LAUSR

Dear Editor, The loss of PINK1/Parkin-dependent mitochondrial clearance causes loss of dopaminergic neurons in the substantia nigra and contributes to the pathogenesis of Parkinson’s disease (PD). Several kinases were reported to regulate the ubiquitin E3 ligase activity of Parkin through phosphorylation but the involvement of protein tyrosine phosphatase (PTPase) for Parkin activity remains elusive. Although the roles of Src homology 2 domain-containing tyrosine phosphatase-2 (SHP2) in development, hematopoiesis and cancer immunology have been intensively reported, knowledge of regulation and function of SHP2 in neuronal diseases remains scant. We previously showed that SHP2 maintains mitochondrial homeostasis through dephosphorylating ANT1 at Tyr-191 during NLRP3 inflammasome activation in macrophages. This previous study prompted us to investigate whether SHP2 regulates mitophagy and mitochondrial quality in neurons and, if so, whether targeting SHP2 could be a novel strategy for neuronal protection in PD. As shown in Fig. 1a, b and Supplementary Fig. 1a–b, CCCPinduced mitochondria ubiquitination, reduction of mitochondrial mass as well as TOM20 ubiquitination and degradation were attenuated by SHP2 knockdown. Mitophagic flux examined by mtKeima also suggests that SHP2 positively regulates mitophagy (Fig. 1c, d, Supplementary Fig. 1c–f). Next, the mitochondrial translocation of Parkin and TOM20 degradation was remarkably decreased after SHP2 knockdown (Fig. 1e, Supplementary Fig. 2a–b). Parkin ubiquitination induced by CCCP treatment was also significantly inhibited by SHP2 knockdown and augmented after SHP2 overexpression (Fig. 1f, Supplementary Fig. 2c). Coimmunoprecipitation assay showed that both endogenous SHP2 (Fig. 1g) and exogenously SHP2 interacted with Parkin (Supplementary Fig. 3a). SHP2 and Parkin colocalization in the mitochondria was validated by Structured Illumination Microscopy (SIM) and immunoblot of mitochondrial and cytosolic fractions (Supplementary Fig. 3b–d). The PTP domain of SHP2 was shown to interact with Parkin (Supplementary Fig. 3e). Moreover, the interaction of SHP2 and Parkin as well as its involvement in mitophagy was also confirmed in primary neuron cells (Supplementary Fig. 4). These findings demonstrate that SHP2-Parkin interaction is required for Parkin-mediated mitophagy. Since the major activity of SHP2 relied on its PTPase activity, a PTPase gain-of-function SHP2 mutant (SHP2-D61A) and a loss-offunction SHP2 mutant (SHP2-C459S) were overexpressed in HeLa cells together with EGFP-Parkin. Mitochondrial translocation of LC3B was remarkably suppressed by SHP2-C459S and promoted by SHP2-D61A (Supplementary Fig. 5a). TOM20 clearance was evidently decreased by the overexpression of SHP2-D61A compared to vector control or SHP2-C459S (Fig. 1h, Supplementary Fig. 5b). These results suggest that the PTPase activity of SHP2 is indispensable for mitophagy regulation. Since it functions as a PTPase, SHP2 might regulate Parkin activity through the Tyr dephosphorylation of Parkin. Consistent with a previous report, the Ser phosphorylation of Parkin was enhanced upon CCCP treatment. However, in contrast, the Tyr phosphorylation of Parkin was reduced (Supplementary Fig. 5c). Noticeably, SHP2 knockdown abolished these changes in Ser and Tyr phosphorylation (Supplementary Fig. 5d). By using the purified SHP2 and Parkin, we confirmed that SHP2 could directly bind and dephosphorylate tyrosine of Parkin (Supplementary Fig. 5e–h). Next, we wondered whether SHP2 could be a potential target to boost Parkin-mediated mitophagy. A SHP2 enzyme activity screen system was employed for identifying compounds that could promote the catalytic activity of SHP2 (Supplementary Fig. 6a). A lactone ring structure-containing compound library (Chengdu, Biopurify) was screened and lovastatin was found to be able to elevate SHP2 activity both in a cell-free system and in cells (Fig. 1i, Supplementary Fig. 6b–c). A surface plasmon resonance (SPR) assay confirmed their interaction (KD= 36 μM, Fig. 1j), which was further demonstrated by the increased thermal stabilization of SHP2 (Supplementary Fig. 6d–e). To examine whether lovastatin could protect mitochondria in neurons, rotenone was utilized to mimic the pathological conditions of mitochondria in PD. Lovastatin dose-dependently increased cell viability in rotenone-treated SH-SY5Y cells (Supplementary Fig. 7a). ROS generation and mitochondrial membrane potential collapse were also suppressed by lovastatin (Supplementary Fig. 7b–c). Elevated mitophagy levels in the cells were also evidenced by red fluorescence from mt-Keima in cells treated with lovastatin (Supplementary Fig. 7d). Finally, as shown in Fig. 1k for the transmission electron microscopy (TEM) images, rotenone treatment led to significant mitochondria swollen and cristae disruption. In the lovastatin-treated group, the damaged mitochondria localized near a lysosome in the autophagolysosome and were surrounded by a double membrane, suggesting that damaged mitochondrion was removed via mitophagy. To clarify the relationship between SHP2 enzyme activity and the ability of lovastatin to promote mitophagy, SHP2 localization after lovastatin treatment was examined. SHP2 as well as Parkin translocation to mitochondria was increased after lovastatin treatment (Supplementary Fig. 8a–c). Furthermore, lovastatin treatment triggered mitochondrial protein degradation as well as Parkin ubiquitination (Supplementary Fig. 9a–b). A significant interaction between SHP2 and Parkin was observed after lovastatin treatment (Fig. 1l, Supplementary Fig. 9c). However, its ability to promote mitochondrial protein clearance and the neuroprotective effect of lovastatin was reversed after SHP2 knockdown (Supplementary Fig. 9d–e). To assess the protective effect of lovastatin on PD, a 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD murine model (MPTP-PD) was employed. Lovastatin and levodopa were administered as depicted in Supplementary Fig. 10a-b, and a