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Human Molecular Genetics, 2003, Vol. 12, No. 20 2625-2635
DOI: 10.1093/hmg/ddg283
© 2003 Oxford University Press

Alpha-synuclein degradation by serine protease neurosin: implication for pathogenesis of synucleinopathies

Atsushi Iwata1,2, Mieko Maruyama1, Takumi Akagi3, Tsutomu Hashikawa3, Ichiro Kanazawa4, Shoji Tsuji2 and Nobuyuki Nukina1,*

1Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, 2-1 Hirosawa Wako-shi, Saitama 351-0198, Japan, 2Department of Neurology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo Bunkyo-ku, Tokyo 113-8655, Japan, 3Laboratory for Neural Architecture, RIKEN Brain Science Institute, 2-1 Hirosawa Wako-shi, Saitama 351-0198, Japan and 4National Center for Psychiatry and Neurology, 4-1-1 Ogawahigashi-cho Kodaira-shi, Tokyo 187-8551, Japan

Received June 2, 2003; Revised July 31, 2003; Accepted August 13, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Accumulation of insoluble alpha-synuclein aggregates in the brain is characteristic of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Although numerous studies on the aggregation properties of alpha-synuclein have been reported, little is known about its degradation so far. In view of proteolytic degradation, we have found that the serine protease neurosin (kallikrein-6) degrades alpha-synuclein and co-localizes with pathological inclusions such as Lewy bodies and glial cytoplasmic inclusions. In vitro study showed that neurosin prevented alpha-synuclein polymerization by reducing the amount of monomer and also by generating fragmented alpha-synucleins that themselves inhibited the polymerization. Upon cellular stress, neurosin was released from mitochondria to the cytosol, which resulted in the increase of degraded alpha-synuclein species. Down-regulation of neurosin caused accumulation of alpha-synuclein within cultured cells. Thus we concluded that neurosin plays a significant role in physiological alpha-synuclein degradation and also in the pathogenesis of synucleinopathies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Protein aggregation and filament formation are the hallmarks of many neurodegenerative diseases. The formation of these disease-specific amyloid structures is thought to play an important role in the pathogenesis of these diseases (1–3). Whether or not cell death is a direct effect of the accumulation of the aggregated proteins is still controversial; however, most studies suggest that aggregated proteins can affect cell viability through several pathways (4,5). For example, recent studies have shown that aggregation of polyglutamine-containing proteins or alpha-synuclein causes proteasome dysfunction, and this might lead to neurodegeneration (6–8).

Accumulation of insoluble alpha-synuclein in the brain is observed in Parkinson's disease (PD), Dementia with Lewy bodies and multiple system atrophy (MSA) (9–13). These synucleinopathies show distinct clinical symptoms from each other; however, they appear to share a common cell death mechanism related to alpha-synuclein aggregation. Alpha-synuclein is a small acidic synaptic protein composed of 140 amino acid residues, including seven incomplete repeats of 11 amino acids and a core sequence of KTKEGV at the amino terminus (14). In vitro generated alpha-synuclein, particularly the mutant A53T protein, forms aggregates under certain conditions (15–17). In vivo studies have shown that mice and flies generated to over-express alpha-synuclein within their neurons develop neurological disorders that mimic PD (18–21).

Although much has been learned about in vitro protein aggregate formation, the mechanism of in vivo alpha-synuclein aggregate formation is still unclear, because simple over-expression of alpha-synuclein rarely forms aggregate in cultured cells. It can be speculated that, unlike other proteins prone to aggregate, such as long polyglutamine stretch-containing proteins, efficient degradation of alpha-synuclein prevents its aggregation under normal conditions. Alternatively, post-translational modification of alpha-synuclein might be needed for its aggregation. For instance, it has been reported that insoluble alpha-synuclein is nitrated or phosphorylated, and these modifications accelerate the accumulation of aggregates in vitro (22,23).

The level of alpha-synuclein messenger RNA is not altered in synucleinopathies (24,25), suggesting that, rather than increased protein expression, protein modification or impairment of its degradation might be related to the pathogenesis of the disease. So far, very little has been known about the degradation of alpha-synuclein. Some studies have suggested that there might be a degradation pathway for alpha-synuclein other than the ubiquitin proteasome pathway (26,27). To shed light on this issue, we searched for the protease that degrades alpha-synuclein and found that alpha-synuclein is degraded by the serine protease neurosin in vitro and in vivo, and neurosin also accumulates in alpha-synuclein inclusions in diseased brain.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fragmented alpha-synucleins are found in mouse brain
While analyzing the expression of alpha-synuclein in normal mouse brain, we found that there were very small but significant amounts of anti-alpha-synuclein antibody reactive low molecular weight bands in immunoblots (Fig. 1A). We confirmed the presence of these bands using a panel of antibodies and also made sure that these bands were not artifacts using various sets of protease inhibitors.



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  		Figure 1. (A) Fragmented alpha-synucleins are present in mouse brains. C57/BL6 mouse brain lysates were run on SDS–PAGE, transferred to a PVDF membrane and immunoblotted with various anti-alpha-synuclein antibodies. Every antibody recognized fragmented alpha-synucleins (arrow). A long period of exposure was required to detect fragmented alpha-synucleins, because they were not abundant. S1 (raised against recombinant human alpha-synuclein), EQV-1 (raised against 61–75 amino acids of human alpha-synuclein), N-19 (raised against 1–19 amino acids of human alpha-synuclein) and MC-36 (raised against 116–130 amino acids of human alpha-synuclein). Arrowhead, full length alpha-synuclein; arrow, fragmented alpha-synucleins. (B) Serine protease specific inhibitors inhibit alpha-synuclein degradation. Recombinant alpha-synuclein was incubated with HEK-293 cell lysate at 37°C for 2 h. Various protease inhibitors were added to the reaction mixture. Five micrograms per milliliter aprotinin, 2 mM PMSF, 5 µg/ml leupeptin, 5 mM AEBSF and Complete (Roche) inhibited the degradation, while 5 mM EDTA, 5 µg/ml pepstatin A and 5 mM EGTA had no effect. (C) Kallikrein degrades alpha-synuclein. Kallikrein inhibitor inhibited the protease activity of HEK-293 cell lysate in a dose-dependent manner. Purified kallikrein and the cell lysate digested recombinant alpha-synuclein similarly. (D) Peptide antibodies against human neurosin specifically recognized transfected neurosin. Two anti-peptide antibodies were generated against sequences of human neurosin and their specificity was evaluated. Lysates from SH-SY5Y cells transfected with plasmid encoding human neurosin cDNA were immunoblotted with NS-5105 and NS-5106 antibodies, both antibodies showed high activity and specificity against human neurosin (-, mock vector transfection, +, neurosin cDNA transfection). The protein of the same molecular weight was also recognized by the antibodies once endogenous neurosin was enriched as shown in the following experiment. (E–G) Neurosin exists in alpha-synuclein inclusions in synucleinopathy brain. Postmortem brain sections from (E) sporadic Parkinson's disease, (F) Neurodegeneration with brain iron accumulation type 1 (type1 NBIA) and (G) multiple system atrophy were double stained using anti-alpha-synuclein monoclonal antibody LB-509 (Alexa 488: green) and anti-neurosin NS-5106 antibody (Alexa 546: red). Note that the core of the Lewy body is preferably stained with anti-neurosin, while the peripheral rim is preferably stained with anti-alpha-synuclein antibody. Glial cytoplasmic inclusions of MSA were stained homogeneously with NS-5106 as well as LB-509. Bar=20 µm.

 
Neurosin degrades alpha-synuclein
We hypothesized that the low molecular weight bands were degraded alpha-synuclein and started exploring the biological significance of these species. According to a previous report, a serine protease neurosin (kallikrein-6) immunoreactivity was present in Lewy bodies (28). To determine whether kallikreins can specifically degrade alpha-synuclein, recombinant alpha-synuclein was incubated with HEK-293 cell lysates together with various protease inhibitors. Serine protease inhibitors such as aprotinin, phenylmethane sulfonyl fluoride (PMSF), leupeptin and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) had an inhibitory effect on alpha-synuclein degradation activity, while ethylenediaminetetraacetic acid (EDTA), pepstatinA and ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) had no effect (Fig. 1B), suggesting that the protease was a serine protease. Furthermore, when kallikrein inhibitor was added to the reaction, it inhibited the degradation activity dose-dependently, and also purified kallikrein itself could degrade alpha-synuclein (Fig. 1C). From these results, we hypothesized that neurosin (kallikrein-6) is an alpha-synuclein degrading enzyme.

Neurosin is present in Lewy bodies and glial cytoplasmic inclusions
For further experiments, antibodies were generated against human neurosin (Fig. 1D). To see if neurosin was involved in synucleinopathies, we stained postmortem brain sections with the antibodies. Anti-neurosin antibody clearly stained both Lewy bodies in PD, neurodegeneration with brain iron accumulation type 1 (type1 NBIA) and glial cytoplasmic inclusions in multiple system atrophy (MSA; Fig. 1E–G). Interestingly, anti-neurosin antibody stained the Lewy bodies preferably at the core of the inclusion, while staining by alpha-synuclein antibody was mainly at the peripheral part of the structure. In contrast, both antibodies stained glial cytoplasmic inclusions homogeneously. Thus, we supposed that neurosin is involved in the development of synucleinopathies through the alpha-synuclein degradation process.

Alpha-synuclein fragments produced by neurosin inhibit alpha-synuclein polymerization
To investigate the role of neurosin in alpha-synuclein degradation, we performed in vitro alpha-synuclein polymerization experiment using recombinant proteins. When incubated at 37°C at a concentration of 10 µg/ml, purified alpha-synuclein monomer started to form SDS-resistant polymers from the time point of 8 h (Fig. 2A). Forty-eight hours was enough to generate 95% of the dimers produced by 96 h, so that for further experiments a time point of 48 h was selected to evaluate the polymerization.



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  		Figure 2. (A) Recombinant alpha-synuclein forms SDS-resistant polymers by in vitro incubation. Recombinant monomer alpha-synuclein was incubated at 37°C for the indicated intervals. SDS-resistant alpha-synuclein dimer appeared at the 8th h, and the level of polymers increased with time. The average band intensity of dimer alpha-synuclein from three independent experiments is shown at the bottom (dimer intensity at 96 h is 1.0). Arrow, dimer; arrowhead, monomer alpha-synuclein. (B) Recombinant alpha-synuclein polymerization is inhibited by neurosin. Recombinant monomer alpha-synuclein was incubated with the indicated amount of recombinant neurosin. Polymerization of alpha-synuclein was inhibited dose dependently. The average band intensity of dimer alpha-synuclein from three independent experiments is shown at the bottom (without neurosin=1.0). *P<0.001 versus without neurosin; **P<0.05 versus without neurosin. Arrow, dimer; arrowhead, monomer alpha-synuclein. (C) Neurosin protease activity is inactivated by heat treatment. Recombinant alpha-synuclein was incubated with neurosin for 1 h and heated at 95°C or kept at 4°C for 5 min. Then, the fragmented alpha-synucleins were added to V5 tagged recombinant alpha-synuclein for 8 h and then blotted with anti-V5 antibody. Heat-treated fragments have no residual proteolytic activity on V5 tagged alpha-synuclein (heat +), while fragments without heat treatment still has residual activity (heat -). MC-36 blot is shown to detect all the synuclein immunoreactive species in the mixture which shows increase in MC-36 immunoreactive low molecular weight band by remaining proteolytic activity. (D) Incubation of monomer alpha-synuclein and predigested alpha-synuclein. The indicated amounts of monomer and predigested alpha-synuclein were incubated. The total amount of alpha-synuclein including the fragments was adjusted to 200 ng. Polymerization of alpha-synuclein was inhibited by addition of predigested alpha-synuclein. The average band intensity of dimer alpha-synuclein from three independent experiments is shown at the bottom (dimer intensity without fragments=1.0); *P<0.001 versus no fragments; **P<0.05 versus no fragments. Arrow, dimer; arrowhead, monomer alpha-synuclein. The right panel shows monomer and fragmented alpha-synuclein without incubation. (E) Fragmented alpha-synuclein reduces polymerization. Two hundred nanograms of monomer alpha-synuclein were incubated with the indicated amount of predigested alpha-synuclein. Addition of predigested alpha-synuclein inhibited the polymerization of alpha-synuclein. The average band intensity of dimer alpha-synuclein from three independent experiments is shown at the bottom (dimer intensity without fragments=1.0), *P<0.001 versus without fragments. Arrow, dimer; arrowhead, monomer alpha-synuclein. (F) Electron microscopic view of the effect of neurosin on alpha-synuclein polymerization. Monomer alpha-synuclein was incubated with or without recombinant neurosin for 48 h. Left panel shows polymerized alpha-synuclein forming ‘sphere’-like structure (arrow), while neurosin-digested synuclein did not form these structures (right). Scale bar=100 nm. (G) Neurosin has less effect on degrading A53T mutant alpha-synuclein. Monomer wild-type, A30P and A53T mutants of alpha-synuclein, and beta-synuclein were incubated with neurosin. At low level of neurosin, A53T mutant was less digested compared with wild-type alpha-synuclein. The relative intensity of monomer alpha-synuclein is shown at the bottom of the figure (1.0=without neurosin for each different synucleins). The blots were done by MC-37 antibody which recognizes carboxyl terminus of alpha- and beta-synuclein equally. Arrow, fragmented; arrowhead, full-length synuclein. *P<0.001 vs monomer intensity without eurosin.

 
To investigate the effect of neurosin in the polymerization process, recombinant neurosin was added to the alpha-synuclein monomer solution, and the mixture was incubated at 37°C for 48 h. Neurosin dose-dependently inhibited the polymerization of alpha-synuclein (Fig. 2B). To determine whether the inhibition effect is due to the presence of proteolytic fragments, or due to a reduction in the level of monomer, we mixed full-length alpha-synuclein with various concentrations of fragmented alpha-synuclein and incubated at 37°C for 48 h. For these experiments, we generated fragmented alpha-synuclein without protease activity by heat treatment of neurosin-digested alpha-synuclein. Heat treatment could completely abolish the residual proteolytic activity (Fig. 2C). Addition of those fragmented alpha-synucleins inhibited the polymerization, both when fragmented alpha-synucleins were added to achieve a total alpha-synuclein amount of 200 ng (both monomer and fragments; Fig. 2D) and when they were added to 200 ng of monomeric alpha-synuclein (Fig. 2E). Thus we concluded that neurosin has an inhibitory effect on alpha-synuclein polymerization from reducing the monomer substrate by proteolytic degradation as well as producing fragmented alpha-synucleins which themselves have an inhibitory effect on polymerization.

Electron microscopic studies were done to evaluate the effect further. Polymerized alpha-synuclein formed ‘sphere’-like structures (Fig. 2F, left panel), but those structures were rarely seen when neurosin was added to the polymerization mixture (Fig. 2F, right panel).

The ability of neurosin to cleave various synucleins was tested. Low concentrations of neurosin digested A53T mutant synuclein less efficiently than the wild-type alpha-synuclein (Fig. 2G). This result led us to the conclusion that neurosin has less proteolytic activity on pathological mutant of alpha-synuclein, suggesting that the impairment of neurosin degradation pathway might be involved in the disease process.

Neurosin is released from mitochondria to cytosol during cellular stress and regulates alpha-synuclein degradation
To study sub-cellular localization of neurosin, immunohistochemical study on HEK-293 cells was performed and revealed that part of the cytoplasmic anti-neurosin staining was co-localized with cytochrome C staining (Fig. 3A). In sub-cellular fractionation experiments, neurosin was found in the mitochondrial and microsomal fractions but not in the nuclear or cytosolic fractions (Fig. 3B), suggesting that nuclear staining in Figure 3A may be non-specific.



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  		Figure 3. (A) Mitochondria have neurosin immunoreactivity. HEK-293 cells were stained with anti-neurosin and anti-cytochrome C antibodies. Cytoplasmic neurosin immunoreactivity co-localized with cytochrome c immunoreactivity (Alexa 546, red; arrows). Nuclear staining is non-specific by the neurosin antibody NS-5106 (Alexa 488, green; arrowhead), which is suggested from the next blot showing no neurosin band in the nuclear fraction. (B) Sub-cellular fractionation of HEK-293 cells. HEK-293 cell lysate was fractionated using differential centrifugation into nuclear, microsomes, mitochondrial and cytosolic fractions and blotted with anti-neurosin NS-5105 antibody. The microsomal and mitochondrial fractions were rich in neurosin, while the nuclear and cytosolic fractions contained very low levels of neurosin. Arrow, neurosin. (C) Stress induced cytosolic release of neurosin. After exposure of SH-SY5Y cells to various stresses, cytosolic fractions were obtained. At 6 h after exposure to stress, neurosin (blotted with NS-5105, arrow) was present in the cytosol. Ultraviolet stimulation had most prominent effect on neurosin release. The anti-actin blot shows equivalency in the amount of protein loaded in each lane. UV40, UV 40 J/m2; UV1000, UV 1000 J/m2; H2O2, 600 µM hydrogen peroxide; SNAP, 25 mM S-nitroso-N-acetylpenicillamine; 2ME, 3 mM 2-mercaptoethanol; tunicamycin, 10 mg/ml tunicamycin; MMS, 100 µg/ml methyl methanesulfonate; sorbitol, 300 mM sorbitol. (D) Neurosin is released from mitochondria by UV irradiation. SH-SY5Y cells were stimulated by 40 J/m2 ultraviolet light. Before stimulation (upper panels) neurosin was localized at mitochondria and co-localized with cytochrome c. Arrows in the upper panels show colocalization of neurosin (NS-5106) and cytochrome c at mitochondria. Arrows in the lower panels show that cytochrome c was released from mitochondria after the stimulation, and neurosin was no longer localized to mitochondria. Bar=20 µm. (E) Time course of neurosin release. SH-SY5Y cells were exposed to 40 J/m2 ultraviolet light and cells were collected at indicated intervals and cytosolic fractions were immunoblotted by anti-neurosin NS-5106 or anti-synuclein S1 antibodies. Release of neurosin into cytosol was observed at 4 h post-stimulation and increased with time; alpha-synuclein fragmentation (arrowhead) was also increased (arrow) in accordance with the cytosolic release of neurosin. (F) Down-regulation of neurosin causes up regulation of alpha-synuclein. Small interference RNA corresponding to 88–107 bp of the open reading frame of neurosin was transfected into SH-SY5Y cells. Cell lysates were collected 2 and 4 days after transfection. RNA interference down-regulated neurosin (NS-5106, arrow), and the amount of alpha-synuclein (S1) was increased (arrowhead). Control (randomized) siRNA did not cause down-regulation of the protein. GAPDH blot shows equal amount of the proteins loaded. *P<0.01 vs 0 day.

 
The physiological role of neurosin was investigated in cells under various stresses. In agreement with previous reports (29–31), the amount of cytosolic neurosin was very low under normal condition. However, levels of cytosolic neurosin were increased under stress. Stimulation of cells with ultra violet (UV) light most effectively released neurosin from the mitochondria to the cytosol (Fig. 3C). The release was confirmed by immunohistochemistry (Fig. 3D).

To examine this phenomenon more closely, cells were exposed to UV light and collected 0, 4 and 8 h later. Increase in the levels of fragmented alpha-synuclein was shown to accompany the release of neurosin into the cytosolic fraction (Fig. 3E).

To see whether neurosin regulates alpha-synuclein degradation, RNA interference was performed in order to reduce cellular neurosin levels. Down-regulation of neurosin in SH-SY5Y cells by siRNA led to an increase in the expression levels of intact alpha-synuclein, while control siRNA had no effect on neurosin or alpha-synuclein levels (Fig. 3F). The experiment confirmed the role of neurosin in alpha-synuclein degradation.

Neurosin and alpha-synuclein co-localize in vivo
To investigate whether neurosin could interact with alpha-synuclein under physiological conditions, we immunoprecipitated these proteins from mouse brain lysate. Anti-alpha-synuclein antibody successfully immunoprecipitated neurosin and, conversely, anti-neurosin antibody immunoprecipitated alpha-synuclein (Fig. 4A). Furthermore, alpha-synuclein rich fractions were prepared from mouse brain using a sucrose gradient method (32,33) and tested for the co-existence of neurosin in these fractions. Neurosin was demonstrated to exist in these fractions (Fig. 4B). To confirm the co-localization in situ, mouse brain striatum was stained with the antibodies to alpha-synuclein and neurosin to show that these two proteins co-localized (Fig. 4C). The result suggests that both alpha-synuclein and neurosin co-localize at axon terminus. Thus we concluded that these proteins could interact in the normal brain.



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  		Figure 4. (A) Neurosin and alpha-synuclein co-immunoprecipitated from mouse brain. Mice brain lysate was immunoprecipitated with normal rabbit serum, S1 anti-alpha-synuclein antibody, and NS-5105, NS-5106 anti-neurosin antibodies. Alpha-synuclein and neurosin co-immunoprecipitated. Anti-neurosin antibodies seem to be less efficient for immunoprecipitation compared to S1 antibodies, however, neurosin to alpha-synuclein ratio among three immunoprecipitation seems to be the same. Arrow, neurosin; arrowhead, alpha-synuclein. (B) Neurosin exists in the alpha-synuclein rich fraction from mouse brain. Alpha-synuclein rich fractions were purified from mice brain by differential centrifugation followed by sucrose density gradient fractionation. Light and heavy indicates the differentiated fractions. Immunoblotting showed the co-existence of synucleins, neurosin and in the fraction. Arrow, neurosin; arrowhead, alpha-synuclein and beta-synuclein. (C) Neurosin and alpha-synuclein immunoreactivity co-localize in mouse brain. Mouse brain striatum was stained with N-19 antibody (Alexa 488: green) and NS-5106 antibody (Alexa 546, red). Synuclein and neurosin co-localized at the axon terminus. Arrows indicate co-localization of the two proteins. Bar=20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Neurosin degrades alpha-synuclein
Alpha-synuclein is a very abundant protein in the central nervous system; however, its physiological role is still ambiguous. Several studies have suggested that, when alpha-synuclein is present in the cytoplasm, it acts as a regulator of signal transduction (34–36), and when bound to membrane at the synaptic terminus, it regulates synaptic vesicle release (37,38). These studies have provided significant information about the protein; however, very little is known about its production and degradation (26,27).

The presence of low molecular weight bands reactive with anti-alpha-synuclein in mouse brain lysates gave us a hint for the alpha-synuclein degradation process. As we investigated the significance of fragmented alpha-synuclein, we performed a literature search and found that an immunohistochemical study had reported protease neurosin (kallikrein-6) immuno-reactivity in Lewy bodies (28). Neurosin is a serine protease preferentially expressed in brain, especially in neurons and oligodendrocytes (29,30,39–41). Serine proteases are known to recognize a lysine residue. Interestingly, alpha-synuclein is a lysine-rich protein, characterized by repeats of the sequence KTKEGV. An inhibition study of alpha-synuclein degrading activity from HEK-293 cells by panels of protease inhibitors clearly showed that the activity is inhibited by serine protease inhibitors. Kallikrein inhibitor also showed dose-dependent inhibition of this degradation, suggesting that these fragments were generated from synuclein by protease of the kallikrein family, especially neurosin. Moreover, we tried to prepare this degrading activity-enriched fraction from the cell lysates and found neurosin in the final concentrated fraction (data not shown).

These findings encouraged us to generate anti-neurosin antibodies, and we found that these antibodies stained alpha-synuclein inclusions in Lewy bodies in PD and neurodegeneration with brain iron accumulation type1, as well as glial cytoplasmic inclusions in MSA, suggesting that the protease might have a significant role in the synucleinopathies. Interestingly, in Lewy bodies, neurosin immunoreactivity seemed to be located at the core of the inclusion, suggesting that neurosin might be involved early in the disease process.

Neurosin has a signal peptide which is thought to be cleaved upon secretion from cells; however, immunohistochemical studies have revealed that a significant amount of neurosin exists in cell bodies (29). Our results also showed that neurosin was present in mitochondria and microsomes in sub-cellular fractionation and immunohistochemically in mitochondria. The neurosin-enriched fraction from the mitochondrial and microsomal fraction also contained alpha-synuclein degrading activity (data not shown), suggesting that neurosin is a major degrading enzyme for alpha-synuclein.

If neurosin is abundant inside mitochondria, while the cytosolic fraction is very low, it seems that the chance for the protease to degrade alpha-synuclein might be low. However, an RNA interference study showed that down-regulation of neurosin increased alpha-synuclein, suggesting that alpha-synuclein is constitutively degraded by neurosin. Moreover, when cells are under a stressed condition, as shown in Figure 3C and D, neurosin is released from mitochondria and accelerates the degradation process. In mouse brain, neurosin exists in the fractions which are rich in synuclein, and the immunostainings of anti-alpha-synuclein and anti-neurosin co-localized. An immunoprecipitation study of brain lysate by anti-synuclein which precipitated neurosin further suggests that alpha-synuclein and neurosin interact directly or both molecules associate with same membrane, and synuclein could interact with neurosin as a substrate in vivo. In the diseased brains of synucleinopathy, neurosin co-localized with alpha-synuclein aggregates. This result also suggests that a very small amount of neurosin could interact with alpha-synuclein in the cytoplasm constitutively and also after release from mitochondria under stress conditions.

Fragmented alpha-synuclein inhibits polymerization of alpha-synuclein
As reported previously, recombinant alpha-synuclein forms polymers upon in vitro incubation (15). Our recombinant protein also formed dimers after 8 h of incubation, and the level of oligomers increased with time. During this polymerization reaction we added neurosin and found a dose-dependent inhibition of the formation of alpha-synuclein polymers. The addition of neurosin was not essential for inhibition of dimer formation; the fragmented alpha-synuclein itself had the effect of inhibiting polymerization. Electron microscopy showed that ‘spheres’ (42) were not formed by neurosin-treated alpha-synuclein, indicating that fragmented alpha-synuclein inhibited fibril formation in its early phase.

The inhibitory role of neurosin for alpha-synuclein aggregation formation might appear to be contradictory to the co-localization with synuclein aggregates. There are two possible explanations for this. One is that the in vitro experiment showed inhibition only when excessive fragments were added to the mixture and the amount of fragmented alpha-synuclein in vivo is not enough to prevent the entire fibrillization process. Another possibility is that the entire polymerization process prevails over the inhibitory processes of various cellular efforts to prevent alpha-synuclein aggregation and neurosin only associated with the preformed aggregates as substrates.

A protective role of neurosin against aggregate formation is interesting because another mitochondrial protein, cytochrome c, was reported to be stimulator of alpha-synuclein aggregation and localizes in Lewy bodies in Parkinson's disease (43). It is possible that in Lewy body-bearing disorders, chronic apoptotic stressors release both protective (neurosin) and accelerating (cytochrome c) factors from mitochondria and both accumulate in Lewy bodies. In this regard, neurosin, located on chromosome 19 (39), may be a candidate gene for risk factors of synucleinopathy.

In summary, we have identified neurosin as an alpha-synuclein degrading enzyme. It exists within mitochondria, is released after cellular stress, and co-localizes with synuclein inclusions in synucleinopathy. Thus we concluded that neurosin may play a significant role in the pathogenesis of synucleinopathy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
cDNAs
Neurosin cDNA was cloned from a human cDNA library using PCR by primers 5'-gcaggagcggccatgaagaag-3' and 5'-cagtggggtggtaggtcggga-3'. Complimentary DNAs for alpha-synuclein and beta-synuclein were obtained as previously described (34). Complementary DNA transfection was done using the calcium phosphate method.

Antibodies
Anti-alpha-synuclein antibodies were raised against peptide sequences of human alpha-synuclein MPVDPDNEAYEMPSE (MC-36) and EGYQDYEPEA (MC-37). Rabbit antibodies against neurosin were also generated by immunizing rabbits with synthetic peptide of human neurosin sequences and affinity-purified peptides. The sequences of the peptides used were LVHGGPCDKTSHPYQAA (NS-5105) and LGKHNLRQRESSQEQSS (NS-5106). To determine the specificity of the antibodies, SH-SY5Y cells were transfected with pcDNA4 vector (Invitrogen, Carlsbad, CA, USA) inserted with neurosin cDNA and the lysate was subjected to immunoblotting. S1 antibodies were generated as previously described (35). Rabbit antibody against the NAC portion of alpha-synuclein EQV-1 was a kind gift from Dr Ueda (44). LB-509 monoclonal antibody was a kind gift from Dr Iwatsubo (10). Antibodies anti-alpha-synuclein N-19 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-cytochrome C (Stressgen, San Diego, CA, USA), anti-V5 (Invitrogen), anti-actin (Chemicon International, Temecula, CA, USA) and anti-GAPDH (Chemicon), Alexa (488, 546) secondary antibodies (Molecular Probes, Eugene, OR, USA) were obtained from commercial sources. Immunohistochemistry and immunoblotting were carried out as previously described (35).

Preparation of mouse brain lysates
C57/BL6 mice were sacrificed and brains were obtained immediately. The brains were homogenized by a teflon homogenizer with 10 strokes in 3 vol/weight ice-cold PBS containing 1% NP-40, 2 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatinA, 5 µg/ml aprotinin and Complete (Roche, Mannheim, Germany) and spun at 20 000g for 10 min at a temperature of less than 4°C to obtain the lysate. The lysate was mixed with SDS–PAGE sample buffer immediately. The total time for obtaining the lysate from the time of decapitation was less than 15 min including the spinning time.

Generation of recombinant synuclein and neurosin
BL21 (DE3) E. coli containing synuclein (alpha wild-type, A30P, A53T, and beta) in pET15b (Novagen, Madison, WI, USA) were induced with 1 mM IPTG. Cells were collected and crude recombinant protein was purified from boiled lysate by nickel chelating column (Amersham Pharmacia Biotech, Uppsala, Sweden). From the eluted crude fraction, alpha-synuclein monomer was purified with an ÄKTA chromatography system (Amersham Pharmacia Biotech) using a Superdex 75 gel filtration column (Amersham Pharmacia Biotech) and stored at -80°C until use. For producing recombinant neurosin, BL21 (DE3) E. coli containing neurosin in pET21a (Novagen, Madison, WI, USA) were induced and purified by nickel chelating column. For V5 tagged synuclein, alpha-synuclein was subcloned to pBAD TOPO (Invitrogen). Recombinant protein was expressed in LMG194 (Invitrogen) with 0.02% arabinose induction and purified by nickel chelating column.

Cell culture and sub-cellular fractionation
HEK-293 and SH-SY5Y cells were grown in Dulbecco's modified Eagle medium (Sigma, St Louis, MO, USA) supplemented by 10% fetal bovine serum or calf serum (BIO Whittaker, Walkersville, MD, USA) at 37°C in 95% air, 5% CO2. For sub-cellular fractionation, cells were homogenized by teflon homogenizer with 10 strokes in 10 mM Tris–HCl pH 6.8, 150 mM MgCl2, 10 mM KCl, 250 mM sucrose and spun at 1000g for 5 min. The pellet was retained as the nuclear fraction; the supernatant was spun at 5000g for 10 min to produce the crude mitochondrial pellet. The resulting supernatant was spun at 100 000g for 1 h to separate the cytosolic fraction (supernatant) from the fraction containing microsomes (pellet). The crude mitochondrial fraction was then washed with 10 mM Tris–HCl pH 6.7, 250 mM sucrose, 150 mM MgCl2 and ultrasonically lysed in 50 mM sodium phosphate buffer and kept as mitochondrial fraction.

Alpha-synuclein in vitro degradation experiment
Recombinant synuclein (alpha wild type, A30P, A53T and beta), 10 µg/ml, was mixed with 0.5 mg/ml HEK-293 lysate or indicated amount of kallikrein (Sigma) in 50 mM sodium phosphate buffer (pH 7.4) at 37°C for 2 h. When indicated, protease inhibitors such as 5 µg/ml aprotinin, 5 mM EDTA, 2 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 5 mM EGTA, Complete (Roche) or 5 mM AEBSF were added. The mixtures were then mixed with SDS–PAGE sample buffer, incubated at 95°C for 5 min. Ten microliters of the reaction mixture were applied to SDS–PAGE and immunoblotting was done with S1 antibodies.

Alpha-synuclein polymerization and polymerization inhibition experiment
For alpha-synuclein polymerization, 10 µg/ml of recombinant alpha-synuclein was incubated at 37°C in 50 mM sodium phosphate buffer, pH 7.4 and incubated at 37°C for the indicated time.

Monomer alpha-synuclein, 10 µg/ml or the indicated amount, was mixed with fragmented alpha-synuclein at concentration of 10 µg/ml or more for 48 h at 37°C. The mixtures were then mixed with SDS–PAGE sample buffer, incubated at 95°C for 5 min. Ten microliters of the reaction mixture were applied to SDS–PAGE and immunoblotting was done with S1 antibodies.

Neurosin heat inactivation experiment
To generate neurosin inactivated fragmented alpha-synuclein, 10 µg/ml recombinant alpha-synuclein was incubated with 1 µg/ml recombinant neurosin for 2 h and heated at 95°C for 5 min. Then 5 µg/ml of recombinant V5 tagged alpha-synuclein was added to the solution and incubated for 8 h at 37°C. The residual proteolytic activity was monitored with the degradation of V5 tagged alpha-synuclein by anti-V5 antibody blot.

Cell stress experiment
SH-SY5Y cells were incubated with 600 µM hydrogen peroxide, 25 mM S-nitroso-N-acetylpenicillamine, 3 mM 2-mercaptoethanol, 10 mg/ml tunicamycin, 100 µg/ml methyl methanesulfonate, 300 mM sorbitol or irradiated with UV light at 40 or 1000 J/m2 using a Stratalinker UV cross-linker (Stratagene, La Jolla, CA, USA). After exposure of SH-SY5Y cells to various stresses, cytosolic fractions were obtained from the cells as described.

RNA interference
Small interference RNA targeted against 88–108 bp of the human neurosin open reading frame (5'-aagacatctcacccctaccaa-3') was generated using siRNA construction kit (Ambion, Austin, TX, USA). BLAST search analysis was done to confirm that the sequence was highly specific to neurosin. Two other different sequences from neurosin was tested for RNAi efficiency, but failed to show any reductions in the level of neurosin. For control siRNA, this sequence was randomized (5'-aaccactaaccgcaccatatc-3'). Two-hundred picomoles of siRNA was transfected using Oligofectamine reagent (Invitrogen) as described by the manufacturer.

Immunoprecipitation
Mouse brain lysates were obtained as described previously. One microgram of antibody or normal rabbit serum was added to 100 µg of lysate and incubated for 4 h at 4°C. Then 20 µl of protein G agarose (Invitrogen) was added and incubated for 2 h. Then the lysates were spun at 20 000g and the pelleted protein G agarose was washed by brain lysis buffer four times, subjected to SDS–PAGE and blotted with NS-5106 and MC-36 antibodies.

Preparation of alpha-synuclein-rich fraction from mouse brain
Mice brains were homogenized in 10 times vol/weight of buffered sucrose (320 mM sucrose, 4 mM HEPES–NaOH, pH 7.3) with Complete (Roche) by teflon homogenizer with 10 strokes and centrifuged for 10 min at 800g. The supernatant was then centrifuged at 9200g for 15 min. The supernatant was centrifuged at 10 200g for 15 min to obtain the pellet, which was then resuspended in buffered sucrose and lysed by adding 9 vol of H2O, homogenized and adjusted to 10 mM HEPES–NaOH, pH 7.2 and kept on ice for 30 min. This was then centrifuged for 20 min at 25 000g. The supernatant was centrifuged at 165 000g for 2 h and then the pellet was resuspended in 40 mM sucrose and subjected to 100–800 mM sucrose gradient in 4 ml scale and spun at 65 000g for 5 h. Out of 500 µl fractions, 20 µl of 200–400 mM bands were subjected as alpha-synuclein-rich fraction to SDS–PAGE and blotted with NS-5106 and MC-37 antibodies.

Image and statistical analysis
Image analysis was done using Scion Image for Windows version 4 (Scion Corporation Frederick, MA, USA). All of the experiments were done in triplicate unless otherwise noted. All the statistical analysis was done using Stat View 5 software (SAS Institute, Cary, NC, USA) by Student's t-test.


   ACKNOWLEDGEMENTS
 
This study was supported in part by grants-in-aid from the Ministry of Health, Labor and Welfare, the Japan Society for the Promotion of Science, and the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   FOOTNOTES
 
* To whom correspondence should be addressed. Email: nukina{at}brain.riken.go.jp Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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