Abstract

Gaucher disease (GD), the most common lysosomal storage disorder (LSD), is caused by defects in the activity of the lysosomal enzyme, glucocerebrosidase, resulting in intracellular accumulation of glucosylceramide (GlcCer). Neuronopathic forms, which comprise only a small percent of GD patients, are characterized by neurological impairment and neuronal cell death. Little is known about the pathways leading from GlcCer accumulation to neuronal death or dysfunction but defective calcium homeostasis appears to be one of the pathways involved. Recently, endoplasmic reticulum stress together with activation of the unfolded protein response (UPR) has been suggested to play a key role in cell death in neuronopathic forms of GD, and moreover, the UPR was proposed to be a common mediator of apoptosis in LSDs (Wei et al. (2008) Hum. Mol. Genet.17, 469–477). We now systematically examine whether the UPR is activated in neuronal forms of GD using a selection of neuronal disease models and a combination of western blotting and semi-quantitative and quantitative real-time polymerase chain reaction. We do not find any changes in either protein or mRNA levels of a number of typical UPR markers including BiP, CHOP, XBP1, Herp and GRP58, in either cultured Gaucher neurons or astrocytes, or in brain regions from mouse models, even at late symptomatic stages. We conclude that the proposition that the UPR is a common mediator for apoptosis in all neurodegenerative LSDs needs to be re-evaluated.

INTRODUCTION

The lysosomal storage disorders (LSDs) are a family of human genetic diseases caused by the defective activity of lysosomal proteins (1). Many of these disorders are characterized by severe neurological impairment (2). Of this family of approximately 40 disorders, Gaucher disease (GD) is the most common (3), although only a relatively small subset of GD patients suffer from neurological disease (4). The acute neuronopathic form of GD is characterized by severe neuronal loss (5) and by astrogliosis (6), but little information is available to delineate the biochemical pathways leading from glucosylceramide (GlcCer) accumulation to neuronal death or dysfunction (1,7).

One pathway which is involved in the pathology of a number of LSDs is altered calcium homeostasis (8). In the case of neuronopathic Gaucher disease (nGD), enhanced agonist-induced calcium release via the ryanodine receptor has been detected in both neuronal culture models (9) and in human brain tissue (10). This results in depletion of endoplasmic reticulum (ER) calcium stores, which could in turn lead to ER stress and activation of the unfolded protein response (UPR) (11). The UPR prevents accumulation of unfolded proteins in the ER by decreasing the protein-folding load, increasing the ER protein-folding capacity, and increasing degradation of misfolded proteins through ER-associated protein degradation or through autophagy (12–14); however, if ER stress is too massive, the UPR will eventually lead to cell death.

UPR activation has been observed in several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases (15), as well as in two neurodegenerative LSDs, GM1 gangliosidosis (16) and infantile neuronal ceroid lipofuscinoses (INCL) (17). In addition, UPR activation was demonstrated in fibroblasts derived from nGD patients and was suggested to be a common mediator for apoptosis in neurodegenerative LSDs (18).

To determine the possible role of the UPR in nGD, we have examined UPR activation in both neuronal culture models of nGD and in neuronal tissues obtained from mouse models of nGD. We did not obtain any evidence for UPR activation and therefore suggest that recent assumptions (18) that the UPR is a common mediator of apoptosis in neurodegenerative LSDs needs to be re-assessed.

RESULTS

Unfolded protein response in Gaucher neurons and astrocytes

We previously demonstrated that incubation of cultured hippocampal neurons for 4–9 days with 200 µm conduritol B-epoxide (CBE), an active site-directed inhibitor of glucocerebrosidase (GlcCerase) (19,20), results in an approximately five-fold increase in intracellular GlcCer levels with no effect on neuronal viability (9). To determine whether the UPR is activated in this nGD model, we examined changes in levels of a number of proteins typically used to indicate activation of the UPR; thus, BiP is an ER chaperone and a regulator of the UPR (21,22), CHOP is a transcription factor that mediates ER stress-induced apoptosis (23), and XBP1 is a potent UPR transcription factor that is spliced by removal of a short intron from its mRNA upon UPR activation (24). No changes were observed by western blot analyses of BiP in neurons incubated with CBE concentrations as high as 600 µm (Fig. 1A). Similarly, no changes were observed by reverse transcriptase–polymerase chain reaction (RT–PCR) in mRNA levels of BiP, CHOP or XBP1 after 5 (Fig. 1B), 8 or 15 (not shown) days of CBE-treatment (200 µm), and no XBP1 mRNA splicing was detected (Fig. 1B). In addition, we systematically examined mRNA levels by quantitative real-time RT-PCR (qPCR) after 8 days of CBE treatment, including analyses of GRP58, a chaperone involved in glycoprotein folding, and Herp (HERPUD1), a ubiquitin-like membrane protein, both of which are induced by ER stress (21,25). No changes were observed in mRNA levels of BiP, CHOP, Herp or GRP58 (Fig. 1C).

Figure 1.

Analysis of unfolded protein response markers in conduritol B-epoxide (CBE)-treated neurons. (A) Hippocampal neurons were treated for 4 days with CBE and levels of BiP analyzed by western blotting. Tubulin was used as loading control. (B) Hippocampal neurons were treated for 5 days with 200 µm CBE and mRNA levels were measured by RT-PCR. Thapsigargin (10 µm, 2 h) treatment of neurons acted as a positive control for XBP1 splicing. GAPDH served as reference gene. (C) Hippocampal neurons were treated for 8 days with 200 µm CBE and mRNA levels were measured by quantitative PCR. Results are shown as fold-change of mRNA levels in CBE-treated versus untreated neurons. Values are means ± SD of results obtained from three independent cultures. Cycle threshold values were normalized using HPRT.

Figure 1.

Analysis of unfolded protein response markers in conduritol B-epoxide (CBE)-treated neurons. (A) Hippocampal neurons were treated for 4 days with CBE and levels of BiP analyzed by western blotting. Tubulin was used as loading control. (B) Hippocampal neurons were treated for 5 days with 200 µm CBE and mRNA levels were measured by RT-PCR. Thapsigargin (10 µm, 2 h) treatment of neurons acted as a positive control for XBP1 splicing. GAPDH served as reference gene. (C) Hippocampal neurons were treated for 8 days with 200 µm CBE and mRNA levels were measured by quantitative PCR. Results are shown as fold-change of mRNA levels in CBE-treated versus untreated neurons. Values are means ± SD of results obtained from three independent cultures. Cycle threshold values were normalized using HPRT.

The lack of UPR activation might be consistent with the normal viability of CBE-treated neurons. Indeed, early studies suggested that neuronal viability is not affected for at least 15 days incubation with CBE (26); however, CBE-treated neurons did show pronounced changes in axonal (26) [but not dendritic (27)] branching. Later studies showed significant changes in agonist-induced calcium release in CBE-treated neurons, although basal levels of calcium release were normal (9). We therefore examined whether the UPR was induced in nGD neurons upon disruption of calcium homeostasis. Rat hippocampal neurons were incubated with thapsigargin, which depletes ER calcium stores (28), and as a result leads to UPR activation and apoptosis (11,29). No differences were observed in neuronal death between control and CBE-treated neurons (6 days) after thapsigargin treatment (76.7% dead cells in control neurons and 69.3% dead cells in CBE-treated neurons after 2 h incubation with 7 µm thapsigargin, and 25.9% and 26.6% dead cells, respectively, using 8 µm thapsigargin). These results imply that there is no difference in the response of CBE-treated compared with control neurons to an inducer of ER stress, strongly suggesting that the UPR is not altered in this neuronal model of nGD even under conditions of ER stress.

A recent study demonstrated glial pathology in neuronal forms of GD (6). We therefore examined whether the UPR is induced in astrocytes, glial cells which play an active role in brain function and in neurodegenerative diseases (30,31). Cultured astrocytes were incubated with [4,5-3H]sphinganine, a precursor of sphingolipid synthesis, which can be used to metabolically label glycosphingolipids including GlcCer (32). Significant [3H]GlcCer accumulation was observed (Fig. 2A) after CBE-treatment (200 µm, 5 days), and no changes were observed in astrocyte viability (Fig. 2B). BiP protein levels were unchanged (Fig. 3A), as were mRNA levels of BiP, CHOP and XBP1 (Fig. 3B and C), Herp and GRP58 (Fig. 3C). The above findings do not lend support to the notion that the UPR is activated in cultured nGD neurons or glia.

Figure 2.

Conduritol B-epoxide (CBE)-treated astrocytes. (A) Astrocytes were incubated with 225 pmol of [4,5-3H] sphinganine for 6 h, and then treated with or without 200 µm CBE for 5 days. The area of the thin layer chromatography plate corresponding to [3H]GlcCer is shown. (B) Cell viability of CBE-treated (200 µm, 5 days) astrocytes was compared with control astrocytes. Results are means ± SD of two independent experiments.

Figure 2.

Conduritol B-epoxide (CBE)-treated astrocytes. (A) Astrocytes were incubated with 225 pmol of [4,5-3H] sphinganine for 6 h, and then treated with or without 200 µm CBE for 5 days. The area of the thin layer chromatography plate corresponding to [3H]GlcCer is shown. (B) Cell viability of CBE-treated (200 µm, 5 days) astrocytes was compared with control astrocytes. Results are means ± SD of two independent experiments.

Figure 3.

Analysis of unfolded protein response markers in CBE-treated astrocytes. (A) Astrocytes were treated for 5 days with 200 µm CBE and levels of BiP analyzed by western blotting. Tubulin was used as loading control. The experiment was repeated two times with similar results. (B) Astrocytes were treated for 5 days with 200 µM CBE and mRNA levels were measured by RT-PCR. Thapsigargin (Tg) treatment of neurons acted as a positive control for XBP1 splicing. GAPDH served as reference gene. (C) Astrocytes were treated for 5–7 days with 200 µm CBE and mRNA levels were measured by quantitative PCR. Results are shown as fold-change of mRNA levels in CBE-treated versus -untreated astrocytes. Values are means ± SD of results obtained from three independent cultures. Cycle threshold values were normalized using HPRT.

Figure 3.

Analysis of unfolded protein response markers in CBE-treated astrocytes. (A) Astrocytes were treated for 5 days with 200 µm CBE and levels of BiP analyzed by western blotting. Tubulin was used as loading control. The experiment was repeated two times with similar results. (B) Astrocytes were treated for 5 days with 200 µM CBE and mRNA levels were measured by RT-PCR. Thapsigargin (Tg) treatment of neurons acted as a positive control for XBP1 splicing. GAPDH served as reference gene. (C) Astrocytes were treated for 5–7 days with 200 µm CBE and mRNA levels were measured by quantitative PCR. Results are shown as fold-change of mRNA levels in CBE-treated versus -untreated astrocytes. Values are means ± SD of results obtained from three independent cultures. Cycle threshold values were normalized using HPRT.

Unfolded protein response in Gaucher brain

Within the past year, a mouse model of nGD has become available (33). In the Gbaflox/flox;Nestin-Cre mouse (hereafter referred to as the Nestin-flox−/− mouse), GlcCerase deficiency is restricted to neural and glial progenitor cells, and the mice exhibit rapid motor dysfunction associated with severe neurodegeneration and apoptotic cell death, and develop paralysis by 21 days of age (33). We examined UPR activation at two different stages of development, the presymptomatic (9 days) and symptomatic stages (21 days). Western blot analyses revealed that levels of BiP were unaltered in the cortex (Fig. 4A) at both of these stages, and also unaltered in the cerebellum and hippocampus after 21 days (not shown). RT-PCR revealed no changes in XBP1 levels or in XBP1 splicing in the cerebellum (Fig. 4B), cortex, brain stem and thalamus (not shown). We next performed an extensive qPCR analysis of mRNA levels of BiP, GRP58, Herp and CHOP in five different brain areas of 9-day and 21-day-old mice. No changes were observed in mRNA levels in the first three markers (Table 1). In some cases, levels of CHOP were elevated to a limited extent of between 1.42-fold and 1.90-fold, but these changes only occurred in some brain areas and were not consistent between the different mice examined (Table 1). Moreover, CHOP can be induced upon elevation of cytosolic calcium levels in a UPR-independent manner (34).

Figure 4.

Analysis of unfolded protein response markers in nestin-flox mice. (A) BiP was measured by western blot analysis in cortical tissue from nestin-flox−/− mice and compared with +/− mice. Tubulin was used as loading control. Duplicates from different mice are shown. (B) RT-PCR analysis of XBP1 splicing in cerebella obtained from nestin-flox mice. Thapsigargin (Tg) served as positive control for XBP1 splicing and HPRT served as the reference gene. This experiment was repeated three times with similar results.

Figure 4.

Analysis of unfolded protein response markers in nestin-flox mice. (A) BiP was measured by western blot analysis in cortical tissue from nestin-flox−/− mice and compared with +/− mice. Tubulin was used as loading control. Duplicates from different mice are shown. (B) RT-PCR analysis of XBP1 splicing in cerebella obtained from nestin-flox mice. Thapsigargin (Tg) served as positive control for XBP1 splicing and HPRT served as the reference gene. This experiment was repeated three times with similar results.

Table 1.

Quantitative polymerase chain reaction analysis of unfolded protein response markers in Nestin-flox mice

 Ratio (nestin-flox−/− versus +/−)
 
 BiP GRP58 Herp CHOP 
9 days-old 
 Cortex 0.77 ± 0.21 0.91 ± 0.16 0.95 ± 0.08 1.08 ± 0.20 
 Hippocampus 1.04 ± 0.26 0.90 ± 0.25 1.09 ± 0.13 0.86 ± 0.19 
 Thalamus 0.86 ± 0.19 0.79 ± 0.13 0.85 ± 0.15 0.83 ± 0.26 
 Cerebellum 0.89 ± 0.66 0.85 ± 0.55 1.00 ± 0.91 1.42 ± 0.37 
 Brain stem 0.85 ± 0.42 0.76 ± 0.33 0.63 ± 0.31 1.63 ± 0.48 
21 days-old 
 Cortex 1.12 ± 0.21 0.97 ± 0.18 1.19±0.11 1.30 ± 0.12 
 Hippocampus 1.09 ± 0.10 1.18 ± 0.07 1.23 ± 0.10 1.04 ± 0.09 
 Thalamus 1.18 ± 0.11 1.26 ± 0.12 0.78 ± 0.14 1.61 ± 0.12 
 Cerebellum 0.96 ± 0.33 1.34 ± 0.16 0.94 ± 0.13 1.90 ± 0.08 
 Brain stem 1.00 ± 0.31 0.93 ± 0.17 1.07 ± 0.17 1.52 ± 0.12 
 Ratio (nestin-flox−/− versus +/−)
 
 BiP GRP58 Herp CHOP 
9 days-old 
 Cortex 0.77 ± 0.21 0.91 ± 0.16 0.95 ± 0.08 1.08 ± 0.20 
 Hippocampus 1.04 ± 0.26 0.90 ± 0.25 1.09 ± 0.13 0.86 ± 0.19 
 Thalamus 0.86 ± 0.19 0.79 ± 0.13 0.85 ± 0.15 0.83 ± 0.26 
 Cerebellum 0.89 ± 0.66 0.85 ± 0.55 1.00 ± 0.91 1.42 ± 0.37 
 Brain stem 0.85 ± 0.42 0.76 ± 0.33 0.63 ± 0.31 1.63 ± 0.48 
21 days-old 
 Cortex 1.12 ± 0.21 0.97 ± 0.18 1.19±0.11 1.30 ± 0.12 
 Hippocampus 1.09 ± 0.10 1.18 ± 0.07 1.23 ± 0.10 1.04 ± 0.09 
 Thalamus 1.18 ± 0.11 1.26 ± 0.12 0.78 ± 0.14 1.61 ± 0.12 
 Cerebellum 0.96 ± 0.33 1.34 ± 0.16 0.94 ± 0.13 1.90 ± 0.08 
 Brain stem 1.00 ± 0.31 0.93 ± 0.17 1.07 ± 0.17 1.52 ± 0.12 

Results are expressed as a ratio of mRNA levels in nestin-flox−/− mice versus +/− mice. Values are means ± SEM (n = 2 for 9-day-old mice, and n = 3 for 21-day-old mice). Cycle threshold values were normalized to levels of HPRT or TBP.

Finally, we examined whether the UPR is altered in two other mouse models of nGD, the Gba mouse (35) and the L444P mouse (36). The Gba mouse dies soon after birth owing to skin permeability dysfunction (37). No changes were observed in BiP, CHOP and XBP1 levels in either the cortex from day 18 embryonic Gba mice or in hippocampal neurons cultured from day 18 embryos (not shown). The L444P mouse, which carries a mutation that most commonly leads to nGD, does not accumulate significant levels of GlcCer (36), and does not exhibit changes in UPR markers (BiP, CHOP and XBP1) in brain regions of 3-month-old mice (as determined by western blot analysis of BiP in the cortex, hippocampus, thalamus, cerebellum and brain stem, not shown) or in cultured neurons from embryonic mice (as determined by RT-PCR of BiP, CHOP and XBP-1 in 5-day-old cultures, not shown).

DISCUSSION

The major finding of the current study is that we find no evidence for UPR activation in various models of nGD. This result is somewhat unexpected as a recent report by Wei et al. (18) suggested that the UPR is activated in nGD, and furthermore proposed that the UPR is a common mediator of apoptosis in both neurodegenerative and non-neurodegenerative LSDs. Indeed, other studies demonstrated UPR activation in two neurodegenerative LSDs, the GM1 gangliosidosis (16) and INCL (17,38,39) (Table 2).

Table 2.

Summary of published data regarding unfolded protein response (UPR) activation in neuronal models of neurodegenerative lysosomal storage disorders

 GM1 gangliosidosis INCL Sialidosis nGD 
Defective enzyme β-galactosidase Palmitoyl-protein thioesterase-1 (PPT1) Neuraminidase GlcCerase 
Accumulating substrate GM1 Palmitoylated proteins Glycoproteins containing sialic acid residues GlcCer 
Model used to investigate UPR activation Spinal cord tissue from β-gal−/− mice and neurospheres Brain tissue from PPT1-KO mice Spinal cord tissue from Neo1−/− mice Cultured neurons and astrocytes, brain tissue from mouse models 
Proposed mechanism of UPR activation GM1 accumulation leading to depletion of ER Ca2+ stores Abnormal accumulation of palmitoylated proteins within the ER No activation of the UPR No activation of the UPR 
Reference Tessitore et al. (16Zhang et al. (17Tessitore et al. (16This study 
 GM1 gangliosidosis INCL Sialidosis nGD 
Defective enzyme β-galactosidase Palmitoyl-protein thioesterase-1 (PPT1) Neuraminidase GlcCerase 
Accumulating substrate GM1 Palmitoylated proteins Glycoproteins containing sialic acid residues GlcCer 
Model used to investigate UPR activation Spinal cord tissue from β-gal−/− mice and neurospheres Brain tissue from PPT1-KO mice Spinal cord tissue from Neo1−/− mice Cultured neurons and astrocytes, brain tissue from mouse models 
Proposed mechanism of UPR activation GM1 accumulation leading to depletion of ER Ca2+ stores Abnormal accumulation of palmitoylated proteins within the ER No activation of the UPR No activation of the UPR 
Reference Tessitore et al. (16Zhang et al. (17Tessitore et al. (16This study 

ER, endoplasmic reticulum; INCL, infantile neuronal ceroid lipofuscinoses; nGD, neuronopathic Gaucher disease; GlcCer, glucosylceramide; GlcCerase, glucocerebrosidase.

There are two noteworthy differences between the study by Wei et al. (18). and the current study. First, in the study by Wei et al., ER stress and the UPR were examined in human skin fibroblast cell lines derived from patients characterized with type 2 (the acute neuronopathic form) nGD, whereas in our study, primary tissues obtained from neuronal models of nGD were used including cultured neurons and glia and also tissues from mouse models of nGD. The use of neuronal cells and brain tissues is of more relevance for understanding which biochemical pathways are triggered in a neurological disease than skin fibroblast cell lines, particularly in light of the altered calcium homeostasis observed in the neuronal nGD models (9,10,40,41), as a result of GlcCer accumulation (40) which could potentially lead to UPR activation (11,14). Secondly, there is no evidence that fibroblasts accumulate significant levels of storage materials in LSDs. This is particularly relevant in diseases such as the gangliosidoses, since gangliosides are not found at high levels in non-neuronal tissues, and evidence for UPR activation in fibroblasts derived from GM1 and GM2 gangliosidoses patients (18) is therefore unlikely to be the result of ganglioside storage. Likewise, in order to conclude that GlcCer storage is the cause of UPR activation in the Gaucher skin fibroblast cell lines, significant levels of accumulation, such as those shown in neuronal cells, need to be demonstrated.

Moreover, the skin fibroblast cell lines used by Wei et al. were obtained from patients with the L444P mutation, whereas our study used mainly either cultured cells treated with CBE (20) or brain tissues derived from the Nestin-flox−/− or Gba−/− mouse, both of which do not contain any GlcCerase. However, analysis of neurons cultured from the L444P mouse, or brain regions from 3-month old L444P mouse, did not reveal any evidence for UPR activation. Thus, we conclude that the UPR is not activated in neuronopathic models of nGD as a result of either GlcCer-induced altered calcium homeostasis or protein misfolding.

Unfortunately, we were not able to determine if the UPR is activated in human neuronopathic Gaucher brain tissue, because of the scarcity of this tissue. In addition, infant or juvenile control brains would have been obtained from individuals that either succumbed to a fatal disease, or to a trauma, in which the UPR might be activated, rendering problematic comparison with infantile or juvenile neuronopathic Gaucher brains.

Despite the lack of evidence of UPR activation in nGD, there is some data supporting UPR activation in two other LSDs (Table 2). In both of these cases, the UPR was examined in neuronal tissues [and in neurospheres obtained from neuronal tissues in the case of the GM1 gangliosidosis (16)], but in contrast, was not observed in spinal cord tissue from a neuraminidase-deficient mice, a model of sialidosis. Together with our data on neuronal models of nGD, it seems reasonable to conclude that the UPR is not a common pathway activated in neurodegenerative LSDs, and that the UPR is not activated merely as a result of a lysosomal insult. Rather, the extent and type of accumulated substrate, and perhaps even the type of mutation, presumably determines disease outcome by triggering a unique cascade of specific cellular events, which ultimately results in cell dysfunction and cell death. Our data do not support the assumption that UPR activation secondary to lysosomal dysfunction underlies pathogenesis in all neurodegenerative LSDs (18), but rather imply that different signalling pathways, as yet unknown, are activated in different diseases, which may lead to the unique pathophysiology described for each disease.

MATERIALS AND METHODS

Materials

CBE was from Matreya (Pleasant Gap, PA) and thapsigargin was from Sigma (St Louis, MO).

Hippocampal cultures

Hippocampal neurons were cultured from embryonic day 16–17 mice [C57BL/6J, Gba (35,42) or L444P (36)] or from embryonic day 17–18 Wistar rats (Harlan) essentially as described (26,42,43), except that neurons were cultured in Neurobasal medium containing B27 supplement (Gibco) and Glutamax. To prevent proliferation of non-neuronal cells, 5 µm of cytosine arabinoside (Sigma) was added 2–3 days after plating.

Glial cultures

Cortices from postnatal day 0–2 ICR mice (Harlan) were dissociated by trypsinization (2.5%, w/v) and DNase treatment (Sigma, 1% w/v, 15 min, 37°C) (43). Tissue was washed in Mg/Ca-free Hank's balanced salt solution and dissociated by repeated passage through a Pasteur pipette. Cells were plated on 60 mm plates (Falcon) in minimal essential medium (plus glutamine) with 10% horse serum and penicillin-streptomycin (100 µg/ml). Medium was replaced every 2–3 days. After cells reached confluency (after 7–10 days in culture), proliferation was stopped by addition of cytosine arabinoside (5 µm).

Cell pellets were either used immediately for RNA or protein extraction, or snap-frozen in liquid N2 and stored at −80°C.

Nestin-flox/flox mice

Flox/flox Cre−/− females were crossed with wild-type/flox Cre+/− males to generate flox/flox Cre+/− mice (the Nestin-flox−/− Gaucher mouse) and wild-type/flox Cre+/− mice, which served as healthy controls. Genotyping was performed by PCR using genomic DNA extracted from mouse tails or embryonic brains as previously described (33). For characterization of the Gba allele, primer 1 (forward: 5′-GTACGTTCATGGCATTGCTGTTCACT-3′) and primer 2 (forward: 5′-ATTCCAGCTGTCCCTCGTCTCC-3′) were used. A 1.9 kb fragment was generated from the wild-type allele, a 2.1-kb fragment from the floxed allele, and a 0.7-kb fragment from the null allele. For detection of Cre, the following primers were used–forward: 5′-ACGAGTGATGAGGTTCGCAA-3′; reverse: 5′-AGCGTTTTCGTTCTGCCAAT-3′, which generated a 600-bp fragment in the Cre+ mice. The colony was maintained in the Experimental Animal Center of the Weizmann Institute.

RNA extraction and polymerase chain reaction

Total RNA was isolated using the RNeasy mini kit (Qiagen) according to manufacturer's instructions, which included a DNAse step and addition of β-mercaptoethanol. cDNA synthesis was performed using the Reverse-iT first strand synthesis kit (Thermo Scientific) using random decamers. cDNA products were stored at −20°C.

Semi-quantitative RT-PCR was performed using Taq DNA polymerase master mix red (Genetec), with cDNA (amount equivalent to 2.5 ng of total RNA), and primer concentrations of 2 nm, in a reaction volume of 25 µl. All reactions were performed in duplicate. The thermal cycling parameters were as follows: step 1, 94°C for 4 min; step 2, 94°C for 30 s, 60°C for 30 s, 72°C for 50 s. Step 2 was repeated for 22–30 cycles. PCR products were separated by electrophoresis on a 1.5–2% agarose gel, except for XBP1 PCR products, which were separated on a 3% agarose gel. GAPDH or HPRT served as reference genes. Primer sequences are given in Table 3.

Table 3.

Primers used for polymerase chain reaction

Gene Primer sequence Reference 
Primers used for semi-quantitative RT-PCR 
BiP F: 5′-CTGGGTACATTTGATCTGACTG-3′ – 
 R: 5′-GCATCCTGGTGGCTTTCCAGCCAT-3′  
CHOP F: 5′-CATACACCACCACACCTGAAAG-3′ Kieran et al. (46
 R: 5′-CCGTTTCCTAGTTCTTCCTTGC-3′  
XBP1 F: 5′-AAACAGAGTAGCAGCGCAGACTGC-3′ Koh et al. (47
 R: 5′-GGATCTCTAAAACTAGAGGCTTGGTG-3′  
GAPDH F: 5′-TTAGCACCCCTGGCCAAGG-3′ Brann et al. (48
 R: 5′-CTTACTCCTTGGAGGCCATG-3′  
HPRT F: 5′-TGCTCGAGATGTCATGAAGG-3′ Hardman et al. (49
 R: 5′-AATCCAGCAGGTCAGCAAAG-3′  
Primers used for qPCR 
BiP F: 5′-TCATCGGACGCACTTGGAA-3′ Hetz et al. (50
 R: 5′-CAACCACCTTGAATGGCAAGA-3′  
CHOP F: 5′-GTCCCTAGCTTGGCTGACAGA-3′ Hetz et al. (50
 R: 5′-TGGAGAGCGAGGGCTTTG-3′  
Herp F: 5′-CATGTACCTGCACCACGTCG-3′ Hetz et al. (50
 R:5′- GAGGACCACCATCATCCGG-3′  
GRP58 F: 5′-GAGGCTTGCCCCTGAGTATG-3′ Hetz et al. (50
 R: 5′-GTTGGCAGTGCAATCCACC-3′  
TBP F: 5′-TGCTGTTGGTGATTGTTGGT-3′ – 
 R: 5′-CTGGCTTGTGTGGGAAAGAT-3′  
Actin F: 5′-TCAGGAGGAGCAATGATCTTG-3′ – 
 R: 5′-CGTTGACATCCGTAAAGACCT-3′  
HPRT F: 5′-TGCTCGAGATGTCATGAAGG-3′ Hardman et al. (49
 R: 5′-AATCCAGCAGGTCAGCAAAG-3′  
Gene Primer sequence Reference 
Primers used for semi-quantitative RT-PCR 
BiP F: 5′-CTGGGTACATTTGATCTGACTG-3′ – 
 R: 5′-GCATCCTGGTGGCTTTCCAGCCAT-3′  
CHOP F: 5′-CATACACCACCACACCTGAAAG-3′ Kieran et al. (46
 R: 5′-CCGTTTCCTAGTTCTTCCTTGC-3′  
XBP1 F: 5′-AAACAGAGTAGCAGCGCAGACTGC-3′ Koh et al. (47
 R: 5′-GGATCTCTAAAACTAGAGGCTTGGTG-3′  
GAPDH F: 5′-TTAGCACCCCTGGCCAAGG-3′ Brann et al. (48
 R: 5′-CTTACTCCTTGGAGGCCATG-3′  
HPRT F: 5′-TGCTCGAGATGTCATGAAGG-3′ Hardman et al. (49
 R: 5′-AATCCAGCAGGTCAGCAAAG-3′  
Primers used for qPCR 
BiP F: 5′-TCATCGGACGCACTTGGAA-3′ Hetz et al. (50
 R: 5′-CAACCACCTTGAATGGCAAGA-3′  
CHOP F: 5′-GTCCCTAGCTTGGCTGACAGA-3′ Hetz et al. (50
 R: 5′-TGGAGAGCGAGGGCTTTG-3′  
Herp F: 5′-CATGTACCTGCACCACGTCG-3′ Hetz et al. (50
 R:5′- GAGGACCACCATCATCCGG-3′  
GRP58 F: 5′-GAGGCTTGCCCCTGAGTATG-3′ Hetz et al. (50
 R: 5′-GTTGGCAGTGCAATCCACC-3′  
TBP F: 5′-TGCTGTTGGTGATTGTTGGT-3′ – 
 R: 5′-CTGGCTTGTGTGGGAAAGAT-3′  
Actin F: 5′-TCAGGAGGAGCAATGATCTTG-3′ – 
 R: 5′-CGTTGACATCCGTAAAGACCT-3′  
HPRT F: 5′-TGCTCGAGATGTCATGAAGG-3′ Hardman et al. (49
 R: 5′-AATCCAGCAGGTCAGCAAAG-3′  

Real-time quantitative RT-PCR (qPCR) was performed using the SYBR Green PCR Master Mix (Finnzyme), and an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with cDNA (equivalent to 8.75 ng of total RNA for CHOP analysis and 2.5 ng for the other genes). The primer concentration was 13 nm in a reaction volume of 20 µl. Each reaction was preformed in triplicate. The thermal cycling parameters were as follows: step 1, 95°C for 10 min; step 2, 95°C for 15 s, 60°C for 30 s and 68°C for 30 s. Step 2 was repeated for 40 cycles and was followed by a dissociation step. The relative amounts of mRNA were calculated from the CT values using hypoxanthine-guanine phosphoribosyltransferase (HPRT) or TATA box binding protein (TBP) for normalization. Normalization quality was assessed using geNorm software with respect to the reference genes HPRT, actin and TBP. The standard error of the ratio was estimated using the bootstrap method (http://www.R-project.org) (44) in the R statistical package (45). Primer sequences are listed in Table 3.

Western blotting

Brain tissue or cells pellets were lysed in ∼6 volumes of RIPA buffer (50 mm Tris pH 7.4, 150 mm NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm EDTA) supplemented with protease and phosphatase inhibitor cocktails (1:100, Sigma). Following homogenization, samples were centrifuged at 14 000gav for 20 min at 4°C, and the supernatant was collected. Protein was quantified using the BCA protein assay reagent (Pierce Chemical Co.). About 10 or 20 µg of protein in sample buffer were electrophoresed on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were incubated with primary antibodies (rabbit anti-BiP (Abcam) 1:10 000 dilution, or mouse anti-tubulin (Sigma) at 1:10 000 dilution for cultured cells or 1:500 000 dilution for tissues) and then incubated with a horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch). Bound antibodies were detected using the SuperSignal West Pico chemiluminescent substrate (Thermo scientific).

Lipid analysis

Metabolic labeling of astrocytes with [4,5-(3)H]sphinganine was performed as described (9,32).

Viability

Astrocytes were analyzed by flow cytometry using the PI signal detector (FL2) in a FACSort flow cytometer (Becton Dickinson & Co.).

FUNDING

This work was supported by the Children's Gaucher Research Fund.

ACKNOWLEDGEMENTS

We would like to thank Eyal Kamhi for his advice regarding qPCR experiments and Dr Marc Offman for performing the bootstrap statistical analysis. A.H. Futerman is the Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute of Science.

Conflict of Interest statement. The authors declare no conflict of interest.

REFERENCES

1
Futerman
A.H.
van Meer
G.
The cell biology of lysosomal storage disorders
Nat. Rev. Mol. Cell Biol.
 , 
2004
, vol. 
5
 (pg. 
554
-
565
)
2
Platt
F.M.
Walkley
S.
Lysosomal Disorders of the Brain
 , 
2004
Oxford
Oxford University Press
3
Beutler
E.
Grabowski
G.A.
Scriver
C.R.
Sly
W.S.
Childs
B.
Beaudet
A.L.
Valle
D.
Kinzler
K.W.
Vogelstein
B.
Gaucher disease
The Metabolic and Molecular Bases of Inherited Disease
 , 
2001
, vol. 
Vol. II
 
McGraw-Hill Inc.
(pg. 
3635
-
3668
)
4
Jmoudiak
M.
Futerman
A.H.
Gaucher disease: pathological mechanisms and modern management
Br. J. Haematol.
 , 
2005
, vol. 
129
 (pg. 
178
-
188
)
5
Sidransky
E.
Tayebi
N.
Stubblefield
B.K.
Eliason
W.
Klineburgess
A.
Pizzolato
G.P.
Cox
J.N.
Porta
J.
Bottani
A.
DeLozier-Blanchet
C.D.
The clinical, molecular, and pathological characterisation of a family with two cases of lethal perinatal type 2 Gaucher disease
J. Med. Genet.
 , 
1996
, vol. 
33
 (pg. 
132
-
136
)
6
Wong
K.
Sidransky
E.
Verma
A.
Mixon
T.
Sandberg
G.D.
Wakefield
L.K.
Morrison
A.
Lwin
A.
Colegial
C.
Allman
J.M.
, et al.  . 
Neuropathology provides clues to the pathophysiology of Gaucher disease
Mol. Genet. Metab.
 , 
2004
, vol. 
82
 (pg. 
192
-
207
)
7
Jeyakumar
M.
Dwek
R.A.
Butters
T.D.
Platt
F.M.
Storage solutions: treating lysosomal disorders of the brain
Nat. Rev. Neurosci.
 , 
2005
, vol. 
6
 (pg. 
713
-
725
)
8
Ginzburg
L.
Kacher
Y.
Futerman
A.H.
The pathogenesis of glycosphingolipid storage disorders
Semin. Cell Dev. Biol.
 , 
2004
, vol. 
15
 (pg. 
417
-
431
)
9
Korkotian
E.
Schwarz
A.
Pelled
D.
Schwarzmann
G.
Segal
M.
Futerman
A.H.
Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons
J. Biol. Chem.
 , 
1999
, vol. 
274
 (pg. 
21673
-
21678
)
10
Pelled
D.
Trajkovic-Bodennec
S.
Lloyd-Evans
E.
Sidransky
E.
Schiffmann
R.
Futerman
A.H.
Enhanced calcium release in the acute neuronopathic form of Gaucher disease
Neurobiol. Dis.
 , 
2005
, vol. 
18
 (pg. 
83
-
88
)
11
Kaufman
R.J.
Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls
Genes Dev.
 , 
1999
, vol. 
13
 (pg. 
1211
-
1233
)
12
Ron
D.
Walter
P.
Signal integration in the endoplasmic reticulum unfolded protein response
Nat. Rev. Mol. Cell Biol.
 , 
2007
, vol. 
8
 (pg. 
519
-
529
)
13
Mori
K.
Tripartite management of unfolded proteins in the endoplasmic reticulum
Cell
 , 
2000
, vol. 
101
 (pg. 
451
-
454
)
14
Kaufman
R.J.
Orchestrating the unfolded protein response in health and disease
J. Clin. Invest.
 , 
2002
, vol. 
110
 (pg. 
1389
-
1398
)
15
Lindholm
D.
Wootz
H.
Korhonen
L.
ER stress and neurodegenerative diseases
Cell. Death Differ.
 , 
2006
, vol. 
13
 (pg. 
385
-
392
)
16
Tessitore
A.
del
P.M.M.
Sano
R.
Ma
Y.
Mann
L.
Ingrassia
A.
Laywell
E.D.
Steindler
D.A.
Hendershot
L.M.
d'Azzo
A.
GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis
Mol. Cell
 , 
2004
, vol. 
15
 (pg. 
753
-
766
)
17
Zhang
Z.
Lee
Y.C.
Kim
S.J.
Choi
M.S.
Tsai
P.C.
Xu
Y.
Xiao
Y.J.
Zhang
P.
Heffer
A.
Mukherjee
A.B.
Palmitoyl-protein thioesterase-1 deficiency mediates the activation of the unfolded protein response and neuronal apoptosis in INCL
Hum. Mol. Genet.
 , 
2006
, vol. 
15
 (pg. 
337
-
346
)
18
Wei
H.
Kim
S.J.
Zhang
Z.
Tsai
P.C.
Wisniewski
K.E.
Mukherjee
A.B.
ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neurodegenerative lysosomal storage disorders and are alleviated by chemical chaperones
Hum. Mol. Genet.
 , 
2008
, vol. 
17
 (pg. 
469
-
477
)
19
Legler
G.
Bieberich
E.
Active site directed inhibition of a cytosolic beta-glucosidase from calf liver by bromoconduritol B epoxide and bromoconduritol F
Arch. Biochem. Biophys.
 , 
1988
, vol. 
260
 (pg. 
437
-
442
)
20
Premkumar
L.
Sawkar
A.R.
Boldin-Adamsky
S.
Toker
L.
Silman
I.
Kelly
J.W.
Futerman
A.H.
Sussman
J.L.
X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease
J. Biol. Chem.
 , 
2005
, vol. 
280
 (pg. 
23815
-
23819
)
21
Ni
M.
Lee
A.S.
ER chaperones in mammalian development and human diseases
FEBS Lett.
 , 
2007
, vol. 
581
 (pg. 
3641
-
3651
)
22
Lee
A.S.
The glucose-regulated proteins: stress induction and clinical applications
Trends Biochem. Sci.
 , 
2001
, vol. 
26
 (pg. 
504
-
510
)
23
Harding
H.P.
Zhang
Y.
Zeng
H.
Novoa
I.
Lu
P.D.
Calfon
M.
Sadri
N.
Yun
C.
Popko
B.
Paules
R.
, et al.  . 
An integrated stress response regulates amino acid metabolism and resistance to oxidative stress
Mol. Cell
 , 
2003
, vol. 
11
 (pg. 
619
-
633
)
24
Yoshida
H.
Matsui
T.
Yamamoto
A.
Okada
T.
Mori
K.
XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor
Cell
 , 
2001
, vol. 
107
 (pg. 
881
-
891
)
25
Kokame
K.
Agarwala
K.L.
Kato
H.
Miyata
T.
Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress
J. Biol. Chem.
 , 
2000
, vol. 
275
 (pg. 
32846
-
32853
)
26
Schwarz
A.
Rapaport
E.
Hirschberg
K.
Futerman
A.H.
A regulatory role for sphingolipids in neuronal growth: inhibition of sphingolipid synthesis and degradation have opposite effects on axonal branching
J. Biol. Chem.
 , 
1995
, vol. 
270
 (pg. 
10990
-
10998
)
27
Schwarz
A.
Futerman
A.H.
Inhibition of sphingolipid synthesis, but not degradation, alters the rate of dendrite growth in cultured hippocampal neurons
Brain Res. Dev. Brain Res.
 , 
1998
, vol. 
108
 (pg. 
125
-
130
)
28
Treiman
M.
Caspersen
C.
Christensen
S.B.
A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPases
Trends Pharmacol. Sci.
 , 
1998
, vol. 
19
 (pg. 
131
-
135
)
29
Price
B.D.
Mannheim-Rodman
L.A.
Calderwood
S.K.
Brefeldin A, thapsigargin, and AIF4- stimulate the accumulation of GRP78 mRNA in a cycloheximide dependent manner, whilst induction by hypoxia is independent of protein synthesis
J. Cell. Physiol.
 , 
1992
, vol. 
152
 (pg. 
545
-
552
)
30
Giaume
C.
Kirchhoff
F.
Matute
C.
Reichenbach
A.
Verkhratsky
A.
Glia: the fulcrum of brain diseases
Cell Death Differ.
 , 
2007
, vol. 
14
 (pg. 
1324
-
1335
)
31
Maragakis
N.J.
Rothstein
J.D.
Mechanisms of disease: astrocytes in neurodegenerative disease
Nat. Clin. Pract. Neurol.
 , 
2006
, vol. 
2
 (pg. 
679
-
689
)
32
Hirschberg
K.
Zisling
R.
van Echten-Deckert
G.
Futerman
A.H.
Ganglioside synthesis during the development of neuronal polarity: major changes occur during axonogenesis and axon elongation, but not during dendrite growth or during synaptogenesis
J. Biol. Chem.
 , 
1996
, vol. 
271
 (pg. 
14876
-
14882
)
33
Enquist
I.B.
Lo Bianco
C.
Ooka
A.
Nilsson
E.
Mansson
J.E.
Ehinger
M.
Richter
J.
Brady
R.O.
Kirik
D.
Karlsson
S.
Murine models of acute neuronopathic Gaucher disease
Proc. Natl Acad. Sci. USA
 , 
2007
, vol. 
104
 (pg. 
17483
-
17488
)
34
Copanaki
E.
Schurmann
T.
Eckert
A.
Leuner
K.
Muller
W.E.
Prehn
J.H.
Kogel
D.
The amyloid precursor protein potentiates CHOP induction and cell death in response to ER Ca2+ depletion
Biochim. Biophys. Acta
 , 
2007
, vol. 
1773
 (pg. 
157
-
165
)
35
Tybulewicz
V.L.
Tremblay
M.L.
LaMarca
M.E.
Willemsen
R.
Stubblefield
B.K.
Winfield
S.
Zablocka
B.
Sidransky
E.
Martin
B.M.
Huang
S.P.
, et al.  . 
Animal model of Gaucher's disease from targeted disruption of the mouse glucocerebrosidase gene
Nature
 , 
1992
, vol. 
357
 (pg. 
407
-
410
)
36
Mizukami
H.
Mi
Y.
Wada
R.
Kono
M.
Yamashita
T.
Liu
Y.
Werth
N.
Sandhoff
R.
Sandhoff
K.
Proia
R.L.
Systemic inflammation in glucocerebrosidase-deficient mice with minimal glucosylceramide storage
J. Clin. Invest.
 , 
2002
, vol. 
109
 (pg. 
1215
-
1221
)
37
Holleran
W.M.
Ginns
E.I.
Menon
G.K.
Grundmann
J.U.
Fartasch
M.
McKinney
C.E.
Elias
P.M.
Sidransky
E.
Consequences of beta-glucocerebrosidase deficiency in epidermis. Ultrastructure and permeability barrier alterations in Gaucher disease
J. Clin. Invest.
 , 
1994
, vol. 
93
 (pg. 
1756
-
1764
)
38
Kim
S.J.
Zhang
Z.
Hitomi
E.
Lee
Y.C.
Mukherjee
A.B.
Endoplasmic reticulum stress-induced caspase-4 activation mediates apoptosis and neurodegeneration in INCL
Hum. Mol. Genet.
 , 
2006
, vol. 
15
 (pg. 
1826
-
1834
)
39
Kim
S.J.
Zhang
Z.
Lee
Y.C.
Mukherjee
A.B.
Palmitoyl-protein thioesterase-1 deficiency leads to the activation of caspase-9 and contributes to rapid neurodegeneration in INCL
Hum. Mol. Genet.
 , 
2006
, vol. 
15
 (pg. 
1580
-
1586
)
40
Pelled
D.
Shogomori
H.
Futerman
A.H.
The increased sensitivity of neurons with elevated glucocerebroside to neurotoxic agents can be reversed by imiglucerase
J. Inherit. Metab. Dis.
 , 
2000
, vol. 
23
 (pg. 
175
-
184
)
41
Lloyd-Evans
E.
Pelled
D.
Riebeling
C.
Bodennec
J.
de-Morgan
A.
Waller
H.
Schiffmann
R.
Futerman
A.H.
Glucosylceramide and glucosylsphingosine modulate calcium mobilization from brain microsomes via different mechanisms
J. Biol. Chem.
 , 
2003
, vol. 
278
 (pg. 
23594
-
23599
)
42
Bodennec
J.
Pelled
D.
Riebeling
C.
Trajkovic
S.
Futerman
A.H.
Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide
FASEB J.
 , 
2002
, vol. 
16
 (pg. 
1814
-
1816
)
43
Kaech
S.
Banker
G.
Culturing hippocampal neurons
Nat. Protoc.
 , 
2006
, vol. 
1
 (pg. 
2406
-
2415
)
44
Canty
A.
Ripley
B.D.
Boot: Bootstrap R (S-Plus) Functions
2007
 
R package version 1.2–28. Available at http://www.R-project.org.
45
R-DevelopmentCoreTeam
R Foundation for Statistical Computing
 , 
2007
Vienna, Austria
46
Kieran
D.
Woods
I.
Villunger
A.
Strasser
A.
Prehn
J.H.
Deletion of the BH3-only protein puma protects motoneurons from ER stress-induced apoptosis and delays motoneuron loss in ALS mice
Proc. Natl Acad. Sci. USA
 , 
2007
, vol. 
104
 (pg. 
20606
-
20611
)
47
Koh
E.H.
Park
J.Y.
Park
H.S.
Jeon
M.J.
Ryu
J.W.
Kim
M.
Kim
S.Y.
Kim
M.S.
Kim
S.W.
Park
I.S.
, et al.  . 
Essential role of mitochondrial function in adiponectin synthesis in adipocytes
Diabetes
 , 
2007
, vol. 
56
 (pg. 
2973
-
2981
)
48
Brann
A.B.
Tcherpakov
M.
Williams
I.M.
Futerman
A.H.
Fainzilber
M.
Nerve growth factor-induced p75-mediated death of cultured hippocampal neurons is age-dependent and transduced through ceramide generated by neutral sphingomyelinase
J. Biol. Chem.
 , 
2002
, vol. 
277
 (pg. 
9812
-
9818
)
49
Hardman
M.J.
Emmerson
E.
Campbell
L.
Ashcroft
G.S.
Selective estrogen receptor modulators accelerate cutaneous wound healing in ovariectomized female mice
Endocrinology
 , 
2008
, vol. 
149
 (pg. 
551
-
557
)
50
Hetz
C.
Lee
A.H.
Gonzalez-Romero
D.
Thielen
P.
Castilla
J.
Soto
C.
Glimcher
L.H.
Unfolded protein response transcription factor XBP-1 does not influence prion replication or pathogenesis
Proc. Natl Acad. Sci. USA
 , 
2008
, vol. 
105
 (pg. 
757
-
762
)