Accumulation of the Authentic Parkin Substrate Aminoacyl-tRNA Synthetase Cofactor, p38/JTV-1, Leads to Catecholaminergic Cell Death

Introduction

Parkinson's disease (PD) is one of the most common neurodegenerative disorders characterized by the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the presence of intracellular inclusions, named Lewy bodies (Dawson and Dawson, 2003). Deficiency of these neurons causes progressive motor impairments, including tremor, rigidity, and bradykinesia. Although the majority of PD is sporadic, the identification of familial PD-linked mutations in genes encoding α-synuclein (Polymeropoulos et al., 1997), DJ-1 (Bonifati et al., 2003), PINK-1 (Valente et al., 2004), LRRK2 (Paisan-Ruiz et al., 2004; Zimprich et al., 2004), and parkin (Kitada et al., 1998) has provided tremendous insight into the pathogenesis of PD (Cookson, 2005). The finding that parkin is an ubiquitin E3 ligase provided an important link between protein aggregation and the ubiquitin-proteasome system (UPS) in the pathogenesis of PD (Shimura et al., 2000; Zhang et al., 2000). Mutations in parkin appear to be very prevalent and account for up to 50% of familial PD (von Coelln et al., 2004a). Parkin is an E3 ligase that is responsible for the addition of poly-ubiquitin chains on specific substrates (von Coelln et al., 2004a), which is recognized by the proteasome for degradation (Sakata et al., 2003). It is thought that autosomal-recessive juvenile parkinsonism (AR-JP)-linked parkin mutants lead to a loss of the ubiquitin ligase activity of parkin and thus fail to ubiquitinate parkin substrates, leading to their accumulation (Cookson, 2003). Accumulation of one or more of the putative substrates of parkin is ultimately thought to be toxic to catecholaminergic neurons (Dong et al., 2003). Identification of authentic substrates has important implications for not only AR-JP but also sporadic PD because parkin is S-nitrosylated in idiopathic PD. S-nitrosylation of parkin inhibits its ubiquitination and protective function (Chung et al., 2004; Yao et al., 2004); thus, accumulation of parkin substrates may also contribute to the neurodegeneration in sporadic PD. A number of parkin substrates have been identified including, CDCrel-1, CDCrel-2, synphilin-1, glycosylated α-synuclein, β-tubulin, cyclin E, synaptotamin XI (SytXI), parkin-associated endothelin-like receptor (Pael-R), and p38/JTV-1 subunit of the multi-tRNA synthetase complex (Zhang et al., 2000; Chung et al., 2001; Imai et al., 2001; Shimura et al., 2001; Choi et al., 2003; Corti et al., 2003; Huynh et al., 2003; Ren et al., 2003; Staropoli et al., 2003; Jiang et al., 2004). CDCrel-1, CDCrel-2, Pael-R, and cyclin E appear to be upregulated in AR-JP brains (Imai et al., 2001; Choi et al., 2003; Staropoli et al., 2003), but none of these substrates have been reported to be upregulated in parkin knock-out (KO) mice. Parkin-deficient mice with targeted disruption of parkin exon 3, which have subtle behavioral deficits and enhanced dopamine metabolism without loss of dopaminergic neurons (Goldberg et al., 2003; Itier et al., 2003), surprisingly, showed similar levels of CDCrel-1, synphilin-1, and α-synuclein (Goldberg et al., 2003; Palacino et al., 2004). Moreover, there seems to be reductions in mitochondrial-associated proteins in parkin exon 3-deleted mice, which leads to potential mitochondrial defects (Palacino et al., 2004). We recently reported that targeted disruption of parkin exon 7 creates a parkin null pheno-type that leads to a reduced number of locus ceruleus neurons, deficits of norepinephrine in the olfactory bulb and spinal cord, and a marked reduction of the norepinephrine-modulated startle response (von Coelln et al., 2004b). We analyzed the levels of known parkin substrates in the brains of parkin exon 7 null mice and AR-JP patients, and here we report the discovery that p38/JTV-1 accumulates in parkin exon 7 null mice and AR-JP brains. Moreover, it accumulates in sporadic PD brains, and adenoviral (AV)-mediated overexpression of p38 leads to selective DA neuronal cell death.

Materials and Methods

cDNA, cell culture, and antibodies. Full-length human p38 cDNA was cloned into the mammalian expression vector pCMV-2A-FLAG (Stratagene, La Jolla, CA). Expression plasmids for FLAG-tagged human parkin and V5-tagged human heat shock protein 70 (Hsp70) were kindly provided by R. Takahashi (RIKEN Brain Science Institute, Tokyo, Japan), and hemagglutinin (HA)-tagged mouse C-terminus of hsc70-interacting protein (CHIP) was kindly provided by S. Hatakeyama (Kyushu University, Fukuoka-shi, Japan). Human full-length parkin and its deletion mutants and ubiquitin cDNAs were cloned into pRK5-HA vector as described previously (Chung et al., 2001). A plasmid containing β-galactosidase cDNA was used as a control in all experiments. The integrity of all constructs was confirmed by sequencing.

Human neuroblastoma SK-N-MC and SH-SY5Y cells were purchased from American Type Culture Collection (Manassas, VA). They were maintained in modified Eagle's medium and DMEM supplemented with 10% (v/v) heat-inactivated fetal calf serum at 37°C in a humidified 5% CO₂/95% air atmosphere, respectively. Human neuroblastoma SK-N-MC and SH-SY5Y cells were transiently transfected with the target vector by the Lipofectamine method according to the instructions of the manufacturers (Invitrogen, Carlsbad, CA).

Adult neural stem cells were isolated from the brains of adult parkin null mice and their littermates and cultured as described previously (Zhao et al., 2003).

To prepare the stable transformed cells, SH-SY5Y cells were transfected with pcDNA3-HA-p38. Selections were started 2 d later using a medium containing 700 μg/ml geneticin (Invitrogen). Individual clones were isolated, and their characterization was examined by Western blotting with an antibody against anti-HA.

To generate a peptide antigen of p38, a peptide containing the last 18 amino acids of the C terminal of p38 was synthesized and cross-linked to keyhole limpet hemocyanin. The conjugated peptide was then used to immunize a New Zealand white rabbit (JH745–748) (Cocalico Biologicals, Reamstown, PA). Antisera were purified by affinity chromatography using the same peptide immobilized on SulfoLink gel matrix (Pierce, Rockford, IL) according to the protocol of the manufacturer. Antibody specificity was confirmed by the ability to preabsorb the immunostaining with excess purified p38 protein and with p38 knock-out mice. p38 monoclonal antibody was kindly provided by S. Kim (University of Seoul, Seoul, Korea), and Pael-R monoclonal antibody was kindly provided by R. Takahashi (RIKEN Brain Science Institute, Tokyo, Japan).

In vitro pull-down assay and immunoprecipitation. The p38 cDNA was cloned into the pGEX-KT vector (Amersham Biosciences, Piscataway, NJ) to produce the fusion protein glutathione S-transferase (GST)-p38. Expression and purification of GST-p38 and GST alone (as a control) using glutathione-Sepharose 4B (Amersham Biosciences) was performed as recommended by the manufacturer. In vitro translation of parkin wild-type (WT) and 77-465 were performed with rabbit reticulocyte lysate (Promega, Madison, WI) and ³⁵S-methionine according to instructions of the manufacturer. The glutathione-Sepharose beads with bound GST-fusion proteins and in vitro-translated parkin WT and 77-465 were incubated in bead-binding buffer [50 mm K-phosphate, pH 7.5, 100 mm KCl, and 10% glycerol (v/v)/0.1% Triton X-100] at 4°C for 2 h. The beads were washed four times with bead-binding buffer without glycerol and Triton X-100. Beads were then heated for 5 min at 95°C in SDS-sample buffer and analyzed by SDS-PAGE. Bands were visualized by autoradiography.

For coimmunoprecipitation from cell cultures, SH-SY5Y cells were transfected with 2 μg of each plasmid. After 48 h, cells were washed with cold PBS and harvested in immunoprecipitation buffer (1% Triton X-100, 2 μg/ml aprotinin, and 100 μg/ml PMSF in PBS). The lysate was then rotated at 4°C for 1 h, followed by centrifugation at 14,000 rpm for 20 min. The supernatants were then combined with 50 μl of protein G Sepharose (Amersham Biosciences) preincubated with antibodies against HA or myc (Roche, Indianapolis, IN), followed by rotating at 4°C for 2 h. The protein G Sepharose was pelleted and washed three times using immunoprecipitation buffer or buffer with additional 500 mm NaCl, followed by three washes with PBS. The precipitates were resolved on SDS-PAGE gel and subjected to Western blot analysis. Immunoblot signals were visualized with enhanced chemiluminescence (Amersham Biosciences).

For coimmunoprecipitation of the endogenous proteins from human brain, frontal cortex gray matter was homogenized in 4 vol of ice-cold PBS containing 320 mm sucrose and 0.1% Triton X-100 with protease inhibitor cocktail (Sigma, St. Louis, MO). The tissue homogenate was centrifuged at 37,000 × g at 4°C for 20 min. The supernatant was used for immunoprecipitation with one of the following antibodies: anti-HA (Roche), anti-myc (Roche), anti-p38, or anti-parkin. Immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analysis with an anti-p38 monoclonal antibody. Immunoblot signals were visualized with enhanced chemiluminescence (Amersham Biosciences). For mapping of the binding region between parkin and p38, the myc-tagged parkin deletion constructs were generated as described previously (Chung et al., 2001), and the myc-tagged p38 deletion fragments were generated as described previously (Kim et al., 2003). We transfected each of the plasmids encoding the deletion fragments of parkin and p38 into SH-SY5Y cells. p38 and parkin were precipitated with the anti-HA antibody (Roche), and the coprecipitation of their counterparts was determined by Western blotting with the corresponding antibodies. Detection was performed with enhanced chemiluminescence (Amersham Biosciences).

In vivo ubiquitination assay. For in vivo ubiquitination assay from cell cultures, SH-SY5Y cells were transiently transfected with 2 μg of pRK5-myc-tagged parkin or myc-tagged parkin mutants, pCMV-FLAG-p38, and 2 μg of pMT123-HA-ubiquitin plasmids for 24 h and then treated with the proteasome inhibitor MG132 (5 μm) (Sigma) for 18 h. Total cell lysates were prepared by harvesting the cells after washing with PBS, followed by solubilizing the pellets in 200 μl of 2% SDS, followed by sonication. The lysates were then rotated at 4°C for 1 h, diluted to 1 ml with TBS, and then boiled and sonicated. A total of 50 μl of the samples were used as input and for immunoprecipitation. Immunoprecipitation was performed with an antibody against FLAG. The precipitates were subjected to Western blotting with anti-HA or anti-FLAG antibodies.

Assessment of cell viability. To examine the effect of p38 on cell death, we cotransfected SK-N-MC cells with either control plasmid and HA tagged-p38 or HA tagged-p38 together with FLAG-parkin and HA tagged-p38 together with Arg42Pro parkin mutant and incubated for 24 h, followed by treatment with tumor necrosis factor-α (TNFα) (PeProtech, Rocky Hill, NJ) for 4 h in the presence of 10 μg/ml cycloheximide (Sigma) and monitored cell death by trypan blue staining.

Preparation of human and mouse brain tissue. Frontal cortical tissue from five control brains and eight PD and diffuse Lewy body (DLB) brains with high Lewy body burden that were age matched with similar postmortem intervals as described previously (Chung et al., 2004) were used to analyze parkin substrates in PD and DLB brains. In a separate cohort of brains frontal cortex tissue from four control brains and four AR-JP brains that were age matched with similar postmortem intervals as previously described (Moore et al., 2005) were used to compare parkin substrate levels in AR-JP brains. Parkin null mice were generated by Lexicon Genetics (The Woodlands, TX) as originally described (von Coelln et al., 2004b).

Detergent-soluble and -insoluble fractions were prepared from human brain tissue and mouse brain tissue by homogenization of samples in lysis buffer [10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 0.5% Nonidet P-40, 10 mm Na-β-glycerophosphate, Phosphate Inhibitor Mixture I and II (Sigma), and Complete Protease Inhibitor Mixture (Roche)], by using a Diax 900 homogenizer (Heidolph, Cinnaminson, NJ). After homogenization, samples were rotated at 4°C for 30 min for complete lysis, then the homogenate was centrifuged (10,000 × g, 4°C, 20 min), and the resulting pellet and supernatant fractions were collected. The pellet fractions was washed once in lysis buffer containing detergent, and the resulting pellet was solubilized in lysis buffer containing 1% SDS. Fractions were quantitated using the BCA kit (Pierce) with BSA standards and analyzed by Western blot.

Western blotting was performed with an anti-parkin (PRK8 mouse monoclonal), anti-p38, anti-CDCrel-1 (Zhang et al., 2000), anti-Pael-R monoclonal antibody, anti-α-synuclein (BD Biosciences, Franklin Lakes, NJ), anti-synaptotagmin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-tubulin (Sigma), anti-synphilin-1 (Biodesign, Kennebunk, ME), and anti-cyclin E (Santa Cruz Biotechnology) antibodies. Detection was performed with enhanced chemiluminescence (Amersham Biosciences).

Animals and stereotaxic injection of p38. All procedures were performed in compliance with the guidelines set forth by the Laboratory Animal Manual of the National Institute of Health Guide to the Care and Use of Animals and were approved by the Johns Hopkins Medical Institute Animal Care Committee. Female BC57 mice were bred and maintained at the animal facility of the Johns Hopkins School of Medicine. They were housed in groups of three per cage in a temperature- and humidity-controlled room with a 12 h light/dark cycle. Food and water were available ad libitum.

Animals weighing 30 g were anesthetized with phentobarbital (60 mg/kg, i.p.). An injection cannula (26.5 gauge) was placed stereotaxically into the SN (anteroposterior, –3.1 mm from bregma; mediolateral, 1.3 mm; dorsoventral, 4.3 mm) according to the atlas of Paxinos et al. (1985). Injections of adenoviral vectors of p38 were made in 2.5 μl of virus solution. The viral suspensions were freshly prepared just before use and were kept on ice before and during the injection. The infusion was done at a rate of 0.2 μl/min. The cannula was left in place for 5 min before slowly withdrawing it to avoid reflux along the injection track. The wound was closed with suture, and the animals were allowed to recover before they were returned to their cage. The entire procedure was well tolerated by the animals.

Immunohistochemistry. Animals were deeply anesthetized (80 mg/kg phentobarbital, i.p.) and transcardially perfused with PBS, followed by ice-cold 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4. Brains were promptly removed and postfixed in 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4, for 2 h at room temperature. Each brain was cryoprotected in 30% sucrose until equilibrated and then stored at –80°C until processed.

Brain tissue was cut into 40 μm sections on a HM440E microtome (Microm, Walldorf, Germany), and the sections were collected in the phosphate buffer and incubated in 0.1 m PBS, pH 7.4, containing 1% BSA and 0.2% Triton X-100. After washing in the rinsing buffer containing the PBS and 0.5% BSA, the sections were incubated with antisera against tyrosine hydroxylase (TH) (1:2000, polyclonal; Novus Biologicals, Littleton, CO) overnight at 4°C with shaking. After washes in the rinsing buffer, the sections were incubated for 1 h in the appropriate biotinylated secondary antisera, washed with the rinsing buffer, and then further incubated for 1 h in the avidin–biotin complex. Antigens were visualized by reaction with 0.05% 3,3′-diaminobenzidine in the presence of 0.003% hydrogen peroxide. After two washes in the phosphate buffer, the sections were mounted on gelatin-coated slides, dehydrated through graded ethyl alcohols, cleared in xylene, and coverslipped with Permount. To verify whether a reduction in TH cell count necessarily implies cell death, some TH sections were counterstained for Nissl (0.2% cresyl violet for 2 min, followed by dehydration with serial concentration of EtOH), and, for each TH section, an adjacent section was stained with cresyl violet.

For immunostaining, human brain tissue fixed with Formalin and then paraffin embedded sections (10 μm) were deparaffinized, treated with H₂O₂, blocked with 3% normal goat serum in TBS, and incubated with anti-p38 polyclonal antibody (1:50) overnight. Subsequently, the tissue was incubated with biotinylated secondary antibodies (anti-rabbit IgG for 1 h), and immunoreactions were visualized with the ABC complex (Vector Laboratories, Burlingame, CA) and DAB (Vector Laboratories).

For double immunofluorescence labeling with α-synuclein and p38, postmortem brain tissues from PD cases and controls were fixed in 4% paraformaldehyde overnight, cryoprotected, and frozen. Free-floating 40-μm-thick sections were blocked with 4% normal goat serum in TBS and incubated with anti-α-synuclein monoclonal antibody (1:5000) (Transduction Laboratories, Lexington, KY) and anti-p38 polyclonal antibody (1:500) overnight at 4°C. Then, the tissue was incubated with cyanine 3-conjugated goat anti-mouse IgG (red color) (Jackson ImmunoResearch, West Grove, PA) and Alexa Fluor goat anti-rabbit IgG (green color) for 4 h. Tissue sections were examined with a Zeiss (Oberkochen, Germany) laser confocal microscope.

Cell counting of SNpc. TH-immunopositive cells in the SNpc were counted as described previously (Yuan et al., 2005). In brief, every fourth section throughout the entire SNpc were scanned under light microscopy, and TH-immunoreactive cells were counted. A cell, when intact, round with clear nucleus, was considered. In the Nissl-stained sections, counting of the cells showing light blue cytoplasm and dark blue nucleus in the SNpc was performed on the adjacent one of the TH-stained section. The number of neurons was expressed as the average of the counts obtained from representative sections.

Statistical analysis. Densitometric analysis of protein bands was analyzed on an AlphaImager 2000 densitometer (Alpha Inotech, Wohlen, Switzerland). Data are expressed as mean ± SEM. The results were statistically evaluated for significance by applying the unpaired two-tailed test. Differences were considered significant when p < 0.05.

Results

Discussion

The major findings of the current study are that the aminoacyl-tRNA synthetase (ARS) cofactor p38/JTV-1 is an authentic parkin substrate and that overexpression of p38 in dopaminergic neurons leads to cell death. p38 accumulates in the ventral midbrain/hindbrain of parkin knock-out mice, AR-JP brains, and PD/DLBD brains with evidence of nitrosative stress. Moreover, we confirm and extend previous observations that parkin interacts with p38, leading to its ubiquitination and subsequent proteasomal degradation (Corti et al., 2003). The majority of p38 degradation seems to be mediated through the ubiquitin proteasome system and not through chaperone-mediated autophagy (CMA) because p38 does not contain a CMA recognition motif (Cuervo, 2004).

Parkin interacts with p38 through the RING finger 1 domain. The RING-IBR-RING domain binds to specific coenzymes, such as UbcH7, UbcH8, Hsp70, CHIP (Zhang et al., 2000; Imai et al., 2002), and substrates. Other substrates, such as CDCrel-1, synphilin-1, and Pael-R, bind the R2 RING finger domain (Zhang et al., 2000; Chung et al., 2001; Imai et al., 2001). Several studies show that the RING-IBR-RING domain is required for ubiquitin ligase activity, which suggests that binding of p38 to this region is important for the ability of parkin to ubiquitinate the protein. p38 contains two functional domains, such as leucine zipper domain at its N terminus, which is involved in macromolecular assembly of ARSs (Quevillon et al., 1999; Ahn et al., 2003) and a GST-homology domain at the C terminus. We did not observe an interaction with parkin in these functional domains; instead, mapping studies indicate that parkin mainly interacts with the 82–162 domain of p38. We also discovered that p38 may be capable of forming a protein complex with parkin, CHIP, and Hsp70. CHIP/Hsp70 appear to facilitate the function of parkin (Imai et al., 2002). However, we did not demonstrate the role of the complex of Hsp70, CHIP, and parkin in p38 turnover, so we assume that the Hsp70/CHIP chaperone system may play an important role in p38 biology, such as the regulation of ubiquitination, turnover, and cell death.

The wide variety of parkin mutations discovered in AR-JP patients is hypothesized to result in a loss of the ubiquitin ligase activity of parkin. Because wild-type parkin binds and ubiquitinates p38, targeting it for degradation to the proteasome, we studied the effect of two mutations in parkin, a missense point mutation (R42P) and a nonsense point mutation (Q311Stop), on their relative ability to bind, ubiquitinate, and degrade p38. Coimmunoprecipitation studies showed that both mutants are defective in binding p38; whereas the R42P mutant decreases the ability of parkin to bind p38, the Q311Stop mutant shows enhanced binding. In addition, the ubiquitination profile of p38 in the presence of these parkin mutants is also disrupted. The Q311Stop mutation abolishes the ability of parkin to ubiquitinate p38, whereas the R42P mutation increases the ubiquitin modification on p38 compared with wild-type parkin. Although these familial-associated mutations are observed to have different effects on the function of parkin, both mutants failed to enhance the degradation of p38. The increased accumulation of p38 with the Q311Stop mutant may be explained by its diminished ability to ubiquitinate p38. Furthermore, the observed enhanced interaction between p38 and the Q311Stop mutant suggests a role for the C-terminal portion of parkin in substrate binding and release. The R42P point mutation lies in the ubiquitin-like domain of parkin, which is predicted to interact with the proteasome (Sakata et al., 2003). Thus, this mutation may result in a disrupted targeting of ubiquitinated p38 to the proteasome, accounting for the accumulation of substrate with the R42P mutant. Thus, whereas the Q311Stop mutant may result in the accumulation of non-ubiquitinated p38, the R42P mutant, which retains its ubiquitin ligase activity, may result in the accumulation of ubiquitinated p38, both of which could be potentially toxic to the cell.

Mutations in parkin are thought to be the most common cause of inherited PD, with the frequency of the mutations estimated at 50% in families with AR-JP (Lucking et al., 2000). As noted above, parkin mutations are thought to result in loss of the ubiquitin E3 ligase activity of parkin. It has been proposed that the loss-of-function mutations of parkin might impair the removal of parkin substrates, leading to their accumulation followed by ubiquitin-proteasome system dysfunction-induced neurodegeneration (Kahle and Haass, 2004). This hypothesis has been questioned recently because parkin exon 3 knock-outs fail to accumulate substrate (Goldberg et al., 2003). However, in the current study, we find that p38 is upregulated in ventral midbrain/hindbrain extracts of both young- and old-aged parkin null mice, whereas none of the other reported parkin substrates are upregulated, including CDCrel-1, α-synuclein, synphilin-1, Pael-R, β-tubulin, cyclin E, and synaptotagmin XI. These results confirm and extend previous observations in parkin exon 3 null mice in which CDCrel-1, α-synuclein, and synphilin-1 were unaltered (Goldberg et al., 2003) and suggest that p38 may be an authentic parkin substrate. Consistent with the notion that p38 is an authentic parkin substrate, we found that p38 is upregulated in the brains of patients with parkin mutations. Similar to our observations in the exon 7 parkin knock-out mice, we failed to observe a significant upregulation of CDCrel-1, α-synuclein, synphilin-1, Pael-R, β-tubulin, cyclin E, and synaptotagmin XI. This contrasts with previous reports, in which other investigators have found an upregulation of O-linked glycosylated α-synuclein, cyclin E, and Pael-R (Imai et al., 2001; Shimura et al., 2001; Staropoli et al., 2003). Methodologic and/or regional brain differences could account for these disparities because we took special care to obtain age-matched and postmortem-matched control human brains for our comparison of substrate levels in AR-JP. Recent reports suggest a dual ubiquitin ligase function for parkin, lysine 48-linked proteasome-dependent and lysine 63-linked proteasome-independent ubiquitination of substrate (Doss-Pepe et al., 2005; Lim et al., 2005). One of the earliest reported substrates of parkin, synphilin-1, has been shown to be primarily ubiquitinated via K63-linkage, promoting the formation of Lewy body-like inclusions (Lim et al., 2005). This phenomenon provides a reasonable explanation for the lack of accumulation of synphilin-1 in parkin null mice compared with wild type in our study and previously published reports (Goldberg et al., 2003). Thus, other reported substrates that have unchanged steady-state levels in parkin null mice and AR-JP brains may indeed be ubiquitinated by parkin but via the K63-ubiquitin linkage and therefore not targeted to the proteasome for degradation. Conversely, significant accumulation of p38 in the parkin null mice and AR-JP brains, as well as in cell culture with proteasome inhibitors, suggests a K48-linked proteasome-dependent ubiquitination by parkin. Additional analyses of the linkage-specific ubiquitination mediated by parkin may clarify the role of the known substrates in the pathogenesis of parkin-associated PD.

Recently, we reported that S-nitrosylation-mediated inhibition of parkin activity could be a potential link between the genetic and sporadic forms of PD (Chung et al., 2004). In brains from patients with PD and DLBD, there is a dramatic increase in the total level of S-nitrosylated proteins, including parkin (Chung et al., 2004). In PD and DLBD brains with enhanced S-nitrosylation, p38 also accumulates, providing another piece of evidence that the function of parkin is compromised by S-nitrosylation in vivo, thus linking genetic and sporadic forms of PD through the S-nitrosylation of parkin and the accumulation of p38. We also showed that p38 accumulates in Lewy bodies of PD patients, confirming a previous observation (Corti et al., 2003). These results together with the upregulation of p38 in parkin exon 7 knock-out mice, AR-JP brains, and PD and DLBD brains with S-nitrosylated parkin and the accumulation of p38 in Lewy bodies argue in favor of p38 being an authentic parkin substrate.

There is growing evidence that overexpression of a variety of parkin substrates may exert cytotoxicity and that overexpression of parkin protects against substrate toxicity (Imai et al., 2001; Dong et al., 2003; Ren et al., 2003; Staropoli et al., 2003). Indeed, parkin is thought to be a multipurpose neuroprotectant (Feany and Pallanck, 2003). We also observed that p38 has a toxic effect in cell culture and that parkin protects against toxicity induced by p38 itself and the enhancement of TNFα toxicity by p38, whereas the familial-associated parkin mutant R42P fails to protects against p38 toxicity. Moreover, adenovirus-p38 overexpression in the SNc of mouse induced a significant loss of dopamine neurons.

The molecular mechanism by which p38 leads to cell death is not known. p38 is an important component of the multi-tRNA synthetase complex present in mammalian systems (Quevillon et al., 1999). p38-deficient mice revealed that this cofactor is a key scaffold for the assembly of the multi-tRNA synthetase complex (Kim et al., 2002). Because p38 is indispensable for the maintenance of the multi-ARS complex (Kim et al., 2002), the uncontrolled intracellular accumulation of p38 in parkin null mice and PD patients may alter the activity and the level of the multi-ARS complex and could lead to deleterious consequences, such as cellular dysfunction and ultimately cell death.