bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2022‒12‒25
twelve papers selected by
Marco Tigano
Thomas Jefferson University

  1. J Mol Cell Cardiol. 2022 Dec 17. pii: S0022-2828(22)00573-9. [Epub ahead of print]175 44-48
      Mitochondrial dysfunction in heart triggers an integrated stress response (ISR) through phosphorylation of eIF2α and subsequent ATF4 activation. DAP3 Binding Cell Death Enhancer 1 (DELE1) is a mitochondrial protein recently found to be critical for mediating mitochondrial stress-triggered ISR (MSR)-induced eIF2α-ATF4 pathway activation. However, the specific role of DELE1 in heart at baseline or in response to mitochondrial stress remains largely unknown. In this study, we report that DELE1 is dispensable for cardiac development and function under baseline conditions. Conversely, DELE1 is essential for mediating an adaptive response to mitochondrial dysfunction-triggered stress in the heart, playing a protective role in mitochondrial cardiomyopathy.
    Keywords:  Dele1; Integrated stress response; Mitochondrial cardiomyopathy; Mitochondrial stress
  2. J Biol Chem. 2022 Dec 14. pii: S0021-9258(22)01240-6. [Epub ahead of print] 102797
      Twinkle is the ring-shaped replicative helicase within the human mitochondria with high homology to bacteriophage T7 gp4 helicase-primase. Unlike many orthologs of Twinkle, the N-terminal domain (NTD) of human Twinkle has lost its primase activity through evolutionarily acquired mutations. The NTD has no demonstrated activity thus far; its role has remained unclear. Here, we biochemically characterize the isolated NTD and C-terminal domain with linker (CTD) to decipher their contributions to full-length (FL) Twinkle activities. This novel CTD construct hydrolyzes ATP, has weak DNA unwinding activity, and assists DNA Polymerase γ (Polγ)-catalyzed strand-displacement synthesis on short replication forks. However, CTD fails to promote multi-kilobase length product formation by Polγ in rolling-circle DNA synthesis. Thus, CTD retains all the motor functions but struggles to implement them for processive translocation. We show that NTD has DNA binding activity, and its presence stabilizes Twinkle oligomerization. CTD oligomerizes on its own, but the loss of NTD results in heterogeneously-sized oligomeric species. The CTD also exhibits weaker and salt-sensitive DNA binding compared to FL Twinkle. Based on these results, we propose that NTD directly contributes to DNA binding and holds the DNA in place behind the central channel of the CTD like a 'doorstop', preventing helicase slippages and sustaining processive unwinding. Consistent with this model, mitochondrial single-stranded DNA binding protein (mtSSB) compensate for the NTD loss and partially restore kilobase length DNA synthesis by CTD and Polγ. The implications of our studies are foundational for understanding the mechanisms of disease-causing Twinkle mutants that lie in the NTD.
    Keywords:  DNA polymerase; Twinkle; helicase; mitochondria; mitochondrial diseases; replication; replisome
  3. Proc Natl Acad Sci U S A. 2022 Dec 27. 119(52): e2215799119
      Capturing mitochondria's intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to two-dimensional (2D) single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) to a benzo-fused cyanine dye, we report a mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, and markedly reduced phototoxicity. PKMO enables super-resolution (SR) recordings of IM dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell three-dimensional (3D) STED nanoscopy of mitochondria for 3D analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers, allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.
    Keywords:  STED nanoscopy; cristae; mitochondria
  4. Nature. 2022 Dec 21.
      R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability1, which has been linked to the action of endonucleases such as XPG2-4. However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1 (refs. 5-7), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref. 8), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2 (ref. 9) and in BRCA1-mutated cancer cells10. These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer.
  5. Aging Cell. 2022 Dec 22. e13752
      Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine also known as a mitokine; however, its role in mitochondrial homeostasis and cellular senescence remained elusive. We show here that knocking down GDF15 expression in human dermal fibroblasts induced mitochondrial dysfunction and premature senescence, associated with a distinct senescence-associated secretory phenotype. Fibroblast-specific loss of GDF15 expression in a model of 3D reconstructed human skin induced epidermal thinning, a hallmark of skin aging. Our results suggest GDF15 to play a so far undisclosed role in mitochondrial homeostasis to delay both the onset of cellular senescence and the appearance of age-related changes in a 3D human skin model.
    Keywords:  GDF15; lipofuscin; mitochondria; mitokine; senescence; skin aging
  6. J Extracell Vesicles. 2022 Dec;11(12): e12295
      Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.
    Keywords:  age-related macular degeneration; complement factor H; extracellular vesicles; human induced pluripotent stem cells; photoreceptors; retina; retinal pigment epithelium
  7. Cell Rep. 2022 Dec 20. pii: S2211-1247(22)01718-1. [Epub ahead of print]41(12): 111826
      Cancer therapies trigger diverse cellular responses, ranging from apoptotic death to acquisition of persistent therapy-refractory states such as senescence. Tipping the balance toward apoptosis could improve treatment outcomes regardless of therapeutic agent or malignancy. We find that inhibition of the mitochondrial protein BCL-xL increases the propensity of cancer cells to die after treatment with a broad array of oncology drugs, including mitotic inhibitors and chemotherapy. Functional precision oncology and omics analyses suggest that BCL-xL inhibition redirects the outcome of p53 transcriptional response from senescence to apoptosis, which likely occurs via caspase-dependent down-modulation of p21 and downstream cytostatic proteins. Consequently, addition of a BCL-2/xL inhibitor strongly improves melanoma response to the senescence-inducing drug targeting mitotic kinase Aurora kinase A (AURKA) in mice and patient-derived organoids. This study shows a crosstalk between the mitochondrial apoptotic pathway and cell cycle regulation that can be targeted to augment therapeutic efficacy in cancers with wild-type p53.
    Keywords:  Aurora kinase; BAX; BCL-2; BCL-xL; CP: Cancer; p21; p53; patient-derived organoids; senescence; senogenic; senolytic
  8. STAR Protoc. 2022 Dec 20. pii: S2666-1667(22)00838-3. [Epub ahead of print]4(1): 101958
      Current approaches, such as fixed-cell imaging or single-snapshot imaging, are insufficient to capture cytoskeleton-mediated mitochondrial fission. Here, we present a protocol to capture actin-mediated mitochondrial fission using high-resolution time-lapse imaging. We describe steps starting from cell preparation and mitochondria labeling through to live-cell imaging and final analysis. This approach is also applicable for analysis of multiple cytoskeleton-mediated organelle events such as vesicle trafficking, membrane fusion, and endocytic events in live cells. For complete details on the use and execution of this protocol, please refer to Shimura et al. (2021).1.
    Keywords:  Cell Biology; Microscopy; Molecular Biology
  9. J Pediatr Neurosci. 2021 Oct-Dec;16(4):16(4): 319-322
      Mitochondrial disorders (MIDs) are frequently multisystemic in nature and cause significant morbidity and mortality. Accurate assessment of mitochondrial disease prevalence has been difficult in the past. Primary MIDs are due to mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA)-located genes. Here we report cases of two siblings who presented to the pediatric emergency department with status epilepticus. Initially, the elder sibling was treated for metabolic encephalopathy and viral encephalitis, during his admission to the hospital. On treatment with multiple antiepileptic drugs, the status epilepticus subsided. A provisional diagnosis of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes was made. Magnetic resonance imaging showed diffusion restriction in the left temporal lobe, insular cortex, and left lentiform nucleus, which completely resolved on follow-up after 1 month. His sudden demise in May 2019 due to status epilepticus, and a similar case presentation in his younger sibling, prompted us to do a genetic analysis test. The exome sequence revealed FASTKD2 mutation, a rare variant. This case report helps in increasing the awareness among the clinicians about the clinical presentation of FASTKD2 mutation case.
    Keywords:  FASTKD2 mutation; MELAS; status epilepticus
  10. Nat Methods. 2022 Dec 22.
      Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch's-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.
  11. Fac Rev. 2022 ;11 35
      To maintain genome fidelity and prevent diseases such as cancer, our cells must constantly detect, and efficiently and precisely repair, DNA damage. Paradoxically, DNA-damaging agents in the form of radiation and chemotherapy are also used to treat cancer. Olivieri et al. used a CRISPR-based screen to identify genes that, when disrupted, lead to sensitivity or resistance to 27 different DNA-damaging agents used in the lab and/or in the clinic to treat cancer patients1. Their results reveal multiple new genes and connections that regulate these critical DNA damage repair pathways, with implications for basic and clinical research as well as cancer therapy.
    Keywords:  CRISPR; DNA damage; DNA repair; DNA-damaging agents