bims-nenemi 2022-10-30 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2022–10–30
fourteen papers selected by
Marco Tigano, Thomas Jefferson University

Targeting Mitochondrial Function with Chemoptogenetics.
Increased Mobile Zinc Regulates Retinal Ganglion Cell Survival via Activating Mitochondrial OMA1 and Integrated Stress Response.
Human mtRF1 terminates COX1 translation and its ablation induces mitochondrial ribosome-associated quality control.
Visualization of BOK pores independent of BAX and BAK reveals a similar mechanism with differing regulation.
Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish.
The RIG-I receptor adopts two different conformations for distinguishing host from viral RNA ligands.
The Efficient Antiviral Response of A549 Cells Is Enhanced When Mitochondrial Respiration Is Promoted.
SHP-1 knockdown suppresses mitochondrial biogenesis and aggravates mitochondria-dependent apoptosis induced by all trans retinal through the STING/AMPK pathways.
The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization.
Tolerance to replication stress requires Dun1p kinase and activation of the electron transport chain.
Cellular senescence is immunogenic and promotes anti-tumor immunity.
Mitochondrial double-stranded RNAs as a pivotal mediator in the pathogenesis of Sjӧgren's syndrome.
Melatonin suppresses the antiviral immune response to EMCV infection through intracellular ATP deprivation caused by mitochondrial fragmentation.
Pooled image-base screening of mitochondria with microraft isolation distinguishes pathogenic mitofusin 2 mutations.

Biomedicines. 2022 Oct 01. pii: 2459. [Epub ahead of print]10(10):

Targeting Mitochondrial Function with Chemoptogenetics.

Amy Romesberg, Bennett Van Houten.

  Mitochondria are ATP-generating organelles in eukaryotic cells that produce reactive oxygen species (ROS) during oxidative phosphorylation (OXPHOS). Mitochondrial DNA (mtDNA) is packaged within nucleoids and, due to its close proximity to ROS production, endures oxidative base damage. This damage can be repaired by base excision repair (BER) within the mitochondria, or it can be degraded via exonucleases or mitophagy. Persistent mtDNA damage may drive the production of dysfunctional OXPHOS components that generate increased ROS, or OXPHOS components may be directly damaged by ROS, which then can cause more mtDNA damage and create a vicious cycle of ROS production and mitochondrial dysfunction. If mtDNA damage is left unrepaired, mtDNA mutations including deletions can result. The accumulation of mtDNA mutations has been associated with conditions ranging from the aging process to cancer and neurodegenerative conditions, but the sequence of events leading to mtDNA mutations and deletions is yet unknown. Researchers have utilized many systems and agents for generating ROS in mitochondria to observe the downstream effects on mtDNA, ROS, and mitochondrial function; yet, there are various drawbacks to these methodologies that limit their precision. Here, we describe a novel chemoptogenetic approach to target oxidative damage to mitochondria and mtDNA with a high spatial and temporal resolution so that the downstream effects of ROS-induced damage can be measured with a high precision in order to better understand the mechanism of mitochondrial dysfunction in aging, cancer, and neurodegenerative diseases.

Keywords:  base excision repair; chemoptogenetics; mitochondria; mitochondrial DNA; mitochondrial dysfunction; reactive oxygen species

DOI:  https://doi.org/10.3390/biomedicines10102459
Antioxidants (Basel). 2022 Oct 10. pii: 2001. [Epub ahead of print]11(10):

Increased Mobile Zinc Regulates Retinal Ganglion Cell Survival via Activating Mitochondrial OMA1 and Integrated Stress Response.

Jiahui Tang, Zhe Liu, Jiaxu Han, Jingfei Xue, Liyan Liu, Jicheng Lin, Caiqing Wu, Qi Zhang, Siting Wu, Canying Liu, Haishun Huang, Yuanyuan Fu, Min Li, Yehong Zhuo, Yiqing Li.

  Retinal ganglion cells (RGCs), the projection neurons of the eye, are irreversibly lost once the optic nerve is injured, which is a critical mechanism of glaucoma. Mobile zinc (Zn2+) levels rapidly increase in retinal interneuron amacrine cells and Zn2+ is then transferred to RGCs via the Zn2+ transporter protein ZnT-3, triggering RGC loss in optic nerve injury. Zn2+ chelation and ZnT-3 deletion promote long-term RGC survival. However, the downstream signaling pathways of Zn2+ in RGCs remains unknown. Here, we show that increased levels of Zn2+ upregulate the expression and activity of mitochondrial zinc metallopeptidase OMA1 in the retina, leading to the cleavage of DELE1 and activation of cytosolic eIF2α kinase PKR, triggering the integrated stress response (ISR) in RGCs. Our study identified OMA1 and ISR as the downstream molecular mechanisms of retinal Zn2+ and potential targets for preventing the progression of Zn2+-associated neuronal damage.

Keywords:  ISR; mitochondria; optic nerve injury; retinal ganglion cells; zinc

DOI:  https://doi.org/10.3390/antiox11102001
Nat Commun. 2022 Oct 27. 13(1): 6406

Human mtRF1 terminates COX1 translation and its ablation induces mitochondrial ribosome-associated quality control.

Franziska Nadler, Elena Lavdovskaia, Angelique Krempler, Luis Daniel Cruz-Zaragoza, Sven Dennerlein, Ricarda Richter-Dennerlein.

Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We address this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for decades, and lead to a comprehensive picture of translation termination in human mitochondria.

DOI: https://doi.org/10.1038/s41467-022-34088-w
Cell Death Differ. 2022 Oct 26.

Visualization of BOK pores independent of BAX and BAK reveals a similar mechanism with differing regulation.

Raed Shalaby, Arzoo Diwan, Hector Flores-Romero, Vanessa Hertlein, Ana J Garcia-Saez.

BOK is a poorly understood member of the BCL-2 family of proteins that has been proposed to function as a pro-apoptotic, BAX-like effector. However, the molecular mechanism and structural properties of BOK pores remain enigmatic. Here, we show that the thermal stability and pore activity of BOK depends on the presence of its C-terminus as well as on the mitochondrial lipid cardiolipin. We directly visualized BOK pores in liposomes by electron microscopy, which appeared similar to those induced by BAX, in line with comparable oligomerization properties quantified by single molecule imaging. In addition, super-resolution STED imaging revealed that BOK organized into dots and ring-shaped assemblies in apoptotic mitochondria, also reminiscent of those found for BAX and BAK. Yet, unlike BAX and BAK, the apoptotic activity of BOK was limited by partial mitochondrial localization and was independent of and unaffected by other BCL-2 proteins. These results suggest that, while BOK activity is kept in check by subcellular localization instead of interaction with BCL-2 family members, the resulting pores are structurally similar to those of BAX and BAK.

DOI: https://doi.org/10.1038/s41418-022-01078-w
Life Sci Alliance. 2023 Jan;pii: e202201514. [Epub ahead of print]6(1):

Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish.

Barbara Uszczynska-Ratajczak, Sreedevi Sugunan, Monika Kwiatkowska, Maciej Migdal, Silvia Carbonell-Sala, Anna Sokol, Cecilia L Winata, Agnieszka Chacinska.

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a -/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to use localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

DOI: https://doi.org/10.26508/lsa.202201514
Mol Cell. 2022 Oct 13. pii: S1097-2765(22)00950-9. [Epub ahead of print]

The RIG-I receptor adopts two different conformations for distinguishing host from viral RNA ligands.

Wenshuai Wang, Anna Marie Pyle.

  RIG-I is an essential innate immune receptor for detecting and responding to infection by RNA viruses. RIG-I specifically recognizes the unique molecular features of viral RNA molecules and selectively distinguishes them from closely related RNAs abundant in host cells. The physical basis for this exquisite selectivity is revealed through a series of high-resolution cryo-EM structures of RIG-I in complex with host and viral RNA ligands. These studies demonstrate that RIG-I actively samples double-stranded RNAs in the cytoplasm and distinguishes them by adopting two different types of protein folds. Upon binding viral RNA, RIG-I adopts a high-affinity conformation that is conducive to signaling, while host RNA induces an autoinhibited conformation that stimulates RNA release. By coupling protein folding with RNA binding selectivity, RIG-I distinguishes RNA molecules that differ by as little as one phosphate group, thereby explaining the molecular basis for selective antiviral sensing and the induction of autoimmunity upon RIG-I dysregulation.

Keywords:  Cryo-EM structure; PRR; RIG-I receptor; antiviral; autoimmunity; host-pathogen; innate immunity; interferon; pattern recognition receptor; unfolded and well folded conformations

DOI:  https://doi.org/10.1016/j.molcel.2022.09.029
Pathogens. 2022 Oct 11. pii: 1168. [Epub ahead of print]11(10):

The Efficient Antiviral Response of A549 Cells Is Enhanced When Mitochondrial Respiration Is Promoted.

Grégorie Lebeau, Daed El Safadi, Aurélie Paulo-Ramos, Mathilde Hoareau, Philippe Desprès, Pascale Krejbich-Trotot, Florian Chouchou, Marjolaine Roche, Wildriss Viranaicken.

  When exposed to a viral infection, the attacked cells promptly set up defense mechanisms. As part of the antiviral responses, the innate immune interferon pathway and associated interferon-stimulated genes notably allow the production of proteins bearing antiviral activity. Numerous viruses are able to evade the interferon response, highlighting the importance of controlling this pathway to ensure their efficient replication. Several viruses are also known to manipulate the metabolism of infected cells to optimize the availability of amino acids, nucleotides, and lipids. They then benefit from a reprogramming of the metabolism that favors glycolysis instead of mitochondrial respiration. Given the increasingly discussed crosstalk between metabolism and innate immunity, we wondered whether this switch from glycolysis to mitochondrial respiration would be beneficial or deleterious for an efficient antiviral response. We used a cell-based model of metabolic reprogramming. Interestingly, we showed that increased mitochondrial respiration was associated with an enhanced interferon response following polyriboinosinic:polyribocytidylic acid (poly:IC) stimulation. This suggests that during viral infection, the metabolic reprogramming towards glycolysis is also part of the virus' strategies to inhibit the antiviral response.

Keywords:  ISGs; OXPHOS; antiviral response; metabolic reprogramming; mitochondrial respiration

DOI:  https://doi.org/10.3390/pathogens11101168
Mol Med. 2022 Oct 22. 28(1): 125

SHP-1 knockdown suppresses mitochondrial biogenesis and aggravates mitochondria-dependent apoptosis induced by all trans retinal through the STING/AMPK pathways.

Xiaonan Zhuang, Jun Ma, Gezhi Xu, Zhongcui Sun.

   BACKGROUND: Oxidative stress-caused damage to the retinal pigment epithelium (RPE) underlies the onset and progression of age-related macular degeneration (AMD). Impaired mitochondrial biogenesis sensitizes RPE cells to mitochondrial dysfunction, energy insufficiency and death. Src-homology 2 domain-containing phosphatase (SHP)-1 is important in regulating immune responses and cell survival. However, its roles in cell survival are not always consistent. Until now, the effects of SHP-1 on RPE dysfunction, especially mitochondrial homeostasis, remain to be elucidated. We sought to clarify the effects of SHP-1 in RPE cells in response to atRAL-induced oxidative stress and determine the regulatory mechanisms involved.
METHODS: In the all trans retinal (atRAL)-induced oxidative stress model, we used the vector of lentivirus to knockdown the expression of SHP-1 in ARPE-19 cells. CCK-8 assay, Annexin V/PI staining and JC-1 staining were utilized to determine the cell viability, cell apoptosis and mitochondrial membrane potential. We also used immunoprecipitation to examine the ubiquitination modification of stimulator of interferon genes (STING) and its interaction with SHP-1. The expression levels of mitochondrial marker, proteins related to mitochondrial biogenesis, and signaling molecules involved were examined by western blotting analysis.
RESULTS: We found that SHP-1 knockdown predisposed RPE cells to apoptosis, aggravated mitochondrial damage, and repressed mitochondrial biogenesis after treatment with atRAL. Immunofluoresent staining and immunoprecipitation analysis confirmed that SHP-1 interacted with the endoplasmic reticulum-resident STING and suppressed K63-linked ubiquitination and activation of STING. Inhibition of STING with the specific antagonist H151 attenuated the effects of SHP-1 knockdown on mitochondrial biogenesis and oxidative damage. The adenosine monophosphate-activated protein kinase (AMPK) pathway acted as the crucial downstream target of STING and was involved in the regulatory processes.
CONCLUSIONS: These findings suggest that SHP-1 knockdown potentiates STING overactivation and represses mitochondrial biogenesis and cell survival, at least in part by blocking the AMPK pathway in RPE cells. Therefore, restoring mitochondrial health by regulating SHP-1 in RPE cells may be a potential therapeutic strategy for degenerative retinal diseases including AMD.

Keywords:  Adenosine monophosphate-activated protein kinase; Age-related macular degeneration; All trans retinal; Mitochondrial biogenesis; Retinal pigment epithelium; Src-homology 2 domain-containing phosphatase-1; Stimulator of interferon genes

DOI:  https://doi.org/10.1186/s10020-022-00554-w
Cell Death Dis. 2022 Oct 26. 13(10): 899

The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization.

Quanfu Li, Yunkai Chu, Shengze Li, Liping Yu, Huayun Deng, Chunhua Liao, Xiaodong Liao, Chihyu Yang, Min Qi, Jinke Cheng, Guoqiang Chen, Lei Huang.

Mitophagy is a vital process that controls mitochondria quality, dysregulation of which can promote cancer. Oncoprotein mucin 1 (MUC1) targets mitochondria to attenuate drug-induced apoptosis. However, little is known about whether and how MUC1 contributes to mitochondrial homeostasis in cancer cells. We identified a novel role of MUC1 in promoting mitophagy. Increased mitophagy is coupled with the translocation of MUC1 to mitochondria, where MUC1 interacts with and induces degradation of ATPase family AAA domain-containing 3A (ATAD3A), resulting in protection of PTEN-induced kinase 1 (Pink1) from ATAD3A-mediated cleavage. Interestingly, MUC1-induced mitophagy is associated with increased oncogenicity of cancer cells. Similarly, inhibition of mitophagy significantly suppresses MUC1-induced cancer cell activity in vitro and in vivo. Consistently, MUC1 and ATAD3A protein levels present an inverse relationship in tumor tissues of breast cancer patients. Our data validate that MUC1/ATAD3A/Pink1 axis-mediated mitophagy constitutes a novel mechanism for maintaining the malignancy of cancer cells, providing a novel therapeutic approach for MUC1-positive cancers.

DOI: https://doi.org/10.1038/s41419-022-05345-z
Biochim Biophys Acta Mol Cell Res. 2022 Oct 22. pii: S0167-4889(22)00174-4. [Epub ahead of print] 119382

Tolerance to replication stress requires Dun1p kinase and activation of the electron transport chain.

Shreya Nagar, Riddhi Mehta, Pritpal Kaur, Roshini T Liliah, Ales Vancura.

  One of the key outcomes of activation of DNA replication checkpoint (DRC) or DNA damage checkpoint (DDC) is the increased synthesis of the deoxyribonucleoside triphosphates (dNTPs), which is a prerequisite for normal progression through the S phase and for effective DNA repair. We have recently shown that DDC increases aerobic metabolism and activates the electron transport chain (ETC) to elevate ATP production and dNTP synthesis by repressing transcription of histone genes, leading to globally altered chromatin architecture and increased transcription of genes encoding enzymes of tricarboxylic acid (TCA) cycle and the ETC. The aim of this study was to determine whether DRC activates ETC. We show here that DRC activates ETC by a checkpoint kinase Dun1p-dependent mechanism. DRC induces transcription of RNR1-4 genes and elevates mtDNA copy number. Inactivation of RRM3 or SGS1, two DNA helicases important for DNA replication, activates DRC but does not render cells dependent on ETC. However, fitness of rrm3Δ and sgs1Δ cells requires Dun1p. The slow growth of rrm3Δdun1Δ and sgs1Δdun1Δ cells can be suppressed by introducing sml1Δ mutation, indicating that the slow growth is due to low levels of dNTPs. Interestingly, inactivation of ETC in dun1Δ cells results in a synthetic growth defect that can be suppressed by sml1Δ mutation, suggesting that ETC is important for dNTP synthesis in the absence of Dun1p function. Together, our results reveal an unexpected connection between ETC, replication stress, and Dun1p kinase.

Keywords:  Dun1p; Replication stress; Ribonucleotide reductase; dNTPs; electron transport chain

DOI:  https://doi.org/10.1016/j.bbamcr.2022.119382
Cancer Discov. 2022 Oct 27. pii: CD-22-0523. [Epub ahead of print]

Cellular senescence is immunogenic and promotes anti-tumor immunity.

Ines Marin, Olga Boix, Andrea Garcia-Garijo, Isabelle Sirois, Adria Caballe, Eduardo Zarzuela, Irene Ruano, Camille Stephan-Otto Attolini, Neus Prats, Jose Alberto Lopez-Dominguez, Marta Kovatcheva, Elena Garralda, Javier Munoz, Etienne Caron, Maria Abad, Alena Gros, Federico Pietrocola, Manuel Serrano.

Cellular senescence is a stress response that activates innate immune cells, but little is known about its interplay with the adaptive immune system. Here, we show that senescent cells combine several features that render them highly efficient in activating dendritic cells (DCs) and antigen-specific CD8 T cells. This includes the release of alarmins, activation of interferon signaling, enhanced MHC class I machinery, and presentation of senescence-specific self-peptides that can activate CD8 T cells. In the context of cancer, immunization with senescent cancer cells elicits strong anti-tumor protection mediated by DCs and CD8 T cells. Interestingly, this protection is superior to immunization with cancer cells undergoing immunogenic cell death. Finally, the induction of senescence in human primary cancer cells also augments their ability to activate autologous antigen-specific tumor-infiltrating CD8 lymphocytes. Our study indicates that senescent cancer cells can be exploited to develop efficient and protective CD8-dependent anti-tumor immune responses.

DOI: https://doi.org/10.1158/2159-8290.CD-22-0523
Mol Ther Nucleic Acids. 2022 Dec 13. 30 257-269

Mitochondrial double-stranded RNAs as a pivotal mediator in the pathogenesis of Sjӧgren's syndrome.

Jimin Yoon, Minseok Lee, Ahsan Ausaf Ali, Ye Rim Oh, Yong Seok Choi, Sujin Kim, Namseok Lee, Se Gwang Jang, Seonghyeon Park, Jin-Haeng Chung, Seung-Ki Kwok, Joon Young Hyon, Seunghee Cha, Yun Jong Lee, Sung Gap Im, Yoosik Kim.

  Sjӧgren's syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (I-IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. Here, we investigate the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating I-IFNs and other glandular phenotypes of SS. We find that mt-dsRNAs are elevated in the saliva and tears of SS patients (n = 73 for saliva and n = 16 for tears) and in salivary glands of non-obese diabetic mice with salivary dysfunction. Using the in-house-developed 3D culture of immortalized human salivary gland cells, we show that stimulation by exogenous dsRNAs increase mt-dsRNAs, activate the innate immune system, trigger I-IFNs, and promote glandular phenotypes. These responses are mediated via the Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT) pathway. Indeed, a small chemical inhibitor of JAK1 attenuates mtRNA elevation and immune activation. We further show that muscarinic receptor ligand acetylcholine ameliorates autoimmune characteristics by preventing mt-dsRNA-mediated immune activation. Last, direct suppression of mt-dsRNAs reverses the glandular phenotypes of SS. Altogether, our study underscores the significance of mt-dsRNA upregulation in the pathogenesis of SS and suggests mt-dsRNAs as propagators of a pseudo-viral signal in the SS target tissue.

Keywords:  Janus kinase 1; MT: Non-coding RNAs; Sjӧgren’s syndrome; acetylcholine; autoimmunity; innate immune response; mitochondrial double-stranded RNA; protein kinase R; spheroid culture

DOI:  https://doi.org/10.1016/j.omtn.2022.09.020
Heliyon. 2022 Oct;8(10): e11149

Melatonin suppresses the antiviral immune response to EMCV infection through intracellular ATP deprivation caused by mitochondrial fragmentation.

Mariko Kikuchi, Miki Kadena, Haruka Fukamachi, Takashi Takaki, Shohei Matsui, Sumire Hoashi-Takiguchi, Hirobumi Morisaki, Nataša Trtić, Mina Mori, Mie Kurosawa, Momoe Itsumi, Takahiro Funatsu, Atsuo Sakurai, Seikou Shintani, Hiroki Kato, Takashi Fujita, Yasubumi Maruoka, Hirotaka Kuwata.

  Melatonin, a sleep hormone derived from the pineal gland, has an anti-inflammatory effect on the immune system in addition to modulating the brain nervous system. Previous studies have shown that melatonin suppresses signaling pathways downstream of multiple pattern recognition receptors on the innate immune cells during pathogen infection, but the specific mechanism of suppression has not been well understood. Using an encephalomyocarditis virus (EMCV) infection model in macrophages, we investigated the effects of melatonin on the antiviral response in innate immunity and found that melatonin attenuated the uptake of viral particles into macrophages. Furthermore, melatonin suppressed cytoskeletal regulation by decreasing ATP production by mitochondria. Finally, in an in vivo infection experiment, we also found that melatonin administration partially exacerbated the infection in the mouse brain. These results suggest that melatonin may have an inhibitory effect on excessive inflammation by suppressing cytoskeletal regulation in the innate immune system, but also suggest that suppression of inflammation may lead to insufficient protection against EMCV infection in vivo.

Keywords:  Antiviral immunity; Cytokines; Inflammation; Innate immunity; Melatonin; Mitochondria; Neuro-immunology

DOI:  https://doi.org/10.1016/j.heliyon.2022.e11149
Commun Biol. 2022 Oct 25. 5(1): 1128

Pooled image-base screening of mitochondria with microraft isolation distinguishes pathogenic mitofusin 2 mutations.

Alex L Yenkin, John C Bramley, Colin L Kremitzki, Jason E Waligorski, Mariel J Liebeskind, Xinyuan E Xu, Vinay D Chandrasekaran, Maria A Vakaki, Graham W Bachman, Robi D Mitra, Jeffrey D Milbrandt, William J Buchser.

Most human genetic variation is classified as variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a method termed "Raft-Seq". We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator Mitofusin 2, even when the cellular phenotype is subtle. We also show that our platform achieves its efficacy using multiple cellular features, which can be configured on-the-fly. Raft-Seq enables a way to perform pooled screening on sets of mutations in biologically relevant cells, with the ability to physically capture any cell with a perturbed phenotype and expand it clonally, directly from the primary screen.

DOI: https://doi.org/10.1038/s42003-022-04089-y