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Human Molecular Genetics Advance Access originally published online on December 20, 2005
Human Molecular Genetics 2006 15(2):337-346; doi:10.1093/hmg/ddi451
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Published by Oxford University Press 2005

Palmitoyl-protein thioesterase-1 deficiency mediates the activation of the unfolded protein response and neuronal apoptosis in INCL

Zhongjian Zhang1, Yi-Ching Lee1, Sung-Jo Kim1, Moonsuk S. Choi1, Pei-Chih Tsai1, Yan Xu2, Yi-Jin Xiao2, Peng Zhang3, Alison Heffer1 and Anil B. Mukherjee1,*

1Section on Developmental Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, The National Institutes of Health, Bethesda, MD 20892-1830, USA, 2Department of Cancer Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA and 3Walter Reed Army Institute of Research, Silver Spring, Maryland, MD 20910-7500, USA

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

Received October 11, 2005; Accepted December 8, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Numerous proteins undergo modification by palmitic acid (S-acylation) for their biological functions including signal transduction, vesicular transport and maintenance of cellular architecture. Although palmitoylation is an essential modification, these proteins must also undergo depalmitoylation for their degradation by lysosomal proteases. Palmitoyl-protein thioesterase-1 (PPT1), a lysosomal enzyme, cleaves thioester linkages in S-acylated proteins and removes palmitate residues facilitating the degradation of these proteins. Thus, inactivating mutations in the PPT1 gene cause infantile neuronal ceroid lipofuscinosis (INCL), a devastating neurodegenerative storage disorder of childhood. Although rapidly progressing brain atrophy is the most dramatic pathological manifestation of INCL, the molecular mechanism(s) remains unclear. Using PPT1-knockout (PPT1-KO) mice that mimic human INCL, we report here that the endoplasmic reticulum (ER) in the brain cells of these mice is structurally abnormal. Further, we demonstrate that the level of growth-associated protein-43 (GAP-43), a palmitoylated neuronal protein, is elevated in the brains of PPT1-KO mice. Moreover, forced expression of GAP-43 in PPT1-deficient cells results in the abnormal accumulation of this protein in the ER. Consistent with these results, we found evidence for the activation of unfolded protein response (UPR) marked by elevated levels of phosphorylated translation initiation factor, eIF2{alpha}, increased expression of chaperone proteins such as glucose-regulated protein-78 and activation of caspase-12, a cysteine proteinase in the ER, mediating caspase-3 activation and apoptosis. Our results, for the first time, link PPT1 deficiency with the activation of UPR, apoptosis and neurodegeneration in INCL and identify potential targets for therapeutic intervention in this uniformly fatal disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Numerous proteins undergo post-translational modification by palmitic acid (S-acylation) for anchorage to membranes, a process critical for their diverse biological functions including vesicular transport, signal transduction and maintenance of cellular architecture (reviewed in 1Go). These proteins include the transferrin receptor (2Go), nitric oxide synthase (3Go), neuronal growth-associated protein-43 (GAP-43; 4Go), acetylcholine esterase (5Go), postsynaptic density protein-95 (PSD-95; 6Go) and the synaptosomal-associated protein-25 (7Go). A critical function of palmitoylation of signaling proteins is to confine them to cellular membranes (reviewed in 8Go). However, although palmitoylation plays essential roles in enabling many proteins to manifest their functions, cycles of protein-palmitoylation and depalmitoylation have been suggested to control their distribution between the membrane and cytoplasm and/or between subdomains of the plasma membrane modulating the coupling of specific signaling proteins to cell surface receptors or intracellular effectors (9Go).

Although palmitoylation and depalmitoylation of proteins may occur non-enzymatically, in 1993, the purification and characterization of palmitoyl-protein thioesterase-1 (PPT1; 10Go) significantly advanced our knowledge that clarified the importance of this lipid modification in cellular functions. This was followed by molecular cloning and characterization of the cDNA as well as the gene for PPT1 and its chromosomal localization (11Go). Soon after these discoveries, it was reported that inactivating mutations in the PPT1 gene are the genetic basis of infantile neuronal ceroid lipofuscinosis (INCL; 12Go). PPT1 catalyzes the cleavage of thioester linkages in S-acylated (palmitoylated) proteins facilitating their degradation and/or recycling. Thus, the lack of PPT1 activity leads to INCL pathogenesis, although the molecular mechanism(s) by which PPT1 deficiency mediates neurodegeneration, until now, remained unexplained.

Although INCL is a rare disease (1 in 100 000 births), it belongs to the most common (1 in 12 500 births) group of heritable, neurodegenerative storage disorders of childhood collectively known as neuronal ceroid lipofuscinoses (NCLs) or more commonly called Batten disease (13Go,14Go). Rapidly progressive brain atrophy, retinal blindness and accumulation of autofluorescent lipopigments in neurons as well as in other cell types are some of the characteristic pathological manifestations of all NCL types. On the basis of age of onset, cellular ultrastructure and composition of the storage material, NCLs are classified into four major subtypes: infantile (INCL), late-infantile (LNCL), juvenile (JNCL) and adult onset (ANCL). Recent reports indicate that mutations in at least six different genes underlie the various forms of NCLs known to date (15Go,16Go).

Among various types of NCLs, INCL is the most lethal and devastating disease. Although normal at birth, children afflicted with this disease undergo complete retinal degeneration and blindness by 2 years of age and by age 4, an isoelectric electroencephalogram attests to brain death. These children remain in a vegetative state for several more years and death occurs around 10–12 years of age (13Go–16Go). At autopsy, characteristic autofluorescent intracellular storage material, known as granular osmiophilic deposits, is found in the brain as well as in other tissues (17Go). Increased levels of apoptosis in the brain biopsy tissues (18Go) and in cultured cells (19Go) from INCL patients and in the brains of PPT1-knockout (PPT1-KO) mice (20Go,21Go) have been reported. Moreover, it has recently been reported that with increased apoptosis there is actual loss of neuronal cells in the brains of PPT1-KO mice (21Go), demonstrating that apoptosis is the cause of neurodegeneration. However, the molecular mechanism(s) of apoptosis in INCL remains poorly understood. Here, we provide compelling evidence that links PPT1 deficiency with abnormal accumulation of S-acylated proteins in the endoplasmic reticulum (ER) activating unfolded protein response (UPR) associated with elevated levels of phosphorylated translation initiation factor, eIF2{alpha}, glucose-regulated protein-78 (Grp-78/Bip), activated caspase-12 leading to caspase-3 activation, apoptosis and neurodegeneration. Our results explain at least one of the major molecular mechanisms of apoptosis leading to neurodegeneration in INCL and identify potential targets for developing novel therapeutic strategies for this uniformly fatal disease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Autofluorescence, ER abnormality and apoptosis in PPT1-KO mice
Intracellular accumulation of autofluorescent material in the neurons and in other cell types of INCL patients (13Go,18Go,19Go) as well as in those of the PPT1-KO mice (20Go,21Go) has been previously reported. However, a correlation between increased accumulation of autofluorescent material with advancing age and neuronal apoptosis leading to clinical signs of neurological impairment has not been documented. This information is important if we are to understand why children afflicted with INCL as well as the PPT1-KO mice are normal at birth and neurological symptoms do not appear until after several months of postnatal life. Thus, we sought to determine whether increased autofluorescence and apoptosis correlate with the appearance of clinical signs of neurological impairment. Accordingly, we analyzed the brain tissues of 1-, 3- and 6-month-old PPT1-KO mice and of their WT littermates for autofluorescence and apoptosis and tested these animals for clasping behavior, a characteristic sign of neurological impairment. We found that in the brains of 1-month-old PPT1-KO mice, autofluorescence is virtually undetectable (Supplementary Material, Fig. S1). We also analyzed the brains of PPT1-KO and WT littermates at 1, 3 and 6 months of age by transmission electron microscopy for apoptosis. The results show that in the brains of 1-month-old PPT1-KO and WT littermates, virtually no apoptotic cells could be detected (Fig. 1A). However, by 3 months of age, a low level of autofluorescence is clearly visible in the brains of PPT1-KO mice (Supplementary Material, Fig. S1) and at this age, EM analysis shows the presence of occasional apoptotic cells (Fig. 1A). At 6 months of age, the brains of the PPT1-KO mice show high levels of autofluorescence (Supplementary Material, Fig. S1) and more frequent apoptosis (Fig. 1A). Most of these apoptotic bodies are intracellular, which is consistent with recent reports that apoptotic cells are rapidly removed by phagocytes to prevent inflammation and autoimmunity. We then performed higher magnification ultrastructural analysis of the brain cells of PPT1-KO mice and their WT littermates. The results show gross morphological abnormalities in the ER of the brain neurons of PPT1-KO mice (Fig. 1B, lower panel) and such abnormalities are not detectable in the WT mouse brain (Fig. 1B, upper panel). At this age, the PPT1-KO mice also began manifesting the clasping behavior (data not shown), which is absent in the WT littermates. Taken together, these results suggest that in the PPT1-KO mice the accumulation of autofluorescent material, ER abnormality and apoptosis correlate with advancing age and the appearance of clasping behavior attesting to neurological impairment.



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  		Figure 1. Ultrastructure analysis by transmission electron microscopy of the brain tissues from PPT1-KO and WT littermates. (A) Brain sections of WT littermates at 1, 3 and 6 months of age (upper row) and brain sections of 1-, 3- and 6-month-old PPT1-KO mice (lower row). Note the apoptotic bodies (APB) engulfed by phagocytes. Multiple APBs are visualized in the brain of a 6-month-old PPT1-KO mouse. (B) Ultrastructure of the brains of WT and PPT1-KO mice. Note the grossly swollen ER in the neuron from the PPT1-KO mouse.

 
PPT1-KO brains show elevated levels of GAP-43, a known palmitoylated protein
It has been reported that S-acylation (palmitoylation) of many important polypeptides is essential for their biological function (22Go). It has also been reported that post-translational fatty-acylation of proteins takes place in the ER–Golgi intermediate compartment allowing membrane anchorage of these proteins (23Go). Although S-acylation is recognized as an important process, these proteins must undergo depalmitoylation before they could be degraded by lysosomal proteases (24Go). Thus, lack of protein depalmitoylation in PPT1-deficient cells is likely to prevent degradation of these polypeptides causing abnormal accumulation. Further, it has been recently suggested that abnormal accumulation in the post-ER compartments may cause retrograde transport of the accumulated material to the ER (25Go). This may result in structural abnormality as well as stress in the ER activating the UPR (26Go–28Go). Recent reports indicate that a neuronal protein, GAP-43, undergoes palmitoylation on multiple cysteine residues (29Go). Thus, we sought to determine whether forced expression of this protein in PPT1-deficient cells results in abnormal intracellular accumulation. Accordingly, we first determined the levels of GAP-43-mRNA and protein in the brains of PPT1-KO and WT littermates at 1, 3 and 6 months of age by real-time PCR and western blot analyses, respectively. The results show that GAP-43-mRNA levels in the brains of PPT1-KO and those of 3-month-old WT mice are not significantly different (P=0.056). However, GAP-43-mRNA levels in the brains of 1- and 6-month-old PPT1-KO mice are significantly (P<0.005) lower than those of their WT littermates (Fig. 2A). Most interestingly, the levels of GAP-43 protein in the brains of the PPT1-KO mice are markedly higher than those of WT littermates (Fig. 2B). This mismatch between the mRNA and protein levels may indicate an increased proportion of S-acylated GAP-43 in the total protein pool in the brains of PPT1-deficient mice.



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  		Figure 2. The expression of a model S-acylated protein, GAP-43 in WT and PPT1-KO mice and in cultured cells, respectively. (A) Relative GAP-43-mRNA levels in the brains of PPT1-KO and WT mice determined by real-time PCR. (B) Western blot analysis of GAP-43 protein levels. (C) Palmitoylated GAP-43 levels (autoradiograph) in neurospheres from WT (left lane) and PPT1-KO (right lane) mice. (D) Induced expression of GFP-GAP-43 in fetal fibroblasts from WT (upper panels) and PPT1-KO (lower panels) littermates. Cells were transfected with GFP-GAP-43 cDNA construct, and fluorescence was analyzed. The cells were also stained with rhodamine-conjugated Grp-78 antibody to determine the location of the ER. Note in the PPT1-KO cell, the GFP-GAP-43 fluorescence (green) merges with Grp-78 (red), demonstrating co-localization of GFP-GAP-43 with the canonical ER marker, Grp-78/Bip, whereas in the WT cell the GFP-GAP-43 fluorescence is diffusely distributed throughout the cell.

 
Abnormal accumulation of palmitoylated GAP-43 in the ER
To understand the reason(s) for the above findings, we sought to determine whether the elevated levels of GAP-43 protein in the PPT1-KO brains reflect a higher proportion of palmitoylated GAP-43 in the total protein pool of the PPT1-deficient cell. Accordingly, we used cultured PPT1-KO and WT neurospheres, labeled them with 3H-palmitic acid and determined the levels of palmitoylated GAP-43 by immunoprecipitation, electrophoresis and autoradiography. The results show that the density of the 3H-palmitate-labeled GAP-43 protein band from PPT1-KO neurospheres is markedly higher than that of the WT neurospheres (Fig. 2C). These results clearly show that in PPT1-deficient cells, either the rate of GAP-43 palmitoylation is more rapid or palmitoylated GAP-43 is not degraded or recycled at a rate that occurs in WT cells. We then sought to delineate whether in PPT1-deficient cells forced expression of GAP-43, a protein known to undergo palmitoylation, causes abnormal accumulation of such protein in the ER. For this, we transfected fetal fibroblasts derived from PPT1-KO mice and from their WT littermates with a green fluorescent protein (GFP)-GAP-43-cDNA construct and analyzed the cellular distribution of GFP fluorescence. We also performed immunocytochemical analysis of the canonical ER-marker-protein, Grp-78/Bip, in the same cells expressing GFP-GAP-43. Our results show that although a low level of GFP fluorescence is diffusely distributed in the cytosol and in the membrane of WT cells (Fig. 2D, upper panels), the strong green fluorescence in the PPT1-KO cells is localized predominantly over the presumptive site of the ER (Fig. 2D, lower panels). Co-localization of GFP fluorescence (green) with Grp-78/Bip (red) confirmed that GAP-43, a model palmitoylated protein, accumulates in the ER (Fig. 2D, lower panels). We also obtained similar results by transfecting the PPT1-KO cells with GFP constructs containing cDNAs of other known palmitoylated proteins such as CD9, CD81 and PSD-95 (data not shown). Taken together, these results suggest that PPT1 deficiency leads to abnormal accumulation of S-acylated proteins in the ER, which is likely to mediate ER stress that leads to the activation of the UPR.

Evidence for ER stress and activation of the UPR in PPT1-KO brain
To determine whether abnormal accumulation of palmitoylated proteins in the brains of PPT1-KO mice mediates ER stress, we performed immunohistochemical analyses of the brain tissues from PPT1-KO mice and of their WT littermates to determine the expression of the canonical ER-stress-marker, Grp-78/Bip (26Go,27Go,30Go). It should be noted that ER stress causes elevation of Grp-78 protein levels in cells. Our results show that the number of cells intensely positive for Grp-78 protein markedly increases in the brain of PPT1-KO mice between 3 and 6 months of age but not in those of the WT littermates (Fig. 3A, lower panels). However, in the brains of 1-month-old PPT1-KO mice, only a light Grp-78 staining is detectable. Similarly, in the brain tissues from WT mice of all three age groups, a light Grp-78 staining is appreciable (Fig. 3A, upper panels). To determine how early in developing PPT1-KO mouse brain evidence of ER stress is detectable, we analyzed Grp-78 protein expression by western blot analysis of protein lysates from neurospheres derived from PPT1-KO and WT fetuses at 15th day of gestation. The results show a markedly elevated level of Grp-78 in the PPT1-KO neurospheres compared with those of the WT littermates (Fig. 3B). Consistent with these results, analysis of the PPT1-KO and WT neurospheres by EM shows that there is structural abnormality in the ER of PPT1-KO neurospheres and not in WT neurospheres (data not shown). Taken together, these results suggest that in the brain of PPT1-KO mice, ER stress is initiated quite early in fetal development.



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  		Figure 3. Brain cells in PPT1-KO mice undergo ER stress. (A) Histochemical detection of Grp-78 protein in the cortex of WT (upper panels) and PPT1-KO (lower panels) mice; scale bar, 20 µm. (B) Western blot analysis for the expression of Grp-78/Bip in neurospheres derived from 15-day fetuses from WT (left lane) and PPT1-KO (right lane) mice.

 
Activation of UPR in the brains of PPT1-KO mice
In response to ER stress, all eukaryotic cells respond through the activation of the UPR that represents a set of signaling pathways for the maintenance of homeostasis (26Go,28Go,31Go). As a result, a program of gene expression is initiated to remedy cellular damage. A key event in this process is the phosphorylation of translation initiation factor, eIF2{alpha}, which mediates transient suppression of global translation and promotes up-regulation of selected stress-induced genes to facilitate recycling and/or degradation of unfolded proteins in the ER (26Go–28Go,30Go–32Go). Accordingly, we determined the levels of phosphorylated eIF2{alpha} in the brains of 1-, 3- and 6-month-old PPT1-KO mice and in those of their WT littermates. The results show increased levels of phosphorylated eIF2{alpha} in the brains of PPT1-KO mice compared with those of their WT littermates of all three age groups (Fig. 4A, upper panels). Most interestingly, we found that the levels of phosphorylated eIF2{alpha} in cultured neurospheres from PPT1-KO fetuses are also significantly higher than in those of WT littermates (Fig. 4B), suggesting that in the brains of PPT1-KO mice the UPR is activated during early fetal development. It has been reported that phosphorylation of eIF2{alpha} stimulates the mRNA expression of selected stress-induced genes such as activated transcription factor-3 (ATF3) and basic leucine zipper-containing transcription factor, X-box-binding protein 1 (XBP1) (33Go). Accordingly, we determined the levels of ATF3- and XBP1-mRNAs in the brains of 1-, 3- and 6-month-old PPT1-KO mice and of WT littermates. The results show that although in the brains of WT mice the ATF3- and XBP1-mRNA levels are low at all age groups studied, these levels are markedly elevated in the brains of 3- and 6-month-old PPT1-KO mice (Fig. 4C and D).



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  		Figure 4. Activation of UPR in PPT1-KO mice. (A) Western blot analyses for non-phosphorylated and phosphorylated eIF2{alpha} protein in the brains of WT littermates and in those of PPT1-KO mice. (B) Western blot analysis demonstrating increased level of phosphorylated eIF2{alpha} in the brains of PPT1-KO (right lane) compared with that of WT (left lane) neurospheres. (C) Age-dependent elevation of ATF3-mRNA expression in the brains of PPT1-KO and WT mice. (D) Relative expression of XBP1-mRNA in the brains of PPT1-KO mice and their WT littermates by quantitative real-time PCR. (E) PP1-mRNA levels in the brains of PPT1-KO mice and their WT littermates. (F) PP1 activities in the brains of PPT1-KO mice and in those of their WT littermates.

 
Reduced eIF2{alpha}-phosphatase activity causes elevated levels of phosphorylated eIF2{alpha}
The elevated levels of phosphorylated eIF2{alpha} in the PPT1-KO brains may either be due to elevated levels of phosphorylation or reduced levels of dephosphorylation. It has been reported that in mammals there are four eIF2{alpha} kinases (34Go) that phosphorylate this transcription initiation factor. These are PERK, GCN2, PKR and HRI. Thus, we first sought to determine whether there is increased expression of these kinases in the brain of PPT1-KO mice and analyzed the mRNA levels by real-time PCR. The results show that PKR-mRNA levels are not appreciably different in the PPT1-KO brains compared with those of the WT littermates at all age groups (Supplementary Material, Fig. S2A). However, GCN2-mRNA levels in the brains of PPT1-KO mice of all three age groups compared with those of WT littermates (Supplementary Material, Fig. S2B) are significantly lower (P<0.005). Interestingly, although PERK- and HRI-mRNA levels in the brains of 1- and 3-month-old PPT1-KO mice are not significantly different from their WT counterparts (P>0.05), at 6 months of age these levels in the brains of PPT1-KO mice (Fig. 2C and D) are significantly lower (P<0.005). Although the reason for these differences in mRNA levels are not clear, it is possible that these lower levels reflect increased neuronal death in the brains of 6-month-old PPT1-KO mice. As reduced dephosphorylation may cause elevated levels of phosphorylated eIF2{alpha}, we quantitated the protein phosphatase-1 (PP1)-mRNA levels by real-time PCR as this enzyme catalyzes the dephosphorylation of eIF2{alpha} (35Go). The results show that PP1-mRNA expression is significantly reduced (P<0.005) in the brains of PPT1-KO mice (Fig. 4E). Consistent with these results, we found that PP1 activity appears to be reduced in the brains of PPT1-KO mice compared with that of WT littermates (Fig. 4F). These results strongly suggest that down-regulation of PP1-catalyzed dephosphorylation rather than increased phosphorylation is the most likely reason for the elevated levels of phospho-eIF2{alpha} in the brains of PPT1-KO mice.

Activation of caspases-12 and caspase-3 in the brains of PPT1-KO mice
It has been reported that irreversible damage caused by prolonged ER stress triggers an apoptotic program that includes the activation of UPR-specific, cysteine proteinase in the ER, caspase-12 (36Go,37Go). There are several pathways to apoptosis, and a family of caspases plays pivotal roles in this form of programmed cell death (reviewed in 38Go). These proteinases are generally classified into two groups: the initiator caspases, which include caspases-8, -9 and -12 and the effector caspases represented by caspases-3, -6 and -7 (39Go). Thus, we first sought to determine whether the expression and activation of any of the initiator caspases are elevated in the brains of PPT1-KO mice. The results of real-time PCR show that caspase-12-mRNA levels are markedly elevated in the brains of PPT1-KO mice in an age-dependent manner compared with those of the WT littermates (Fig. 5A). Western blot analyses show that the levels of pro (inactive)- as well as cleaved (active)-caspase-12 proteins are elevated in the brains of the PPT1-KO mice but not in those of the WT littermates (Fig. 5B). These results together with the finding of structural abnormalities in the ER, elevated levels of phosphorylated eIF2{alpha} and increased levels of Grp-78 suggest that the cells in the PPT1-KO mice undergo ER stress, which activates caspase-12, a resident cysteine protease in the ER that leads to apoptosis.



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  		Figure 5. Expression and activation of ER-resident caspases leading to the activation of effector caspase, caspase-3. (A) Caspase-12 mRNA levels in the brains of PPT1-KO mice determined by real-time PCR. (B) Western blot analysis of caspase-12 protein from brain lysates of PPT1-KO and WT mice; arrows indicate the pro- (inactive) and cleaved- (active) caspases. ß-Actin was used as loading standard for proteins. (C) Caspase-3 mRNA expression. (D) Caspase-3 activity.

 
As activation of the initiator caspase, caspase-12 in the ER (36Go,37Go) may lead to the activation of the executioner caspase, caspase-3 (36Go,40Go), we determined the levels of caspase-3 mRNA as well as its enzyme activity in the brains of 1-, 3- and 6-month-old PPT1-KO mice and in those of their WT littermates. Our results show an age-dependent elevation of caspase-3-mRNA expression in the brains of PPT1-KO mice (Fig. 5C). Consistent with these results, caspase-3 enzyme activities are significantly elevated (P<0.005) compared with those of the WT littermates (Fig. 5D). These results indicate that caspase-3 is activated in the brains of PPT1-KO mice mediating apoptosis and consequent neurodegeneration.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In this study, we have uncovered that: (a) the ER in the brain cells of PPT1-KO mice are structurally abnormal, (b) proteins that undergo palmitoylation, such as GAP-43, abnormally accumulate in the PPT1-KO mouse brains, (c) forced expression of GAP-43 in PPT1-deficient cells results in abnormal accumulation in the ER, (d) over-expression of other palmitoylated proteins, such as CD-9, CD-81 and PSD-95, also cause abnormal accumulation in the ER and (e) PPT1 deficiency leads to the activation of UPR, which is associated with elevated levels of phosphorylated eIF2{alpha}, increased expression of Grp-78/Bip, ATF3, XBP1 and activation of the ER-resident cysteine protease, caspase-12, leading to caspase-3 activation and apoptosis. The results of this study, for the first time, link PPT1 deficiency with the activation of the UPR and define at least one of the major molecular mechanisms of apoptosis in the PPT1-KO mice, which leads to neurodegeneration.

How might PPT1 deficiency activate UPR in the PPT1-KO mice? It has been reported that protein palmitoylation, a common post-translational lipid modification, plays critical roles in neuronal cells (6Go,41Go,42Go). 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 (43Go). Further, palmitoylation not only alters protein structure but also increases hydrophobicity, which allows them to anchor onto biological membranes (22Go) including the membranes of the ER. Consistent with these reports, it has been recently demonstrated that a mammalian palmitoyl transferase, HIP14, which catalyzes the palmitoylation of several important neuronal proteins (23Go,42Go–44Go), is predominantly localized to the ER–Golgi membranes and to cytoplasmic vesicles (45Go). HIP14 also catalyzes its own palmitoylation (43Go) for membrane anchorage. Thus, it is reasonable to assume that in the absence of depalmitoylation in PPT1-KO mice, HIP-14 remains activated on ER–Golgi membranes, further increasing the levels of S-acylated proteins in this organelle. This notion is consistent with the results of our present experiments demonstrating that PPT1-KO brains contain higher levels of GAP-43 than 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. Most importantly, the results of forced expression of GFP-GAP-43 in cells from PPT1-KO mice and their WT littermates show that the intense GFP-GAP-43 fluorescence in PPT1-deficient cells co-localizes 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.

Recent reports indicate that PPT1 plays critical roles in regulating palmitate turnover on PSD-95, a scaffolding neuronal protein that requires palmitoylation for membrane anchorage, essential for its function (6Go). Moreover, vesicular trafficking of PSD-95 is dependent on palmitoylation/depalmitoylation cycle and disruption of either of these processes may lead to the disruption of normal protein trafficking through the ER–Golgi compartment (6Go). Indeed, transfection of cells from PPT1-KO and WT mice with GFP constructs containing cDNAs of several known palmitoylated proteins such as GAP-43 (Fig. 2D) as well as CD9, CD81 and PSD-95 (data not shown) indicate that these proteins also abnormally accumulate in the ER of PPT1-KO but not in that of the WT cells. The ER is the site for synthesis, post-translational modification and delivery of biologically active proteins to their proper target sites (26Go). It has been demonstrated that S-acylation occurs in the ER (23Go) and the lipid-modified proteins anchor to the membranes of the ER, Golgi and cytoplasmic vesicles. Thus, the lack of PPT1 may lead to abnormal accumulation in these organelles due to the fact that thioester linkages in S-acylated proteins are not cleaved and S-acylated proteins remain membrane-bound. Therefore, 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.

An alternative possibility is that due to PPT1 deficiency, palmitate residues in S-acylated proteins are not removed and as a result these proteins are not degraded by lysosomal proteases. Further, it has been recently proposed that abnormal accumulation of S-acylated proteins in PPT1-deficient cells may occur from autophagy and failure of lysosomal degradation of these proteins (24Go). This may cause congestion in various post-ER compartments causing retrograde transport of S-acylated proteins from post-ER compartments to the ER mediating ER stress and the activation of the UPR. Consistent with this hypothesis, it has been proposed that retrograde transport from post-ER compartments to the ER may occur as a result of lysosomal dysfunction (25Go), although the mechanism of such retrograde transport is not yet clearly understood. Indeed, the results of a recent study show that in another lysosomal storage disease that causes neurodegeneration and GM1 gangliosidosis, abnormal accumulation of GM1 gangliosides in the ER leads to the activation of the UPR-mediating apoptosis (46Go). As a majority of more than 40 lysosomal storage diseases develop neurodegeneration (reviewed in 47Go), it is tempting to speculate that activation of UPR secondary to lysosomal dysfunction may underlie pathogenesis in those lysosomal storage diseases that lead to neurodegeneration.

The results of our present study, for the first time, show that in the absence of depalmitoylation resulting from inactivating mutations in the PPT1 gene, the UPR is activated and neuronal apoptosis results. Increased apoptosis and actual neuronal loss in the brains of PPT1-KO mice with increasing age (21Go) lead to neurodegeneration. Although PPT1 is expressed in virtually all cell types, the nervous system is known to express numerous proteins that require S-acylation for their membrane anchorage and biological function (2Go–8Go). Thus, it is conceivable that the absence of PPT1 has the most devastating effect on the nervous system compared with other organs in the PPT1-KO mice and in INCL patients. The results of our study also identify several potential targets for intervention in INCL. On the basis of the results described above, we propose that reduction of ER stress by small molecules and suppression of caspase-12 activation may provide novel approaches for developing effective therapeutic strategies in this devastating disease. Indeed, through a series of elegant experiments it has been recently reported that salubrinal, an agent that selectively inhibits de-phosphorylation of eIF2{alpha}, a key component of the UPR, is cytoprotective (32Go,48Go), providing the ‘proof of principle’ that alleviation of ER stress may be a novel approach to developing effective therapeutic agents for neurodegenerative diseases such as INCL in which ER stress and activation of the UPR play critical pathogenic role.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
PPT1-KO mice and genotyping
PPT1-KO mice were generated by gene targeting in ES cells as previously described (20Go). All mice were maintained and housed in a germ-free facility and animal procedures were carried out in accordance with institutional guidelines after the NICHD Animal Care and Use Committee approved an animal study protocol.

Cell culture and reagents
Mouse neurospheres were isolated from the brains of 15-day-old fetuses of PPT1-KO and WT littermates. The cells were cultured in NeuroCult NSC Basal Medium (Stem Cell Technologies) containing NeuroCult NSC proliferation supplements and human epidermal growth factor were added to a final concentration of 20 ng/ml and incubated at 37°C under an atmosphere of 5% CO2 and 95% air.

Transmission electron microscopy
The brain tissues were fixed in 2.5% glutaraldehyde in sodium phosphate buffer. After 2 h, the tissues were washed with Millonig's phosphate buffer once and kept in the same buffer until final processing. Thin sections of the brain tissues were stained with lead citrate and uranyl acetate and examined with an LEO 912 electron microscope by JFE Enterprises (Brookeville, MD, USA).

RNA isolation and quantitative PCR analysis
Total RNA from the brains of PPT1-KO mice and their WT littermates was isolated using TriZol reagent (Invitrogen) and further purified by QIAGEN RNeasy Mini Kit and treated with DNAse (DNAse I, 30 U/µg total RNA) (QIAGEN), then reverse transcribed using SuperScript III First-Strand Synthesis System (Invitrogen). Expression of mRNA was quantitated by using SYBR Green PCR Master Mix, performed with ABI Prism 7000 Sequence Detection System (Applied Biosystems) with cDNA equivalent to 100 ng of total RNA for caspase-12, caspase-3 and 10 ng of total RNA for other genes. The primers used for these genes are presented in Table 1. The results were analyzed using ABI Prism Software version 1.01 (Applied Biosystems). The final data were normalized to ß-actin standard and presented as fold change in PPT1-KO mice and compared with those of WT mice. Experiments were done in triplicate for the PPT1-KO and WT mice.


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  		Table 1. Primers used for quantitative real-time PCR
 
Western blot analyses
Mouse brains 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 and phosphatase-inhibitor cocktails (Sigma-Aldrich). Twenty micrograms of total protein from each sample was resolved by SDS–PAGE, electrotransferred to polyvinylidene fluoride (PVDF) membrane (Immobilon P, Millipore Corp.) and immunodetected as described previously (19Go). The primary antibodies against the following polypeptides were used: anti-KDEL (1 : 1000; Stressgen), anti-eIF2{alpha} (1 : 1000; a generous gift from Dr T.E. Dever, NICHD, NIH), anti-phospho-eIF2{alpha} (1 : 1000; Upstate Biotechnology), anti-caspase-12 (1 : 1000; Cell Signaling Technology) and anti-Gap-43 (1 : 1000; Chemicon). The second antibodies used in the study are: goat anti-rabbit IgG, rabbit anti-mouse IgG and donkey anti-goat IgG (Santa Cruz Biotechnology). Chemiluminescent detection was performed by using ECL system (Amersham-Pharmacia Biotech) according to the manufacturer's instructions.

Immunohistochemistry
The brain tissues were either fixed in 3.7% paraformaldehyde or flash-frozen, and histological sections prepared. The sections were overlaid with anti-KDEL antibody (1 : 500) and incubated for 30 min in a humidified chamber. After incubation, the slides were washed three times with 1x phosphate-buffered saline (PBS) and were further incubated with anti-mouse biotinylated secondary antibody (1 : 500; Vector Laboratories) for 1 h. The washed sections were then incubated with ABC complex by following the manufacturer's protocol (Vector Laboratories).

GFP-GAP-43 fusion protein expression by fluorescence microscopy
For GFP-GAP-43 fusion protein, cDNA of GAP-43 was amplified by Platinum Pfx enzyme (Invitrogen) PCR amplification. The primers used for this reaction are as follows: GAP-43-F, 5'-AAGCTTATGCTGTGCTGTATGAGAAGAACCAAACAG-3'; GAP-43-R, 5'-CCGCGGGGCATGTTCTTGGTCAGCCTCGGGGTCTTC-3'. This PCR product was subcloned into the pCR-BluntII-TOPO vector (Invitrogen). This full-length GAP-43 cDNA was excised by HindIII and SacII and then re-ligated into the pEGFP-N1 vector (Invitrogen) that was predigested with HindIII and SacII and purified by agarose gel electrophoresis. Fetal mouse fibroblasts from PPT1-KO and WT littermates were established and maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum and were transfected with pEGFP-N1-GAP-43 using Lipofectamine 2000 reagent (Invitrogen). After 24 h of incubation, transfected cells were washed three times with PBS and fixed in 3.7% formaldehyde solution for 15 min. Nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride (Sigma). Fluorescence was visualized with the Axioskop2 plus fluorescence microscope (Carl Zeiss), and the image was processed with the AxioVision 4.2 software (Carl Zeiss) and the Photoshop 7.0 program (Adobe).

3H-Palmitate labeling of proteins and immunoprecipitation
Cells were metabolically labeled prior to immunoprecipitation with [3H]-palmitic acid (200 µCi/ml palmitate; Amersham Pharmacia Biotech) in NeuroCult medium (Stem Cell Technologies) for 3 h. Labeled cells were washed two times with ice-cold PBS and resuspended in RIPA lysis buffer (10 mM NaPO4, pH 7.2, 1 M NaCl, 40 mM NaF, 10 mM EDTA, 0.5% Triton X-100). After extracting for 15 min at 4°C, insoluble material was removed by centrifugation at 10 000g for 15 min. For immunoprecipitation, the samples were then incubated with GAP-43 antibodies (anti-rabbit, 1 : 100 dilution, Abcam) for overnight at 4°C. After addition of protein A Sepharose beads (Pharmacia), samples were incubated for 1 h at 4°C. Immunoprecipitates were washed three times with buffer containing 50 mM Tris–HCl, pH 7.2, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl and 0.3% Triton X-100, boiled in SDS–PAGE sample buffer (80 mM Tris, pH 6.8, 10% glycerol, 5% SDS, 150 mM dithiothreitol, 0.005% bromophenol blue) for 5 min. For autoradiography, protein samples were separated by SDS–PAGE and dried using vacuum dryer. Moreover, gel was exposed to BAS-IIIs Imaging plate (Fuji) for 2 weeks, and the image was processed using the BAS-1500 Reader (Fuji) with the Image Reader V1.4E and Image Gauge V3.0 programs (Fuji).

Caspase-3 assay
The DEVD-cleaving activity of active caspase-3 was measured by using Caspase-3 Assay Kit from PharMingen (San Diego, CA, USA) following the manufacturer's protocol. N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcourmarin was used as the fluorogenic substrate in the assay. AMC fluorescence is quantified by UV spectrofluorometer using an excitation wavelength of 380 nm.

PP1 assay
The protein serine/threonine phosphatase-1 activity was measured by using Protein Serine/Threonine Phosphatase (PSP) Assay System (New England BioLabs, Beverly, MA, USA) following the manufacturer's protocol. The 32P-labeled MyBP was used as the substrate in the assay. The protein serine/threonine phosphatase-1 activity was determined by measuring the release of inorganic phosphate. The radioactivity in the assay was counted by using liquid scintillation analyzer (Perkin-Elmer, Model 2800TR).

Statistical analysis.
Data are expressed as mean of at least three determinations±standard deviation (SD). Statistical analyses were performed by Student's t-test using Excel Office 2000 (Microsoft) and a P-value of <0.05 was considered statistically significant.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank S.L. Hofmann for the generous gift of the PPT1-KO mice generated in her laboratory and for critical review of the manuscript with helpful comments; H. Gainer, R. Youle, J.Y. Chou, I. Owens, S.W. Levin, J.-Y. Wang and O. Rennert for critical review of the manuscript; T.E. Dever for eIF2{alpha} monoclonal antibodies and S. Everett and R. Dreyfuss for expert assistance on photomicrography. This research was supported in part by the Intramural Research Program of the NIH (NICHD).

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

  1. Schmidt, M.F.G. (1989) Fatty acylation of proteins. Biochim. Biophys. Acta, 988, 411–426.[Medline]

  2. Jing, S. and Townbridge, I.S. (1987) Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site. EMBO J., 6, 327–331.[ISI][Medline]

  3. Robinson, L.J., Busconi, L. and Michel, T. (1995) Agonist-modulated palmitoylation of endothelial nitric oxide synthase. J. Biol. Chem., 270, 995–998.[Abstract/Free Full Text]

  4. Sudo, Y., Valenzuela, D., Beck-Sickinger, A.G., Fishman, M.C. and Strittmatter, S.M. (1992) Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43. EMBO J., 11, 2095–2102.[ISI][Medline]

  5. Hess, D.T., Slater, T.M., Wilson, M.C. and Skene, P.J.H. (1992) The 25 kDa synaptosomal-associated protein SNAP-25 is the major methionine-rich polypeptide in rapid axonal transport and a major substrate for palmitoylation in adult CNS. J. Neurosci., 12, 4634–4641.[Abstract]

  6. El-Husseini, A.-D., Schnell, E., Dakoji, S., Sweeney, N., Zhou, Q., Prange, O., Gauthier-Campbell, C., Aguilera-Moreno, A., Nicoll, R.A. and Bredt, D.S. (2002) Synaptic strength regulated by palmitate cycling on PSD-95. Cell, 108, 849–863.[CrossRef][ISI][Medline]

  7. Randall, W.R. (1994) Cellular expression of a cloned, hydrophilic, murine acetylcholinesterase: evidence of palmitoylated membrane-bound forms. J. Biol. Chem., 269, 12367–12374.[Abstract/Free Full Text]

  8. Dunphy, J.T. and Linder, M.E. (1998) Signalling functions of protein palmitoylation. Biochim. Biophys. Acta, 1436, 245–261.[Medline]

  9. Yeh, D.C., Duncan, J.A., Yamashita, S. and Michel, T. (1999) Depalmitoylation of endothelial nitric oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca2+-calmodulin. J. Biol. Chem., 274, 33148–33154.[Abstract/Free Full Text]

  10. Camp, L.A. and Hofmann, S.L. (1993) Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras. J. Biol. Chem., 268, 22566–22574.[Abstract/Free Full Text]

  11. Camp, L.A., Verkruyse, L.A., Afendis, S.J., Slaughter, C.A. and Hofmann, S.L. (1994) Molecular cloning and expression of palmitoyl-protein thioesterase. J. Biol. Chem., 269, 23212–23219.[Abstract/Free Full Text]

  12. Vesa, J., Hellsten, E., Verkruyse, L.A., Camp, L.A., Rapola, J., Santavuori, P., Hofmann, S.L. and Peltonen, L. (1995) Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature, 376, 584–587.[CrossRef][Medline]

  13. Goebel, H.H. and Wisniewski, K.E. (2004) Current state of clinical and morphological features in human NCL. Brain. Pathol., 14, 61–69.[ISI][Medline]

  14. Wisniewski, K.E., Connell, F., Kaczmarski, W., Kaczmarski, A., Siakotos, A., Becerra, C.R. and Hofmann, S.L. (1998) Palmitoyl-protein thioesterase deficiency in a novel granular variant of LINCL. Pediatr. Neurol., 18, 119–123.[CrossRef][ISI][Medline]

  15. Haltia, M. (2003) The neuronal ceroid-lipofuscinoses. J. Neuropathol. Exp. Neurol., 62, 1–3.[ISI][Medline]

  16. Hofmann, S.L., Atashband, A., Cho, S.K., Das, A.K., Gupta, P. and Lu, J.Y. (2002) Neuronal ceroid lipofuscinoses caused by defects in soluble lysosomal enzymes (CLN1 and CLN2). Curr. Mol. Med., 2, 423–437.[CrossRef][Medline]

  17. Wisniewski, K.E., Zhong, N. and Philippart, M. (2001) Pheno/genotypic correlations of neuronal ceroid lipofuscinoses. Neurology, 57, 576–581.[Abstract/Free Full Text]

  18. Riikonen, R., Vanhanen, S.L., Tyynela, J., Santavuori, P. and Turpeinen, U. (2000) CSF insulin-like growth factor-1 in infantile neuronal ceroid lipofuscinosis. Neurology, 54, 1828–1832.[Abstract/Free Full Text]

  19. Zhang, Z., Butler, J.D., Levin, S.W., Wisniewski, K.E., Brooks, S.S. and Mukherjee, A.B. (2001) Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood. Nat. Med., 7, 478–484.[CrossRef][ISI][Medline]

  20. Gupta, P., Soyombo, A.A., Atashband, A., Wisniewski, K.E., Shelton, J.M., Richardson, J.A., Hammer, R.E. and Hofmann, S.L. (2001) Disruption of PPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout mice. Proc. Natl Acad. Sci. USA, 98, 13566–13571.[Abstract/Free Full Text]

  21. Bible, E., Gupta, P., Hofmann, S.L. and Cooper, J.D. (2004) Regional and cellular neuropathology in the palmitoyl protein thioesterase-1 null mutant mouse model of infantile neuronal ceroid lipofuscinosis. Neurobiol. Dis., 16, 346–359.[CrossRef][ISI][Medline]

  22. Resh, M.D. (1999) Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta, 1451, 1–16.[Medline]

  23. McLaughlin, R.E. and Denny, J.B. (1999) Palmitoylation of GAP-43 by the ER–Golgi intermediate compartment and Golgi apparatus. Biochim. Biophys. Acta, 1451, 82–92.[Medline]

  24. Lu, J.-Y., Verkruyse, L.A. and Hofmann, S.L. (1996) Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase. Proc. Natl Acad. Sci. USA, 93, 10046–10050.[Abstract/Free Full Text]

  25. Ron, D. and Oyadomari, S. (2004) Lipid phase perturbations and the unfolded protein response. Dev. Cell, 7, 287–288.[CrossRef][ISI][Medline]

  26. Schroder, M. and Kaufman, R.J. (2005) The mammalian unfolded protein response. Annu. Rev. Biochem., 74, 739–789.[CrossRef][ISI][Medline]

  27. Rao, R.V. and Bredesen, D.E. (2004) Misfolded proteins, endoplasmic reticulum stress and neurodegeneration. Curr. Opin. Cell Biol., 16, 653–662.[CrossRef][ISI][Medline]

  28. Harding, H.P., Calfon, M., Urano, F., Novoa, I. and Ron, D. (2002) Transcriptional and translational control in the mammalian unfolded protein response. Annu. Rev. Cell Dev. Biol., 18, 575–599.[CrossRef][ISI][Medline]

  29. Gauthier-Campbell, C., Bredt, D.S., Murphy, T.H. and El-Husseini, A.-D. (2004) Regulation of dendritic branching and filopodia formation in hippocampal neurons by specific acylated protein motifs. Mol. Biol. Cell, 15, 2205–2217.[Abstract/Free Full Text]

  30. Lee, A.S. (2001) The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem. Sci., 26, 504–510.[CrossRef][ISI][Medline]

  31. Hinnebush, A. (2000) In Sonnenberg, N., Hershey, J.W.B. and Mathews, M.B. (eds), Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 185–244.

  32. Boyce, M., Bryant, K.F., Jousse, C., Long, K., Harding, H.P., Scheuner, D., Kaufman, R.J., Ma, D., Coen, D.M., Ron, D. and Yuan, J. (2005) A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science, 307, 935–939.[Abstract/Free Full Text]

  33. Jiang, H.Y., Wek, S.A., McGrath, B.C., Lu, D., Hai, T., Harding, H.P., Wang, X., Ron, D., Cavener, D.R. and Wek, R.C. (2004) Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response. Mol. Cell. Biol., 24, 1365–1377.[Abstract/Free Full Text]

  34. Dever, T.E. (2002) Gene-specific regulation by general translation factors. Cell, 108, 545–556.[CrossRef][ISI][Medline]

  35. Brush, M.H., Weiser, D.C. and Shenolikar, S. (2003) Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Mol. Cell. Biol., 23, 1292–1303.[Abstract/Free Full Text]

  36. Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B.A. and Yuan, J. (2000) Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature, 403, 98–103.[CrossRef][Medline]

  37. Nakagawa, T. and Yuan, J. (2000) Cross-talk between two cysteine protease families: activation of caspase-12 by calpainin apoptosis. J. Cell. Biol., 150, 887–894.[Abstract/Free Full Text]

  38. Riedl, S.J. and Shi, Y. (2004) Molecular mechanisms of caspase regulation during apoptosis. Nat. Rev. Mol. Cell. Biol., 5, 897–907.[CrossRef][ISI][Medline]

  39. Morishima, N., Nakanishi, K., Takenouchi, H., Shibata, T. and Yasuhiko, Y. (2002) An endoplasmic reticulum stress-specific caspase cascade in apoptosis: cytochrome c-independent activation of caspase-9 by caspase-12. J. Biol. Chem., 277, 34287–34294.[Abstract/Free Full Text]

  40. Hetz, C., Russelakeiro, M., Maundrell, K., Castilla, J. and Soto, C. (2003) Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein. EMBO J., 22, 5435–5445.[CrossRef][ISI][Medline]

  41. Rocks, O., Peyker, A., Kahms, M., Verveer, P.J., Koerner, C., Lumbierres, M., Kuhlmann, J., Waldmann, H., Wittinghofer, A. and Bastiaens, P.I. (2005) An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science, 307, 1746–1752.[Abstract/Free Full Text]

  42. Fukata, M., Fukata, Y., Adesnik, H., Nicoll, R.A. and Bredt, D.S. (2004) Identification of PSD-95 palmitoylating enzymes. Neuron, 44, 987–996.[CrossRef][ISI][Medline]

  43. Huang, K., Yanai, A., Kang, R., Arstikaitis, P., Singaraja, R.R., Metzler, M., Mullard, A., Haigh, B., Gauthier-Campbell, C., Gutekunst, C.A., Hayden, M.R. and El-Husseini, A.-D. (2004) Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins. Neuron, 44, 977–986.[CrossRef][ISI][Medline]

  44. Singaraja, R.R., Hadano, S., Metzler, M., Givan, S., Wellington, C.L., Warby, S., Yanai, A., Gutekunst, C.A., Leavitt, B.R., Yi, H., Fichter, K. et al. (2002) HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis. Hum. Mol. Genet., 11, 2815–2828.[Abstract/Free Full Text]

  45. Martin, J.E. and Shaw, G. (1998) Phenotype analysis in neurological models of human disease. Neuropathol. Appl. Neurobiol., 24, 83–87.[ISI][Medline]

  46. Tessitore, A., del P. Martin, M., Sano, R., Ma, Y., Mann, L., Ingrassia, A., Laywell, E.D., Steindler, D.A., Hendershot, L.M. and d'Azzo, A. (2004) GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis. Mol. Cell, 15, 753–766.[CrossRef][ISI][Medline]

  47. Proia, R.L. and Wu, Y.-P. (2004) Blood to brain to the rescue. J. Clin. Invest., 113, 1108–1110.[CrossRef][ISI][Medline]

  48. Lu, P.D., Jousse, C., Marciniak, S.J., Zhang, Y., Novoa, I., Scheuner, D., Kaufman, R.J., Ron, D. and Harding, H.P. (2004) Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2. EMBO J., 23, 169–179.[CrossRef][ISI][Medline]


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