Parkin Cleaves Intracellular α-Synuclein Inclusions via the Activation of Calpain

Mutations in the α-synuclein and parkin genes cause heritable forms of Parkinson's disease. In the present study, we examined the possible functional relationship between the parkin and α-synuclein genes in a conditionally immortalized embryonic hippocampal cell (H19-7) line. Whereas transient transfection of α-synuclein into neuronal H19-7 cells caused the formation of its intracytoplasmic inclusions and a significant cell death, the combined overexpression of parkin restored the α-synuclein-induced decrease in cell viability to control levels. In addition, the overexpression of parkin was found to generate selective cleavage of α-synuclein. Furthermore, the cytoprotective effect of parkin on α-synuclein-induced cell death was not inhibited in the presence of a proteasome inhibitor. Interestingly, the overexpression of parkin induced the activation of an intracellular cysteine protease, calpain, but not caspase, and the cytoprotective effect of parkin on α-synuclein cytotoxicity was significantly inhibited by the presence of calpain-specific inhibitors. In conclusion, our results suggest that parkin accelerates the degradation of α-synuclein via the activation of the nonproteasomal protease, calpain, leading to the prevention of α-synuclein-induced cell death in embryonic hippocampal progenitor cells.


Introduction
Parkinsons disease (PD) 1 is a common neurodegenerative disorder involving the deterioration of dopaminergic neurons in the pars compacta region of the substantia nigra (1). PD patients suffer from rigidity, slowness of movement, tremor and lack of balance. The etiology of PD is poorly understood, but genetic factors are believed to play an important role in its pathophysiology (2,3). It has been reported that familial PD involves mutations in three different genes. Firstly, two missense mutations of the α-synuclein gene have been linked to familial PD (4). α-Synuclein is a major component of Lewy bodies (LBs), the pathological hallmark of PD and dementia with LBs (5).
Secondly, the ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) gene has also been linked to some cases of familial PD. A missense mutation in UCH-L1 was identified in two members of the same kin having early-onset autosomal dominant and typical PD (6). UCH-L1 appears to be involved in scavenging the ubiquitin peptide after the ubiquitin molecule has tagged the damaged molecules for proteasomal degradation.
Thirdly, parkin is the causative gene of early-onset autosomal recessive juvenile Parkinsonism (AR-JP) (7,8). Recently parkin has been reported to be an ubiquitinprotein ligase E3 (9,10). Various mutations of parkin have been reported in AR-JP patients, including exon deletion, exon multiplication and point mutation (11). Parkin consists of two functionally different domains; the C-terminal RING box, which recruits a specific E2 enzyme, and the N-terminal ubiquitin-like (Ubl) domain required for the recognition of the target protein, which is to be ubiquitinated before proteasomal degradation. Various mutations of parkin are believed to be involved in the impairment of the normal metabolism of target proteins by proteasome, and the accumulations of unknown protein may cause AR-JP.
While missense mutations in α-synuclein or UCH-L1 cause rare autosomal dominant forms of PD, mutations of parkin are a relatively common cause of AR-JP.
Proteolytic cleavage of α-synuclein by parkin and calpain As is the case in conventional idiopathic PD, the neuropathologic changes of parkinlinked AR-JP are largely confined to the brainstem and include the loss of selected neurons and local gliosis. However, α-synucleinand ubiquitin-positive neuronal inclusions, LBs, are generally absent in parkin-linked AR-JP (11)(12)(13).
Based on the mutation in UCH-L1 and parkin, it is suggested that the impaired proteasomal degradation of abnormal proteins seems to underlie the pathogenesis of PD.
In addition, the previous report that the mRNA expression patterns of parkin and αsynuclein are remarkably similar (14), suggested that these two proteins may be involved in common pathways, which contribute to the pathophysiology of PD. The substrate proteins for parkin remain largely unknown, with the exception of CDCrel-1 (15) and the Pael receptor (16). In PD, parkin is co-localized in some LBs (17) and all axonal spheroids (18), which contain aggregated α-synuclein. The association between parkin and α-synuclein in LB suggests that parkin interacts with α-synuclein, and this interaction may play a role in the pathogenesis of PD. On the basis of the above reports, the objective of the present study was to investigate the functional relationship between parkin and α-synuclein in immortalized hippocampal progenitor cells.

Measurement of calpain and caspase-3 activity using a fluorescent substrate peptide-
The activities of calpain and caspase-3 can be determined using the highly fluorescence

Results
Parkin selectively binds to α-synuclein in hippocampal progenitor H19-7 cells-The parkin gene has been reported to be expressed in a various stages of the CNS developmental process (21). In both the rat and the mouse, parkin begins to be with the Myc peptide was identified (Fig. 1A). However, we had previously observed that the endogenous protein level of α-synuclein is too low to be detected in H19-7 cell (22).
Secondly, in order to clarify the relationship between parkin and α-synuclein, we checked whether parkin binds to α-synuclein in a specific way in H19-7 cells. In a previous study, we observed that the exogenous addition of bacterially recombinant αsynuclein proteins leads to their intracellular transport, subsequently causing a significant necrotic-like cell death in the H19-7 cell (22). The neuronal cell death appeared to be correlated with the Rab5A-specific endocytosis of α-synuclein that subsequently caused the formation of Lewy body-like intracytoplasmic inclusions. This was further supported by the fact that the expression of GTPase-deficient Rab5A resulted in a significant decrease of its cytotoxicity as a result of incomplete endocytosis of α-synuclein (22). To determine whether exogenous α-synuclein interacts with parkin, 10 µM of α-synuclein was added to the cells for 1 hr, and then immunoprecipitation was performed using the anti-parkin antibody. Western blot analysis with anti-α-synuclein antibodies showed evidence of the expression of αsynuclein with an approximate 19-kDa molecular size in the immunoprecipitated protein complexes, indicating that intracellular parkin binds to α-synuclein (Fig. 1B).
In order to determine whether intracellular-transfected α-synuclein also binds to parkin, two mammalian expression vectors encoding α-synuclein and Myc-tagged parkin (pcDNA3.1-Myc/His), respectively, was co-transfected into the H19-7 cells in a transient manner. After 48 hr, immunoprecipitation was performed with an anti-Myc antibody, and the precipitates were analyzed by Western blot analysis using the anti-αsynuclein antibody. As shown in Fig. 1C, parkin selectively binds to α-synuclein.
These findings suggest that parkin is functionally linked to α-synuclein in the embryonic hippocampal cell lines.
Proteolytic cleavage of α-synuclein by parkin and calpain Parkin binds to α-synuclein through its Ubl domain-To explore the domain in parkin that is associated with its interaction with α-synuclein, binding assays were performed using several deletion mutants of parkin ( Fig. 2A). Plasmids, encoding a series of deletion parkin mutants with Myc-tag, were co-transfected into H19-7 cells using a plasmid to express wild type α-synuclein, followed by immunoprecipitation with antiα-synuclein IgG. Apart form the whole complete protein, only the peptide, with its Nterminal portion of parkin remaining intact, still retained the binding activity to αsynuclein, whereas the peptide with its C-terminal part intact did not retain this binding activity ( Interestingly, when α-synuclein was co-expressed with parkin, simultaneously, parkin significantly suppressed α-synuclein-induced cell death (Fig. 3). Meanwhile, the overexpression of parkin alone has not any remarkable effect on cell viability. These data indicated that the overexpression of α-synuclein leads to neuronal cell death, while parkin selectively blocks the cytotoxicity of α-synuclein. Based on the previous findings that α-synuclein is degraded by 26S-proteasome (22), and parkin acts as ubiquitin-protein ligase E3 (9, 10, 23), the protective effect of parkin on α-synucleins toxicity could be exerted by means of the removal of α-synuclein, by parkin-mediated ubiquitination of α-synuclein and subsequent proteasome-mediated degradation.
Proteolytic cleavage of α-synuclein by parkin and calpain Previously we have shown that the H19-7 cells were not found to contain any significant levels of α-synuclein (22), which is consistent with previous report in which α-synuclein only begins to be expressed after the post-natal period in the rat central nervous system (24). To ascertain whether parkin controls the degradation of These data suggested that the cytoprotective effect of parkin on α-synuclein may be mediated by the activation of some other proteasome-independent proteolytic machinery.
In addition, the effect of parkin on the ubiquitin-proteasome-dependent proteolytic pathways was assessed by using the poly-ubiquitinated green fluorescent protein (GFP)-based reporter system as a proteasomal target protein (26). with Ub G76V GFP plus parkin, the mean fluorescence intensity of the cells significantly decreased ( Fig. 6a and b). As described above, it is presumed that parkin activates the ubiquitination process of ubiquitin-GFP fusion proteins as an E3 protein ligase, or that it affects the proteolytic enzyme activity of either proteasome or other protease(s). The possibility as to whether parkin induces the activation of the proteolytic proteasomal machinery was tested in the presence of proteasome inhibitor. Compared with control cells transfected parkin alone, the addition of Clasto-Lactacystin β-lactone did not block the parkin-induced degradation of ubiquitin-GFP, and a significantly reduced level of Ub G76V GFP's fluorescence was observed ( Fig. 6c and d). This result shows that parkin appears to stimulate a novel proteasome-independent degradation process of the ubiquitinated-GFP fusion protein.
To assess the presence of ubiquitinated-GFP fusion protein, Western blot analysis was performed using anti-GFP antibodies. As shown in Figure 6C, most of Ub-X-GFP proteins were greatly ubiquitinated, and only small amounts of cleaved GFP protein were expressed as a result of de-ubiquitination process. In consistent with Fig 6B,  Western blot analysis with anti-PARP antibody. When intracellular calpain activity was suppressed by calpeptin, the cleavage of intact 116 kDa-PARP into its 32 kDafragment was significantly suppressed (Fig. 7). This finding suggested that the parkininduced novel fragmentation of PARP occurred via the activation of calpain, but not that of caspase. In support of this conclusion, when cells were pretreated with a typical, Proteolytic cleavage of α-synuclein by parkin and calpain cell-permeable inhibitor of caspase, zVAD, the generation of the parkin-mediated novel PARP fragment was greatly enhanced in H19-7 cells, whereas stausporineinduced PARP cleavage was completely blocked by zVAD, but not by calpeptin (Fig.   7).
To Overall, our data suggest that parkin activates the proteasome-independent cysteine protease, calpain, but not caspase, in neuronal H19-7 cells.

Parkin induce the degradation α-synuclein via the activation of calpain in H19-7 cells-
In previous studies, we and Eberz et al. reported that calpain degrades α-synuclein in vitro (30,31). Next, we evaluated whether parkin-induced calpain activation is involved in the degradation of α-synuclein in vivo. The H19-7 cells were transfected with a plasmid encoding parkin or/and α-synuclein, respectively. Intracellular levels of α-synuclein and its degradation were measured by Western blot analysis using anti-αsynuclein IgG. As shown in Fig. 9, compared to that with α-synuclein alone, co-  (Fig. 10A) or calpastatin peptide (Fig. 10B). As a control, the addition of each calpain inhibitor alone did not produce any significant effect on cell viability (Fig. 10). These results clearly show that parkin potentiates the degradation of α-synuclein, via the activation of calpain, which sequentially rescues the neuronal hippocampal cells from a α-synuclein-induced neuronal cell death.

Discussion
The presynaptic protein, α-synuclein, is a major component of heavily- proteases, but not by caspase-3, during β-lapachone-mediated apoptosis (27,37). In accordance with the previous findings, degenerated dopaminergic neurons located in the substantia nigra area of PD patients are particularly sensitive to oxidative stresses, and are prone to exhibit an elevated internal Ca 2+ concentration (38). Calpains role in the degeneration process was suggested after increased expression levels of m-calpain were found in the mesencephalon region of PD patient (39). Furthermore, we and Eberz et al.
When we determined that parkin might lead to the degradation of α-synuclein via the activation of intracellular calpain in vivo, we detected that the production of the forms had no effect (45). In an independent study, when two proteins, not known to be involved in disease, were incubated under conditions where they formed amyloid fibrils, only the pre-fibrillar intermediates, and not the mature fibrils, caused cytotoxicity (46).