PINK1 Knockout Cell Line (HEK293)

AIMS:PTEN induced putative kinase 1 (PINK1)-mediated mitophagy process is tightly associated with various age-dependent diseases in mammals. The roles of miRNAs (miRNAs) in the PINK1-mediated mitophagy process are not fully understood. Here we discovered that miR-34a-5p suppresses PINK1 expression directly though two post-transcriptional non-classical binding modes, resulting in inhibition of PINK1-mediated mitophagy process. MAIN METHODS:For in vivo experiments, brains were dissected from 8 weeks old and 40 weeks old C57BL/6 male mice to measure miR-34a-5p expression and PINK1 expression. For in vitro experiments, overexpression of miR-34a-5p mimics in HEK293 cells was performed to investigate the effect of miR-34a-5p on PINK1 expression and its regulatory mechanism, parkin recruitment and mitophagy process. KEY FINDINGS:The level of miR-34a-5p was upregulated and the level of PINK1 mRNA was downregulated in brains of aged mice. Both the 3'-untranslated region (3'UTR) and the Coding DNA sequence (CDS) of PINK1 mRNA were bound to the non-seed region of miR-34a-5p, rather than the seed region, resulting in a decrease in PINK1 expression. Endogenous miR-34a-5p knockout increased PINK1 expression. Further results indicated that miR-34a-5p inhibits mitophagy process by reduction of PINK1. miR-34a-5p hinders phosphorylated Ser65-ubiquitin (pS65-Ub) accumulation, prevents the mitochondrial recruitment of Parkin, attenuates ubiquitination and delays the clearance of damaged mitochondria. SIGNIFICANCE:We firstly found that miR-34a-5p suppresses PINK1 directly and further regulates mitophagy through non-canonical modes. This finding hints at a crucial role of miR-34a-5p implicated in accelerating the pathogenesis of age-related neurological diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disorder marked by deteriorating dyspnea and declining pulmonary function. Despite its rising prevalence and incidence, therapeutic options remain limited. PTEN-induced kinase 1 (PINK1), known for its role in PINK1/Parkin-dependent mitophagy, contributes to the pathogenesis of various lung diseases. In this study, we elucidate a previously unrecognized mechanism of PINK1, beyond its canonical mitophagy function, during pulmonary fibrosis. We established a bleomycin (BLM)-induced pulmonary fibrosis model in Pink1 knockout (Pink1) mice and treated BEAS-2B cells with transforming growth factor-beta 1 (TGF-β1) to simulate the microenvironment of pulmonary fibrosis. A significant elevation in PINK1 expression was observed in vivo and in vitro systems. While PINK1/Parkin-dependent mitophagy was activated, mitophagy mediated by BCL2-interacting protein 3 (BNIP3) and FUN14 domain-containing 1 (FUNDC1) was suppressed. Further experiments in carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated PINK1 knockout (KO) HEK293 cells and YFP-Parkin-expressing HeLa cells demonstrated that PINK1 deficiency enhanced BNIP3- and FUNDC1-mediated mitophagy, whereas PINK1 overexpression inhibited it. Moreover, dual BNIP3/FUNDC1 knockdown significantly reversed the anti-apoptotic effect of PINK1 KO. We conclude that PINK1 deficiency promotes the clearance of damaged mitochondria via BNIP3/FUNDC1 upregulation, preserving mitochondrial homeostasis, mitigating alveolar epithelial injury, and attenuating fibrosis. Thus, PINK1 may inhibit BNIP3- and FUNDC1-mediated mitophagy besides driving PINK1-dependent mitophagy during pulmonary fibrosis.

Mutations in PINK1 (PTEN-induced putative kinase 1) are associated with autosomal recessive early-onset Parkinson's disease. Full-length PINK1 (PINK1-l) has been extensively studied in mitophagy; however, the functions of the short form of PINK1 (PINK1-s) remain poorly understood. Here, we report that PINK1-s is recruited to ribosome fractions after short-term inhibition of proteasomes. The expression of PINK1-s greatly inhibits protein synthesis even without proteasomal stress. Mechanistically, PINK1-s phosphorylates the translation elongation factor eEF1A1 during proteasome inhibition. The expression of the phosphorylation mimic mutation eEF1A1S396E rescues protein synthesis defects and cell viability caused by PINK1 knockout. These findings implicate an important role for PINK1-s in protecting cells against proteasome stress through inhibiting protein synthesis.

Ubiquitin (Ub), a central regulator of protein turnover, can be phosphorylated by PINK1 (PTEN-induced putative kinase 1) to generate S65-phosphorylated ubiquitin (pUb). Elevated pUb levels have been observed in aged human brains and in Parkinson's disease, but the mechanistic link between pUb elevation and neurodegeneration remains unclear. Here, we demonstrate that pUb elevation is a common feature under neurodegenerative conditions, including Alzheimer's disease, aging, and ischemic injury. We show that impaired proteasomal activity leads to the accumulation of sPINK1, the cytosolic form of PINK1 that is normally proteasome-degraded rapidly. This accumulation increases ubiquitin phosphorylation, which then inhibits ubiquitin-dependent proteasomal activity by interfering with both ubiquitin chain elongation and proteasome-substrate interactions. Specific expression of sPINK1 in mouse hippocampal neurons induced progressive pUb accumulation, accompanied by protein aggregation, proteostasis disruption, neuronal injury, neuroinflammation, and cognitive decline. Conversely, Pink1 knockout mitigated protein aggregation in both mouse brains and HEK293 cells. Furthermore, the detrimental effects of sPINK1 could be counteracted by co-expressing Ub/S65A phospho-null mutant but exacerbated by over-expressing Ub/S65E phospho-mimic mutant. Together, these findings reveal that pUb elevation, triggered by reduced proteasomal activity, inhibits proteasomal activity and forms a feedforward loop that drives progressive neurodegeneration.