extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Chemistry

text:
The inhibitory effect of pyrroloquinoline quinone on the amyloid formation and cytotoxicity of truncated alpha-synuclein

Background Parkinson's disease (PD) involves the selective damage of dopaminergic neuron cells resulting from the accumulation and fibril formation of alpha-synuclein. Recently, it has been shown that not only full-length alpha-synuclein, but also C-terminal truncated forms exist in the normal brain, as well as Lewy bodies, which are cytoplasmic inclusions in PD. It is known that truncated alpha-synuclein has a much higher ability to aggregate and fibrillate than full-length alpha-synuclein. Since the fibrils and precursor oligomers of alpha-synuclein are cytotoxic to the neuron, inhibitors that prevent the formation of oligomers and/or fibrils might open the way to a novel therapeutic approach to PD. However, no inhibitor for truncated alpha-synuclein has been reported yet. Results In this study, we first characterized the aggregation and cytotoxicity of C-truncated alpha-synuclein119 and alpha-synuclein133 which have been found in both the normal and the pathogenic brain. Alpha-synuclein119 aggregated more rapidly and enhanced significantly the fibril formation of alpha-synuclein. Although both of alpha-synuclein119 and alpha-synuclein133 showed a high cytotoxicity, alpha-synuclein133 showed a similar aggregation with full-length alpha-synuclein and no acceleration effect. We showed that PQQ dramatically inhibits the fibril formation of C-terminal truncated alpha-synuclein110119, and 133 as well as the mixtures of full-length alpha-synuclein with these truncated variants. Moreover, PQQ decreases the cytotoxicity of truncated alpha-synuclein. Conclusions Our results demonstrate that PQQ inhibits the amyloid fibril formation and cytotoxicity of the C-truncated alpha-synuclein variants. We believe that PQQ is a strong candidate for a reagent compound in the treatment of PD.

Background
Parkinson' disease (PD) is a common neurodegenerative movement disorder affecting approximately 1% of the population above 65 years of age; it is a progressive neurodegenerative disease which develops slowly. The PD is caused by the specific and progressive degeneration of dopaminergic neurons in the substantia nigra. The neurodegeneration is accompanied by the presence of cytoplasmic inclusions, termed Lewy bodies (LBs). These inclusions are the hallmark pathological feature of PD [1][2][3][4]. The hypothesis according to which α-synuclein (α-Syn) plays a causative role in PD pathogenesis is strongly supported, since the major fibrillar protein component of LBs in both sporadic and familial PD is α-Syn, and three different α-Syn missense mutations (A30P, A53T and E46K) and the duplication and triplication of loci cause autosomal-dominant PD [5][6][7][8][9]. Several in-vitro studies have suggested that α-Syn's propensity to oligomerize and form fibrils may play a crucial role in its toxicity [10,12]. Current treatments are only symptomatic and do not stop or delay the progressive loss of neurons; there is as yet no preventative therapy available for PD. α-Synuclein (140 aa) is a natively unfolded protein that is enriched in the presynaptic terminal of the neurons in the brain. The primary sequence of α-Syn is subdivided into three regions: the amphiphatic N-terminal region (residues 1-60), the highly hydrophobic central region called "NAC" (61-95), and the acidic C-terminal region (96-140) [13]. The C-terminal region is important for the high thermostability of α-Syn and for the chaperon activity [14]. Moreover, the C-terminal region regulates the amyloid aggregation and fibril formation of α-Syn. It is known from patients suffering from α-synucleinopathy that the C-terminal-truncated forms of α-Syn consist in LBs [15][16][17][18]. Several in vitro studies have shown the propensity of α-Syn to aggregate into amyloid fibrils, a process that is accelerated by the truncation of its Cterminal. In addition, when full-length α-Syn is mixed with C-terminal-truncated variants, the truncated α-Syn accelerate the aggregation of the full-length protein [17,[19][20][21]. Recently, truncated α-Syn has been identified in the normal brain [18]. This indicates that the truncation of the C-terminal of α-Syn has some relevance to the pathogenesis of PD. Although many truncated variants have been characterized, some of the truncated α-Syn variants which exist in both the normal and the pathogenic brain, such as α-Syn119 and α-Syn133, have not yet been investigated through in vitro aggregation and fibril formation studies.
Neurodegenerative diseases, such as Parkinson' disease, Alzheimer's disease and prion disease, share the feature that the causative proteins change their conformation from the natural to the β-strand-rich conformation, acquiring oligomeric status, and subsequently forming supramolecular assemblies and amyloids. Considering that the formation of amyloid fibrils as well as their precursor oligomers is cytotoxic, agents that prevent the formation of the oligomers and/or fibrils might open the way to a novel therapeutic approach to these neurodegenerative diseases [22]. Therefore, considerable effort has been made to discover a molecule which prevents the amyloid formation of the causative proteins in these diseases, i.e., α-Syn [11,23], amyloid β [24,25] and prion protein [26], respectively.
Previously, we studied the amyloid fibril formation mechanism of α-Syn and the development of a strategy for its prevention [27]. Also, we have reported that the antioxidant pyrroloquinoline quinone (PQQ) (Fig. 1), which is a noncovalently bound bacterial cofactor in the oxidative metabolism of alcohols, prevents the amyloid fibril formation and aggregation of full-length α-Syn in vitro in a PQQ-concentration-dependent manner [28]. PQQ forms a conjugate with WT α-Syn, and this PQQconjugated α-Syn is also able to prevent α-Syn amyloid fibril formation. The fact that PQQ shows anti-fibrilforming activity for α-Syn and other amyloid proteins and has never been reported, nor has a molecule which is able to prevent the amyloid formation of truncated α-Syn ever been described; this encouraged us to further investigate the effects of PQQ on the fibrillization and cytotoxicity of C-terminal-truncated α-Syn.
In this study, we first characterized the aggregation and fibrillation of truncated α-Syn119 and 133 as well as mixtures of full-length α-Syn with these truncated variants. We were able to demonstrate that PQQ dramatically inhibits the fibril formation of C-terminal-truncated α-Syn110, α-Syn119, and α-Syn133. Moreover, PQQ decreased the cytotoxicity of the truncated variants. Our results suggest PQQ has a strong potential as a relevant leading compound in the design of new amyloid inhibitors.

Effect of truncation on the aggregation and fibril formation of α-synuclein
The binding of TfT to protein fibrils is accompanied by a characteristic increase in fluorescence intensity in the vicinity of 482 nm. (Fig. 2) compares the fibril formation patterns of full-length and truncated α-Syn110, α-Syn119, and α-Syn133, as monitored by TfT fluorescence. In agreement with earlier studies, the truncated α-Syn110 showed a much faster rate of fibril formation than fulllength α-Syn. Also the fibril formation of truncated α-Syn119 was faster than that of full-length α-Syn. Meanwhile, the fibril formation of truncated α-Syn133 was slower than that of full-length α-Syn. The maximal values of fluorescence intensity in truncated α-Syn110, α-Syn119, and α-Syn133 were 3, 1.5 and 0.6-fold of fulllength α-Syn and the lag time of truncated α-Syn was 4.8, 14.3 and 36.1 h respectively (full-length α-Syn, 21.4 h). The light scattering from protein solutions reflects the quantity of aggregates; i.e., this contained large soluble oligomers, amorphous aggregates and amyloid fibrils. The light scattering study showed that the aggregation ability of truncated α-Syn110 was higher than that of fulllength α-Syn, but that the aggregation ability of truncated α-Syn119 and α-Syn133 was at same level as that of the full-length protein (Fig. 3).

Truncated α-synuclein enhanced the fibril formation of αsynucleins
These truncated variants co-exist with full-length α-Syn in both the normal and the pathogenic brain. To investigate the effect of truncated α-Syn on the fibril formation of α-Syn, the fibrillation of a mixture of truncated and full-length α-Syn was monitored by TfT fluorescence and light scattering analysis. When truncated α-Syn110 and α-Syn119 coexisted with full-length α-Syn at a ratio of 1:1, the fibrillation rate increased and the lag time for fibrillation was reduced significantly, compared with those of full-length α-Syn (( Fig. 2) and Table 1).
Also, we performed the experiments to show the effect of truncated α-Syn with full length one. In order to show that the presence of truncated α-Syn enhances the fibril formation of α-Syn, various samples with different ratio of full length and α-Syn119 were prepared, fixing the final molar ratio at 70 μM α-Syn. If the presence of truncated α-Syn enhances the fibril formation of α-Syns, the amount of fibril should be higher than the sum of the amount of fibril formed with each α-Syn sample with each concentration. We used the amount of amyloid after 80 hours of incubation of the sample with 70 μM full length α-Syn alone as the control (100%). While the sample containing 35 μM of α-Syn119 alone resulted 120% of   The observed amyloid formation was not the simple sum of the expected amount of amyloid forming from each α-Syn. The experiments were carried out with different mixing ratio, and similarly, the amount of fibril estimated from TfT fluorescence was not the simple sum and higher than those from the each α-Syn samples (Fig.  4). It is obvious that co-existence of full length α-Syn and truncated synuclein enhanced the total fibril formation of α-Syns. The presence of α-Syn110 also enhanced the total amyloid formation of α-Syn, but the effect was weaker than α-Syn119. However, truncated α-Syn133 has no effect on fibrillation of α-Syn.
The light-scattering assay showed that the aggregation of full-length of α-Syn was increased 3-fold by mixing with α-Syn110. When α-Syn119 was mixed with fulllength α-Syn, the OD 500 was slightly increased (Fig. 5). But α-Syn133 did not enhance the aggregation of fulllength α-Syn. It indicated that α-Syn110 and α-Syn119 enhanced the aggregation of full-length α-Syn, but not α-Syn133.

PQQ prevents the fibril formation of truncated α-synuclein
We have previously reported that PQQ prevents the aggregation and fibril formation of full-length α-Syn, and here we investigated whether or not PQQ also prevents the fibril formation of truncated α-Syn. It has been reported, in agreement with this study, that C-terminal truncation accelerates α-Syn fibril and oligomer formation. Also, truncated α-Syn promotes the fibril formation of full-length α-Syn. However, the presence of PQQ in truncated α-Syn solutions resulted in the dramatic pre-  The lag time analysis of fibril formation was calculated from fitting-curve of TfT fluorescence was provided by PRISM (GraphPad Software)  vention of fibril formation in our study (Fig. 6). Upon the addition of 280 μM PQQ, the fibrils formed in 70 μM truncated α-Syn110, α-Syn119, and α-Syn133 decreased to less than 50, 27, and 14.8% of the fibrils formed in the absence of PQQ, respectively. The higher the concentration of PQQ, the less truncated α-Syn fibrils were formed (Fig. 7). This indicates that the inhibitory effect is PQQdose-dependent. In addition, fibril formation in the mixture of truncated and full-length α-Syn was markedly inhibited (Fig. 8). In the presence of PQQ, moreover, the light scattering did not increase significantly, indicating that PQQ prevents the formation of truncated α-Syn aggregates (( Fig. 3) and (Fig. 5)). These results indicate that PQQ prevents the amyloid fibril formation and aggregation of truncated α-Syn as well as full-length α-Syn in a PQQ-concentration-dependent manner.

PQQ decreases the cytotoxicity of truncated α-synuclein
Previous studies have shown that aggregation and fibrillization of amyloid protein is toxic to neural cells [10,12,29,30]. We evaluated the effect of PQQ on the cytotoxicity of truncated variants to cultured PC12 cells. PC12 is a rat kidney cell line frequently used in the study of normally developing nervous systems. Full-length and truncated α-Syn samples were added to PC12 cells and incubated in the presence or absence of PQQ. These samples were then further cultured for four days with shaking at 37°C. We then evaluated the cytotoxicity of full-length and truncated α-Syn by adenylate kinase assay (AK assay), which measures quantitatively the release of AK from the damaged cells. The released AK converts ADP to ATP, which is measured by the bioluminescence assay, thus allowing the accurate and sensitive determination of cytotoxicity. Upon incubation of PC12 cells with 14 μM α-Syns (full-length αSyn, α-Syn110, α-Syn119, and α-Syn133), the bioluminescence increased to 1500, 3600, 28200, and 7300 RLUs, respectively. This indicates that the C-terminal-truncated α-Syn is highly toxic to PC12 cells, as opposed to the full-length α-Syn (Fig. 9). Addition of 28 μM α-Syn results in 1.4-to 1.8-fold higher bioluminescence than with 14 μM α-Syn, indicating that the cytotoxicity truncated α-Syn is dose-dependent. The cytotoxicity evaluation results of α-Syn119 based on MTT assay are in good agreement with those from the   adenylate kinase assay, as α-Syn119 is more toxic than wild-type, full-length α-Syn (Fig. 10). However, surprisingly, when PC12 cells to which truncated α-Syn was added were incubated with 200 μM PQQ, the bioluminescence decreased to 30-70% of that of the samples not treated with PQQ. The significant decrease in release of AK from damaged cells, suggests that the presence of PQQ greatly decreases the cytotoxicity of truncated α-Syn.
Recent studies have suggested that the oligomeric intermediate of fibrillation has a higher toxicity than the fibril. For the therapeutic application of PQQ, it is therefore important to investigate the effect of PQQ on both the oligomer formation and cytotoxicity of truncated α-Syn. We investigated the formation of oligomers of truncated synuclein119 with and without PQQ by dot blotting analysis using the anti-oligomer-specific antibody A11 [31]. We incubated 200 μM truncated synuclein119 in the absence or presence of a four-fold molar excess of PQQ and sampled at several times. In the absence of PQQ, the staining intensity of the α-Syn119 increased during the 12-hour time course (Fig. 11), reflecting the accumulation of oligomer. In contrast, the amount of oligomer did not change in the presence of PQQ. The cytotoxicity of these incubated samples, measured by adding them to U2-OS cells and culturing for 24 hours, increased in an oligomer dose-dependent manner. In the presence of PQQ, the cytotoxicity of a 12-hour incubated α-Syn119 was only   10%, compared to the 30% cytotoxicity of the α-Syn119 incubated 12 hours without PQQ. This clearly indicates that the cytotoxicity of α-Syn is related the oligomer formation by the truncated α-Syn119, and PQQ prevents the cytotoxicity of truncated α-Syn119 by inhibiting the formation of oligomers.

Discussion
In this work, we prepared recombinant truncated α-Syn110, α-Syn119, and α-Syn133 and investigated the aggregation and fibril formation of these proteins in vitro. Also, we investigated the inhibitory effect of PQQ on the fibril formation and cytotoxicity of C-terminal-truncated α-Syn. It is the first attempt to characterize the aggregation and fibril formation of α-Syn119 and α-Syn133. It is possible that these truncated α-Syn variants play an important role in the pathogenesis of PD and related diseases, because C-terminal-truncated α-Syn119 and α-Syn133 have been found in both the normal and the pathogenic brain [18]. It is commonly understood that rapid aggregation and fibril formation occurs upon the truncation of the acidic C-terminal of α-Syn, which contains 10 glutamates and 5 aspartates. By truncation of negative charge acids, the repulsion between C-terminals of α-Syn was decreased and the possibility of interaction between hydrophobic regions contained NAC was increased. As a result, the aggregation and/or fibril formation were accelerated. In fact, α-Syn110 and α-Syn119 did show high aggregation and fibrillization speeds and rates compared to full-length α-Syn in our present study.
Unexpectedly the result of TfT fluorescence assay indicated that α-Syn133 slowed down the fibrillation in comparison with full-length α-Syn, whereas, α-Syn133 has almost equivalent aggregation with full-length α-Syn was observed. We understood that the speed of fibril formation of α-Syn133 decreased whereas the final quantity of fibril and aggregates are in the same level as those for wild type. Considering that the fibril elongation process is influenced by the status of soluble oligomers, the decreased kinetics in fibril formation of α-Syn133 might suggest the change of oligomeric status of α-Syn133.
Some studies have investigated the C-terminal-truncated α-Syn seeding mechanism, in which the truncated fragments nucleate full-length α-Syn aggregation through the formation of hybrid protofibrils and then fibrils [17,20,32,33]. This shortens the lag-time of fibril formation. When truncated α-Syn110 and α-Syn119, but not α-Syn133, coexisted with full-length α-Syn, it dramatically enhanced the total fibril formation of α-Syn. It indicated that co-existence of full length and truncated synuclein119 have synergistic effect on aggregation and fibrillation. Alzheimer's disease results mainly from the accumulation of amyloid β (1-42), though the total amount is lower than that of amyloid β (1-40). Likewise, it is presumed that the induction of the aggregation of fulllength α-Syn by a small amount of C-terminal-truncated α-Syn is critical to its pathogenesis. Therefore, it is important to develop an inhibitor for the aggregation and fibrillization of truncated forms of α-Syn as well as fulllength α-Syn.
PQQ exists in several plants and animals, and has been drawing attention as a novel nutritional factor due to its positive effect on cellular growth and its potential as a key pharmacological compound. Pharmacological research has addressed the issue of PQQ as a potential antioxidant and redox modulator. We have previously reported that PQQ prevents the fibril formation of full-length α-Syn and also influences the conformational change of α-Syn [28].
In our present study, PQQ significantly decreased the aggregation and fibril formation of truncated α-Syn as well as full-length α-Syn. Furthermore, in the presence of PQQ, aggregation and fibril formation in mixtures of Cterminal-truncated and full-length α-Syn were inhibited. This is the first report of an inhibitor for the aggregation and fibril formation of C-terminal-truncated α-Syn. In the presence of 4-fold molar PQQ, the fibril formation of full-length α-Syn and α-Syn133 was prevented to 10% of the fibrils formed in the absence of PQQ. However, in the case of α-Syn110 with 4-fold molar PQQ, fibril formation decreased only by 50%. The fibril formation of α-Syn119 decreased to 30% under the conditions of the experiment, as monitored by TfT fluorescence assay. This indicates that PQQ inhibits fibrillization in all of these C-terminaltruncated α-Syns, but that the potency of its inhibitory activity is different in each case (Fig. 7).
We have previously reported on the UV spectrum of α-Syn incubated with PQQ, which showed remarkable differences from the typical absorbance of PQQ or α-Syn alone. This suggests that PQQ forms a conjugate with α-Syn and that PQQ has reactive quinone groups, just as the oxidized form of baicalein [23]. Therefore, it is highly probable that PQQ binds with α-Syn via a Schiff base. Likewise, it is conceivable that PQQ binds to C-terminaltruncated α-Syn via a Lys residue. Although the number of Lys residues in C-terminal-truncated α-Syn is equal to that in full-length α-Syn, the inhibitory effect of PQQ is different between full-length α-Syn and truncated α-Syn. Based on the results of the TfT fluorescence assay, it is considered that the difference in the efficacy of PQQ's inhibitory activity derives from the speed of aggregation and fibrillization of each variant of truncated α-Syn. Since the aggregation and fibril formation of truncated α-Syn is faster than those of full-length α-Syn, in the case of truncated α-Syn, the reaction rate of binding with PQQ may be lower. As a result, until fibril formation begins, the instances of PQQ binding with truncated α-Syn will be fewer than with full-length α-Syn. We have reported that not only PQQ, but also PQQ-conjugated α-Syn lowered fibril formation [28]. This "feedback effect" gives PQQ considerable leverage in its inhibition of full-length α-Syn aggregation. In the case of C-terminal-truncated α-Syn, however, the effect is reduced by the ever decreasing quantity of PQQ-conjugated α-Syn.
It was known that amyloid proteins are toxic to culture cell and the expression of amyloid proteins was induced the damage of neurons in many neurodegeneration models. It is therefore important to evaluate the effect of PQQ on the cytotoxicity of amyloid proteins to develop an adequate therapy strategy. In this study, we measured the cytotoxicity of C-terminal-truncated α-Syn by adenylate kinase assay, MTT and ATP assay. The results show that C-terminal-truncated α-Syn was much more toxic to PC12 cells in a dose-dependent manner than full-length α-Syn. Especially the cytotoxicity of truncated α-Syn119 was distinctly different from that of other α-Syn variants. Under the experimental conditions, the luminescence intensity was 20-fold higher than that of full-length α-Syn. Interestingly truncated α-Syn133 is more toxic to PC12 cells than full-length α-Syn and α-Syn110. As we mentioned above, the decreased kinetics in fibril formation of α-Syn133 might suggest the change of oligomeric status of α-Syn133. In this respect, our interpretation that the increased toxicity of α-Syn133 owes to the change in the oligomeric status is in consistent with the experimental observation. We believe that truncation of the seven C-terminal residues affected the oligomeric status, consequently change the fibrillation kinetics.
Recently some studies suggested the intermediate of fibrillation, oligomer, has a high toxicity than fibril. It was known that some inhibitor of α-Syn aggregation increased the cytotoxicity of α-Syn despite the aggregation and fibril formation was significantly decreased [11]. These reports suggested that inhibitors induced the stable state of oligomer, intermediate of aggregation, and increased the cytotoxicity more. Other studies showed cytotoxicity of amyloid proteins was dependent on the conformations of oligomers as followed references [34,35], not the property of fibrillation. Thus, these studies supported that the fibrillation tendency of each variant dose not directly correlate their cytotoxicity effects. We showed that formed oligomer of truncated α-Syn119 was related to cytotoxicity of α-Syn and PQQ could inhibit the cytotoxicity of truncated synuclein119 by preventing the oligomer formation. In the progress of fibril formation, oligomerization is essential step for the fibrillation. From the results of kinetics of truncated synuclein in the presence of PQQ, the lag-time of fibrillation was delayed by PQQ which means that the formation of seeds for elongation of fibril, were prevented by PQQ. Together with results of cytotoxicity, it suggested that PQQ also could decrease the formation of truncated synuclein oli-gomers. This suggests that PQQ is a strong candidate for future anti-PD reagent compounds.

Conclusions
Here, we showed that α-Syn110 and α-Syn119 has a high propensity of aggregation and enhanced the fibrillation of α-Syn. Although both of α-Syn119 and α-Syn133 showed a high cytotoxicity to culture cell, α-Syn133 showed a similar aggregation with full-length α-Syn and no acceleration effect. PQQ dramatically inhibits fibril formation in C-terminal-truncated α-Syn, as well as in mixtures of full-length α-Syn with truncated variants. Moreover, PQQ decreases the cytotoxicity of truncated α-Syn. Together with other findings on the inhibitors of amyloid proteins, this suggests that inhibitors other than PQQ, which also bind to target proteins, could prevent the aggregation and fibrillation of truncated α-Syn in a similar manner. In any case, PQQ is a strong candidate for a reagent compound in the treatment of PD and related diseases.

Chemicals
Thioflavin T (TfT) was purchased from Sigma. Pyrroloquinoline quinone was kindly donated by Mitsubishi Gas Chemical Company, Inc.
Expression vectors containing full-length α-Syn and truncated variants were thus constructed. The proteins used in this study were expressed in Escherichia coli BL21 (DE3). α-Syn expression was induced by the addition of 0.3 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG). The cells were disrupted using a French press (Ohtake Works, Inc.), and the supernatant was collected by centrifugation (18,500 g, 10 min, 4°C). After boiling the collected supernatant at 100°C for 20 min, it was centrifuged (18,500 g, 10 min, 4°C) and dialyzed against buffer A (20 mM Tris-HCl, pH 8.0). After further centrifugation (12,000 g, 20 min, 4°C), the supernatant was loaded onto a Resource Q column (for full length α-Syn, truncated α-Syn119 and α-Syn133) or a Resource S column (for truncated α-Syn110) (GE Healthcare Bio-Science Corp.) and eluted with a 0-0.5 M NaCl gradient in buffer A. The eluted fractions were analyzed by SDS-PAGE, and the fractions containing α-Syn were dialyzed against PBS buffer (8.1 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , 137 mM NaCl and 2.7 mM KCl, pH 7.3). The concentrations of the proteins were determined by means of a DC protein assay kit (Bio-Rad).

Preparation of aggregated solutions and TfT fluorescence & light-scattering assay
Purified α-Syn was concentrated to ca. 3.0 mg/ml using Amicon Ultra-15 filters (Millipore) in PBS buffer, pH 7.3, centrifuged (150,000 g, 1 h, 4°C) to remove any aggregates, and adjusted to 2.0 mg/ml. PQQ was dissolved in the same buffer at the defined concentration. The reaction solution contained 70 μM full-length or truncated α-Syn in the presence or absence of PQQ. Mixtures of fulllength α-Syn and truncated α-Syn with or without 280 μM PQQ were also prepared. These samples were incubated on a 96-well plate in 200 μl of PBS buffer, pH 7.3, with 0.02% NaN 3 at 37°C with shaking at 700 rpm. Fibril formation was monitored by thioflavin T (TfT) fluorescence. Aliquots of 5 μl were removed from the incubated samples and added to 250 μl of 25 μM TfT in PBS buffer, pH 7.3. TfT fluorescence was recorded at 486 nm with excitation at 450 nm using an ARVO MX 1420 multilabel counter (PerkinElmer). The lag time analysis of fibril formation was calculated from fitting-curve of ThT fluorescence was performed by PRISM (GraphPad Software). To compare the total aggregation of full-length and truncated α-Syn, the light scattering was studied. After incubation, 60-μM samples were measured at 500 nm. Usually, the formation of aggregations is monitored at 330 nm. However, PQQ has a typical absorbance at around 340 nm; so, the light scattering was monitored at 500 nm

Cell culture and cytotoxicity analyses of truncated αsynuclein
PC12 cells (ATCC CRL1721) were routinely cultured in RPMI-1640 medium (Sigma) containing 5% fetal calf serum, 10% horse serum and 1% penicillin-streptomycin, and maintained at 37°C in a humidified incubator with 5% CO 2 /95% room air. For the cytotoxicity studies, the medium was removed and fresh medium was gently added before plating.
The cytotoxicity was measured by adenylate kinase assay. In brief, PC12 cells were plated at a density of 10,000 cells per well on CC3-coated 96-well plates (Nunc) in 100 μl of Opti-MEM medium. After 24 h of incubation, full-length or truncated α-Syn was added at the indicated concentration, and 200 μM PQQ was also added in half of the wells. The cells were incubated for an additional 4 days with shaking at 500 rpm, 37°C. The release of adenylate kinase from the damaged cells was measured using a ToxiLight ® Bioassay Kit obtained from LONZA [36,37]. Fifty μl of culture medium were removed from the each well and added to 100 μl of the reaction substrate. After incubation at room temperature for 5 min, the luminescence was measured on an ARVO MX 1420 multilabel counter (PerkinElmer). The bioluminescence of the control cells treated with PBS or PQQ instead α-Syn was also measured in the same way. PQQ had no effect on the AK assay as the luminescence with PBS was identical to that with PBS plus PQQ. The results shown in (Fig. 9) are after subtraction of these background values.
The cytotoxicity was measured by MTT and ATP assay. In brief, PC12 or U2-OS cells were plated at a density of 10000 cells per well on CC3-coated 96-well plates in 100 μl of fresh medium. After 24 h of incubation, full-length or truncated α-Syn119 was added at final concentration of 20 μM and cells were incubated for an additional 24 h. In MTT assay, cellular redox activity of tetrazolium formazan, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium were measured Cell Counting Kit-8 (DOJINDO) [38]. After replacement to fresh medium, 10 μl of stock WST-8 was added and the incubation was continued for another 2~3 h. Absorbance values at 450 nm were determined with an ARVO MX 1420 multilabel counter. The absorbance of the cells treated with PBS instead α-Syn was also measured in the same way as control (100%). In ATP assay, cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) following the manual. After replacement to 100 μl fresh medium, 100 μl of stock solution was added and the incubation was continued for 5 min. The luminescence values were determined with an ARVO MX 1420 multilabel counter. The luminescence of the cells treated with PBS instead α-Syn was also measured in the same way as control (100%).

Dot blot analysis
Purified α-Syn119 was concentrated using Amicon Ultra-15 filters (Millipore) in PBS buffer, pH 7.3, centrifuged (150,000 g, 1 h, 4°C) to remove any aggregates, and adjusted to 3.0 mg/ml in the absence or presence of 800 μM PQQ. These samples were incubated in a 2 ml tube, in 1.2 ml of PBS buffer, pH 7.3, at 37°C with shaking at 700 rpm. The incubated α-Syn119 was sampled in several times. 1 μl of each sample applied to a nitrocellulose membrane, blocked with 10% skim milk in PBS containing 0.01% Tween 20 (TBS), at room temperature for 1 h, washed three times for 5 min each with TBS and incubated for 1 hr at room temperature with the anti-oli-gomer antibody A11 (0.1 μg/ml in TBS). The membranes were washed three times for 5 min each with TBS, incubated with anti-rabbit IgG diluted 1:10,000 in TBS and incubated for 1 hour at room temperature. The blots were washed three times with TBS and developed with ECL chemiluminescence kit from Amersham-Pharmacia (Piscataway, NJ).


extfieldsofstudy: Biology Medicine

text:
Multiplex SILAC Analysis of a Cellular TDP-43 Proteinopathy Model Reveals Protein Inclusions Associated with SUMOylation and Diverse Polyubiquitin Chains

Transactive response (TAR) DNA-binding protein 43 (TDP-43) is a major protein component within ubiquitin-positive inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Although TDP-43 is a nuclear DNA/RNA-binding protein, in pathological conditions, TDP-43 has been reported to redistribute to the cytoplasm where it is cleaved and forms insoluble, ubiquitinated, and phosphorylated inclusions. Here we present a cellular model in which full-length human TDP-43 or a splicing isoform (TDP-S6) that lacks the C terminus is overexpressed in a human cell line and mouse primary neurons. Whereas recombinant and endogenous TDP-43 was primarily localized in the nucleus, the shorter TDP-S6 formed highly insoluble cytoplasmic and nuclear inclusions reminiscent of disease-specific pathology. Western blot analysis of detergent-insoluble extracts showed an increase in high molecular weight immunoreactive species for TDP-S6 compared with TDP-43, consistent with ubiquitination or ubiquitin-like modifications. We used a multiplex stable isotope labeling with amino acids in cell culture approach to compare the detergent-insoluble proteome from mock-, TDP-43-, and TDP-S6-transfected cells. TDP-S6 overexpression caused a concomitant increase in both ubiquitin

a C terminus recapitulates many of the cellular and biochemical features associated with disease pathology and that the interplay of ubiquitination and SUMOylation may have an important role in TDP-43 regulation. Molecular & Cellular Proteomics 9:705-718, 2010.
Frontotemporal lobar degeneration (FTLD) 1 is a progressive neurodegenerative disease characterized by prominent behavioral abnormalities, personality changes, and language dysfunction, and it co-occurs with the development of parkinsonism and motor neuron disease in some patients (1,2). Like other neurodegenerative diseases, FTLD is characterized by the abnormal accumulation of ubiquitinated intracellular protein aggregates (3,4). In addition to the aggregation of tau in some forms of FTLD, more than half of cases are marked by ubiquitin-positive inclusions and are subclassified as FTLD-U. Transactive response (TAR) DNA-binding protein 43 (TDP- 43) has been identified as a major protein component of inclusions in FTLD-U and amyotrophic lateral sclerosis (ALS) (5). TDP-43 aggregation is also observed in hereditary inclusion body myopathy and Paget disease of the bone with frontotemporal dementia (6) as well as in some cases of Alzheimer and Parkinson diseases (7,8). This indicates that a common underlying mechanism may broadly define a spectrum of neurodegenerative disorders termed "TDP-43 proteinopathies" (9,10).
TDP-43 is a 414-amino acid protein that contains two RNA recognition motifs (RRM1 and RRM2) and a glycine-rich Cterminal domain. It is highly conserved in human, mouse, fly, and worm and is expressed in all tissues, including the brain (11)(12)(13). First characterized to bind and repress the promoter activity of TAR DNA in the human immunodeficiency virus 1 long terminal repeat region (14), TDP-43 was later found to regulate splicing of the transcripts of apolipoprotein A-II (15) and cystic fibrosis transmembrane conductance regulator (16). Therefore, TDP-43 can act both as a transcriptional repressor and as a splicing regulator. Although physiological TDP-43 resides mainly in the nucleus, pathology-relevant TDP-43 redistributes from the nucleus to the cytoplasm where it is cleaved and forms phosphorylated and ubiquitinated inclusions (5,(17)(18)(19). The degree to which TDP-43 is posttranslationally modified and whether other modifications initiate nuclear to cytoplasmic translocation and aggregation remain unknown.
The primary transcripts of TDP-43 in mouse and human undergo multiple alternative splicing events in which 10 splice variants (S1-S10) have been documented (11). With the exception of full-length TDP-43, all alternatively spliced isoforms are expressed as truncated proteins that lack the C-terminal glycine-rich domain. Isoforms that lack this domain are no longer capable of enhancing the skipping of exon 9 of the cystic fibrosis transmembrane conductance regulator gene via interactions with heterogeneous nuclear ribonucleoprotein A/B (11,20). To date, the vast majority of sporadic and familial TARDBP gene variants found in ALS cases (21)(22)(23)(24)(25) are reported to have missense mutations resulting in single amino acid substitutions located in the glycine-rich domain. This establishes an intriguing relationship between ALS disease etiology and the function of the TDP-43 C-terminal region. Whether TDP-43 splice variants that lack this C terminus contribute to translocation and aggregation requires further investigation.
In this study, we overexpressed human TDP-43 and TDP-S6, a splice variant lacking the glycine-rich C terminus, in human embryonic kidney 293 (HEK-293) cells and mouse hippocampal neurons. When compared with TDP-43, TDP-S6 showed both nuclear and cytoplasmic location and extensive aggregation. Biochemical analysis revealed that TDP-S6 was almost completely Sarkosyl-insoluble. Moreover, both insoluble TDP-43 and TDP-S6 displayed varying levels of posttranslational modifications that included ubiquitination and/or ubiquitin-like modifications, phosphorylation, and proteolytic cleavage. To assess protein differences among the detergentinsoluble extracts from mock-, TDP-43-, and TDP-S6-transfected cells, a multiplex stable isotope labeling with amino acids in culture (SILAC) strategy was used in combination with LC-MS/MS (26). This quantitative proteomics approach metabolically introduces a mass difference into proteins by incorporation of heavy isotopic forms of arginine and lysine. We identified and characterized a novel association between the small ubiquitin (Ub)-like modifier-2/3 (SUMO-2/3) and TDP-43. Additional targeted proteomics analysis found multiple polyubiquitin structures in the TDP insoluble fractions. Our data indicate that overexpression of a truncated TDP-43 splice variant recapitulates many of the features associated with disease pathology and that SUMOylation and ubiquitination may play important roles in regulating TDP-43 functions.
Primary Neuronal Cultures-Primary hippocampal neuron cultures were prepared from wild type C57BL/6 mice (Charles River Laboratories, Wilmington, MA) at embryonic day E18. The embryos were dissected, and the hippocampus was isolated in dissection buffer (Hanks' balanced salt solution, 10 mM HEPES, 1% penicillin/streptomycin). After mild trypsinization with 0.25% trypsin and 0.01% deoxyribonuclease in dissection buffer for 15 min at 37°C, the tissue fragments were rinsed twice with dissection buffer and twice with plating medium (buffered minimum Eagle's medium essential medium (Invitrogen), 0.6% glucose (Invitrogen), 2 mM L-glutamine (Mediatech Inc., Manassas, VA), 10% heat-inactivated horse serum (Invitrogen), 1% penicillin/streptomycin). The tissue was then subjected to mechanical dissociation by repeated aspiration through a fire-polished Pasteur pipette in dissociation medium. Viable cells were determined by trypan blue exclusion. Neurons were plated at a density of 50,000 cells/cm 2 on poly-L-lysine (Sigma)-coated coverslips (Propper Manufacturing Co., Long Island City, NY) and maintained in Neurobasal medium (Invitrogen) containing B-27 supplement (Invitrogen), 2 mM L-glutamine, and 1% penicillin/streptomycin at 37°C under 5% CO 2 .
Subcellular Fractionation-To examine the solubility of TDP-43 and TDP-S6, sequential extractions were performed as described previously (5) with slight modification. Cells (ϳ1 ϫ 10 7 ) were washed twice, collected with ice-cold PBS buffer, and then lysed in Sarkosyl buffer (10 mM Tris, pH 7.5, 5 mM EDTA, 1 mM DTT, 0.5 M NaCl, 10% sucrose, 1% N-lauroylsarcosine, 10 mM ␤-glycerophosphate, 10 mM sodium orthovanadate, 10 mM tetrasodium pyrophosphate, 50 mM sodium fluoride). Resulting lysates were spun at 180,000 ϫ g for 30 min at 22°C to generate the detergent-soluble samples. Insoluble pellets were washed three times with additional Sarkosyl buffer, then extracted with urea buffer (30 mM Tris, pH 8.5, 7 M urea, 2 M thiourea, 2% sodium dodecyl sulfate), and centrifuged at 25,000 ϫ g for 30 min at 22°C. Protease inhibitors (Roche Applied Science) were added to all buffers prior to use. Protein concentration was determined for Sarkosyl fractions by bicinchoninic acid (BCA) protein assay (Pierce) according to the manufacturer's instructions. For urea fractions, protein concentration was determined likewise and/or by estimating Coomassie Blue G-250 staining intensity of a small fraction of the urea extracts following electrophoresis in polyacrylamide gels using titrated BSA as a standard (30).
Western Blotting-Immunoblotting was performed according to standard procedures. Briefly, samples in Laemmli sample buffer were separated by SDS-PAGE and transferred overnight to PVDF Immobilon-P membranes (Millipore, Billerica, MA). To ensure both equal loading and complete transfer of proteins from the gel, membranes were reversibly stained with Ponceau S (Diasys Europe Ltd., Workingham, UK). Blots were blocked for 1 h at room temperature using 1ϫ Blocking Buffer (USB Corp., Cleveland OH), probed with primary antibody in TBS with 0.1% Tween 20 overnight at 4°C, and incubated for 1 h at room temperature with secondary antibodies (1:20,000) conjugated to fluorophores (Molecular Probes, Eugene OR; Rockland, Gilbertsville PA). Blots were dried, scanned, and quantified with an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE).
SILAC and LC-MS/MS-Cells were cultured in Dulbecco's modified Eagle's medium (deficient in L-lysine and L-arginine) supplemented with 5% dialyzed fetal calf serum (Invitrogen) as described (31). For stable isotopic labeling, arginine and lysine were added in light (Arg0/Lys0; Sigma), medium (Arg6/Lys4), or heavy forms (Arg10/ Lys8; Cambridge Isotope Laboratories, Andover, MA) to a final concentration of 0.26 mM. Cells were cultured for seven passages to ensure full labeling and then transfected with pcDNA3.1-HA (mock), HA-TDP-43, and HA-TDP-S6 plasmids, respectively. After 2 days, the cells were harvested, equally mixed, and lysed to prepare the Sarkosyl-insoluble, urea-soluble fraction as described above. The fraction was reduced with 10 mM DTT, alkylated with 50 mM iodoacetamide for 30 min in the dark, and then resolved on a 10% polyacrylamide-SDS gel. After staining with Coomassie Blue, one gel lane was cut into five gel bands, and bands were subjected to in-gel digestion (12.5 g/ml trypsin). Extracted peptides were loaded onto a C 18 column (75-m inner diameter, 10 cm long, ϳ300 nl/min flow rate, 5-m resin from Michrom Bioresources, Auburn, CA) and eluted during a 10 -30% gradient (Buffer A: 0.4% acetic acid, 0.005% heptafluorobutyric acid, 5% ACN; Buffer B: 0.4% acetic acid, 0.005% heptafluorobutyric acid, 95% ACN). The eluted peptides were detected by Orbitrap (350 -1500 m/z; 1,000,000 automatic gain control target; 1000-ms maximum ion time; resolution, 60,000 full-width at half-maximum) followed by five data-dependent MS/MS scans in the linear ion trap quadrupole (2 m/z isolation width, 35% collision energy, 5,000 automatic gain control target, 200-ms maximum ion time) on a hybrid mass spectrometer (Thermo Finnigan, San Jose, CA).
Acquired MS/MS spectra were extracted and searched against the human reference database from the National Center for Biotechnology Information (December 13, 2007) using the SEQUEST Sorcerer algorithm (version 2.0, SAGE-N) (32). Searching parameters included mass tolerance of precursor ions (Ϯ50 ppm) and product ion (Ϯ0.5 m/z); partially tryptic restriction; fixed mass shift for modification of carboxamidomethylated Cys (ϩ57.0215 Da); dynamic mass shifts for oxidized Met (ϩ15.9949), Lys (ϩ4.02511 for 2 H 2 or ϩ8.01420 for 13 C 6 15 N 2 ), and Arg (ϩ6.02013 for 13 C 6 or ϩ10.00827 for 13 C 6 15 N 4 ); five maximal modification sites; and three maximal missed cleavages. Only b and y ions were considered during the database match. To compare medium and heavy labeled samples, searches were performed with static modifications ϩ4.02511 on Lys and ϩ6.02013 on Arg and dynamic modifications of ϩ3.98909 on Lys and ϩ3.98814 on Arg to account for the mass difference between medium and heavy labeled peptides. To evaluate the false discovery rate (FDR), all original protein sequences were reversed to generate a decoy database that was concatenated to the original database (a total of 53,830 protein entries) (33,34). The FDR was estimated by the number of decoy matches (n d ) and total number of assigned matches (n t ). FDR ϭ 2 ϫ n d /n t , assuming that mismatches in the original database were the same as in the decoy database. To remove false positive matches, assigned peptides were grouped by a combination of trypticity (fully, partial, and non-tryptic) and precursor ion charge state (1ϩ, 2ϩ, 3ϩ, and 4ϩ). Each group was first filtered by mass accuracy (10 ppm for high resolution MS) and by dynamically increasing XCorr (minimal 1.8) and ⌬Cn (minimal 0.05) values to reduce protein FDR to less than 0.2%. The identified proteins/peptides are listed in the supplemental data (supplemental Tables S1-S6 and S8) with accession number, matched peptide number, sequencing coverage, mass shift, and matching scores. Every peptide in the tables was linked to assigned MS/MS spectra, precursor ion mass, and charge state. If peptides were matched to multiple members of a protein family, the matched members were clustered into a single group (supplemental Tables S7 and S9).
SILAC Quantification and Bioinformatics Analysis-Quantitative pairwise comparisons of control, TDP-S6-, and TDP-43-transfected cells were carried out according to reported methods (35, 36) with slight modification. (i) For, ion extraction from MS scans, the ion currents for identified peptides were extracted in MS survey scans of high resolution (60,000) based on the isotopic ion selected for MS/MS sequencing. A number of parameters were defined, including precursor m/z, charge state, retention time, ion peak width, height, area, and noise level. The noise level was derived by averaging signal intensity of all ions in the MS scan after removing outliers that were at least two S.D. away from the mean. The intensity of ions was presented by the peak height and normalized according to the noise intensity under the assumption that the noise level of MS scans reflects, at least partially, variable ionization efficiency. The peaks used in the analysis had a minimal intensity of 2 signal-to-noise ratio. (ii) Ion matching among light, medium, and heavy isotopes was allowed with a tolerance of 10 ppm. If a sequenced peptide could not be matched, we estimated that the maximum ion current for undetected signal was equal to the noise level and used it to derive the peptide ratio. (iii) For data integration, the ratio of every peptide was transformed into logarithmic (log 2 ) values that were averaged over all peptides of a particular protein to determine the protein ratio. If a protein was quantified by both matched peptide ratios and unmatched peptide ratios, only the matched data were averaged. The results are shown in supplemental Table S7 with the number of peptides quantified for every protein. (iv) For data normalization, according to the null hypothesis, the histogram of all protein log 2 ratios was fitted to a Gaussian distribution to evaluate systematic bias (according to the mean) and experimental variation (based on S.D.). The data were then normalized by subtracting the mean in every protein ratio. The S.D. values in all SILAC comparisons were less than 0.4. (v) For data filtering, we selected the cutoff of log 2 ratios that were outside a 95% confidence interval (ϳ2 S.D.) from the mean of the Gaussian distribution. Finally, the quantified proteins were manually examined with respect to MS/MS assignment, ion peak matching, and ion intensity.
Semiquantitative Proteomics by Spectral Counts-To compare differences between the detergent-soluble and -insoluble proteome, we compared the identified proteins in both samples based on spectral counts. The spectral counts were first normalized to ensure that average spectral counts per protein was the same in the two data sets (37). A G test was used to judge statistical significance of protein abundance difference (38). Briefly, the G value of each protein was calculated as shown in Equation 1, where S tc and S si are the detected spectral counts of a given protein in the total cell lysate and in the Sarkosyl-insoluble fraction, respectively, and "ln" is the natural logarithm. Although theoretical distribution of the G values is complex, these values approximately fit to the 2 distribution (1 degree of freedom), allowing the calculation of related p values.
Quantitative Analysis of Polyubiquitin Chains and Other Targeted Proteins by LC/Selective Reaction Monitoring (SRM)-The analysis of polyUb linkages, ubiquitin E1 enzyme, and proteasome subunit Rpn2 was performed with metabolically heavy labeled cells as internal standards (ϩ8.01420 for Lys and ϩ10.00827 for Arg) using a previously reported protocol (36,39). The labeled cells were spiked into transfected cells followed by protein extraction. Sarkosyl-insoluble fractions were resolved on a one-dimensional SDS gel. The gel regions above 80 kDa, which contained the vast majority of polyUb species, were used for in-gel trypsin digestion that produced a pair of light and heavy GG-linked ubiquitin peptides corresponding to every polyUb linkage. Digested peptides were analyzed by the same LC system as above in which peptide ion pairs of interest were selected for fragmentation and quantified by related product ion pairs, a process termed SRM or multiple reaction monitoring. The detailed LC/ SRM parameters are shown in supplemental Table S10.

Expression, Localization, and Biochemical
Properties of Recombinant TDP-43 and TDP-S6 -We cloned and overexpressed HA-tagged (at the N terminus) human TDP-43 and TDP-S6 in HEK-293 cells (Fig. 1). Human TDP-S6 is generated via an additional splicing event within exon 6 and encodes a 295-amino acid protein in which the first 277 amino acids are identical to those of TDP-43 (Fig. 1A). Although mouse TDP-S6 has been shown to have altered nuclear structure in mammalian cells (40), the human TDP-S6 transcript, identified in HEK-293 cells (11), has not yet been characterized. West-ern blotting of total cell lysate prepared from the transfected cells indicated that both recombinant proteins were expressed and recognized by the HA antibody. TDP-43 also displayed two higher molecular mass species at ϳ50 kDa ( Fig. 1, B and C, asterisk). After treatment with an increasing amount of alkaline phosphatase, the upper band disappeared, and the middle band became significantly weaker, whereas the intensity of unmodified TDP-43 remained stable. This supports that TDP-43 is preferentially phosphorylated on the C terminus on at least two different residues (Fig. 1C), consistent with current identification of two phosphorylation sites near the C terminus of TDP-43 in diseased tissues (41,42).
Subcellular localization of full-length TDP-43 and TDP-S6 was assessed by immunofluorescence confocal microscopy. Overexpressed TDP-43 in HEK-293 cells had primarily nuclear localization ( Fig. 2A, upper panel), consistent with that of endogenous TDP-43 in untransfected cells ( Fig. 2A, upper panel, left bottom corner). In contrast, TDP-S6 formed aggregates that were mainly localized to the cytoplasm with some inclusions in the nucleus ( Fig. 2A, middle and bottom panels). Observed puncta were of variable size, ranging from fine granular deposits to large, aggresome-like inclusions. To assess whether the TDP-S6 phenotype observed in HEK-293 cells could be recapitulated in neurons, we transfected primary mouse hippocampal neurons with TDP-43 or TDP-S6 constructs (Fig. 2B). Consistent with results in HEK-293 cells, TDP-43 in neurons was nuclear, whereas the majority of TDP-S6 formed granular deposits in the cytoplasm of the soma and neurites (Fig. 2, B and C). Enolase was stained as a neuron-specific marker.
To evaluate the solubility of overexpressed TDP-43 or TDP-S6, HEK-293 cells were transfected with either construct and then mixed equally to eliminate experimental variability in subsequent processing. The mixed cells were sequentially extracted with Sarkosyl-containing buffer and urea and then analyzed by immunoblotting. The differences in molecular mass between HA-TDP-43 (47 kDa), native TDP-43 (43 kDa), and HA-TDP-S6 (37 kDa) allowed analysis of all three proteins in a single lane (Fig. 3A). In addition, phosphorylated TDP-43 isoforms were also resolved. Although almost all of HA-TDP-S6 was insoluble, 60 Ϯ 7% of unmodified HA-TDP-43 and virtually none of the native unmodified TDP-43 were present in the insoluble fraction (Fig. 3, A and B). The degree of TDP-S6 and TDP-43 enrichment in the insoluble fraction was also repeatable using a second preparation of cells (data not shown). Based on these findings, biochemical insolubility of TDP-S6 is consistent with the inclusions observed by immunofluorescence (Fig. 2). The overexpression of HA-TDP-43 may account for the differences in solubility between the recombinant and native proteins as has been recently reported (43). Interestingly, the phosphorylated species of recombinant HA-TDP-43 (91 Ϯ 4%) and endogenous TDP-43 (44 Ϯ 1%) were more enriched in the insoluble fraction than the unmodified forms, respectively (60 Ϯ 7 and Ͻ5%), supporting previous findings linking TDP-43 phosphorylation with aggregation and insolubility (5). To ensure that overexpression of TDP-43 or TDP-S6 did not dramatically bias the global distribution of proteins to the insoluble fraction, the percentage of total protein in each fraction (detergent-soluble and -insoluble) was calculated from mock-, HA-TDP-43-, and HA- TDP-S6-transfected cells. Approximately 96% of all cellular proteins were detergent-soluble irrespective of the recombinant protein expressed, indicating that overexpression of our target proteins did not cause a gross increase in the total amount of protein within the insoluble fraction.
To further characterize proteins enriched in the Sarkosylinsoluble fraction, we identified proteins in the total cell lysate and in the Sarkosyl-insoluble sample using a label-free LC-MS/MS approach. The relative abundance of proteins in the two samples was compared by spectral counts, and the difference was statistically evaluated by G test (38). Among 1265 proteins profiled (supplemental Table S9), the level of 267 proteins was significantly altered with corresponding p values below 0.01. Listed in Fig. 3C are proteins with large changes, including components associated with the proteasome, caveolae, aggresomes, and a number of nuclear complexes. Specifically, flotillin-1 and -2 are markers for caveolae/ lipid rafts that are highly enriched in cholesterol and are thus more resistant to detergent extraction (44). Vimentin is a protein marker of aggresomes that is often associated with proteasome proteins (45). Finally, particular DNA/RNA-interacting proteins were found to be intrinsically detergent-insoluble. Therefore, the protocol of Sarkosyl-based differential extraction is capable of enriching both aggregated proteins and other detergent-insoluble complexes.
Further analysis of detergent-soluble and insoluble fractions from transfected cells revealed that high molecular mass TDP-43-immunoreactive species were more abundant in fractions corresponding to TDP-S6, rather than TDP-43, overexpression. This observation is consistent with ubiquitination, ubiquitin-like modifications, and/or protein polymerization typically associated with protein insolubility (Fig. 4). To this end, immunoblotting of Sarkosyl-insoluble fractions with a ubiquitin-specific antibody demonstrated an increase in global protein ubiquitination with TDP-43 (Ն250 kDa) and more dramatically with TDP-S6 overexpression (Fig. 4). TDP-S6 also displayed two short TDP-43-immunoreactive species (ϳ30 and ϳ20 kDa) that were not detected by an HA antibody. In addition, a third fragment (ϳ37 kDa) was identified in TDP-S6 urea fractions by both TDP-43 and HA antibodies. Although TDP-S6 lacks the extreme C terminus, the proteolytic fragmentation observed here is in agreement with reported C-terminal TDP-43 fragmentation in FTLD-U cases (19). Full-length TDP-43 was also cleaved but at a much lower level (observed on overexposed images; data not shown). These results show that an increase in phosphorylation, ubiquitination, and proteolysis is related to the insolubility of TDP proteins. Taken together, the microscopic and biochemical data for TDP-S6 in cell culture are highly consistent with that of pathologic TDP-43 observed in disease tissue (5,9,18,19).
Quantitative Analysis of Insoluble TDP-43 and TDP-S6 Proteome Using Multiplex SILAC-As both recombinant TDP-43 and TDP-S6 are highly enriched in detergent-insoluble extracts, we used a multiplex SILAC approach to evaluate proteins that co-enrich in the detergent-insoluble proteome of cells overexpressing TDP-43 or TDP-S6 (46) (Fig. 5A). Human HEK-293 cells were fully labeled with light, medium, or heavy arginine and lysine amino acids. The three labeled cell populations were transfected with HA-TDP-43, HA-TDP-S6, or a mock plasmid. After incubation for 2 days, the cells were harvested, equally mixed, and subjected to sequential protein extraction. Although pooling the labeled cells minimizes experimental variation during protein extraction and subsequent steps in the SILAC analysis, it is possible for biological variation to occur during cell labeling and DNA transfection. Therefore, we performed a second independent analysis as a biological replicate with the labeled isotopes "swapped" in HEK-293 cells prior to transfection (Fig. 5A). The detergentinsoluble extracts were then resolved on an SDS gel, excised into gel bands, digested with trypsin, and analyzed by LC-MS/MS on a high resolution Orbitrap mass spectrometer (Fig.  5B). After database searching and stringent filtering mainly by mass accuracy (Յ10 ppm) and SEQUEST scores (XCorr and ⌬Cn), a total of 2670 proteins that were clustered into 1117 groups were identified and quantified (supplemental Tables S1-S7). The corresponding false discovery rate was calculated to be less than 0.2% according to the target-decoy strategy (33,34).
To evaluate the quality of the quantitative data, we analyzed the results based on the null hypothesis as described previously (35). The protein ratios in all pairwise comparisons were converted into log 2 values, and the resulting histogram of all values was fitted to a Gaussian distribution (Fig. 5C). The majority of proteins fit the curve well, indicating that they did not change under the conditions analyzed. Although the fitted mean (Ϫ0.01) suggested little systematic bias introduced by sample handling (e.g. a slightly different amount of starting cells), the S.D. (0.31) provided a good measure of the magnitude of variations in the analysis. In all six pairwise comparisons of light, medium, and heavy labeling in the two experiments, the value of the S.D. was stable (0.28 Ϯ 0.03; Fig. 5D), indicating that the variations in our SILAC analyses were consistent. Proteins were considered changed if their values fell outside more than 2 S.D. (0.6; ϳ95% confidence interval) and showed consistency in experiments 1 and 2. A large number of proteins were removed when comparing results from experiments 1 and 2, suggesting that the biological replicate is essential to reduce false positives in the SILAC assay. The filtered proteins were further validated by manual examination of paired ion currents in raw files, resulting in the acceptance of six proteins with altered expression (Table I).
Validation and Subcellular Co-localization of SUMO-2/3 and Ubiquitin-The list of proteins with altered levels in the Sarkosyl-insoluble fraction included overexpressed TDP proteins (TDP-43 or TDP-S6), ubiquitin, and SUMO-2/3 (Table I). In gel band 1 (Ͼ180 kDa; Fig. 5B), the MS results (Fig. 6, A and B) indicated that TDP-S6-expressing cells had more TDP in the insoluble proteome compared with TDP-43-expressing cells, consistent with the high molecular mass immunoreactivity in Western blotting (Fig. 4B). As expected, native TDP-43 from mock-transfected cells was not observed at this high molecular mass range. SILAC also confirmed our Western blot results for ubiquitin (Fig. 4B), which increased concomitantly with TDP levels within this molecular mass range. However, although TDP-S6 showed significant ubiquitin enrichment compared with mock-transfected cells when summed over all molecular mass regions (gel bands [1][2][3][4][5], no enrichment was observed for ubiquitin in the TDP-43 insoluble fraction (Table  I). In contrast, the significant increase in total ubiquitin enrichment for TDP-S6 may be a reflection of its aggregation observed in cell culture. Interestingly, SUMO-2/3 was identified as a novel component enriched in both the TDP-43 and TDP-S6 insoluble proteome (summed over all molecular mass regions), suggesting the presence of both polyubiquitination and SUMOylation in the insoluble (Fig. 6C) proteome. The elevation of SUMO-2/3 modifications was correlated to the degree of TDP protein insolubility (Fig. 3), suggesting that insoluble TDP proteins are direct targets of SUMOylation.
To assess whether TDP-43 is covalently modified by SUMO-2/3, we performed a deSUMOylation assay using SENP2 (47). An increasing amount of SENP2 was added to either detergent-soluble or -insoluble samples from cells overexpressing TDP-43. A Western blot with anti-SUMO-2/3 showed a decrease in high molecular mass SUMO-2/3 immunoreactivity with increasing enzyme concentration (Fig. 6D). In addition, three specific bands (ϳ55, ϳ65, and ϳ75 kDa) on the blots were recognized by SUMO-2/3, TDP-43, and HA antibodies. Because unmodified HA-TDP-43 has a molecular mass of ϳ45 kDa and SUMO-2/3 is ϳ10 kDa in size, these bands likely represent mono-, di-, and triSUMOylated forms of TDP-43. The tri-and diSUMOylated HA-TDP-43 showed decreasing signals when incubated with titrated SENP2. In contrast, the monoSUMOylated band was increased at 1 M SENP2, possibly due to processing of tri-and diSUMOylated bands. This increase was appropriately reduced with increased SENP2 concentration (5 M). Thus, HA-TDP-43 is directly modified by SUMO-2/3 in detergent-insoluble fractions.
To assess whether SUMO-2/3 co-localizes with TDP-43 or TDP-S6 in HEK-293 cells, we performed immunofluorescence confocal microscopy (Fig. 7A). Endogenous SUMO-2/3 in untransfected cells was diffusely expressed mainly in the nucleus with some accumulation in nuclear bodies as described previously (48). In cells expressing full-length TDP-43, the majority of endogenous SUMO-2/3 was diffusely localized throughout the nucleus, although co-localization was also observed in certain SUMO-2/3 bodies when TDP-43 expression levels were higher (Fig. 7A, arrowheads). In TDP-S6-expressing cells, SUMO-2/3 was sequestered within TDP-S6 nuclear inclusions. Notably, SUMO-2/3 did not co-localize with cytoplasmic aggregates of TDP-S6. Similar results were obtained in cells co-expressing green fluorescent protein-tagged SUMO-2 and TDP constructs (data not shown). These data strongly support that SUMOylation is associated with TDP protein insolubility and localization in the nuclei but not in the cytoplasm.
Ub is attached to protein substrates in monomeric form or as polymers (polyUb) assembled through the N-terminal amino group and the side chains of all seven lysine residues (Lys-6, Lys-11, Lys-27, Lys-29, Lys-33, Lys-48, and Lys-63). These diverse polyUb structures may regulate downstream signaling specificity and determine the consequence of ubiquitination (36,47). Whereas the Lys-63 polyUb linkage is proposed to mediate nonproteolytic events, including protein trafficking and inclusion formation (49), the Lys-48 linkage and other atypical polyUb linkages mediate proteasomal degradation (36). To elucidate the role of different polyUb linkages with TDP protein aggregates, we examined transfected cells with Lys-48 or Lys-63 polyUb linkage-specific antibodies (29) compared with untransfected cells and found that both linkages were clearly present in the cytoplasmic and nuclear TDP inclusions (Fig. 7, B and C). These results indicate that mixed polyUb chains (Lys-48 and Lys-63) are specifically associated with TDP protein aggregates.
To confirm the presence of Lys-48 and Lys-63 polyUb linkages observed in TDP-S6 cytoplasmic aggregates, we analyzed the levels of polyUb linkages in the Sarkosyl-insoluble proteome by targeted mass spectrometry (36,39). Metabolically heavy labeled proteins were used as internal standards for their unlabeled counterparts, allowing the quantitation of native proteins in each sample. Trypsin cleaves endogenous polyUb chains and their corresponding internal standards to generate linkage-specific peptides tagged with two Gly residues (51). During the LC/SRM analysis, heavy and light peptides co-eluted and were fragmented to produce product ion pairs (Fig. 8, A and B), which were used as surrogates to provide sensitive measurements of relative protein abundance. Among all polyUb linkages, Lys-63 increased ϳ3.3-fold in the insoluble fraction of TDP-S6-expressing cells over that of mock-transfected cells. In addition, Lys-48 link-

TABLE I List of proteins that were altered in cells overexpressing TDP-43 or TDP-S6
The log 2 ratios summed over all molecular mass regions (gel bands [1][2][3][4][5] are shown as the mean of two biological replicates ϮS.E. If the proteins were quantified only in one experiment, the S.E. value was not available. NC, not changed (the measured difference was not statistically significant ages (ϳ2.0-fold), Lys-11 linkages (ϳ1.5-fold), and Lys-29 linkages (ϳ1.3-fold) were also measured in this sample (Fig. 8C), whereas the other four potential linkages mediated by three lysines (Lys-6, Lys-27, and Lys-33) and the N terminus were not detected. In the TDP-S6 insoluble fraction, all measured linkages other than Lys-29 were significantly increased. In comparison, within the TDP-43 insoluble fraction, Lys-63 was the only polyUb linkage significantly increased (ϳ 2.2-fold) over the level in mock-transfected cells. This is in good agreement with the localization of Lys-63-linked polyUb and nuclear TDP-43 bodies by immunofluorescence in HEK-293 cells expressing a high level of TDP-43 (Fig. 7C). These data further support the involvement of multiple polyUb chains in TDP protein aggregation and indicate that the Lys-63 polyUb linkage may be the most significant form associated with TDP-43 or TDP-S6 overexpression.

DISCUSSION
The abnormal accumulation of phosphorylated and ubiquitinated species in protein inclusions is observed in a wide variety of neurodegenerative diseases (3,4). In Alzheimer and Parkinson diseases, these intracellular inclusions are called neurofibrillary tangles and Lewy bodies and are composed of pathologically altered forms of tau and ␣-synuclein, respectively (3,52). In FTLD-U and ALS, TDP-43 has been identified as the major component of tau-and ␣-synuclein-negative inclusions where it is both phosphorylated and ubiquitinated (5) mary neurons. The full-length protein was expressed almost exclusively in the nucleus where it co-localized with endogenous TDP-43. In sharp contrast, the shorter TDP-S6 formed highly insoluble cytoplasmic and nuclear inclusions reminiscent of disease-specific pathology. Moreover, overexpression of both TDP-43 and TDP-S6 resulted in the enrichment of specific post-translational modifications within their respective insoluble protein extracts. These post-translational modifications included ubiquitination, phosphorylation, and TDP-43-specific C-terminal fragmentation. Based on our multiplex SILAC proteomics approach, we identified the significant enrichment of SUMO-2/3 and Ub within the Sarkosyl-insoluble, urea-soluble extracts.
In our cellular model, endogenous Ub showed strong association with the TDP aggregates in both biochemical analysis and immunostaining, supporting the hypothesis that aggregation-prone TDP-43 is a major ubiquitinated species.
Although TDP-S6-expressing cells displayed inclusions of various sizes, only a fraction of recombinant TDP-43-expressing cells formed small nuclear aggregates dependent on high level expression (Fig. 7). Quantitative MS analysis revealed that several main polyUb chains were present in the insoluble extracts from these cells with Lys-63 linkages being the most up-regulated. These findings are reminiscent of previous characterization of tau-and SOD1-positve inclusions (49) and are consistent with the idea that Lys-63-linked chains may facilitate the formation of inclusions and direct subsequent clearance by the autophagy pathway.
Recent studies have suggested that ubiquitinated inclusions in FTLD-U brains are primarily composed of C-terminal fragments of TDP-43 and that these fragments may comprise the primary pathologic species of TDP-43 aggregates by serving as a seed for aggregation (53). Moreover, caspase-3 activation has been implicated in the proteolytic processing of TDP-43 into three C-terminal fragments similar to those seen in disease tissues (54). Although TDP-S6 lacks the final 137 amino acids of full-length TDP-43, the splice variant still contains all three purported caspase-3 cleavage sites at amino acids 10 -13 (DEND), 86 -89 (DETD), and 216 -219 (DVMD), corresponding to the 42-, 35-, and 25-kDa fragments, respectively, in the full-length protein (54). Interestingly, the fragmentation of TDP-S6 resulted in three lower molecular mass species that approximate the molecular mass expected of caspase-3 cleavage products in this HA-tagged construct (37,30, and 20 kDa). These data suggest that TDP-S6 cleavage preferentially occurs in cells and potentially as a result of a caspase-3-mediated mechanism. Alternatively, these fragments may instead be derived from the cleavage of sequestered endogenous TDP-43 in TDP-S6-expressing cells, but further studies using targeted MS analysis will be necessary to validate enzyme-specific TDP-43 cleavage.
Currently, it remains unknown whether aberrant splicing of the TDP-43 transcript results in the production of pathologic TDP isoforms in ALS or FTLD-U. However, two TDP-43 splicing isoforms have been identified previously in human brain and spinal cord tissue (55). One of these isoforms, which closely resembles TDP-S6, was identified in two ALS cases. This novel transcript lacked all of exon 3 and a significant portion of exon 6, resulting in expression of a protein product (ϳ28 kDa) without a glycine-rich C terminus (55). In this study, TDP-S6, a C-terminal truncated splice variant previously identified in HEK-293 cells (11), was found predominately localized to the cytoplasm and mimicked the aggregation observed in FTLD-U and ALS. It should be noted that although the human TDP-S6 variant formed both cytoplasmic and nuclear inclusions, overexpression of the mouse TDP-S6 in HEK-293 cells resulted only in the formation of nuclear specific speckle-like structures, termed TDP bodies (40). Thus, despite sharing 96% amino acid identity, inconsistency between mouse and human TDP-S6 localization may result from differences in species-specific nuclear/cytoplasmic translocation or different experimental conditions. SILAC analysis of this cellular model revealed novel components in the formation of protein inclusions. SUMO-2/3 was found to be preferentially enriched within the TDP-43 and TDP-S6 insoluble proteomes and was subsequently validated by Western blotting. Interestingly, immunofluorescence confocal microscopy indicated that SUMO-2/3 co-localized mainly with TDP-S6 nuclear inclusions, thereby implicating a unique nuclear association between SUMO-2/3 and TDP-S6. This nuclear association is not completely surprising because TDP-43 has been previously found to associate with promyelocytic leukemia bodies (40), nuclear structures known to co-localize with SUMO (27,48). Moreover, sequence analysis of TDP-43 revealed a canonical SUMO conjugation site motif (⌿KX(D/E); where ⌿ indicates hydrophobic and indicate any residue, amino acids 135-138 of TDP-43) among its potential SUMO-targeted lysines (56). Using an in vitro de-SUMOylation assay, we provide experimental evidence that TDP-43 itself is directly SUMOylated within the insoluble fraction. This is also supported by a recent large scale study wherein TDP-43 was identified as a SUMO-2 conjugate that was accumulated over 7-fold in response to heat shock (57). Notably, protein SUMOylation has also been linked to aggregated proteins in Alzheimer disease, Parkinson disease, and poly(Q) disorders (58). In addition, SUMO-1 has been shown to specifically co-localize with ubiquitinated nuclear neuronal inclusions in FTLD-U tissues (59). Although protein SUMOylation functions in regulating transcription and nuclear transport (60), it has also been shown to cross-talk with ubiquitination because the SUMO tag on proteins may serve as a recognition signal for subsequent ubiquitination and proteasome-mediated degradation (61)(62)(63). More specifically, the recent identification of SUMO-targeted ubiquitin ligases (STUbLs) (64) raises the intriguing possibility that upon SUMOylation misfolded nuclear TDP-S6 is subjected to sequestration in nuclear aggregates and STUbL-dependent ubiquitination and degradation (65). Interestingly, a STUbLmediated mechanism for the cellular regulation of insoluble TDP-S6 is supported by the immunochemical co-localization of polyubiquitin chains with both nuclear and cytoplasmic TDP-S6 inclusions (Fig. 7). The mechanism of TDP-43 SUMOylation underlying disease-specific protein translocation, degradation, or aggregation is worth further investigation.
In addition to Ub and SUMO-2/3, actin-related protein 1 (ARP1) was also found to be enriched in the Sarkosyl-insoluble fractions of TDP-43-and TDP-S6-transfected cells. ARP1 is a subunit of dynactin, a macromolecular complex that interacts with both microtubules and cytoplasmic dynein. ARP1 is also involved in protein transport and vesicular trafficking (66). Interestingly, gene mutation in another subunit of dynactin, p150Glued, has been reported in ALS cases (67), and the related mouse model developed motor neuron disease (68). In addition, overexpression of TDP-S6 reduces N-ethylmaleimide sensitive factor, an ATPase involved in intracellular trafficking, and dynamin 3, a microtubule-associated protein involved in vesicle budding (69). These results suggest that protein transportation may be dysregulated in neurodegenerative diseases. Furthermore, these findings, coupled with an increase in Lys-63 polyUb linkages, further support an association between TDP-43 and protein sorting pathways.
In sum, our data show that the C-terminal domain is necessary for TDP-43 nuclear localization and that without this region TDP-43 becomes primarily sequestered within cytoplasmic aggregates, consistent with a previous report showing that the C terminus is essential for the solubility and cellular localization of TDP-43 (50). Thus, it might be plausible that the genetic mutations in TDP-43 found within the Cterminal region (21)(22)(23)(24)(25) somehow disrupt critical amino acid residues needed for TDP-43 nuclear localization and proper function. Although further studies are needed to evaluate the presence of TDP-S6 or other similar short isoforms in disease tissue, our data raise the intriguing possibility that dysregula-tion of human TDP-43 alternative splicing or preferential proteolytic processing to produce TDP N-terminal fragments can contribute to the pathology of FTLD-U and ALS.