bims-mideyd 2022-12-11 papers

Free Radic Biol Med. 2022 Dec 06. pii: S0891-5849(22)01030-9. [Epub ahead of print]

Lactate protects against oxidative stress-induced retinal degeneration by activating autophagy.

Guang-Ping Zou, Tao Wang, Jin-Xing Xiao, Xiao-Yu Wang, Li-Ping Jiang, Fang-Fang Tou, Zhi-Ping Chen, Xin-Hui Qu, Xiao-Jian Han.

Age-related macular degeneration is the common cause of blindless among the aged, which can mainly attribute to oxidative stress and dysregulated autophagy in retinal pigment epithelium cells. Lactate was reported to act as signaling molecule and exerted beneficial effect against oxidative stress. This study aims to investigate the protective effect of lactate against oxidative stress-induced retinal degeneration. Here, H2O2-induced oxidative stress cell model and sodium iodate-induced mice retinal degeneration model were established. It was found that H2O2 inhibited cell viability in ARPE-19 cells and sodium iodate induced deterioration of retinal pigment epithelium as well as apoptosis in retina. Pretreatment with lactate alleviated oxidative stress-induced cell death and retinal degeneration. Molecularly, lactate activated autophagy by up-regulating the ratio of LC3II/I, increased formation of LC3 puncta and autophagic vacuole. Further, lactate prevented H2O2-induced mitochondrial fission and maintained mitochondrial function by alleviating H2O2-induced mitochondrial membrane potential disruption and intracellular ROS generation. In contrast, application of 3-methyladenine, an inhibitor of autophagy, effectively weakened the protective effect of lactate against oxidative stress in vivo and in vitro. Taken together, all data in this study indicate that lactate protects against oxidative stress-induced retinal degeneration and preserves mitochondrial function by activating autophagy.

Keywords: Autophagy; Lactate; Mitochondrial dynamics; Oxidative stress; Retinal degeneration

DOI: https://doi.org/10.1016/j.freeradbiomed.2022.12.004

Front Physiol. 2022 ;13 1045613

Pigment epithelium-derived factor is an interleukin-6 antagonist in the RPE: Insight of structure-function relationships.

Alexandra Bernardo-Colón, Miriam Lerner, S Patricia Becerra.

Retinal and choroidal inflammatory lesions increase the levels of the pro-inflammatory cytokine interleukin-6 (IL-6). Pigment epithelium-derived factor (PEDF) has anti-inflammatory properties, but it is not known if it can prevent the production of IL-6 by the retinal pigment epithelium. To investigate the anti-inflammatory effects of PEDF in the RPE, we used human ARPE-19 cells stimulated with human recombinant tumor necrosis factor-alpha (TNF-α) to induce overexpression of the IL6 gene. We found that the viability of ARPE-19 cells decreased by 22% with TNF-α at 10 ng/ml, being drastically decreased at ≥50 ng/ml. TNF-α at 5-100 ng/ml elevated the production and secretion of IL-6 protein, as measured by ELISA. To challenge the TNF-α-mediated stimulation of IL-6, we used recombinant human PEDF protein. PEDF at 100 nM recovered the TNF-α-mediated loss of cell viability and repressed IL-6 gene expression as determined by RT-PCR. PEDF at 10-100 nM attenuated the IL-6 protein secretion in a dose dependent fashion (IC50 = 65 nM), being abolished with 100 nM PEDF. To map the region that confers the IL-6 blocking effect to the PEDF polypeptide, we used chemically synthesized peptides designed from its biologically active domains, pro-death 34-mer, and pro-survival 44-mer and 17-mer (H105A), to challenge the IL-6 overproduction. The pro-survival peptides recovered the TNF-α-mediated cell viability loss, and inhibited IL-6 secretion, while the 34-mer did not have an effect, suggesting a role for the pro-survival domain in blocking TNF-α-mediated cell death and IL-6 stimulation. Our findings position PEDF as a novel antagonistic agent of IL-6 production in RPE cells, underscoring its use for the management of retinal disease-related inflammation.

Keywords: alpha; inflammation; interleukin-6 (IL-6); pigment epithelium-derived factor (PEDF); retinal pigment epithelium (ARPE-19); tumor necrosis factor

DOI: https://doi.org/10.3389/fphys.2022.1045613

Neurobiol Dis. 2022 Dec 05. pii: S0969-9961(22)00333-3. [Epub ahead of print]176 105941

DJ-1 promotes energy balance by regulating both mitochondrial and autophagic homeostasis.

Federica De Lazzari, Francesco Agostini, Nicoletta Plotegher, Michele Sandre, Elisa Greggio, Aram Megighian, Luigi Bubacco, Federica Sandrelli, Alexander J Whitworth, Marco Bisaglia.

The protein DJ-1 is mutated in rare familial forms of recessive Parkinson's disease and in parkinsonism accompanied by amyotrophic lateral sclerosis symptoms and dementia. DJ-1 is considered a multitasking protein able to confer protection under various conditions of stress. However, the precise cellular function still remains elusive. In the present work, we evaluated fruit flies lacking the expression of the DJ-1 homolog dj-1β as compared to control aged-matched individuals. Behavioral evaluations included lifespan, locomotion in an open field arena, sensitivity to oxidative insults, and resistance to starvation. Molecular analyses were carried out by analyzing the mitochondrial morphology and functionality, and the autophagic response. We demonstrated that dj-1β null mutant flies are hypoactive and display higher sensitivity to oxidative insults and food deprivation. Analysis of mitochondrial homeostasis revealed that loss of dj-1β leads to larger and more circular mitochondria, characterized by impaired complex-I-linked respiration while preserving ATP production capacity. Additionally, dj-1β null mutant flies present an impaired autophagic response, which is suppressed by treatment with the antioxidant molecule N-Acetyl-L-Cysteine. Overall, our data point to a mechanism whereby DJ-1 plays a critical role in the maintenance of energy homeostasis, by sustaining mitochondrial homeostasis and affecting the autophagic flux through the maintenance of the cellular redox state. In light of the involvement of DJ-1 in neurodegenerative diseases and considering that neurons are highly energy-demanding cells, particularly sensitive to redox stress, our study sheds light on a key role of DJ-1 in the maintenance of cellular homeostasis.

Keywords: Autophagy; DJ-1; Energy balance; Mitochondria; Redox homeostasis

DOI: https://doi.org/10.1016/j.nbd.2022.105941

Methods Mol Biol. 2023 ;2560 287-302

Generation of Human iPSC-Derived Retinal Organoids for Assessment of AAV-Mediated Gene Delivery.

Amy Tso, Bruna Lopes da Costa, Alexandra Fehnel, Sarah R Levi, Laura A Jenny, Sara D Ragi, Yao Li, Peter M J Quinn.

Human retinal organoids derived from induced pluripotent stem cells (iPSCs) serve as a promising preclinical model for testing the safety and efficacy of viral gene therapy. Retinal organoids recapitulate the stratified multilayered epithelium structure of the developing and maturating human retina. As such, retinal organoids are unique tools to model retinal disease and to test therapeutic interventions toward their amelioration. Here, we describe a method for the generation of human iPSC-derived retinal organoids and how they can be utilized for the assessment of recombinant adeno-associated viral (rAAV)-mediated gene delivery.

Keywords: 3D retinal organoids; AMASS (agarose microwell array seeding and scraping); Adeno-associated virus (AAV); Cell tropism; Gene therapy; Induced pluripotent stem cells (iPSCs); Retinal pigment epithelium (RPE); Transgene expression assay

DOI: https://doi.org/10.1007/978-1-0716-2651-1_27