Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on April 27, 2006
Human Molecular Genetics 2006 15(11):1826-1834; doi:10.1093/hmg/ddl105

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Published by Oxford University Press 2006

Endoplasmic reticulum stress-induced caspase-4 activation mediates apoptosis and neurodegeneration in INCL

Sung-Jo Kim, Zhongjian Zhang, Emiko Hitomi, Yi-Ching Lee and Anil B. Mukherjee^*

Section on Developmental Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892-1830, USA

^* To whom all correspondence should be addressed. Tel: +1 3014967213; Fax: +1 3014026632; Email: mukherja{at}exchange.nih.gov

Received March 7, 2006; Accepted April 13, 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Infantile neuronal ceroid lipofuscinosis (INCL), a neurodegenerative storage disorder of childhood, is caused by mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1 cleaves thioester linkages in S-acylated (palmitoylated) proteins and its mutation causes abnormal intracellular accumulation of fatty-acylated proteins and peptides leading to INCL pathogenesis. Although apoptosis is the suggested cause of neurodegeneration in INCL, the molecular mechanism(s) of apoptosis remains unclear. Using the PPT1-knockout (PPT1-KO) mice that mimic INCL, we previously reported that one mechanism of apoptosis involves endoplasmic reticulum (ER) stress-induced caspase-12 activation. However, the human caspase-12 gene contains several mutations, which make it functionally inactive. Thus, it has been suggested that human caspase-4 is the counterpart of murine caspase-12. Here we report that in the human INCL brain ER stress-induced activation of unfolded protein response (UPR) mediates caspase-4 and caspase-3 activation and apoptosis. Moreover, we show that the INCL brain contains high level of growth-associated protein-43 (GAP-43), which is known to undergo palmitoylation. We also demonstrate that transfection of cultured INCL cells with a green fluorescent protein–GAP-43 cDNA construct shows abnormal localization of this protein in the ER. Further, INCL cells manifest evidence of ER stress and UPR (elevated levels of Grp-78/Bip and GADD153), caspase-4 as well as caspase-3 activation and cleavage of poly(ADP)-ribose polymerase, a compelling sign of apoptosis. Most importantly, we show that inhibition of caspase-4 activity protects INCL cells from undergoing apoptosis. Our results provide insight into at least one of the molecular mechanisms of apoptosis in INCL and may allow the identification of potential targets for therapeutic intervention.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, represent a group of newly identified lysosomal storage disorders of childhood (1–6). With a worldwide distribution, NCLs are the most common (one in 12 500 births) autosomal recessive neurodegenerative storage disorders that have no effective treatment (1–6). Rapid degeneration of the retina, brain atrophy and lysosomal accumulation of autofluorescent lipopigments in neurons and other cell types are the characteristic pathological manifestations of NCL. On the basis of age of onset of clinical symptoms, cellular ultrastructure and composition NCLs are classified into four major subtypes. These are infantile NCL (INCL), late-infantile NCL, juvenile NCL and adult types. Mutations in at least seven different genes are the underlying genetic basis of various forms of NCLs recognized to date (3–5). Because of the lack of effective treatment(s), this group of diseases remains uniformly fatal.

INCL is a rare (one in 100 000 births) but devastating disease, caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene (7,8). Many proteins, especially in the nervous system, require S-acylation (palmitoylation) for access to membranes for their function (9). Palmitate residues are added on cysteine residues in polypeptides via a thioester linkage. Although this post-translational modification is critical for the function of many proteins, it is critically important that the palmitate residues be removed once these proteins have manifested their function(s) so that they can undergo degradation or are recycled. PPT1 cleaves thioester linkages in S-acylated (palmitoylated) proteins and facilitates the removal of the palmitate residues.

Although children suffering from INCL are normal at birth, by 2 years of age they undergo complete retinal degeneration leading to blindness and by age 4 electroencephalographic studies manifest lack of any brain activity. These children live in a vegetative state for several more years and death occurs 8–12 years of age (reviewed in 2–5). Although apoptosis is the suggested cause of neurodegeneration in INCL, the molecular mechanism(s) by which lack of PPT1 leads to apoptosis remains unclear. Recently, using PPT1-knockout (PPT1-KO) mice (10), which recapitulate the clinical and pathological features of INCL (10,11), we reported that the brain cells in these mice undergo endoplasmic reticulum stress (ER stress). ER stress activates unfolded protein response (UPR) and the ER-resident cysteine protease, caspase-12 leading to caspase-3 activation and apoptosis (12). However, it has been reported that although murine caspase-12 is an active enzyme, the human caspase-12 contains several mutations, which renders it non-functional (13). It has also been reported that human caspase-4, which is also a resident of the ER, is the counterpart of murine caspase-12 and is activated by ER stress (14). Therefore, we sought to determine whether apoptosis in human INCL is mediated by ER stress and identify the caspase responsible for inducing neuronal apoptosis. Here, we report that neuronal apoptosis in INCL is caused by ER-stress-mediated caspase-4 activation leading to caspase-3 activation and apoptosis. Most importantly, we show that inhibition of caspase-4 activity is cytoprotective raising the possibility that inhibition of this caspase may have therapeutic potential for INCL.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Neuronal apoptosis in INCL brain
Increased levels of neuronal apoptosis have been reported in brain biopsy samples from INCL patients (15). We have also previously reported that cultured lymphoblast cells from INCL patients undergo apoptosis at a high level (16). However, because in both of these studies apoptosis was detected by TUNEL assay, which is not the most reliable assay for detecting apoptosis, we used transmission electron microscopy to confirm the previous results using autopsy INCL brain tissues and those from an age- and sex-matched control. The results show that although apoptotic cells are virtually undetectable in the control brain samples (Fig. 1A, upper panels), numerous apoptotic cells can be readily detected in the brain tissues from the INCL patient (Fig. 1A, lower panels). These results confirm the previous reports that neuronal apoptosis are readily detectable in the brains of INCL patients causing neurodegeneration (15). Consistent with these results, neuronal apoptosis has been reported in the PPT1-KO mice (11,12).

View larger version (67K): [in this window] [in a new window]   Figure 1. Apoptosis in INCL brain. (A) Ultrastructure analysis by transmission electron microscopy of the brain tissues from normal and INCL patient. Note that the apoptotic body was detected in INCL patient samples only. (B) Caspase-4 expression patterns in brain samples. 1, normal hippocampus; 2, normal cortex; 3, INCL hippocampus; 4, INCL cortex. Asterisk denotes non-specific bands. (C) Immunohistochemical detection of cleaved caspase-3 protein in the brain of normal (left panel) and INCL patient (right panel). Scale bar, 20 µm. (D) PARP expression patterns in brain samples. 1, normal hippocampus; 2, normal cortex; 3, INCL hippocampus; 4, INCL cortex.

 
ER stress and the activation of caspases
It has been reported that in cultured human cells in which chemically induced ER stress mediates the activation of an ER-resident cysteine protease, caspase-4 leads to apoptosis. This suggests that caspase-4 may be the human counterpart of murine caspase-12 (17). Thus, we sought to determine whether caspase-4 is activated in the brain of INCL patients. Accordingly, we determined the levels of both pro (inactive)- and cleaved (active)-caspase-4 in the postmortem brain tissues from an INCL patient and that of an age- and sex-matched normal control. Our results show that in the normal brain (hippocampus and cortex) both pro- and cleaved-caspase-4 proteins are virtually undetectable (Fig. 1B, lanes 1 and 2), whereas in the INCL brain the levels of both inactive and active caspase-4 are clearly detectable (Fig. 1B, lanes 3 and 4). We also found that caspase-4 mRNA and protein levels in cultured INCL cells are markedly elevated compared with those of the normal control cells (Supplementary Material, Fig. S1A and B). To determine whether the activation of caspase-4 leads to caspase-3 activation, we performed immunohistochemical determination of cleaved-caspase-3 by using an antibody that specifically detects active caspase-3. The results show that in the normal brain activated caspase-3 is not detectable (Fig. 1C, left panel), whereas it is readily detectable in the INCL brain (Fig. 1C, right panel). Consistent with these results, the pro- (inactive) and cleaved (active)-caspase-3 protein bands in control cells are virtually non-detectable, whereas they are readily detectable in INCL cells (Supplementary Material, Fig. S1C). Because caspase-3 activation mediates the cleavage of poly(ADP)-ribose polymerase (PARP), a sign of apoptosis, we determined whether cleaved PARP is detectable in the brain tissues of INCL patients by western blot analysis. Our results show that a trace amount of uncleaved PARP may be detectable in the hippocampus and in the cortex of the normal brain (Fig. 1D, lanes 1 and 2), whereas both uncleaved and cleaved PARP bands are readily detectable in those of the INCL brain (Fig. 1D, lanes 3 and 4). Similarly, in normal cells, an uncleaved-PARP-protein band is barely detectable, whereas in INCL cells high density uncleaved- as well as cleaved-PARP protein bands are readily detectable (Supplementary Material, Fig. S1D). These results show that in the human INCL-activated caspase-4, caspase-3 and cleaved-PARP are readily detectable.

Stress in the ER in INCL
As it has been reported that the activation of caspase-4 in cultured human cells is precipitated by chemically induced ER stress (17), we sought to determine whether there is evidence of ER stress in the INCL brain and in cultured cells derived from INCL patients. We determined the level of X-box binding protein-1 (XBP1), a critical marker of ER stress and UPR, in normal and INCL brains. The results show that XBP1 protein is undetectable in the normal brain (Fig. 2A, lanes 1 and 2), whereas it is clearly detectable in the INCL brain tissues (Fig. 2A, lanes 3 and 4). We determined the levels of glucose responsive protein-78 (Grp-78/Bip), a canonical ER marker protein the expression levels of which are stimulated by ER-stress-induced UPR (18,19), by western blot analysis. The results show that although trace amounts of Grp-78/Bip is detectable in the hippocampus and the cortex of a normal brain (Fig. 2B, lanes 1 and 2), this protein level is markedly elevated in those of the INCL brain (Fig. 2B lanes 3 and 4). To further confirm the level of Grp-78/Bip at the cellular level, we performed immunohistochemical analysis of the brain tissues from control and INCL patient. Our results show that a faint staining of Grp-78/Bip is detectable in the hippocampus and in the cortex of the normal brain (Fig. 2C, upper panels), whereas the staining in those of the INCL brain is significantly more intense (Fig. 2C, lower lanes). In the ER-stress-mediated apoptosis pathway, C/EBP homologous protein (CHOP), commonly known as growth arrest- and DNA damage-inducible gene 153 (GADD153), plays critical roles (reviewed in 20). Accordingly, using cultured lymphoblasts/fibroblasts from INCL patients and normal subjects, we determined the levels of both Grp-78/Bip and CHOP (GADD153) by western blot analysis. Our results show that the levels of both Grp-78 (Fig. 2D) and GADD153 (Fig. 2E) are markedly elevated in the lymphoblasts/fibroblasts derived from INCL patients. Taken together, these results indicate that cultured cells as well as the neurons in the brain of an INCL patient undergo ER stress, which most likely mediates the activation of the UPR leading to caspase, caspase-4 activation that mediates apoptosis.

View larger version (68K): [in this window] [in a new window]   Figure 2. ER stress in INCL. (A) XBP1 expression patterns in brain samples. 1, normal hippocampus; 2, normal cortex; 3, INCL hippocampus; 4, INCL cortex. (B) Western blot analysis of Grp-78/Bip in brain samples. 1, normal hippocampus; 2, normal cortex; 3, INCL hippocampus; 4, INCL cortex. (C) Immunohistochemical detection of Grp-78/Bip protein in the hippocampus and cortex of normal (upper panels) and INCL patient (lower panels). Scale bar, 20 µm. (D) Western blot analysis of Grp-78/Bip protein in the normal (left lane) and INCL (right lane) lymphoblasts. (E) GADD153 (CHOP) protein expression patterns in the normal (left lane) and INCL (right lane) fibroblasts.

 
Abnormal accumulation of palmitoylated proteins in the ER of INCL cells
It has been reported that caspase-4 is a resident cysteine protease of the ER and that ER stress leads to its activation (17). We recently uncovered that in the brains of PPT1-KO mice growth-associated protein-43 (GAP-43), a highly palmitoylated protein, abnormally accumulates in the ER causing stress in this organelle. The ER stress activates UPR, which activates caspase-12 leading to apoptosis (12). Thus, we sought to determine whether GAP-43 is also abnormally accumulated in the ER of INCL brain and in cultured cells derived from INCL patients. Accordingly, we performed immunohistochemical analysis of the INCL brain sections for GAP-43. The results show that GAP-43 is virtually undetectable in the normal brain sections (Fig. 3A, left panel), whereas it is readily detectable in those of the INCL patient (Fig. 3A, right panel). However, it was difficult to discern the subcellular localization of the GAP-43 staining in the sections of the postmortem brain tissues. Thus, we transfected the normal and INCL fibroblasts with a green fluorescent protein (GFP)–GAP-43 cDNA construct and cytochemically analyzed the cells for the location of GFP fluorescence. We used GRP-78 antibody to determine the location of the ER. Our results show that in normal cells GFP fluorescence is diffusely distributed throughout the cell including the cell membrane and the ER (Fig. 3B, upper panels), whereas in INCL fibroblasts, intense GFP fluorescence is localized exclusively in the perinuclear area, characteristic of the ER–Golgi location (Fig. 3B, lower panels). The location of the GFP fluorescence was confirmed by the merging of GFP (green) with GRP-78/Bip (red) fluorescence. Additionally, we have obtained forced expression of other palmitoylated proteins such as CD9, CD63, CD81 and PSD95 in normal and INCL fibroblasts using GFP constructs and found that these proteins, such as GAP-43, are also abnormally accumulated in the ER of the INCL fibroblasts (Supplementary Material, Fig. S1E). These results show that in INCL cells palmitoylated proteins abnormally accumulate in the ER.

View larger version (51K): [in this window] [in a new window]   Figure 3. Expression pattern of a model S-acylated protein, GAP-43, in normal and INCL patient brain and in cultured cells. (A) Immunohistochemical detection of GAP-43 protein in the cortex of normal (left panels) and INCL patient (right panels) brain sections. Scale bar, 20 µm. (B) Induced expression of GFP–GAP-43 in normal (upper panels) and INCL patient (lower panels) fibroblasts. Cells were transfected with GFP–GAP-43 cDNA construct and fluorescence was analyzed. The cells were also stained with rhodamine-labeled Grp-78/Bip antibody to determine the location of the ER. Note that in the INCL patient cell the GFP–GAP-43 fluorescence (green) merges with Grp-78/Bip (red) demonstrating colocalization of GFP–GAP-43 with the canonical ER marker, Grp-78/Bip, whereas in the normal cell the GFP–GAP-43 fluorescence is diffusely distributed throughout the cell. Scale bar, 20 µm.

 
Caspase-4 mediates apoptosis in INCL
To delineate whether caspase-4 activation leads to caspase-3 activation, we treated the normal and INCL lymphoblasts with caspase-4 and caspase-3 inhibitors, respectively, and determined the effects of these inhibitors on the levels of cleaved (activated)-caspase-3 protein by western blot analysis. The results show that in the untreated normal lymphoblasts both caspase-4 and caspase-3 levels are markedly lower (Fig. 4A, left panel) than those of the untreated INCL lymphoblasts (Fig. 4A, right panel). Further, the levels of both pro and cleaved caspase-3 in the INCL lymphoblasts appear to be markedly higher than those of the normal lymphoblasts (Fig. 4A, right panel). Most interestingly, although treatment of the INCL cells with caspase-3 inhibitor does not prevent caspase-3 cleavage, it inhibits caspase-3 activity. However, treatment of the cells with caspase-4 inhibitor shows virtually no cleaved caspase-3 protein band (Fig. 4A, right panel), suggesting that caspase-4 activation mediates caspase-3 activation. Because activated caspase-4 may indirectly activate caspase-3 via caspase-7 activation, we determined whether in INCL cells caspase-7 is activated. The results show that both in INCL and normal cells cleaved caspase-7 is not detectable, although a clear pro-caspase-7 protein band is readily detectable (data not shown).

View larger version (52K): [in this window] [in a new window]   Figure 4. Effect of caspase-4 inhibitor on cultured cells from INCL patient. (A) Detection of caspase-3, -4 proteins as determined by western blot using normal (left panels) and INCL patient (right panels) lymphoblast cells. (B) Detection of PARP in normal (left panels) and INCL patient (right panels) lymphoblast. (C) Relative cell death rate of normal and INCL patient cells. (D) Expression pattern of GFP–caspase-4 construct in normal and INCL patient fibroblast. Cells transfected with pEGFP-N1–caspase-4 plasmids were examined by fluorescence microscope for the detection of caspase-4 and apoptosis. GFP–caspase-4 (green) and apoptosis marker Annexin-V-Cy3 (red). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.

 
It has been reported that caspase-3 catalyzed cleavage of PARP, a 116 kDa nuclear enzyme, is closely linked to apoptosis (21). Because it appears that caspase-4 activation leads to caspase-3 activation, which cleaves PARP, we sought to determine whether treatment of the cells with caspase-4 inhibitor would prevent PARP cleavage. The results show that treatment of the cells with caspase-4 inhibitor prevents PARP cleavage. As expected, caspase-3 inhibitor also prevented PARP-cleavage (Fig. 4B). Taken together, these results indicate that in INCL cells caspase-4 activation leads to the activation of caspase-3 mediating PARP cleavage, a recognized indicator of apoptosis.

Inhibition of caspase-4 activation is cytoprotective
To determine whether inhibition of caspase-4 protects the INCL cells, we compared the percentage of dead cells before and after treatment with caspase-4 inhibitor. Treatment with dimethyl sulfoxide (DMSO), the diluent for the inhibitor, served as a control. The results show that although the percentage of cell death was the highest in untreated and DMSO-treated cultures, caspase-4 inhibitor treatment dramatically reduced the percentage of cell death (Fig. 4C). To further determine whether the cytoprotective effect of caspase-4 inhibitor is due to the inhibition of apoptosis, we transfected both INCL and normal fibroblasts with a GFP–caspase-4 expression construct (pEGFP-N1-caspase-4) and determined the level of GFP-tagged caspase-4 expression as well as apoptosis after 24 and 48 h of treatment with caspase-4 inhibitor. The pattern of caspase-4–GFP expression in INCL patient fibroblasts was determined by the presence of green fluorescence and apoptosis was detected by annexin-V-cy3 fluorescence (red). The cell nuclei were stained with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue). The results show that although there is virtually no detectable apoptotic cells in normal fibroblast cultures 24 and 48 h after treatment (Fig. 4D, upper panels), a very large percentage of the INCL cells expressing GFP–caspase-4 appear to undergo apoptosis (Fig. 4D, middle panels). Most importantly, treatment of these INCL cells with caspase-4 inhibitor appears to protect the cells from apoptosis (Fig. 4D, lower panels).

	DISCUSSION

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Only a few human ailments are as hopelessly intractable as neurodegenerative diseases (22). Delineating the molecular pathways to neurodegeneration has interested neurobiologists and clinicians alike and these pathways are slowly being uncovered (reviewed in 23). In this study, we uncovered at least one of the molecular mechanisms of neurodegeneration in INCL. An important finding in this study is that PPT1 deficiency leads to abnormal accumulation of S-acylated proteins in the INCL brain and forced expression of proteins, such as GAP-43, CD9, CD63, CD81 and PSD95 that undergo palmitoylation, abnormally accumulate in the ER of cultured INCL cells. Although we have not established a cause and effect relationship between abnormal accumulation of these proteins in the ER and the activation of UPR, caspase-4 activation and apoptosis, it is clear that PPT1 deficiency and ER stress are closely linked. Our results also show that caspase-4, reported to be activated by ER stress (17), mediates activation of caspase-3 and apoptosis. Most importantly, we show that inhibition of caspase-4 activity is protective of INCL cells providing the proof of principle that caspase-4 inhibitor(s) may be beneficial for preventing neuronal death by apoptosis in INCL.

It has been reported that protein palmitoylation, a common post-translational lipid modification, plays critical roles in neuronal cells (24–26). Moreover, specific subcellular distribution of certain palmitoylated proteins is achieved by constitutive de/re-acylation cycles, which drive their rapid exchange between the plasma membrane and the ER–Golgi compartment (27,28). Further, palmitoylation not only alters protein structure but also increases hydrophobicity, which allows them to anchor onto biological membranes (9) including the membranes of the ER. Consistent with these reports, it has been recently shown that a mammalian palmitoyl transferase, HIP14, which palmitoylates several neuronal proteins, is predominantly localized to the ER–Golgi membranes and cytoplasmic vesicles (27–29). HIP14 also catalyzes its own palmitoylation (27) for membrane anchorage. Thus, it is reasonable to assume that in the absence of depalmitoylation in INCL, HIP-14 is likely to remain activated on ER–Golgi membranes, further increasing the levels of S-acylated proteins in these organelles. This notion is consistent with our previous results demonstrating that PPT1-KO brains contain higher levels of GAP-43 compared with those in WT brains. Moreover, the results of ³H-palmitate labeling of proteins reveal that appreciably higher levels of palmitoylated-GAP-43 accumulation occurs in PPT1-KO neurospheres compared with those of WT neurospheres (12). Most importantly, the results of induced expression of GFP–GAP-43 in cells from PPT1-KO mice and their WT littermates show that the GFP–GAP-43-fluorescence in PPT1-deficient cells colocalizes with that of the canonical ER marker protein, Grp-78/Bip, suggesting that in the absence of PPT1, S-acylated proteins tend to accumulate abnormally in the ER.

The ER is the site for synthesis, post-translational modification and delivery of biologically active proteins to their proper target sites (18). Because S-acylation occurs in the ER (29) and because these lipid-modified proteins anchor to the membranes of the ER, Golgi and cytoplasmic vesicles, the absence of PPT1 may lead to abnormal accumulation in these organelles because the thioester linkages in S-acylated proteins are not cleaved and the proteins remain membrane-bound. Thus, it is likely that the influx of nascent S-acylated proteins may exceed the processing capacity of the ER, and as a result, the homeostasis in this organelle is perturbed. These conditions, arising from PPT1 deficiency, may lead to ER stress activating UPR. Further support of this notion comes from a recent report indicating that the activation of the UPR may occur as a result of dysfunction of lysosomes that takes place in G_M1-gangliosidosis, a lysosomal storage disease causing neurodegeneration (30). Because PPT1 is a lysosomal enzyme, it is possible that lysosomal dysfunction in PPT1-KO cells lead to a retrograde transport of storage material from lysosomal compartment to the ER and contributes to the activation of the UPR. It has been recently proposed that retrograde transport from post-ER compartments to the ER may occur as a result of lysosomal dysfunction, although the mechanism for such retrograde transport remains undefined (31). It should be noted that although ER stress activates caspase-4 leading to apoptosis in INCL cells, we recently found that secondary to ER stress, the generation of reactive oxygen species and disruption of calcium homeostasis may lead to mitochondrial caspase-9 pathway of apoptosis (32). Moreover, in cultured neurospheres from PPT1-KO mice also manifest the activation of both caspase-12 and caspase-9 pathways to apoptosis.

	MATERIALS AND METHODS

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Postmortem INCL and normal control brain tissues, cell cultures and transfection
Postmortem brain tissues from an INCL patient, with clinical and pathological diagnosis of INCL were obtained from Human Brain and Spinal Fluid Resource Center, Los Angeles, CA, USA. A sister of this patient who also died of INCL was a compound heterozygote for PPT1 mutations [del 398T in exon 4 and C451T in exon 5; see Table 2 of Das et al. (33)]. Because INCL is an autosomal recessive disease, we assume that the patient was also a compound heterozygote for the same PPT1 mutations as indicated above. Normal human fibroblast cell line and INCL patient fibroblast and lymphoblast cell lines, carrying homozygous PPT1 missense mutation, A364T (R122W), were obtained from Dr Krystyna Wisniewski's laboratory. They were cultured in DMEM and RPMI1640 medium supplemented with 10% FBS at 37°C at an atmosphere of 5% CO₂ and 95% air. For fluorescence microscopic detection, these cell lines were spread into Lab-Tek chamber slide (Nalgen Nunc). All cell lines were transfected with respective cDNA constructs using Lipofectamine 2000 reagent (Invitrogen) according to vendor's protocol.

Treatments
After transfection, cells were treated with fresh medium containing either 2 µMcaspase-3 inhibitor (Z-DEVD-FMK, Biovision), 2 µM caspase-4 inhibitor (Z-LEVD-FMK, Biovision).

GFP-tagged protein expression vector construction
For caspase-4–GFP-tagged protein, cDNA of caspase-4 was redesigned by Platinum Pfx enzyme (Invitrogen) PCR amplification. The primers used for this reaction are as follows: caspase-4-F, 5'-CTC GAG ATG GCA GAA GGC AAC CAC AGA AAA AAG CC-3'; caspase-4-R, 5'-CCG CGG ATT GCC AGG AAA GAG GTA GAA ATA TCT TG-3'; GAP-43-F, 5'-AAG CTT ATG CTG TGC TGT ATG AGA AGA ACC AAA CAG-3';GAP-43-R, 5'-CCG CGG GGC ATG TTC TTG GTC AGC CTC GGG GTC TTC-3'. This PCR product was subcloned into the pCR-BluntII-TOPO vector (Invitrogen). This full-length caspase-4 or GAP-43 were excised and then relegated into the pEGFP-N1 vector (Clontech). The plasmids used for transfection were prepared by using a plasmid mini kit (Qiagen). The consistency between plasmid preparations was monitored by determining the concentration of plasmids by both spectrophotometry and agarose gel electrophoresis.

Western blot analysis
Postmortem human brain tissues were homogenized in protein extracting buffer (50 mM Tris–HCI, 150 mM NaCl, 0.25% SDS, 1 mM EDTA and 1% NP-40) containing protease-inhibitor cocktails (Sigma). Cultured cells from normal subjects and INCL patients were treated in PhosphoSafe extraction reagent (EMD Biosciences). Twenty micrograms of total proteins from each sample were resolved by electrophoresis using 4–15% SDS–polyacrylamide gels (Bio-Rad) under denaturing and reducing conditions. Proteins were then electrotransferred to nitrocellulose membrane (Bio-Rad). The membranes were blocked with 5% non-fat dry milk (Bio-Rad) and then subjected to immunoblot analysis using the methods as described before. The primary antibodies used in the present study are anti-caspase-4 (Sigma or Biovision), anti-caspase-3, anti-caspase-7, anti-PARP (Cell Signaling), anti-Grp78/Bip (KDEL) (Stressgen), anti-XBP1 (Santa Cruz Biotechnology), anti-GADD153/CHOP (Abcam) and anti-ß-actin (Sigma). The secondary antibodies used in the study are goat anti-rabbit IgG (Santa Cruz Biotechnology), donkey anti-goat IgG (Santa Cruz Biotechnology), rabbit anti-mouse IgG (Santa Cruz Biotechnology). Chemiluminescent detection was performed by using Supersignal west pico luminol/enhancer solution (Pierce) according to manufacturer's instructions.

Transmission electron microscopy
The brain tissues were fixed in 2.5% glutaraldehyde in sodium phosphate buffer for 2 h. The tissues were then washed with Millonig's phosphate buffer once and kept in the same buffer at 4°C until final processing. Lead citrate and uranyl acetate were used to stain the thin sections. The stained sections were then examined with an LEO 912 electron microscope by JFE Enterprises (Brookeville, MD, USA).

Immunohistochemistry
The brain tissues were fixed in 3.7% paraformaldehyde. The anti-Grp78/Bip (KDEL) and anti-cleaved caspase-3 (Cell Signaling) antibodies were diluted 1:200 and incubated overnight at 4°C in a humidified chamber. After incubation, the slides were washed three times with 1xPBS and were further incubated with anti-mouse biotinylated secondary antibody (1:500; Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were washed three times with 1xPBS and then incubated with ABC complex by following manufacturer's protocol (Vector Laboratories).

Cell viability test
Cell viability was determined by Trypan blue exclusion. Cell suspension was mixed with 0.4% Trypan blue vital stain solution (Cambrex), and cell viability was determined on a haemocytometer under a microscope.

Fluorescence microscopy
After transfection, the cells were incubated at 37°C in an atmosphere of 5% of CO₂ and 95% air for 24 h. They were washed three times with PBS, pH 7.6 and incubated 3.7% formaldehyde solution for 15 min. Apoptosis detection can be analyzed by Annexin-V-Cy3 apoptosis detection kit (Biovision) according to manufacturer's instructions. Nuclei were stained with DAPI (Sigma). Fluorescence was visualized with the Axioskop2 plus fluorescence microscope (Carl Zeiss), and the image was processed with the AxioVision 4.3 (Carl Zeiss) and the Photoshop 7.0 program (Adobe).

	SUPPLEMENTARY MATERIAL

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Supplementary Material is available at HMG Online.

	ACKNOWLEDGEMENTS

 
We thank J.Y. Chou, I. Owens and S.W. Levin for critical review of the manuscript and S. Everett and R. Dreyfuss for expert assistance on photomicrography. We also thank L.W. Johnston, President, BDSRA, for providing information on the availability of postmortem INCL brain tissue samples. Postmortem INCL brain tissue samples were obtained from the Human Brain and Spinal Fluid Resource Center, VA West Los Angeles Healthcare Center, Los Angeles, CA 90073, USA, which is sponsored by NINDS/NIMH, National Multiple Sciences Society and Department of Veterans Affairs. We also obtained control autopsy brain tissue samples from the Brain and Tissue Bank for Developmental Disorders, University of Maryland, Baltimore, MD 21201, USA. This research was supported in part by the Intramural Research Program of the NIH (NICHD).

Conflict of Interest statement. None declared.

	REFERENCES

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