting low residual activity (Meivar- Levy et al., 1994). Cellular folding rescue and enhancement of N370S GC activity was previously reported (Mu et al, 2008a; Mu et al., 2008b; Offman et al, 2010; Sawkar et al., 2002; Wang et al., 201 lb; Yu et al., 2007). As opposed to L444P GC, N370S GC folding was previously shown to be amenable to rescue with GC specific chemical chaperones, suggesting that the location and nature of these two mutations have different destabilizing effects on the enzyme's native folding and_^ cellular trafficking (Sawkar et al., 2005; Sawkar et al., 2002). In addition, GD patients carrying the N370S GC variant never present neuronopathic GD symptoms typically associated with L444P GC (Michelakakis et al., 1995). To verify whether lacidipine mediated rescue of mutated GC folding is restricted to the L444P GC variant, GD patient-derived fibroblasts carrying N370S GC were cultured in the presence of lacidipine and proteostasis regulators, and GC activities were measured. Diltiazem was used for comparison as previously shown to cause increase in N370S GC folding (Mu et al., 2008a). Similar to what was reported above for L444P GC fibroblasts, lacidipine (20 μΜ) treatment for 72 hrs resulted in an increase in N370S GC activity (1.8-fold, p<0.001; Figure 1G), which was enhanced by the addition of MG-132 and celastrol (3.4- and 2.0-fold, respectively, Figure 1H). Diltiazem (10 μΜ, 1.4-fold; Figure 1G) mediated increase in N370S GC activity was enhanced by the addition of MG-132 and celastrol (3.1- and 1.7-fold, respectively, Figure II). These results suggest that lacidipine rescues the folding and activity of different mutated GC variants and thus functions as a proteostasis regulator in GD patient-derived fibroblasts.

In order to confirm that the increase in activity detected in cells treated with lacidipine results from rescue of mutated GC folding and trafficking to the lysosome, L444P GC glycosylation state and cellular localization were tested. GC glycosylation state was investigated by endoglycosidase H (EndoH) treatment. EndoH hydrolyzes high mannose, immature N-linked glycoproteins. EndoH treatment followed by GC detection by Western blot typically reveals a low MW band corresponding to partially glycosylated, ER-retained GC (EndoH-sensitive) and a high MW band corresponding to fully glycosylated, lysosomal GC (EndoH-resistant) (Maley et al., 1989). The total protein content of cells cultured in media supplemented with lacidipine (20 μΜ), MG-132 (0.6 μΜ), celastrol (0.6 μΜ), or a combination thereof for 48 hrs was subjected to EndoH treatment and GC was detected by western blot. A representative western blot (Figure 2A) and quantification of EndoH-resistant and EndoH-sensitive GC bands (Figure 2B) are reported. In untreated cells, nearly all L444P GC was detected as EndoH-sensitive, as expected (Mu et al., 2008b). However, a band corresponding to EndoH-resistant L444P GC was detected in cells treated with lacidipine and its intensity was comparable to that detected in cells cultured with MG-132 or celastrol (the results obtained from the experiments conducted with MG-132 and celastrol have been previously shown (Mu et al., 2008b), and are reported here for comparison). Interestingly, lacidipine treatment resulted in an about 2-fold increase of total L444P GC, and a decrease of EndoHsensitive fraction to 80% of total GC. Co-treatment with lacidipine and MG-132 was observed to cause a 2.5-fold increase in the EndoH-resistant pool of L444P GC, compared to cells treated with either molecule alone (Figure 2A-B), which correlated with results obtained from GC enzymatic assays.

L444P GC cellular localization was evaluated using immunofluorescence microscopy of L444P GC patient-derived fibroblasts treated with lacidipine (20 μΜ) and MG-132 (0.6 μΜ) for 48 hrs and using antibodies specific for GC, for an ER marker (Calnexin, CNX), and for a lysosomal marker (LAMP-1). Co-localization of GC and CNX (Figure 2C) and GC and LAMP- 1 (Figure 2D) is reported in green. L444P GC was barely detectable in untreated cells due to extensive ERAD (Figure 2C-D), as previously reported (Michelakakis et al., 1995). Analysis of merged images revealed the presence of a large pool of enzyme in the ER (Figure 2C) and in the lysosome (Figure 2D) in lacidipine treated cells, suggesting that lacidipine treatment increases the pool of folded L444P GC that escapes ERAD and traffics to the lysosomes. Moreover, co- treatment with lacidipine and MG-132 further increased the pool of ER and lysosomal GC (Figure 2C-D), demonstrating that these two molecules synergize to rescue L444P GC folding and trafficking, and confirming the results obtained from enzymatic assays (Figure 1 A-B).

It was previously showed that RyRs inhibition creates an environment more amenable to L444P GC proteostasis than that of the ER of untreated GD fibroblasts (Wang et al, 2011b). The results reported here suggest that combining inhibition of RyRs and LTCC enables direct rescue of L444P GC proteostasis. Lacidipine treatment, however, seems to rescue L444P GC folding and activity more efficiently than the other LTCC blockers tested. This suggests that lacidipine is a more potent modulator of intracellular [Ca²⁺] than other Ca²⁺ blockers used here and previously (Mu et al., 2008a; Ong et al., 2010; Wang et al., 2011b), or that cell treatment with lacidipine rescues mutant GC folding by activating other cellular mechanisms that influence the mutated GC folding free energy diagram. The following studies were conducted to investigate these hypotheses. Diltiazem was used as comparisons in these studies because, although it also inhibits LTCC and RyRs and was reported to enhance the folding of mutated GC variants (Mu et al., 2008a), it is shown here to rescue L444P GC folding to a significantly lower extent than lacidipine. In addition, the mechanism involved in diltiazem mediated GC variants folding rescue still remains elusive.

Lacidipine depletes cytosolic free [Ca²⁺] in Gaucher's disease patient-derived fibroblasts.

Glucosylceramide buildup causes [Ca²⁺]ER efflux and elevation of cytosolic [Ca²⁺] in GD cells (Korkotian et al., 1999). Lacidipine and diltiazem, by binding to LTCC and RyRs, are expected to lower cytosolic [Ca²⁺] and increase [Ca²⁺]E_R, respectively. It was investigated whether the larger increase in mutated GC variants activity caused by cell treatment with lacidipine compared to diltiazem correlates with their different effect on intracellular Ca²⁺ mobilization. Cytosolic free [Ca ⁺] was evaluated by monitoring changes in Fura-2 fluorescence (Ong et al., 2010) in L444P, N370S, and wild type GC fibroblasts treated with lacidipine or diltiazem (Figure 3). Lacidipine treatment was observed to deplete cytosolic [Ca²⁺] with higher efficiency than diltiazem treatment in all cell types. In addition, depletion of cytosolic [Ca²⁺] is markedly more enhanced in L444P GC than in N370S GC cells, suggesting a correlation between LTCC blocker mediated Ca²⁺ homeostasis modulation and rescue of mutated GC variants' folding.

Lacidipine treatment upregulates BiP expression in L444P GC fibroblasts.

It was previously reported that the ER luminal chaperone BiP plays a key role in L444P GC folding. Upregulation of BiP expression, in combination with moderate UPR induction through MG-132 treatment, was shown to dramatically enhance the folding of L444P GC (Wang et al., 2011b). It was investigated whether cell treatment with lacidipine influences the expression of ER chaperones and conducted quantitative RT-PCR analyses to measure the expression of the representative chaperones BiP, Calnexin (CNX), and Calreticulin (CRT) in L444P GC fibroblasts treated with lacidipine (20 μΜ), diltiazem (10 μΜ), MG-132 (0.6 μΜ), celastrol (0.6 μΜ), or a combination thereof. BiP expression (Figure 4A) was dramatically upregulated by lacidipine treatment (5.6- fold, pO.01), and lacidipine and MG-132 co-treatment (13.1-fold, pO.01). Diltiazem treatment resulted in a milder increase in BiP expression (1.8- fold), even when used in combination with MG-132 (3.1 -fold). Even though celastrol treatment was observed to cause a modest increase in BiP expression (1.9-fold), supplementing celastrol- containing medium with a LTCC blocker did not influence BiP transcription. CNX (Figure 4B) was mildly upregulated by lacidipine (2.6-fold) as well as by lacidipine and MG-132 treatment (2.8-fold), while a lower increase in CNX expression was observed in cells treated with diltiazem (1.5-fold), and diltiazem and MG-132 (1.8-fold). A modest increase in CNX expression was also observed in cells treated with celastrol and lacidipine (1.6-fold), and with celastrol and diltiazem (1.5-fold), which was actually lower than the increase in CNX expression caused by treatment with celastrol alone. CRT expression was not substantially altered by any of the small molecule treatment, with the exception of lacidipine, which caused a 2.9-fold increase (Figure 4C).

ER chaperone expression in cells treated with lacidipine (20 μΜ), diltiazem (10 μΜ), and MG-132 (0.6 μΜ) was confirmed by Western blot using chaperone specific antibodies (Figure 4D). BiP protein accumulation was enhanced by treatment with lacidipine alone or in combination with MG-1 2 compared to untreated cells, but only slightly enhanced by diltiazem and MG-132 treatment. CNX and CRT protein levels did not seem to be drastically altered. These results are consistent with RT-PCR analyses, and confirm the key role of BiP expression in promoting native folding of L444P GC (Wang et al., 201 lb).

As opposed to what was previously observed investigating RyRs blockers, which do not directly modulate the expression of ER chaperones, despite dramatically enhancing MG-132 mediated L444P GC folding rescue (Wang et al., 201 1b), this data indicates that lacidipine's mechanism of action is based on extensive remodeling of ER chaperone pathways. However, it was found that although cell treatment with lacipidine significantly enhances BiP expression, it does not alter its cellular localization. Particularly, immunofluorescence studies conducted to test BiP localization in the ER and in the Golgi revealed that BiP is still primarily localized in the ER. (Figure 27).

Lacidipine treatment causes modest activation of all three arms of the UPR, but does not induce cytotoxicity in L444P GC patient-derived fibroblasts.

The unfolded protein response (UPR) is a tripartite signal transduction cascade activated in response to the accumulation of misfolded proteins in the ER. UPR induction is mediated by the activation of three integral ER membrane proteins, namely activating transcription factor 6 (ATF6), double-stranded RNA-activated ER kinase (PERK), and the inositol requiring kinase 1 (IRE1) (Schroder and Kaufman, 2005), which lead to the upregulation of UPR related genes, including chaperones and ERAD proteins. The expression of ATF6, PERK, and IREl was monitored to evaluate UPR induction in GD patient-derived fibroblasts treated with lacidipine and diltiazem. Lacidipine was found to activate each one of the three arms of the UPR, and with higher efficiency than diltiazem. The increase in expression of UPR associated genes in cells treated with lacidipine was found to be comparable to that of cells treated with MG-132 and considerably enhanced in cells treated with both molecules. These results correlate with measurements of L444P GC activity reported above, in which maximal increase was obtained upon co-treatment with lacidipine and MG-132 (Figure 1).

Activation of IREl causes X-box binding protein- 1 (Xbp-1) mRNA cleavage (Ron and Walter, 2007). The product of Xbp-1 spliced mRNA acts as an activator of UPR target genes, whereas the product of the unspliced Xbp-1 precursor acts as a repressor (Ron and Walter, 2007). RTPCR experiments followed by gel electrophoresis were conducted to evaluate the accumulation of the spliced and unspliced forms of Xbp-1 in L444P GC fibroblasts treated with LTCC blockers (lacidipine (20 μΜ) or diltiazem (10 μΜ)) and a proteostasis regulator (MG-132 (0.6 μΜ) or celastrol (0.6 μΜ)) for 24 hrs (Figure 5 A). As previously reported (Mu et al., 2008b), treatment of L444P GC fibroblasts with MG-132 enhanced Xbp-1 splicing. The increase in Xbp-1 splicing observed in cells treated with lacidipine was comparable to that mediated by MG-132, indicating a similar effect of these two molecules on the activation of this arm of the UPR. A 2.5-fold increase in spliced Xbp-1 accumulation was observed upon co- treatment with lacidipine and MG-132 compared to treatment with MG-132 only, recapitulating the synergistic effect of lacidipine and MG-132 observed in enzymatic assays (Figure 5A-B). A nearly 2-fold increase in spliced Xbp-1 was detected in L444P GC fibroblasts treated with diltiazem and MG-132 compared to that measured in fibroblasts treated with MG-132 only. Even though cell treatment with celastrol causes increase in Xbp-1 splicing and in L444P GC folding rescue (Mu et al., 2008b), addition of celastrol to the media of lacidipine- or diltiazem- treated cells did not increase Xbp-1 splicing. Taken together, these results suggest a synergistic effect of LTCC blockers lacidipine and diltiazem and the proteostasis regulator MG-132 on the activation of the IRE1 arm of the UPR in L444P GC patient-derived fibroblasts. Among the culturing conditions investigated, the highest the degree of Xbp-1 splicing was observed in cells displaying the maximum increase of L444P GC activity (lacidipine and MG-132 treatment, Figure 1), suggesting a key role of IRE 1 activation in rescuing L444P GC folding.

The second arm of the UPR is mediated by ATF6 activation (Ron and Walter, 2007).

Quantitative RT-PCR was used to evaluate ATF6 expression in cells treated as described above. Lacidipine treatment resulted in ATF6 upregulation (2.1 -fold), which was further enhanced by the addition of MG-132 (3.5-fold), suggesting that the ATF6 arm of the UPR is also activated by treatment with lacidipine, particularly when used in combination with MG-132 (Figure 5C). Diltiazem treatment barely affected ATF6 expression, and the addition of neither MG-132 nor celastrol to diltiazem-supplemented media caused significant changes.

The third branch of the UPR is induced by PERK oligomerization and phosphorylation of the eukaryotic translation initiation factor-2 (eIF2a). eIF2a induces the expression of the transcription factor ATF4 and a subset of ATF4 target genes, including CHOP (Ron and Walter, 2007). Lacidipine treatment caused CHOP upregulation (3.7-fold), which was considerably enhanced by the addition of MG-132 (5.0-fold), indicating that the PERK arm of the UPR is activated in response to lacidipine treatment (Figure 5D). Treatment with diltiazem alone or in combination with a proteostasis regulator did not cause significant changes in CHOP expression (Figure 5D).

Prolonged induction of the UPR and inability of the ER folding capacity to cope with the load of misfolded proteins leads to apoptosis. A number of genes are involved in the regulation of apoptosis induction, including the pro-apoptotic genes encoding for Bcl-2 homologous antagonist (BAK) and BCL2-associated X protein (BAX) (Scorrano et al., 2003), and the anti-apoptotic gene encoding the apoptosis regulator Bcl-2 (Rodriguez et al., 2010), which expression was investigated in cells treated with LTCC blockers and proteostasis regulators as described before. MG-132 and celastrol treatments caused upregulation of the pro- apoptotic proteins BAK and BAX. Specifically, MG-132 induced upregulation of BAK (2.1 - fold, Figure 5E) and celastrol upregulation of BAX (3.1 -fold, Figure 5F). Lacidipine did not significantly alter either BAX or BAK expression, while diltiazem caused upregulation of both BA (1.9-fold) and BAX (2.1-fold). When cells were co-treated with a LTCC and a proteostasis regulator, changes in BAX and BAK expression were observed to inversely correlate with the extent of L444P GC activity increase, suggesting that the mechanism of lacidipine mediated L444P GC folding rescue involves inhibition of apoptosis. For instance, administration of lacidipine to MG-132 treated cells mildly decreased the expression of BAK and BAX observed in cells treated with MG-132 only, even though lacidipine and MG-132 were shown to have a synergic effect on UPR induction (Figure 5A-D) and on the rescue of L444P GC activity (Figure IB). Interestingly, when diltiazem was used in combination with a proteostasis regulator, MG- 132 mediated BAK upregulation was enhanced (2.9-fold), but celastrol mediated BAX upregulation was lowered, which may explain why treatment with celastrol, but not with MG- 132, results in enhancement of L444P GC activity increase mediated by diltiazem.

The expression of the anti-apoptotic Bcl-2 encoding gene was also evaluated (Figure 5G). Bcl-2 contribute to maintaining ER Ca2+ homeostasis by reducing [Ca2+]ER efflux (Eckenrode et al., 2010; Rong et al., 2009), and was found to be upregulated in L444P GC fibroblasts cultured with either lacidipine or diltiazem (1.9-fold), underscoring the therapeutic potential of Ca²⁺ homeostasis modulation in L444P GC fibroblasts. MG-132 treatment lowered Bcl-2 expression (0.8-fold), while celastrol treatment did not seem to affect it. The addition of a proteostasis regulator to lacidipine treated cells resulted in lowered Bcl-2 expression, particularly it brought Bcl-2 expression back to the level detected in untreated cells, while the addition of a proteostasis regulator to diltiazem treated cells resulted in substantial downregulation of Bcl-2 (MG-132 and diltiazem: 2.3-fold; celastrol and diltiazem: 2.0-fold). Similar to what was reported above regarding the expression of pro-apoptotic genes, modulation of Bcl-2 expression correlates with the ability of LTCC blockers to rescue L444P GC folding when used alone or in combination with an UPR-inducing proteostasis modulator.

Next, it was tested whether lacidipine mediated changes in the expression of pro- and anti-apoptotic genes translate in differences in cytotoxicity and cell death, a common marker of cells treated for L444P GC folding rescue through UPR activators, such as MG-132, tunicamycin, and thapsigargin (Wang et al., 201 1b). L444P GC patient-derived fibroblasts treated with Ca²⁺ blockers and proteostasis regulators as described above were tested using the CytoGLO™ Annexin V-FITC Apoptosis Detection Kit to monitor membrane rearrangement (Annexin V binding) and fragmentation (propidium iodide (PI) binding), which occur during early and late apoptosis, respectively (Table 2 and Figure 9). TABLE 2 - Cell toxicity assay (pO.01)

1 Change (%) in number of cells bound to Annexin V/PI compared to untreated cells.

2 Change (%) in Annexin V/PI binding affinity compared to untreated cells.

Treatment of L444P GC fibroblasts with lacidipine (20 μΜ) did not cause cytotoxicity and the apoptosis induction was so not significantly altered compared to untreated cells. MG-132 (0.6 μΜ) and celastrol (0.6 μΜ) resulted in 47.36% and 10.40% increase in Annexin V binding, and 7.40% and 4.70% increase in dead cell population respectively. The addition of lacidipine to MG-132 or celastrol treated cells led to a decrease in Annexin V binding to 40.77% and 8.34% and to a decrease in dead cell population to 4.45% and 4.05%, respectively. This results indicate that lacidipine treatment under conditions observed to rescue mutated GC folding and induce UPR not only does not cause cytotoxicity, but it also partially counteracts the cytotoxic effect of UPR inducing proteostasis regulators.

Interestingly, lacidipine mechanism of L444P GC folding rescue differs significantly from that of RyRs blockers reported previously (Wang et al., 201 lb). While cell treatment with RyRs blockers does not activate the UPR but rescues GD fibroblasts from UPR induced toxicity, lacidipine treatment concurrently activates the UPR and ameliorates UPR induced toxicity in GD fibroblasts.

Lacidipine treatment upregulates GC chromosomal expression in L444P GC fibroblasts.

As briefly alluded to before (Figure 2A-B), the total amount of L444P GC seems to be dramatically increased by lacidipine treatment. Upregulation of GC gene (GBA) as well as of other lysosomal storage disorder associated genes was previously reported in cells treated for the rescue of L444P GC folding through UPR induction (Wang et al, 201 1 b). This finding resonates with the general increase in lipid metabolism that normally occurs during UPR (Schroder and Kaufman, 2005) and was suggested as a potentially therapeutic "side-effect" of mutated GC proteostasis regulation via UPR activation.

Quantitative RT-PCR and Western blot analyses were conducted to understand whether the increase in cellular concentration of L444P GC observed in cells treated with lacidipine is due to upregulation of GC expression in addition to L444P GC enhanced folding and lowered ERAD. L444P GC fibroblasts were treated with lacidipine (20 μΜ), diltiazem (20 μΜ), MG-132 (0.6 μΜ), celastrol (0.6 μΜ), or a combination thereof (Figure 6A). Lacidipine treatment was observed to enhance GC mRNA expression (3.1 -fold) to an extent similar to MG- 132 (3.1 -fold) or celastrol (3.4-fold). Treatment with diltiazem resulted in a lower increase in GC expression (2.5-fold), most likely reflecting diltiazem's milder effect on UPR induction. Interestingly, addition of MG-132 resulted in increase in both lacidipine and diltiazem mediated GC upregulation (4.5- and 4.6-fold respectively), while celastrol slightly lowered it (2.9- and 2.2-fold, respectively). These transcriptional changes were confirmed at the translational level by western blot analyses (Figure 6B). L444P GC fibroblasts were cultured with lacidipine (20 μΜ), MG-132 (0.6 μΜ), and celastrol (0.6 μΜ) for 48 hrs, and bands detected with a GC- specific antibody were quantified using NIH Java Image analysis software (Figure 6C). The L444P GC content of lacidipine-treated cells increased about 50% compared to that of untreated cells, similarly to what was observed in MG-132-treated cells. In addition, the combination of lacidipine and MG-132 caused a 2.5-fold increase in total L444P GC, which is much higher than what was observed cells treated with either one of these molecules, and is in perfect agreement with the results obtained from quantitative RT-PCR. Celastrol treatment slightly increased GC protein accumulation, and the addition of lacidipine did not result in any significant change. In summary, this data indicates that GC chromosomal expression is enhanced upon treatment with lacidipine, and suggests that GC upregulation contributes to L444P GC folding rescue mediated by UPR induction.

Discussion

Ubiquitously expressed voltage-gated LTCCs support inward current of Ca ions. The function of Ca²⁺ ions as an intracellular second messenger has been reported in many cellular processes, ranging from gene expression to cardiac and smooth muscle contraction. Because Ca²⁺ mediates both physiological and pathological events, considerable effort has been devoted to the study of Ca²⁺ channel antagonists, a chemically and pharmacologically heterogeneous group of drugs widely used as therapeutic agents as well as research tools. The prototypical LTCC antagonists are diltiazem (a benzothiazepine), verapamil (a phelylalkylamine) and nifedipine (a 1 ,4-dihydropyridine) (Triggle, 2006). Diltiazem and verapamil are approved FDA approved for the treatment of hypertension and cardiac arrhythmias (Hockerman et al., 1997). They were reported to rescue folding, trafficking and activity of GC variants in patient-derived fibroblasts (Mu et al., 2008a), but failed to rescue mutated GC activity in mice (Sun et al., 2009). In an effort to discover small molecules that efficiently rescue the folding of mutated GC variants by enhancing the cellular folding capacity but without inducing cytotoxicity, we tested a series of 1,4-dihydropyridines, a class of LTCC antagonists known to lower intracellular [Ca²⁺] with higher selectivity than benzothiazepines and phelylalkylamine (Triggle, 2003).

Lacidipine was found to rescue the activity of GC variants carrying the two most common mutations, L444P and N370S, in GD patient-derived fibroblasts. In particular, lacidipine mediates a substantially higher increase in L444P GC activity than what was observed using other LTCC and RyRs blockers to date (Mu et al, 2008a; Ong et al, 2010; Wang et al., 201 1b), and this increase is markedly enhanced by co-treatment with proteostasis modulators MG-132 and celastrol. The nature of LTCC blockers' chemical structure— with lacidipine highly hydrophobic, diltiazem and verapamil charged at physiologic pH (Triggle, 2003) - is likely to influence their cell permeability and explain why treatment of GD fibroblasts with lacidipine resulted in higher depletion of cytosolic [Ca²⁺] and more effective remodeling of L444P GC proteostasis compared to diltiazem and verapamil.

L444P GC fibroblasts treated with lacidipine (and diltiazem for comparison) were used to conduct mechanistic studies and gain a better understanding of the molecular mechanisms involved in L444P GC proteostasis. We demonstrated that lacidipine mediated rescue of mutated GC folding (Figure 1) correlates with its ability to a) lower cytoplasmic [Ca²⁺], thereby counteracting the effect of GC substrate accumulation (Figure 3), b) enhance the ER's folding capacity via substantial upregulation of BiP expression (Figure 4), confirming that BiP plays a key role in the folding of L444P GC, c) induce the UPR (Figure 5) and upregulate GC expression (Figure 6), and d) lower cytotoxicity and limit UPR mediated apoptosis induction (Figure 5 and Table 2). This mechanism is not activated by diltiazem and verapamil. An analogous mode of action was previously reported to explain the synergic effect of two distinct small molecules, a proteostasis regulator (MG-132) and a RyRs blocker (ryanodine), on L444P GC folding rescue. Particularly, MG-132 was reported to induce BiP upregulation and UPR induction and ryanodine to lower intracellular [Ca²⁺] and counteract UPR induced cytotoxicity (Wang et al., 201 1b), suggesting that combining these different mechanisms of proteostasis regulation is an effective strategy to rescue mutated GC folding. In summary, this study sheds light on the cellular pathways involved in mutated GC folding and introduces a novel strategy to rescue mutant GC folding via small molecule treatment that combines remodeling of two general cellular pathways involved in protein homeostasis: protein folding and Ca²⁺ homeostasis.

Experimental Procedures: ERAD Inhibitor Treatment

Reagents and cell culture: Eeyarestatin I was purchased from ChemBridge. MG-132 and kifunensine were from Cayman Chemical. Celastrol was from Alexis Biochemicals. Conduritol B Epoxide (CBE) was from Toronto Research Chemicals. 4-methylumbelliferyl β- D-glucoside (MUG) was from Sigma-aldrich. Cell culture media were from Lonza.

GD patient-derived fibroblasts homozygous for the L444P (1448T>C) mutation (GM10915), and N370S (1226A>G) mutation (GM00852), and Tay-Sachs disease patient- derived fibroblast heterozygous for the G269S (c.805G>A) mutation and a 4 base pair insertion (c.l278insTATC) (GM 13204) were obtained from Coriell Cell Repositories. Fibroblasts were grown at 37°C in 5% C02 in minimal essential medium with Earle's salts, supplemented with 10% heat-inactivated fetal bovine serum and 1% glutamine Pen-Strep. Medium was replaced every 3 or 4 days. Monolayers were passaged with TrypLE Express.

Enzyme activity assays: The intact cell glucocerebrosidase (GC) activity assay was performed as previously described (Mu et al., 2008b). Briefly, 100 μΐ aliquots of 104 cells were plated in each well of a 96-well plate and incubated overnight to allow cell attachment. The medium was replaced with fresh medium containing small molecules (small molecule concentrations and time of incubation are specified in each experiment) and plates were incubated at 37°C. The medium was then aspirated and monolayers were washed with PBS three times. The assay reaction was started by the addition of 50 μΐ of 2.5 mM MUG in 0.2 M acetate buffer (pH 4.0) and stopped by the addition of 150 μΐ of 0.2 M glycine buffer (pH 10.8) to each well after 7 hrs of incubation at 37°C. Liberated 4-methylumbelliferone was measured (excitation 365 nm, emission 445 nm) with a SpectraMax Gemini plate reader (Molecular Device). Non-lysosomal GC activity was evaluated by measuring GC activities in the presence of Conduritol B Epoxide (CBE) at 1 mM final concentration. Relative GC activities were calculated by subtracting the background of non lysosomal activity and normalizing the obtained values by the activity of untreated cells.

β-hexosaminidase A (Hex A) activity assay was performed as previously described

(8,15). Cells were cultured as described for GC activity assays. The assay was performed by lysing the cells with 60 μΐ of 10 mM citrate/phosphate buffer (CP buffer, pH 4.2) containing 0.5% human serum albumin and 0.5% Triton X-100. 30 μΐ aliquots of lysates were mixed with 30 μΐ of 3.2 mM MUGS dissolved in CP buffer and incubated at 37°C for 1 hour. The reaction was stopped by the addition of 200 μΐ of 0.1 M 2-amino-2-methyl-l-propanol (pH 10.5). HexA a activity was evaluated by measuring the solution's fluorescence (excitation 365 nm, emission 450 nm). Relative HexA a activities were calculated by normalizing HexA activities to that of untreated cells.

Quantitative RT-PCR and Xbp-1 splicing: As previously described (Mu et al.,

2008b), cells were incubated with small molecules for 24 hrs before total RNA was extracted using RNAGEM™ reagent (ZyGEM). cDNA was synthesized from total RNA using qScript™ cDNA SuperMix (Quanta Biosciences). Total cDNA amount was measured by NanoDrop 2000 (Thermo Scientific). Quantitative PCR reactions were performed using cDNA, PerfeCTa™ SYBR Green FastMix™ (Quanta Biosciences), and corresponding primers (Table 1) in the CFX96™ Real-Time PCR detection system (Bio-Rad). Samples were heated for 2 min at 95 °C and amplified in 45 cycles of 1 s at 95°C, 30 s at 60°C, and 30 s at 72°C. Analyses were conducted using CFX manager software (Bio-Rad) and the threshold cycle (CT) was extracted from the PCR amplification plot. The ACT value was used to describe the difference between the CT of a target gene and the CT of the housekeeping gene, GAPDH: ACT = CT (target gene) - CT (GAPDH). The relative mRNA expression level of a target gene in treated cells was normalized to that measured in untreated cells: relative mRNA expression level = 2 exp [-(ACT (treated cells) - ACT (untreated cells))]. Each data point was evaluated in triplicate and measured three times.

RT-PCR analysis of Xbp-1 splicing was performed using total cDNA, Taq DNA polymerase, and the Xbp-1 primers listed in Table SI following reverse transcription. PCR products were separated on a 2.5% agarose gel. Spliced Xbp-1 bands were quantified by NIH ImageJ analysis software.

Western blot analysis: Cells were incubated with small molecules for 48 hrs, collected and lysated with the complete lysis-M buffer containing the protease inhibitor cocktail (Roche). Total protein concentrations were determined by Bradford assay (Thermo Scientific) and each sample was diluted to the same protein concentration. EndoH treatment was performed by incubating samples at 95°C for 10 min, followed by incubation with EndoH (New England Biolabs) for 1 hrs at 37°C. Aliquots of cell lysates were separated by 10% SDS-PAGE gel, and Western blot analyses were performed using primary antibodies (rabbit anti-BiP and mouse anti- CRT (Stressgen), mouse anti-CNX and rabbit anti-Glucocerebrosidase (Sigma-Aldrich), or rabbit anti-GAPDH (Santa Cruz Biotechnology)) and appropriate secondary antibodies (HRP- conjugated goat anti-rabbit (Santa Cruz Biotechnology) or goat anti-mouse IgG (Stressgen)). Blots were visualized using Luminata Forte Western HRP substrate (Millipore) and quantified by NIH ImageJ analysis software.

Immunofluorescence microscopy: Fibroblasts were seeded on glass coverslips, cultured in the presence of small molecules for 48 hrs, and fixed with 4% paraformaldehyde for 30 min. Cells were permeabilized with 0.1% Triton-X for 5 min and incubated with 8% BSA for 1 hr. Following incubation for 1 hr with primary antibodies (rabbit anti- -glucocerebrosidase and mouse anti-CNX antibodies, Sigma- Aldrich), cells were washed three times with 0.1% Tween-20 PBS, and then incubated with secondary antibodies for 1 hr (Dylight 488 goat anti- mouse IgG and Dylight 549 goat anti-rabbit IgG from KPL, and FITC anti-LAMP-1 from Biolegend). Images were obtained using an Olympus 1X81 confocal microscope and co- localized using the Fluoview software. Co-localization heatmap images were analyzed using NIH ImageJ analysis software.

Toxicity assay: L444P patient-derived cells were treated with Eerl (2 μΜ and 6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) 16 hrs at 37 °C. Cells were collected and cell toxicity tested with the CytoGLOTM Annexin V-FITC Apoptosis Detection Kit (IMGENEX) according to the manufacturer's instructions and analyzed by flow cytometry (FACSCantoTM II, Beckon Dickingson) with a 488-nm Argon laser.

Results

ERAD Inhibition Enhances Mutated GC Folding, Trafficking, and Activity in GD Patient-derived Fibroblasts

In order to investigate the role of the ERAD pathway on the folding of mutated GC variants, L444P GC fibroblasts were cultured in the presence of ERAD inhibitors (Eerl and Kif) for 5 days, and GC activities were evaluated every 24 h with the intact cell GC activity assay (Mu et al., 2008b). Culturing conditions resulting in maximal rescue of L444P GC activity are reported in Fig. 1 1 (blue lines). L444P GC activity was observed to increase up to 2.0-fold in cells treated with Eerl (8 μΜ final medium concentration, p O.001) for 48 h compared with untreated cells, which corresponds to about 25% of the WT cellular activity (Fig. 1 1A), and is expected to ameliorate GD symptoms (Schueler et al., 2004).

It was hypothesized that Eerl-mediated ERAD inhibition prolongs ER retention of mutated GC, thereby enhancing the pool of GC folding intermediates amenable to folding rescue. Hence, it was asked whether combining ERAD inhibition with enhancement of the cellular folding capacity could further increase the pool of natively folded GC that traffics to the lysosomes. To investigate this question, Eerl-treated cells were cultured in the presence of MG- 132 and celastrol, small molecules previously reported to function as proteostasis regulators and rescue GC folding through a mechanism distinct from ERAD modulation. Experiments were designed to explore the addition of a constant concentration of proteostasis regulator (0.4, 0.6, or 0.8 μΜ) to a range of Eerl concentrations. Co-administration of Eerl (2 μΜ) and MG-132 (0.6 μΜ) for 48 h resulted in a dramatic 4.2-fold increase in L444P GC activity (p<0.001; Fig. 11 A) compared with untreated cells, which corresponds to 52.5% of WT GC activity and is significantly higher than the activity of cells treated only with Eerl (2.0-fold) or MG-132 (2.4- fold) under the same conditions. Co-treatment with Eerl and celastrol was also observed to enhance L444P GC activity rescue(Fig. 11B). Specifically* GD cells treated with Eerl (5 μΜ) and celastrol (0.4 μΜ)ίθΓ 48 h displayed a 3.1-fold increase in L444P GC activity (p O.001), which is higher than what was observed in cells treated only with Eerl (1.3-fold) or celastrol (1.4-fold) under the same conditions.

A similar set of experiments was conducted using Kif, the other ERAD inhibitor selected. Cell treatment with Kif (100 nM) modestly increased L444P GC activity (1.2-fold, 15% of WT activity; p <0.01), whereas co-administration of Kif (50 nM)and MG-132 (0.4 μΜ) led to a dramatic 3.8-fold increase in L444P GC activity (47.5% of WT activity; p <0.01) after 120 h (Fig. 1 1C). Co-treatment with Kif and celastrol was also explored (Fig. 1 1D). Optimal culturing conditions (100 nM Kif and 0.8 μΜ celastrol) resulted in a 2.3-fold increase in L444P GC activity (28.8% of WT activity, p <0.01), again higher than what was measured upon treatment with either molecule alone. Taken together, these results suggest that ERAD limits the folding and trafficking of L444P GC and that ERAD inhibition is a viable strategy to promote native folding and trafficking of this mutated, degradation-prone enzyme variant. In order to confirm that the increase in GC activity measured in cells treated with Eerl and Kif is caused by partial restoration of L444P GC folding and lysosomal trafficking, the L444P GC glycosylation state and its intracellular localization and trafficking was investigated.

The L444P GC glycosylation state was investigated by endoglycosidase H (Endo H) treatment as described previously (Wang et al., 201 1a), using culturing conditions that resulted in maximal GC activity rescue (6 μΜ Eerl, 50 nM Kif, 0.6 μΜ MG-132 for 48 h).The total protein content was subjected to Endo H treatment, which hydrolyzes immature high mannose N-linked glycoproteins. GC detection by Western blot typically reveals a low M_r band corresponding to partially glycosylated, ER-retained GC (Endo H-sensitive) and a high M_r band corresponding to fully glycosylated, lysosomal GC (Endo H-resistant) (Maley et al. 1987). A representative Western blot (Fig. HE) and quantification of EndoH-resistant and Endo Hi- sensitive GC bands (Fig. 1 IF) are reported. In untreated cells, nearly all L444P GC was detected as Endo H-sensitive. A band corresponding to Endo H-resistant L444P GC was detected in cells treated with Eerl, and its intensity was 1.6-fold higher than that detected in cells treated with MG-132 (results obtained using MG-132 were reported previously (Mu et al. 2008b) and are included here for comparison). Kif treatment caused a mild increase in the GC Endo H-resistant pool, corresponding to about 13% of that of MG-132-treated cells. Co-treatment with MG-132 and Eerl (2 μΜ) or Kif resulted in a 2.6-and 1.4-fold increase, respectively, in the Endo H- resistant L444P GC, compared with treatment with MG-132 only, which is in agreement with results obtained from enzymatic assays (Fig. 11, A and C).

L444P GC intracellular localization was evaluated using immunofluorescence microscopy and subcellular fractionations of L444P GC patient-derived fibroblasts treated with small molecules at concentrations corresponding to maximum activity rescue (6 μΜ Eerl, 50 nM Kif, and 0.6 μΜ MG-132) for 48 h. Immunofluorescence microscopy was conducted using antibodies specific for GC, for an ER marker (CNX), and for a lysosomal marker (LAMP-1) to evaluate GC localization in the ER and in the lysosome, respectively. Co-localizations of GC and CNX (Fig. 12 A) and of GC and LAMP-1 (Fig. 12B) are reported, respectively, in pink and purple (merged colors) and analyzed with ImageJ software to provide a co-localization heatmap. L444P GC was barely detectable in untreated cells due to extensive ERAD. In agreement with the results obtained from GC enzymatic assays (Fig. 1 1 A), a significantly larger pool of GC was detected in the ER and in the lysosome upon Eerl treatment. Furthermore, GC accumulation was observed to increase with increasing concentration of Eerl and to be further enhanced by co- treatment with Eerl and MG-132 (Fig. 12, A and B). Kif treatment also enhanced GC localization in the ER and in the lysosomes compared with untreated cells, albeit to a lower extent than Eerl treatment (Fig. 12, A and B). Co-treatment with Kif and MG-132 increased GC accumulation in the ER and in the lysosomes, again supporting the results obtained from GC enzymatic assays (Fig. 1 1C).

Subcellular fractions of cell homogenates were collected upon Percoll density gradient centrifugation, and GC enzyme activity assay for each fraction was performed to evaluate L444P GC intracellular localization. Because β-hexosaminidase A (HexA) trafficking and activity are not altered in GD fibroblasts compared with WT fibroblasts, HexA activity was first evaluated in each fraction to distinguish fractions containing ER and lysosomes. HexA activity was detected in both low density (fractions 1 and 2) and high density (fractions 7 and 8) fractions in untreated L444P GC cells (Fig. 12C, dashed line, right y axis), which comprise the ER and the lysosomes, respectively, as reported previously (Ishii et al., 2007). In untreated cells, L444P GC activity (Fig. 12C, left y axis) was barely detectable in low density fractions (ER) and undetectable in high density fractions (lysosomes). Treatment with MG-132 resulted in a significant increase in L444P GC activity in both low and high density fractions, confirming that MG-132 promotes rescue of mutant GC folding and trafficking (Fig. 12C). A significant increase in GC activity was also detected in Eerl-treated cells, particularly in low density ER fractions, confirming that Eerl functions by inhibiting ERAD and prolonging ER retention. Kif treatment also enhanced GC activity both in the low and high density fractions compared with untreated cells, although to a lower extent than Eerl treatment, in agreement with the results obtained from GC enzymatic assays (Fig. 11 , A and C).

In order to investigate whether ERAD inhibition is a mutation-dependent strategy for the rescue of unstable GC variants, N370S GC fibroblasts were cultured in the presence of Eerl and a proteostasis regulator, and GC activities were evaluated every 24 h for up to 3 days (Fig. 13A and Fig. 18). Eerl treatment (4 μΜ) for 72 h resulted in a 1.25-fold increase in GC activity (15.6% WT activity; p< 0.01). Similar to what was described for L444P GC cells, a lower concentration of Eerl (2 μΜ) combined with MG-132 (0.2 μΜ) further enhanced N370S GC rescue, resulting in a 2.1 -fold increase in activity compared with untreated cells (26.3% of WT activity, < 0.001), which is higher than what observed in the presence of either molecule alone (Eerl, 1.2-fold; MG-132, 1.5-fold). When the same experiment was conducted using celastrol as a proteostasis modulator, co-treatment with Eerl (2 μΜ) and celastrol (0.2 μΜ) resulted in a 1.4- fold increase in activity (17.5% of WT activity, p <0.01), which again is significantly higher than what was observed using either molecule alone (Eerl, 1.2-fold; celastrol, 1.2-fold).

To confirm that the observed increase in GC activity is due to rescue of the enzyme folding and trafficking, N370S GC cellular localization was evaluated by immunofluorescence microscopy in cells treated with Eerl (2 μΜ) and MG-132 (0.2 μΜ). Eerl treatment resulted in an increase in N370S GC accumulation in the ER and in the lysosomes compared with untreated cells. Co-administration of Eerl and MG-132 further enhanced N370S GC concentration both in the ER and in the lysosomes (Fig. 13, B and C), confirming the results obtained from enzymatic assays.

In summary, these results demonstrate that ERAD prevents native folding of mutated, unstable GC and provide compelling evidence that ERAD inhibition is a viable strategy to rescue lysosomal activity of degradation-prone GC variants containing destabilizing, non- inactivating mutations. Interestingly, the activity rescue measured in N370S GC fibroblasts was consistently less pronounced than that observed in L444P GC fibroblasts. It is hypothesized that this difference is due to the different destabilizing effect of the N370S and L444P substitutions. L444P GC is normally completely targeted to ERAD, whereas the N370S GC variant partially escapes degradation and can be detected throughout the secretory pathway (Sawkar et al., 2006). Hence, ERAD is likely to have a more direct and rate-limiting role in L444P GC processing, and, not surprisingly, ERAD inhibition results in more efficient rescue of L444P GC than N370S GC folding.

ERAD Inhibition Enhances HexA Activity in Tay-Sachs Patient-derived Fibroblasts

A number of loss-of-function LSD are caused by destabilizing mutations and degradation of secretory proteins. It was demonstrated that ERAD inhibition enhances folding of mutated GC variants. The question then became whether ERAD inhibition is a general strategy to rescue activity of mutated proteins containing misfolding, non-inactivating associated with the development of LSD. Thus, the folding of HexA, deficiency of which causes Tay-Sachs disease was investigated. Specifically, the focus was placed on one of the most prevalent mutations, the G269S substitution in the HexA a subunit, which destabilizes the protein native structure causing loss of activity to approximately 10% of WT (Tropak et al. 2004). Patient- derived fibroblasts harboring G269S HexA were cultured in the presence of an ERAD inhibitor and a proteostasis modulator, and HexA activity was measured as previously described (Mu et al., 2008b). Administration of Eerl (6 μΜ) for 96 h led to a 1.4-fold increase in G269S HexA a activity (14% of WT HexA activity; p <0.01 ; Fig. 14). The addition of MG-132 (0.2 μΜ) to cells treated with Eerl (2 μΜ) for 96 h caused a further increase in HexA activity (1.7-fold, -17% of WT; p <0.01 ; Fig. 14).

These findings suggest that ERAD inhibition facilitates folding of destabilized enzyme variants prone to degradation. In summary, results from studies in Gaucher and Tay- Sachs disease patient-derived cells indicate that the activity rescue observed upon treatment with ERAD inhibitors is inversely proportional to the loss of lysosomal activity normally associated with each enzyme variant.

ERAD Inhibition via Eerl Treatment Causes Up-regulation of BiP Expression

It was speculated that small molecule-mediated ERAD inhibition, by inducing accumulation of misfolded proteins in the ER, could lead to up-regulation of ER chaperones. The ER luminal chaperone BiP plays a critical role in the rescue of L444P GC folding. Whether the increase in mutated GC activity observed upon ERAD inhibition could be attributed to up- regulation of BiP or other ER chaperones induced in response to the sudden load of misfolded proteins in the ER was considered. Quantitative RT-PCR experiments were conducted to evaluate the expression levels of representative ER chaperones (BiP, CNX, and CRT) in L444P GC fibroblasts treated with Eerl (2 and 6 μΜ), Kif (50 nM), MG-132 (0.6 μΜ), or a combination thereof (Fig. 15, A-C). BiP expression (Fig. 15 A) was mildly up-regulated by Eerl treatment at 2 μΜ (1.6- fold, j!?<0.01)but highly up-regulated at 6 μΜ (9.2-fold) and upon co-treatment with Eerl (2 μΜ) and MG-132 (15.7-fold). Hence, the highest up-regulation of BiP expression was observed at conditions causing maximal rescue of GC activity, namely upon cell treatment with a high concentration of Eerl (6 μΜ) or co-treatment with MG-132 and a low concentration of Eerl (2 μΜ), suggesting a correlation between BiP transcriptional regulation and GC activity rescue. The dramatic increase in BiP expression observed could be part of UPR induction, which was previously shown to facilitate GC folding rescue and is analyzed below in more detail.

Interestingly, cell treatment with if under conditions observed to maximize L444P GC activity rescue only resulted in a moderate increase of BiP expression (2.0-fold, p<0.0\), even when used in combination with MG-132 (2.2-fold, p<0.01). Hence, under the conditions tested, Kif-mediated ERAD inhibition does not lead to ER stress and chaperone up-regulation. The lower increase in BiP expression observed upon treatment with Kif compared with Eerl may not be an indication of the lower rescue of L444P GC activity but rather of the two molecules' different mechanisms of action. In support of this hypothesis is evidence that even the addition of MG-132, which dramatically increases Kif-mediated L444P GC rescue(Fig. 1 1C), does not cause up-regulation of BiP expression.

CNX was mildly up-regulated by treatment with Eerl or Kif, alone or in combination with MG-132 (Fig. 15B). CRT expression was not substantially altered by Eerl treatment, although it was up-regulated (3.5-fold) by co-treatment with MG-132 and Eerl (Fig. 15C).

Western blot analyses (Fig. 15D) were conducted to confirm ER chaperone expression, and bands were quantified with ImageJ software (Fig. 15E). BiP protein accumulation was enhanced by treatment with Eerl in a concentration-dependent fashion (2 μΜ Eerl caused a 1.5-fold increase, and 6 μΜ Eerl resulted in 2.5-fold increase). Co-treatment with Eerl and MG-132 further enhanced BiP accumulation (2.7-fold) compared with untreated cells. Kif treatment, however, caused a very modest increase in BiP accumulation when used alone (1.2-fold) or in combination with MG-132 (1.6-fold). CNX and CRT protein levels did not seem to be considerably altered upon small molecule treatment. Overall, results from Western blot analyses are consistent with RT-PCR experiments, with the exception of CRT expression, for which transcriptional changes are not reflected at the translational level, suggesting that CRT up-regulation by cell treatment with MG-132 and Eerl does not translate into enhanced accumulation of CRT protein.

Induction of UPR Depends on Mechanism of ERAD Inhibition Accumulation of misfolded proteins triggers ER stress, which in turn leads to UPR induction. UPR manifests as a series of attempts to restore a physiologic balance between folded and misfolded proteins in the ER (Schroeder et al., 2005 and Ron et al. 2007). Specifically, UPR induction is regulated by three proximal membrane signal transducers, namely inositol-requiring kinase 1 (IRE1), activating transcription factor 6 (ATF6), and double-stranded RNA-activated ER kinase (PERK). Their activation results in transcriptional modulation of a series of downstream genes involved in enhancement of chaperone capacity, reduction of protein synthesis, and, eventually, induction of apoptosis (Schroeder et al., 2005 and Ron et al. 2007). In order to evaluate UPR induction in cells treated with ERAD inhibitors, the expression of three representative target proteins was measured: X-box-binding protein- 1 (Xbp-1) which is activated by IRE1; activating transcription factor 4 (ATF4), which is part of the PERK signaling cascade; and C/EBP homologous protein (CHOP), which is up-regulated in response to ATF6 activation (Schroeder et al., 2005). Quantitative RT-PCR was conducted to evaluate the expression of Xbp-1, ATF4, and CHOP in cells treated with Eerl (2 and 6 μΜ), Kif (50 nM),MG-132 (0.6 μΜ), or a combination thereof.

Activation of the IRE1 signaling cascade involves splicing of Xbp-1 niRNA. Spliced Xbp-1 mediates induction of UPR genes, whereas the unspliced Xbp-1 precursor functions as a repressor (Ron et al., 2007). To evaluate activation of the IRE1 arm of the UPR, spliced and unspliced Xbp-1 mRNA were quantified by RT-PCR followed by gel electrophoresis (Fig. 16, A and B). MG-132 was previously shown to enhance Xbp-1 splicing and is reported here for comparison. Treatment with Eerl resulted in Xbp-1 splicing in a concentration-dependent fashion (2 μΜ Eerl, 1.3-fold increase in splicing; 6 μΜ Eerl, 2.1 -fold increase). Coadministration of Eerl (2 μΜ) and MG-132 further increased Xbp-1 splicing (3.6-fold compared with MG-132 treatment). Kif treatment did not seem to induce splicing of Xbp-1, and co- treatment with Kif and MG-132 resulted in an increase in spliced Xbp-1 similar to that induced by treatment only with MG-132. In summary, Xbp-1 splicing was induced upon Eerl but not by Kif treatment, suggesting that activation of the IRE1 pathway depends on the specific mechanism of ERAD inhibition.

ATF4 expression was up-regulated 4.4-fold by treatment with Eerl and 6.0-fold by co-treatment with Eerl and MG-132 (p <0.05), a clear indication of the PERK arm's activation in cells treated with Eerl (Fig. 16D). A considerably lower increase in ATF4 expression was observed in cells treated with Kif (2.5-fold, p < 0.01 ). Moreover, co-treatment with Kif and MG- 132 did not result in significant up-regulation of ATF4 expression compared with treatment only with MG-132, again suggesting that UPR activation depends on the mechanism of ERAD inhibition.

CHOP was found to be highly up-regulated upon Eerl treatment (Fig. 16C). Specifically, 2 μΜ Eerl resulted in a 4.2-fold increase in CHOP expression, 6 μΜ Eerl resulted in an 18.5-fold increase, and co-administration of Eerl (2 μΜ) and MG-132 resulted in a 24.0- fold increase (p < 0.01), indicating that Eerl mediated ERAD inhibition causes activation of the ATF6 pathway. Because CHOP plays a role in the induction of apoptotic pathways (Oyadomari et al., 2004), these results also suggest that Eerl treatment might activate UPR-induced apoptosis, which is analyzed below. Treatment with Kif alone or in combination with MG-132 led to 2.2-and 6.5-fold CHOP up-regulation, respectively, which are significantly lower than those observed upon Eerl treatment and do not exceed those observed upon treatment with MG- 132 (Fig. 16C). In summary, it was demonstrated that Eerl but not Kif, when used under conditions that promote rescue of L444P GC activity, is associated with dramatic activation of the UPR.

Up-regulation of the GC-encoding gene (GB A) as well as of other genes encoding for lysosomal proteins associated with the development of LSD was previously reported in cells treated with UPR-inducing proteostasis regulators (Wang et al. 2011b). This was attributed to the general up-regulation of lipid metabolism that occurs during UPR in order to expand the size of the ER and dilute the load of misfolded proteins (Schroeder 2005). Whether ERAD inhibition increases GC accumulation in the ER by up-regulating its transcription in addition to preventing its degradation was then considered. Quantitative RT-PCR was conducted to measure GC expression in L444P GC fibroblasts treated with Eerl (2 and 6 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) (Fig. 16, E-G). Eerl treatment was observed to enhance GC expression in a concentration-dependent fashion (2μΜ Eerl, 2.3-fold; 6 μΜ Eerl, 3.3-fold; p < 0.01). Co- treatment with Eerl (2 μΜ) and MG-132 (0.6 μΜ) resulted in a 4.4-fold increase in GC expression (p < 0.05), which is higher than what was observed using Eerl alone (2.3-fold) but comparable with treatment with MG-132 only (4.0-fold). A lower increase in GC expression was measured in cells treated with Kif (1.9-fold), as expected, considering the modest UPR induction caused by Kif treatment. Similar to what was observed for Eerl, the increase in GC expression observed upon co-treatment with MG-132 (2.8-fold) was lower than that detected in cells treated only with MG-132 (4.0-fold).

GC expression was also investigated by Western blot analyses (Fig. 16, F and G). It is important to notice that changes in protein accumulation detected by Western blot are attributable to both GC transcriptional modulation caused by ERAD inhibition-induced UPR and GC post-translational processing caused by ERAD inhibition-mediated protein rescue. L444P GC content was barely detectable in untreated cells, as expected, due to extensive ERAD (Sawkar et al., 2006), whereas treatment with either ERAD inhibitor enhanced GC accumulation level. GC accumulation increased in Eerl-treated cells in a concentration-dependent fashion (cf. bands corresponding to 2 μΜ Eerl and 6 μΜ Eerl treatments). The addition of MG-132 further enhanced GC accumulation observed in Eerl- and Kif- treated cells.

In summary, ERAD inhibition resulted in both an increase in GC expression and cellular accumulation. GC up-regulation was found to be proportional to the extent of UPR induction measured upon treatment with each specific ERAD inhibitor. However, co-treatment with an ERAD inhibitor and a proteostasis modulator, MG-132, which was demonstrated to have a synergistic effect on the rescue of mutated GC activity (Fig. 1 1 , A and C), did not cause a corresponding synergistic increase in GC transcription, suggesting that rescue of mutated GC cannot be solely attributed to the effect of ERAD inhibitors on GC expression.

If ER stress persists, prolonged UPR activation typically leads to induction of apoptosis (Schroeder et al., 2005). Whether cell treatment with Eer and Kif influenced UPR- induced apoptosis was considered. The Cyto-GLO™ annexin V-FITC apoptosis detection kit was used to detect membrane rearrangement (annexin V binding, a measurement of early apoptosis) and fragmentation (propidium iodide binding, a measurement of late apoptosis) in L444P GC fibroblasts treated with Eerl (2 μΜ), Kif (50 nM), and MG-132 (0.6 μΜ) (Fig. 17, A-C). High annexin V binding and consequently dramatic increase in cell fluorescence were observed upon cell treatment with Eerl compared with untreated cells (Fig. 17A). The addition of MG-132 to Eerl-treated cells resulted in an even higher increase in annexin V binding. However, no increase in annexin V binding in cells treated with Kif compared with untreated cells was detected. Moreover, the addition of Kif and MG-132 resulted in annexin V binding indistinguishable from that observed in cells treated only with MG-132 (Fig. 17B). Measurements of propidium iodide binding, which is taken as an estimate of the dead cell population, showed . A 4.1 % increase in dead cells was observed upon Eerl treatment compared with untreated cells, whereas a negligible increase (0.4%) was observed upon Kif treatment (Fig. 17C). These results demonstrate that although Eerl treatment causes a dramatic increase in mutated GC activity at the cost of significant cell toxicity and apoptosis induction, treatment with Kif facilitates folding without induction of apoptosis.

Discussion

ERAD inhibition was shown to promote folding of the two most common GC variants: L444P GC (Figs. 1 1 and 12), which is typically completely targeted to ERAD, and N370S GC (Fig. 13), a presumably less destabilized variant that is moderately resistant to ERAD and retains partial residual activity (Grace et al., 1994). ERAD inhibition was also observed to rescue folding of Tay-Sachs disease G269S HexA (Fig. 14), which, similar to N370S GC, retains partial activity (Tropak et al., 2004). Interestingly, higher activity rescue was consistently observed in L444P GC cells, which normally display complete loss of activity, compared with N370S GC and G269S Hex A cells. This suggests that the rescue in protein folding caused by treatment with ERAD inhibitors inversely correlates with the stability and residual activity of the mutated substrate.

Experimental evidence suggests that the single-site mutations considered cause destabilization of the protein native, lowest free energy conformation. In order to quantify the stability of mutated variants, in silico analysis of folding free energy changes (ΔΔΰ) between WT and mutant proteins using PoPMuSiC software was conducted (Gilis et al., 2000 and Kwasigroch et al, 2002). AAG values of 0.92 and 4.17 kcal/mol were obtained for N370S and L444P GC, respectively, indicating that both mutations have a destabilizing effect on native folding, the L444P substitution causing significantly higher loss of stability. A similar analysis conducted for the Hex A protein revealed the AAG caused by the G269S substitution to be 0.49 kcal/mol, which is comparable with the ΔΔΰ of N370S GC and considerably lower than that of L444P GC. These values support the experimental results described above, suggesting a correlation between protein stability and degradation.

ERAD inhibition and proteostasis modulation resulted in synergistic rescue of lysosomal activity in patient-derived cells (Figs. 1 1, 13 A, and 14), indicating that a larger pool of unstable proteins that escapes ERAD and can engage the ER folding pathway is rescued by combining ERAD inhibition with up-regulation of the cellular folding capacity. Interestingly, the Eerl activity window was consistently shifted toward lower medium concentrations when Eerl was combined with a proteostasis regulator, implying that ER retention needs to be carefully modulated to meet the capacity of the cellular folding machinery. It remains to be determined whether Eerl treatment results in higher activity rescue than Kif treatment due to higher efficiency of the molecular mechanism involved (p97 versus ER mannosidase inhibition).

Detailed investigations of the molecular mechanism of ERAD inhibition and consequent changes in the cellular folding capacity were conducted in L444P GC fibroblasts treated to block two different steps of the ERAD pathway and prevent early recognition of misfolding intermediates (Kif) or retro translocation of irremediably misfolded substrates (Eerl) (Fig. 10). ER stress and UPR normally observed upon accumulation of misfolded proteins were investigated (Figs. 15 and 16) and seemed to be highly dependent on the specific mechanism of ERAD inhibition. It is speculated that by inhibiting retrotranslocation of irremediably misfolded proteins, Eerl inevitably leads to significant accumulation of misfolded proteins and consequent induction of UPR and apoptosis. Kif, however, by preventing targeting of folding intermediates to the ERAD pathway, is expected to enhance retention of substrates that can still be assisted by the ER chaperone pathway and reach native folding. As a result, Kif-mediated ERAD inhibition, despite promoting significant ER retention and folding of mutated GC, particularly when used in combination with a proteostasis modulator (Figs. 1 1 and 12), does not cause ER stress, as demonstrated by investigating changes in ER chaperone expression (Fig. 15); nor does it cause activation of UPR (Fig. 16) and apoptosis (Fig. 17).

Up-regulation of the GC-encoding gene, which was previously suggested to contribute to the rescue of GC activity mediated by UPR inducing proteostasis regulators was also observed in this study in association with UPR activation (Fig. 16). Kif treatment, for instance, which did not cause significant UPR, was not associated with considerable increase in GC expression. Interestingly, co-treatment with Kif and G-132, despite causing a dramatic increase in GC activity, did not result in UPR induction and GC up-regulation. These results, taken together, suggest that the rescue of GC folding observed in cells treated with ERAD inhibitors, alone or in combination with proteostasis regulators, cannot be solely attributed to UPR activation.

Finally, ERAD inhibitors led to dramatically different levels of apoptosis induction (Fig. 17). Specifically, Kif treatment did not cause cytotoxicity and did not increase MG-132 associated induction of apoptosis. Hence, detailed investigations should be conducted to identify the steps of the ERAD pathway that can be modulated for the rescue of degradation-prone substrates without dramatically compromising protein homeostasis and disrupting the functioning of the folding quality control system.

Experimental Procedures: Combined Eerl and Lacidipine Treatment

Reagents and cell culture

Eeyarestatin I was purchased from ChemBridge. Lacidipine and Conduritol B Epoxide (CBE) were from Toronto Research Chemicals. Fluvastatin was from Enzo Life Sciences. 4-methylumbelliferyl β-D-glucoside (MUG) was from Sigma-Aldrich. Cell culture media were from Lonza.

GD patient-derived fibroblasts homozygous for the L444P (1448T>C) mutation (GM10915) were obtained from Coriell Cell Repositories. Fibroblasts were grown at 37°C in 5% C0₂ in minimal essential medium with Earle's salts, supplemented with 10% heat-inactivated fetal bovine serum and 1% glutamine Pen-Strep. Medium was replaced every 3 or 4 days. Monolayers were passaged with TrypLE Express.

Enzyme activity assay

The intact cell glucocerebrosidase (GC) activity assay was performed as previously described (Mu et al., 2008b). Briefly, 100 aliquots of 10⁴ cells were plated in each well of a 96-well plate and incubated overnight to allow cell attachment. The medium was replaced with fresh medium containing small molecules (small molecule concentrations and time of incubation are specified in each experiment) and plates were incubated at 37°C. The medium was then aspirated and cells were washed with PBS three times. The assay reaction was started by the addition of 50 of 2.5 mM 4-methylumbelliferyl β-D-glucoside (MUG) in 0.2 M acetate buffer (pH 4.0) and stopped after 7 hrs of incubation at 37°C by the addition of 150 μΐ, of 0.2 M glycine buffer (pH 10.8) to each well. Liberated 4-methylumbelliferone was measured (excitation 365 nm, emission 445 nm) with a SpectraMax Gemini plate reader (Molecular Device). Non-lysosomal GC activity was evaluated by measuring GC activities in the presence of Conduritol B Epoxide (CBE) at 1 mM final concentration. Relative GC activities were calculated by subtracting the background of non lysosomal activity and normalizing the obtained values by the activity of untreated cells.

Quantitative RT-PCR

Quantitative RT-PCR was performed as previously described (Wang et al., 201 1c). Cells were incubated with small molecules for 24 hrs before total RNA was extracted using RNAGEM™ reagent (ZyGEM). cDNA was synthesized from total RNA using qScript™ cDNA SuperMix (Quanta Biosciences). Total cDNA amount was measured by NanoDrop 2000 (Thermo Scientific). Quantitative PCR reactions were performed using cDNA, PerfeCTa™ SYBR Green FastMix™ (Quanta Biosciences) and corresponding primers (Table 1 ) in the CFX96™ Real-Time PCR detection system (Bio-Rad). Samples were heated for 2 min at 95°C and amplified in 45 cycles of 1 s at 95°C, 30 s at 60°C, and 30 s at 72°C. Analyses were conducted using CFX manager software (Bio-Rad) and the threshold cycle (Cj) was extracted from the PCR amplification plot. The AC value was used to describe the difference between the CT of a target gene and the CT of the housekeeping gene, GAPDH: ACy- CT (target gene) - CT (GAPDH). The relative mRNA expression level of each target gene in treated cells was normalized to that measured in untreated cells: relative mRNA expression level = 2 exp [-(ACj (treated cells) - ACT (untreated cells))]. Each data point was evaluated in triplicate and measured three times. RT-PCR analysis of Xbp-1 splicing was performed using total cDNA, Taq DNA polymerase and the Xbp-1 primers listed in Table SI following reverse transcription. PCR products were separated on a 2.5% agarose gel. Spliced Xbp-1 bands were quantified by NIH Image J analysis software.

Western blot analysis

Cells were incubated with small molecules for 48 hrs, collected and lysated with the complete lysis-M buffer containing the protease inhibitor cocktail (Roche). Total protein concentrations were determined by Bradford assay (Thermo Scientific) and each sample was diluted to the same protein concentration. Aliquots of cell lysates were separated by 10% SDS- PAGE gel, and Western blot analyses were performed using primary antibodies (rabbit anti-BiP and mouse anti-CRT (Stressgen), mouse anti-CNX and rabbit anti-Glucocerebrosidase (Sigma- Aldrich), or rabbit anti-GAPDH (Santa Cruz Biotechnology)) and appropriate secondary antibodies (HRP-conjugated goat anti-rabbit (Santa Cruz Biotechnology) or goat anti-mouse IgG (Stressgen)). Blots were visualized using Luminata Forte Western HRP substrate (Millipore) and quantified by NIH ImageJ analysis software.

Immunofluorescence Microscopy

Fibroblasts were seeded on glass coverslips, cultured in the presence of small molecules for 48 hrs, and fixed with 4% paraformaldehyde for 30 min. Cells were permeabilized with 0.1% Triton-X for 5 min and incubated with 8% BSA for 1 hr. Following 1 hr of incubation with primary antibodies (rabbit anti-p-glucocerebrosidase and mouse anti-CNX antibodies, Sigma- Aldrich), cells were washed three times with 0.1% Tween-20/PBS, and then incubated with secondary antibodies for 1 hr (Dylight 488 goat anti-mouse IgG and Dylight 549 goat anti -rabbit IgG from KPL, and FITC anti-LAMP-1 from Biolegend). Images were obtained using an Olympus 1X81 confocal microscope and co-localized using the Fluoview software. Colocalization heatmap images were analyzed using NIH ImageJ analysis software.

Toxicity assay

L444P patient-derived cells were treated with Eerl (6 μΜ) and lacidipine (10 μΜ) for 16 hrs at 37 °C. Cells were collected and cell toxicity was tested with the CytoGLO™ Annexin V-FITC Apoptosis Detection Kit (IMGENEX) according to the manufacturer's instructions and analyzed by flow cytometry (FACSCanto™ II, Beckon Dickingson) with a 488-nm Argon laser.

Statistical analysis

All data is presented as mean ± s.d., and statistical significance was calculated using a two-tailed t-test. Inhibition of ERAD and modulation of Ca homeostasis synergize to rescue L444P GC folding, trafficking and activity in cells derived from patients with Gaucher's disease.

The folding of mutated GC variants can be partially rescued by inhibiting specific steps of the ERAD pathway in GD cells. It was questioned whether enhancing the cellular folding capacity via modulation of Ca²⁺ homeostasis would allow further increasing native folding of degradation-prone GC mutants achieved via ERAD inhibition. Thus, simultaneous modulation Ca²⁺ homeostasis and ERAD inhibition in GD cells was attempted. Specifically, LTCC blocker lacidipine that restores Ca²⁺ homeostasis by inhibiting L-type Ca²⁺ channels on the cell membrane and RyRs on the ER membrane and Eeyarestatin I (Eerl) that blocks the ERAD pathway by inhibiting the p97 ATPase were used and the activity and intracellular trafficking of mutated GC were investigated. Experiments were performed by administrating a constant concentration of lacidipine (5, 10, or 20 μΜ) to fibroblasts derived from GD patients homozygous for the L444P GC allele cultured in medium supplemented with a range of Eerl concentrations. GC enzymatic activity was evaluated every 24 hrs for up to 72 hrs with the intact cell GC activity assay (Fig. 20A and Fig. 25). Culturing conditions resulting in maximal rescue of L444P GC activity are reported in Figure 20A. Co-treatment with Eerl (6 μΜ) and lacidipine (20 μΜ) for 48 hrs resulted in a 2.9-fold increase in L444P GC activity compared to untreated cells (p<0.001 ; Fig. 20A), which corresponds to 36.3% of WT activity and is compatible with effective treatment (Schueler et al. 2004). This increase in GC activity is significantly higher than that measured in cells treated only with Eerl (1.6-fold) or lacidipine (1.8-fold) under the same conditions and was still observed after 72 hrs of incubation (Eerl 6 μΜ and lacidipine 20 μΜ, 2.6-fold increase in GC activity; Fig. 25).

In order to verify that the increase in GC activity observed in cells treated with Eerl and lacidipine is due to rescue of L444P GC folding and lysosomal trafficking, L444P GC intracellular localization was investigated. Cells were treated under culturing conditions that gave rise to maximal GC activity increase and analyzed by immunofluorescence microscopy. Specifically, L444P GC patient-derived fibroblasts were cultured with Eerl (6 μΜ), lacidipine (10 μΜ) and a combination thereof for 48 hrs. GC localizations in the ER and in the lysosome were detected with antibodies specific for GC, for an ER marker (CNX), and for a lysosomal marker (LAMP-1). Co-localization of GC and CNX (Fig. 20B) and of GC and LAMP-1 (Fig. 20C) is shown in pink and purple, respectively, in merged images. Heatmaps of co-localization images were obtained with NIH ImageJ software. L444P GC was barely detectable in untreated cells due to extensive ERAD, as expected (Mu et al., 2008b). Treatment with lacidipine or Eerl resulted in increase in the pool of GC that accumulates both in the ER and in the lysosome. The addition of lacidipine to Eerl treatment did not significantly increase GC accumulation in the ER compared to cells treated only with Eerl, but resulted in a significantly larger pool of GC in the lysosome compared to cells treated with either Eerl or lacidipine. These results demonstrate that combining modulation of Ca²⁺ homeostasis and ERAD enhances rescue of GC folding intermediates that escape ERAD and promotes their trafficking through the secretory pathway, thereby leading to the increase in lysosomal GC activity observed with enzymatic assays (Fig. 20A).

Lacidipine treatment attenuates the cytotoxic effect of Eerl-mediated ERAD inhibition in GD patient-derived fibroblasts.

By inhibiting the retrotranslocation of misfolded proteins, Eerl treatment causes accumulation of misfolded intermediates in the ER and, consequently, ER stress and induction of the UPR. UPR is activated to cope with aberrantly accumulating misfolded proteins (Ron et al., 2007). Not surprisingly, moderate UPR induction was repeatedly reported to promote rescue of misfolding-prone GC variants. However, prolonged UPR induction observed upon sustained treatment with Eerl was also observed to cause activation of apoptosis. Cell treatment with lacidipine, on the other hand, was shown not to cause cytotoxicity under conditions observed to rescue folding of mutated GC variants. Because lacidipine treatment also causes moderate UPR induction, it was hypothesized that an anti-apoptotic effect associated with lacidipine treatment protects cells from UPR-induced apoptosis. Therefore, whether lacidipine treatment could counteract the cytotoxic effect of Eerl was evaluated by studying apoptosis in cells co-treated with lacidipine and Eerl.

CytoGLO™ Annexin V-FITC Apoptosis Detection Kit was used to monitor membrane rearrangement (Annexin V binding) and fragmentation (propidium iodide (PI) binding) that occur during early and late apoptosis, respectively. L444P GC fibroblasts were cultured with lacidipine (10 μΜ) and Eerl (6 μΜ) for 16 hrs (Fig. 21A-B). Annexin V binding affinity in cells treated with lacidipine was comparable to that measured in untreated cells, whereas a dramatic increase in Annexin V binding was observed in cells treated with Eerl, reflectirig the onset of apoptosis. The addition of lacidipine to Eerl-treated cells resulted in significant decrease in Annexin V binding compared to cells treated only with Eerl, suggesting that lacidipine treatment partially alleviates Eerl cytotoxic effect (Fig. 21 A). The dead cell population was evaluated by measuring PI binding affinity. A negligible increase (0.3%) in PI binding was observed upon lacidipine treatment compared to untreated cells, while Eerl treatment caused a 10.4% increase (Fig. 2 I B). The addition of lacidipine to Eerl-treated cells reduced the PI binding population to 6.7%, confirming that lacidipine has an anti-apoptotic effect. These results, taken together, establish that lacidipine treatment enhances Eerl-mediated rescue of mutated GC native folding and activity but it counteracts its cytotoxic effect, therefore protecting the cells from UPR induced apoptosis, a particularly appealing property for therapeutic solutions based on the modulation of the proteostasis network.

Lacidipine treatment remodels Eerl-mediated activation of the UPR pathway. Previous studies showed that Eerl, when administered under conditions that result in maximal increase in L444P GC activity, is associated with significant UPR induction and cell apoptosis, whereas lacidipine treatment induces UPR but does not cause apoptosis. Lacidipine prevents apoptosis in cells treated with Eerl (Fig. 21). Therefore, whether lacidipine affects UPR induction in Eerl-treated cells. The UPR is a complex tripartite pathway regulated by three transmembrane signal transducers, namely inositol requiring kinase 1 (IREl), activating transcription factor 6 (ATF6) and double-stranded RNA-activated ER kinase (PERK). Activation of these three sensors leads to transcriptional regulation of a series of UPR target genes that mediate cellular folding (Ron et al., 2007, Schroder et al., 2005). In order to evaluate UPR induction, the expression of three representative UPR target proteins was measured: X-box binding protein-1 (Xbp-1), which is spliced and activated by IREl ; activating transcription factor 4 (ATF4), which is part of the PERK signaling cascade; and C/EBP homologous protein (CHOP), which is upregulated in response to ATF6 activation (Schroder et al., 2005). Quantitative RT-PCR was conducted to evaluate the expression levels of Xbp-1, ATF4, and CHOP in cells treated with lacidipine (10 μΜ) and Eerl (6 μΜ).

The precursor mRNA of Xbp-1 is spliced upon activation of the IREl signaling cascade. Spliced Xbp-1 mRNA functions as an activator of the IREl branch of the UPR, while the unspliced precursor acts as a repressor (Ron et al., 2007). Spliced and unspliced forms of Xbp-1 mRNA were analyzed by RT-PCR followed by gel electrophoresis. Bands corresponding to spliced Xbp-1 mRNA were quantified with NIH ImageJ software to evaluate the activation level of the IREl arm of the UPR (Fig. 22A-B). In agreement with previous work, the amount of spliced Xbp-1 in lacidipine treated cells was similar to that of untreated cells, while a considerable amount of spliced Xbp-1 was observed in cells treated with Eerl, indicating a strong activation of the IREl cascade upon Eerl treatment. In cells treated with both lacidipine and Eerl, the amount of spliced Xbp-1 was found to further increase 1 .7-fold compared to cells treated only with Eerl, suggesting a synergistic effect of lacidipine and Eerl on the induction of the IREl arm. Xbp-1 is an essential pro-survival UPR component and its activation is associated with attenuated apoptosis under ER stress conditions (Gupta et al., 2010). Hence, enhanced splicing of Xbp-1 in lacidipine and Eerl co-treated cells correlates with the decrease in apoptosis induction observed under the same conditions. The expression level of ATF4 was evaluated in order to monitor the activation of the PERK branch. ATF4 transcriptional expression was upregulated 1.8- and 4.4-fold in cells treated with lacidipine and Eerl, respectively, compared to untreated cells, indicating that these two proteostasis modulators have different effects on the induction of this arm of the UPR. Interestingly, the addition of lacidipine to Eerl-treated cells was observed to reduce ATF4 expression to only 2.1 -fold increase compared to untreated cells, indicating that lacidipine suppresses Eerl-mediated activation of the PERK arm (Fig. 22C).

CHOP, a downstream effector of the ATF6 branch, was found to be highly upregulated by both lacidipine and Eerl treatment (6.1- and 18.5-fold, respectively; Fig. 22D). The addition of lacidipine to Eerl-treated cells lowered CHOP upregulation to 14.7-fold. CHOP mediates UPR induced apoptosis activation (Oyadomari et al. 2004). These results confirm that lacidipine treatment suppresses UPR-induced apoptosis caused by Eerl treatment.

In summary, treatment with lacidipine was found to remodel the UPR pathway and lower UPR induced apoptosis caused by treatment with Eerl. Particularly, lacidipine inhibits the activation of PERK and ATF6 arms, which mediate induction of apoptosis, and enhances that of the pro-survival IREl/Xbp-1 arm, thus counteracting the progression of the UPR induced apoptotic cascade. Lacidipine treatment alters the expression of genes involved in the regulation of UPR-induced apoptosis; particularly, it causes upregulation of the anti-apoptotic gene Bcl-2.

Whether the protective effect observed in Eerl treated cells upon lacidipine treatment could be attributed to the upregulation of Bcl-2 was evaluated. The expression level of Bcl-2 was evaluated by performing quantitative RT-PCR in cells cultured with lacidipine (10 μΜ) and Eerl (6 μΜ). Lacidipine treatment resulted in 3.0-fold increase in Bcl-2 expression compared to untreated cells, while Eerl treatment caused a 2.0-fold decrease. Co-treatment with lacidipine and Eerl resulted in considerable upregulation of Bcl-2 expression (4.2-fold) (Fig. 22E). Interestingly, Bcl-2 was shown to prevent apoptosis induction mediated by CHOP (Szegezdi et al. 2006), which expression is lowered in association with the upregulation of Bcl-2 in cells co- treated with lacidipine and Eerl.

GC encoding gene (GBA), as well as other genes encoding for lysosomal proteins that are associated with the development of LSD, is upregulated in cells

treated with proteostasis modulators that induce the UPR. It was speculated that GC is upregulated as part of the general modulation of lipid metabolism that occurs during UPR to expand the size of the ER and dilute the load of misfolded proteins (Schroder et al. 2005). Whether the synergistic effect of Ca²⁺ homeostasis modulation and ERAD inhibition could also be attributed to GC transcriptional upregulation in addition to inhibition of GC degradation was considered. Quantitative RT-PCR was conducted to measure the expression of GC in GD patient-derived fibroblasts treated with lacidipine (10 μΜ) and Eerl (6 μΜ). Co-administration of lacidipine and Eerl resulted in 5.2-fold upregulation of GC expression compared to untreated cells, which is higher than what was observed in cells treated only with lacidipine (2.5-fold) or Eerl (3.3-fold) (Fig. 22F).

GC expression was also evaluated by Western blot (Fig. 22G-H). As shown in Figure 22H, L444P GC content was barely detectable in untreated cells, as expected, due to extensive ERAD (Sawkar et al, 2006), while treatment with either lacidipine or Eerl significantly enhanced GC protein accumulation. Co-treatment with lacidipine and Eerl further enhanced GC accumulation (1.4-fold increase compared to Eerl treatment alone) in agreement with the results obtained from quantitative RT-PCR reflecting GC transcriptional regulation and enzymatic assays, reflecting GC folding rescue.

Among ER resident chaperones, BiP plays a critical role in the folding of mutated GC variants. It was previously demonstrated that the increase in lysosomal GC activity observed upon chemically induced inhibition of ERAD or modulation of Ca²⁺ homeostasis in cells derived from patients with GD is partially due to the upregulation of BiP expression associated with UPR induction. Therefore BiP expression in cells treated with lacidipine and Eerl was evaluated. The total protein content of treated and untreated cells was analyzed by Western blot (Fig. 23 A) using a BiP-specific antibody and bands were quantified with NIH ImageJ software (Fig. 23B). Co-administration of lacidipine and Eerl resulted in 3.5-fold increase in BiP cellular accumulation, which is lower than what was observed in cells treated only with Eerl (4.4-fold). CNX and CRT protein levels were not altered by lacidipine and Eerl treatment. BiP is normally upregulated upon activation of the UPR (Schroder et al. 2005). In summary the decrease in BiP expression observed in cells treated with Eerl and lacidipine reflects lacidipine mediated attenuation of UPR induction.

Chemically induced upregulation of BcI-2 enhances mutated GC activity rescue and cell viability.

Bcl-2 is the prototype of an expanding family of proteins that regulate cell survival and apoptosis in multiple cell types (Chipuk et al., 2010). As discussed above, treatment with lacidipine prevents UPR induced apoptosis and cell death caused by Eerl treatment. The addition of lacidipine to Eerl treated cells results in upregulation of Bcl-2 expression to a considerably higher level than Eerl treatment alone (Fig. 22E). In order to investigate the role of Bcl-2 in cells treated for the rescue of mutated GC folding via UPR induction, Bcl-2 upregulation was chemically induced. Fluvastatin is a small molecule previously reported to prevent H₂0₂-induced apoptosis by upregulating Bcl-2 expression (Xu et al., 2008). Whether fluvastatin could also counteract the apoptotic effect of prolonged UPR induction was also considered. Fluvastatin was administered to cells treated with UPR inducing proteostasis modulators known to rescue native folding of mutated GC, Eerl and MG-132. MG-132 functions by inhibiting proteasomal degradation, inducing UPR and upregulating chaperone expression in GD cells (Mu et al., 2008b). Co-treatment with Eerl and MG-132 was found to dramatically enhance the activity of L444P GC (to 52% of WT activity), but at the cost of even higher induction of apoptosis. Fluvastatin (100 nM) was administered to cells treated with Eerl (2 and 6 μΜ) and MG-132 (0.6 μΜ) and tested Bcl-2 expression, apoptosis and GC activity rescue. Fluvastatin treatment caused dramatic upregulation of Bcl-2 in GD cells (18.4-fold compared to untreated cells) and did not cause any cytotoxicity (Fig. 24A-B). Upregulation of Bcl-2 was also observed upon addition of fluvastatin in Eerl-treated cells. Specifically, fluvastatin treatment caused 4.6-fold increase in Bcl-2 upregulation in cells treated with Eerl 2μΜ and 5.2-fold in cells treated with Eerl 6 μΜ compared to cells treated only with Eerl under the same conditions (Fig. 24A). Fluvastatin treatment reduced apoptosis by 0.9% in cells treated with Eerl 2 μΜ and by 3.7% in cells treated with Eerl 6 μΜ (Fig. 24B). Similar results were obtained upon addition of MG-132. Bcl-2 expression in cells treated with both Eerl and MG- 132 was downregulated (1.2-fold) compared to untreated cells. However, the addition of fluvastatin caused upregulation of Bcl-2 expression (2.8-fold) and decrease in dead cell population (4.0%). These results demonstrate that the upregulation of Bcl-2 expression enhances cellular tolerance to UPR induced stress and cell survival, therefore preventing apoptosis in cells treated for the rescue of mutated GC folding.

To investigate whether chemically induced upregulation of Bcl-2 expression affects mutated GC activity rescue, patient-derived fibroblasts harboring L444P GC were cultured with Eerl (2 and 6 μΜ), MG-132 (0.6 μΜ), and fluvastatin (100 nM) for up to 72 hrs and GC activity was measured every 24 hrs (Fig. 24C and 26). Interestingly, chemically induced upregulation of Bcl-2 expression did not reduce the increase in L444P GC activity mediated by UPR induction.

Compared to treatment with Eerl (2 or 6 μΜ) alone, co-treatment with fluvastatin and Eerl for 48 hrs did not affect L444P GC activity at low Eerl concentration, but resulted in a modest increase at high Eerl concentration (to 2.0-fold). Co-administration of Eerl and MG-132 resulted in a 3.0-fold increase in L444P GC activity as expected, which was further enhanced to 3.3-fold upon the administration of fluvastatin, indicating that upregulating Bcl-2 expression to maintain cell viability enhances Eerl and MG-132 mediated L444P GC activity rescue. Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular embodiments disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is therefore evident that the particular illustrative embodiments disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the present invention. While compositions and methods are described in terms of "comprising," "containing," or "including" various components or steps, the compositions and methods can also "consist essentially of or "consist of the various components and steps. All numbers and ranges disclosed above may vary by some amount. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range is specifically disclosed. In particular, every range of values (of the form, "from about a to about b," or, equivalently, "from approximately a to b," or, equivalently, "from approximately a-b") disclosed herein is to be understood to set forth every number and range encompassed within the broader range of values. Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. Moreover, the indefinite articles "a" or "an," as used in the claims, are defined herein to mean one or more than one of the element that it introduces. If there is any conflict in the usages of a word or term in this specification and one or more patent or other documents that may be incorporated herein by reference, the definitions that are consistent with this specification should be adopted.

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Claims

What is claimed is:

1. A method comprising administering to a subject a therapeutically effective amount of an L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure and a therapeutically effective amount of at least one inhibitor of ER-associated degradation.

2. The method of claim 1 wherein the L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure is selected from the group consisting of lacidipine, lercanidipine, nifedipine, nitrendipine, nicardipine, nimodipine, nisoldipine, manidipine, amlodipine, isradipine, felodipine, cilnidipine, benidipine, and a combination thereof.

3. The method of claim 1 wherein the subject has a lysosomal storage disease.

4. The method of claim 1 wherein the subject has Gaucher's disease.

5. The method of claim 1 wherein the subject is at least one cell.

6. The method of claim 5 wherein the at least one cell comprises at least one fibroblast derived from an individual with a lysosomal storage disease.

7. The method of claim 1 wherein the inhibitor is eeyarestatin I or kifunensine.

8. The method of claim 6 wherein the disease is Gaucher's disease.

9. A method comprising administering to a subject a therapeutically effective amount of at least one inhibitor of ER-associated degradation.

10. The method of claim 9 wherein the subject has Gaucher's disease or Tay-Sachs disease.

1 1. The method of claim 9 wherein the inhibitor is eeyarestatin I or kifunensine.

12. A method for restoring enzymatic activity to a mutated enzyme associated with a lysosomal storage disease comprising:

introducing an L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure to a subject; and

allowing the L-type Ca²⁺ channel blocker with a 1 ,4 dihydropyridine structure to modulate a cellular folding pathway while preventing apoptosis induction so as to at least partially increase the folding of the mutated enzyme.

13. The method of claim 12 wherein the L-type Ca²⁺ channel blocker with a 1 ,4 dihydropyridine structure at least partially lowers the cytoplasmic concentration of Ca²⁺.

14. The method of claim 12 wherein the L-type Ca²⁺ channel blocker with a 1 ,4 dihydropyridine structure at least partially increases the expression of a molecular chaperone BiP/GRP78.

15. The method of claim 12 wherein the L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure at least partially induces an unfolded protein response.

16. The method of claim 12 wherein the L-type Ca channel blocker with a 1,4 dihydropyridine structure at least partially decreases cytotoxicity and at least partially prevents apoptosis induction by modulation of pro- and anti-apoptotic genes.

17. The method of claim 12 wherein the L-type Ca²⁺ channel blocker with a 1,4 dihydropyridine structure is lacidipine.

18. The method of claim 12 wherein the lysosomal storage disease is Gaucher's disease.

19. The method of claim 12 further comprising introducing an inhibitor of ER- associated degradation to the subject and allowing the inhibitor to prolong ER retention of the mutated enzyme so as to at least partially increase the folding of the mutated enzyme.

20. The method of claim 19 wherein the inhibitor is eeyarestatin I.