Lipofuscin in keratinocytes: Production, properties, and consequences of the photosensitization with visible light

Author(s)

Tonolli, Paulo N. ^[1] ; Martins, Waleska K. ^{[1, 2]} ; Junqueira, Helena C. ^[1] ; Silva, Maryana N. ^[2] ; Severino, Divinomar ^[1] ; Santacruz-Perez, Carolina ^[1] ; Watanabe, I ^[3] ; Baptista, Mauricio S. ^[1]

Affiliation

^[1] Univ Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo, SP - Brazil

^[2] Univ Anhanguera Sao Paulo UNIAN SP, Sao Paulo, SP - Brazil

^[3] Univ Sao Paulo, Inst Ciencias Biomet, Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: Free Radical Biology and Medicine; v. 160, p. 277-292, NOV 20 2020.
• Web of Science Citations: 1

Abstract

A dysfunction in the mitochondrial-lysosomal axis of cellular homeostasis is proposed to cause cells to age quicker and to accumulate lipofuscin. Typical protocols to mediate lipofuscinogenesis are based on the induction of the senescent phenotype either by allowing many consecutive cycles of cell division or by treating cells with physical/chemical agents such as ultraviolet (UV) light or hydrogen peroxide. Due to a direct connection with the physiopathology of age-related macular degeneration, lipofuscin that accumulates in retinal pigment epithelium (RPE) cells have been extensively studied, and the photochemical properties of RPE lipofuscin are considered as standard for this pigment. Yet, many other tissues such as the brain and the skin may prompt lipofuscinogenesis, and the properties of lipofuscin granules accumulated in these tissues are not necessarily the same as those of RPE lipofuscin. Here, we present a light-induced protocol that accelerates cell aging as judged by the maximization of lipofuscinogenesis. Photosensitization of cells previously incubated with nanomolar concentrations of 1,9-dimethyl methylene blue (DMMB), severely and specifically damages mitochondria and lysosomes, leading to a lipofuscin-related senescent phenotype. By applying this protocol in human immortalized non-malignant keratinocytes (HaCaT) cells, we observed a 2.5-fold higher level of lipofuscin accumulation compared to the level of lipofuscin accumulation in cells treated with a typical UV protocol. Lipofuscin accumulated in keratinocytes exhibited the typical red light emission, with excitation maximum in the blue wavelength region (similar to 450 nm). Fluorescence lifetime image microscopy data showed that the keratinocyte lipofuscin has an emission lifetime of similar to 1.7 ns. Lipofuscin-loaded cells (but not control cells) generated a substantial amount of singlet oxygen (O-1(2)) when irradiated with blue light (420 nm), but there was no O-1(2) generation when excitation was performed with a green light (532 nm). These characteristics were compared with those of RPE cells, considering that keratinocyte lipofuscin lacks the bisretinoids derivatives present in RPE lipofuscin. Additionally, we showed that lipofuscin-loaded keratinocytes irradiated with visible light presented critical DNA damages, such as double-strand breaks and Fpg-sensitive sites. We propose that the DMMB protocol is an efficient way to disturb the mitochondrial-lysosomal axis of cellular homeostasis, and consequently, it can be used to accelerate aging and to induce lipofuscinogenesis. We also discuss the consequences of the lipofuscin-induced genotoxicity of visible light in keratinocytes. (AU)

• FAPESP's process: 18/23257-0 - Modulation of biochemical and photochemical parallel damages: implications on pro-survival autophagy
• Grantee: Maryana do Nascimento da Silva
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 18/22922-0 - Modulation of autophagy by biochemical and photochemical stress: therapeutic implications
• Grantee: Waleska Kerllen Martins Gardesani
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC