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Protective effect of TAT-delivered -synuclein: relevance of the C-terminal domain and involvement of HSP70

Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; and
^* Department of Biochemistry, University of Padova, Padova, Italy

¹Correspondence: Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy. E-mail: forloni{at}marionegri.it

SPECIFIC AIMS

The goal of this work was to investigate the molecular and structural basis of the protective role of -synuclein (-syn) against oxidative stress. We wanted to determine whether -syn intracellular concentration, the Parkinson’s disease (PD) -linked -syn mutations A30P and A53T, and -syn C-terminal domain were able to modulate -syn-mediated neuroprotection. Moreover, we decided to verify whether -syn protective function involved the up-regulation of the chaperone protein HSP70. We developed an in vitro model of oxidative injury in rat PC12 cells by exposing cells to H₂O₂ and tuned -syn intracellular concentration, adding to cells the fusion proteins TAT--syn (WT, A30P, and A53T).

PRINCIPAL FINDINGS

1. Relevance of -syn intracellular concentration for -syn protective action and influence of the -syn pathogenetic mutations A30P and A53T on -syn protective function
To confirm the role of -syn concentration in modulating its function, we incubated PC12 cells for 72 h with increasing amounts (0.1–6 µM) of the fusion proteins TAT--syn (WT, A30P, and A53T) (TAT is the translocation domain of the HIV-1 Tat protein that let the fusion protein cross the plasma membrane) and then we evaluated cellular viability. We found that at a nanomolar concentration (0.1 µM) no aggregation or toxicity occurred, whereas at micromolar concentrations (1.5 up to 6 µM), TAT--syn aggregated (with the appearance of thioflavin-T positive aggregates) and was intrinsically toxic to cells, with a more pronounced toxic effect of the mutated forms than the wild-type form. Consequently, a protective effect of TAT--syn was possible only on a nanomolar scale; in this situation, we challenged undifferentiated PC12 cells with H₂O₂ 150 µM for 72 h. We found that both the wild-type and the mutated forms of -syn were able to protect cells against oxidative stress, with no relevant differences in extent (Fig. 1 A). The protective mechanism mediated by TAT--syn was tested with other toxic injuries (6-hydroxydopamine [6-OH DA], serum deprivation, and the proteasome inhibitors lactacystin and MG132) to assess its specificity. When PC12 cells were challenged with 6-OH DA 100 µM for 24 h, wild-type and mutated forms of -syn protected to the same extent; when PC12 cells were serum deprived for 24 h, only TAT--syn (WT) 0.1 µM protection was complete whereas the mutated forms of TAT--syn protected partially. With the proteasome inhibitors (lactacystin or MG132, both 10 µM for 24 h), TAT--syn (WT) 0.1 µM gave no protection.

View larger version (30K): [in this window] [in a new window]   Figure 1. TAT--syn (WT, A30P, and A53T) 0.1 µM protect PC12 cells from oxidative stress and up-regulate HSP70 protein level. A) Cells were preincubated with TAT--syn (WT, A30P, and A53T) 0.1 µM for 1 h, then challenged with H2O2 150 µM for 72 h. Cell viability was assessed by erythrosine dye exclusion assay (***P<0.001, Tukey’s post hoc test). B) PC12 cells were exposed to NGF (100 ng/mL) for 6 days, then preincubated with TAT--syn (WT) 0.1 µM for 1 h and challenged with H2O2 100 µM for 72 h. At the end of treatment, cell viability was assessed by Hoechst 33258 staining and microscope scoring of at least 5 different microscopic fields at x10 magnification (***P<0.001, **P<0.05; Tukey’s post hoc test). Results are expressed as % of control ±SE (n=10). Similar results were obtained using TAT--syn (A30P and A53T). C)PC12 cells were preincubated with TAT--syn (WT, A30P, and A53T)) 0.1 µM for 1 h, H2O2 150 µM was added for further 24 h and cells were then lysed and analyzed by Western blot. D) PC12 cells were preincubated with TAT--syn (WT) 0.1 µM for 1 h and after further 24 h they were fixed with 4% paraformaldehyde and HSP70 immunoreactivity was assessed. Left: Control cells. Right: TAT--syn (WT) 0.1 µM-treated cells (x20). Similar results were obtained using TAT--syn (A30P and A53T) 0.1 µM.

2. -Synuclein protective action in differentiated PC12 cells and in SK-N-BE cells
The protective function of TAT--syn at nanomolar concentrations was replicated in NGF-differentiated PC12 cells and in human neuroblastoma SK-N-BE cells. NGF-differentiated PC12 cells were preincubated with TAT--syn (WT, A30P, and A53T) for 1 h, then challenged with H₂O₂ 100 µM for 72 h. We verified a relevant increase of cell survival in TAT--syn preincubated cells even if TAT--syn protection was not complete, probably because NGF-differentiated PC12 cells were more sensitive to the toxic effect of oxidative stress than undifferentiated PC12 cells (Fig. 1B ). We preincubated SK-N-BE cells for 1 h with TAT--syn 0.5 µM, then exposed cells to oxidative challenge (H₂O₂ 75 µM for 24 h). We confirmed the presence of a protection mediated by TAT--syn 0.5 µM (data not shown).

3. Effect of TAT--syn 0.1 µM and 1–10 µM on HSP70 protein level
To better identify the molecules involved in -syn-mediated protection against oxidative stress, we evaluated the possible involvement of the chaperone protein HSP70. We found that 25 h after TAT--syn (WT, A30P, and A53T) 0.1 µM addiction to PC12 cells, the HSP70 protein level was increased, and this was confirmed by Western blot and immunocytochemistry (Fig. 1C, D ). In our experimental model, the treatment with H₂O₂ 150 µM alone did not affect the HSP70 basal level, and no evident differences in HSP70 induction were noticeable in treatment with TAT--syn (WT) vs. TAT--syn (A30P and A53T).

To verify the effect of TAT--syn at micromolar scale on HSP70 protein level, we performed other experiments following the above method by adding to PC12 cells TAT--syn (WT) 1 or 10 µM for 25 h, and we then evaluated the HSP70 protein level by Western blot. We found that TAT--syn (WT) decreased HSP70 immunoreactivity. Finally, we checked by RT-PCR whether TAT--syn (WT) 0.1 or 1 µM addiction affected HSP70 transcription; our results did not support this hypothesis.

4. Relevance of -syn C-terminal domain for cellular protection and HSP70 modulation
To check the hypothesis that -syn C-terminal domain was involved in -syn-mediated protection against oxidative stress, we purified the fusion protein TAT--syn (WT[1-97]) lacking -syn C-terminal domain. We at first incubated PC12 cells for 72 h with increasing amounts of TAT--syn (WT[1-97]) (1.5 up to 6 µM). Cell viability was negatively affected and a strong thioflavin-T positive intracellular aggregation occurred (Fig. 2 A). On the contrary, at nanomolar scale (0.1 µM) we found no intrinsic toxicity of TAT--syn (WT[1-97]) (Fig. 2B ). We performed an oxidative challenge to PC12 cells after 1 h preincubation with TAT--syn (WT[1-97]) 0.1 µM. We did not register any protective effect on cell viability, and cellular survival was similar in the presence or absence of TAT--syn (WT[1-97]), whereas TAT--syn (WT) 0.1 µM confirmed its protective action (Fig. 2B ). We decided to estimate whether the deletion of -syn C-terminal domain influenced -syn-mediated HSP70 up-regulation. After 25 h from TAT--syn (WT[1-97]) 0.1 µM treatment, we found no increase of HSP70 protein basal level by Western blot (Fig. 2C ) or immunocytochemistry (data not shown). Finally, we examined whether TAT--syn (WT[1-97]) 1 or 10 µM affected the HSP70 protein level; after 25 h from TAT--syn (WT[1-97]) 1–10 µM treatment, Western blot showed no increase of HSP70 basal level.

View larger version (26K): [in this window] [in a new window]   Figure 2. TAT--syn (WT[1-97]) fails in preventing H2O2-induced toxicity in PC12 cells and it is unable to up-regulate HSP70 at protein level. A) PC12 cells were incubated with TAT--syn (WT[1-97]) at micromolar concentrations for 72 h, then cellular viability was assessed by erythrosine dye exclusion assay (*P<0.05, ***P<0.001; Tukey’s post hoc test). Top: PC12 control cells; bottom: TAT--syn (WT[1-97]) 3 µM-treated PC12 cells, x20. B) PC12 cells were preincubated with TAT--syn (WT) 0.1 µM or TAT--syn (WT[1-97]) 0.1 µM for 1 h prior to H2O2 150 µM exposure for a further 72 h. Cell viability was then assessed by erythrosine dye exclusion assay. (*P<0.03; Tukey’s post hoc test). C) PC12 cells were preincubated for 1 h with TAT--syn (WT[1-97]) 0.1 µM, H202 150 µM was added for another 24 h, then cells were lysed and analyzed for HSP70 immunoreactivity by Western blot.

CONCLUSIONS AND SIGNIFICANCE

Our simple in vitro model of oxidative stress allowed us to better understand the molecular and structural basis of -syn-mediated protective action. First, we confirmed that -syn function is affected by its intracellular concentration. At micromolar concentrations, -syn aggregation occurs, and this is the key event that triggers -syn toxicity; instead, at nanomolar concentrations the protein is neuroprotective. Our data clearly show that -syn-mediated protective function is not directly influenced by the pathogenetic mutations A30P and A53T, that can indirectly affect -syn protective function by promoting -syn aggregation that likely prevents the protein from performing its positive physiological functions. We demonstrated that this -syn-mediated protective action is not restricted to a particular cell line and/or specific for oxidative stress, as we found -syn-mediated protection also against serum deprivation. On the contrary, we did not find any protection against proteasome inhibition, maybe as directly affects some molecular mediators of -syn protection (or -syn itself), counteracting -syn neuroprotection.

We found that the chaperone protein HSP70 can be up-regulated at the protein level by TAT--syn when added to cells at nanomolar concentration; when TAT--syn was added at aggregating concentrations, there was a decrease in HSP70 immunoreactivity, perhaps due to a sequestration of the chaperone in -syn aggregates. The final effect of HSP70 up-regulation when TAT--syn was added at nanomolar scale probably helps strengthen the cellular capability to resist to oxidative stress, eventually promoting cell survival.

Our experiments allowed us to define the protein domain involved in -syn-mediated protection. In fact, -syn C-terminal domain has proved to be essential both for -syn protective action and for -syn-mediated HSP70 up-regulation (and down-regulation), suggesting that this domain directly or indirectly modulates the interactions with HSP70.

In summary, this work clarifies the absence of a direct influence of the A30P and A53T mutations on -syn-mediated protective action against oxidative stress at nanomolar scale. Then we have defined the protein C-terminal domain as essential for mediating -syn protective function and we pointed out a possible molecular partner (HSP70) for a -syn protective role. Our data confirm that the control of -syn intracellular concentration is significant and that alteration of -syn homeostasis (caused by genetic or environmental factors) may be one of the early triggers of the Parkinson’s disease.

View larger version (17K): [in this window] [in a new window]   Figure 3. Schematic representation of the neurotoxic or neuroprotective function of -synuclein (-syn) depending on its intracellular concentration and solubility. At micromolar scale (upper pathway), aggregation followed by cell death takes place. The Parkinson’s disease-linked -syn mutations (A30P and A53T) negatively affect cell viability, speeding up -syn aggregation process; at aggregating concentration, -syn directly or indirectly down-regulates HSP70 level, while -syn C-terminal domain positively regulates the protein solubility. At nanomolar scale (lower pathway), no aggregation occurs and -syn can perform a neuroprotective function, probably thanks to an increase of HSP70 protein level and to the involvement of other protective molecular intermediates. While the C-terminal domain of the protein (that is required for -syn chaperone-like action) is essential for this protective function, the presence of the pathogenetic mutations A30P and A53T has no direct influence on -syn-mediated protection.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1621fje

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This Article Full Text (PDF) All Versions of this Article: 18/14/1713 04-1621fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ALBANI, D. Articles by FORLONI, G. Search for Related Content PubMed PubMed Citation Articles by ALBANI, D. Articles by FORLONI, G.