bims-tofagi 2024-06-02 papers

Cell Rep. 2024 May 29. pii: S2211-1247(24)00622-3. [Epub ahead of print]43(6): 114294

TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.

Bhaskar Saha, Hallvard Olsvik, Geneva L Williams, Seeun Oh, Gry Evjen, Eva Sjøttem, Michael A Mandell.

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α and is required for TBK1 to interact with and activate a set of ubiquitin-binding autophagy adaptors including NDP52, p62/SQSTM1, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1 following mitochondrial damage. TRIM5α's ubiquitin ligase activity is required for the accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Our data support a model in which TRIM5α provides a mitochondria-localized, ubiquitin-based, self-amplifying assembly platform for TBK1 and mitophagy adaptors that is ultimately necessary for the recruitment of the core autophagy machinery.

Keywords: CP: Cell biology; NBR1; NDP52; Optineurin; TAX1BP1; TBK1; TRIM27/RFP; autophagy; p62; tripartite motif; ubiquitin ligase

DOI: https://doi.org/10.1016/j.celrep.2024.114294

J Mol Biol. 2024 May 29. pii: S0022-2836(24)00226-2. [Epub ahead of print] 168631

FIP200 phosphorylation regulates late steps in mitophagy.

Christopher Eickhorst, Riccardo Babic, Jorrell Rush-Kittle, Leon Lucya, Fatimah Lami Imam, Pablo Sánchez-Martín, David M Hollenstein, Jonas Michaelis, Christian Münch, Chris Meisinger, Dea Slade, Laura Gámez-Díaz, Claudine Kraft.

Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200's interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.

Keywords: ATG16L1; Atg1/ULK1 kinase complex; FIP200; autophagy; mitophagy

DOI: https://doi.org/10.1016/j.jmb.2024.168631

Free Radic Biol Med. 2024 May 28. pii: S0891-5849(24)00498-2. [Epub ahead of print]221 235-244

GSK-3α-BNIP3 axis promotes mitophagy in human cardiomyocytes under hypoxia.

Hezlin Marzook, Anamika Gupta, Manju N Jayakumar, Mohamed A Saleh, Dhanendra Tomar, Rizwan Qaisar, Firdos Ahmad.

Dysregulated autophagy/mitophagy is one of the major causes of cardiac injury in ischemic conditions. Glycogen synthase kinase-3alpha (GSK-3α) has been shown to play a crucial role in the pathophysiology of cardiac diseases. However, the precise role of GSK-3α in cardiac mitophagy remains unknown. Herein, we investigated the role of GSK-3α in cardiac mitophagy by employing AC16 human cardiomyocytes under the condition of acute hypoxia. We observed that the gain-of-GSK-3α function profoundly induced mitophagy in the AC16 cardiomyocytes post-hypoxia. Moreover, GSK-3α overexpression led to increased ROS generation and mitochondrial dysfunction in cardiomyocytes, accompanied by enhanced mitophagy displayed by increased mt-mKeima intensity under hypoxia. Mechanistically, we identified that GSK-3α promotes mitophagy through upregulation of BNIP3, caused by GSK-3α-mediated increase in expression of HIF-1α and FOXO3a in cardiomyocytes post-hypoxia. Moreover, GSK-3α displayed a physical interaction with BNIP3 and, inhibited PINK1 and Parkin recruitment to mitochondria was observed specifically under hypoxia. Taken together, we identified a novel mechanism of mitophagy in human cardiomyocytes. GSK-3α promotes mitochondrial dysfunction and regulates FOXO3a -mediated BNIP3 overexpression in cardiomyocytes to facilitate mitophagy following hypoxia. An interaction between GSK-3α and BNIP3 suggests a role of GSK-3α in BNIP3 recruitment to the mitochondrial membrane where it enhances mitophagy in stressed cardiomyocytes independent of the PINK1/Parkin.

Keywords: BNIP3; FOXO3a; GSK-3alpha; Mitophagy; PINK1; Parkin

DOI: https://doi.org/10.1016/j.freeradbiomed.2024.05.041

Autophagy. 2024 May 31.

Atg44/Mdi1/mitofissin facilitates Dnm1-mediated mitochondrial fission.

Kentaro Furukawa, Manabu Hayatsu, Kentaro Okuyama, Tomoyuki Fukuda, Shun-Ichi Yamashita, Keiichi Inoue, Shinsuke Shibata, Tomotake Kanki.

Mitochondria undergo fission and fusion, and their coordinated balance is crucial for maintaining mitochondrial homeostasis. In yeast, the dynamin-related protein Dnm1 is a mitochondrial fission factor acting from outside the mitochondria. We recently reported the mitochondrial intermembrane space protein Atg44/mitofissin/Mdi1/Mco8 as a novel fission factor, but the relationship between Atg44 and Dnm1 remains elusive. Here, we show that Atg44 is required to complete Dnm1-mediated mitochondrial fission under homeostatic conditions. Atg44-deficient cells often exhibit enlarged mitochondria with accumulated Dnm1 and rosary-like mitochondria with Dnm1 foci at constriction sites. These mitochondrial constriction sites retain the continuity of both the outer and inner membranes within an extremely confined space, indicating that Dnm1 is unable to complete mitochondrial fission without Atg44. Moreover, accumulated Atg44 proteins are observed at mitochondrial constriction sites. These findings suggest that Atg44 and Dnm1 cooperatively execute mitochondrial fission from inside and outside the mitochondria, respectively.

Keywords: Atg44; Dnm1; mitochondrial fission; mitofissin; mitophagy; yeast

DOI: https://doi.org/10.1080/15548627.2024.2360345

Autophagy. 2024 May 27. 1-16

Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue.

Jens O Watzlawik, Xu Hou, Tyrique Richardson, Szymon L Lewicki, Joanna Siuda, Zbigniew K Wszolek, Casey N Cook, Leonard Petrucelli, Michael DeTure, Dennis W Dickson, Odetta Antico, Miratul M K Muqit, Jordan B Fishman, Karima Pirani, Ravindran Kumaran, Nicole K Polinski, Fabienne C Fiesel, Wolfdieter Springer.

The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens.Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.

Keywords: Autophagy; PINK1; Parkinson disease; mitochondria; mitophagy; ubiquitin

DOI: https://doi.org/10.1080/15548627.2024.2356490

Front Neurosci. 2024 ;18 1390215

PINK1 knockout rats show premotor cognitive deficits measured through a complex maze.

Isabel Soto, Vicki A Nejtek, David P Siderovski, Michael F Salvatore.

Cognitive decline in Parkinson's disease (PD) is a critical premotor sign that may occur in approximately 40% of PD patients up to 10 years prior to clinical recognition and diagnosis. Delineating the mechanisms and specific behavioral signs of cognitive decline associated with PD prior to motor impairment is a critical unmet need. Rodent PD models that have an impairment in a cognitive phenotype for a time period sufficiently long enough prior to motor decline can be useful to establish viable candidate mechanisms. Arguably, the methods used to evaluate cognitive decline in rodent models should emulate methods used in the assessment of humans to optimize translation. Premotor cognitive decline in human PD can potentially be examined in the genetically altered PINK1-/- rat model, which exhibits a protracted onset of motor decline in most studies. To increase translation to cognitive assessment in human PD, we used a modified non-water multiple T-maze, which assesses attention, cognitive flexibility, and working memory similarly to the Trail Making Test (TMT) in humans. Similar to the deficiencies revealed in TMT test outcomes in human PD, 4-month-old PINK1-/- rats made more errors and took longer to complete the maze, despite a hyperkinetic phenotype, compared to wild-type rats. Thus, we have identified a potential methodological tool with cross-species translation to evaluate executive functioning in an established PD rat model.

Keywords: PINK1 knockout rat; Parkinson’s disease; cognitive; locomotor; maze; prodromal; trail-making test

DOI: https://doi.org/10.3389/fnins.2024.1390215