Biology subjects

Seabra, M. C.

Publications and source records attributed to Seabra, M. C..

8 recordsLinked to original sources

bioRxiv2024

Enhancing lysosome function via mTOR/TFEB activation reduces lipofuscin-like granules in early Age-related Macular Degeneration

Age-related macular degeneration (AMD) is the most common blinding disease in the western world and is currently incurable. Although the exact causes of AMD are not clear, the primary origin of pathology appears to be in the retinal pigment epithelium (RPE). RPE is responsible for the daily digestion of photoreceptor outer segments (POS), which imposes a heavy continuous burden on the lysosomal network. POS feeding assay in vitro suggested that the accumulation of autofluorescence granules (AFG), similar to lipofuscin in vivo, derives from lysosomal dysfunction. Here we show that synchronous phagocytosis of POS leads to early transient mTOR activation followed by inhibition in late phagosome maturation. One of its substrates, the transcription factor EB (TFEB) increases during phagosome maturation albeit mostly in its inactive phosphorylated form. We questioned whether induction of the mTOR/TFEB axis could improve digestion of POS and hence reduce AFG load. Treatment of POS-fed cells with rapamycin, an mTORC1 inhibitor after the appearance of AFG results in 30% reduction of AFG load. This effect is dependent on active lysosomal enzymes and induction of active dephosphorylated TFEB with consequent activation of GADD34 and lysosomal biogenesis. As a proof of concept, we show that overexpressing a constitutively active form of unphosphorylated TFEB dramatically reduces POS-dependent AFG accumulation. Overall, this study suggests that viral or pharmacological approaches activating the mTOR/TFEB axis in the RPE could be beneficial as cell-protective treatment of early/intermediate cases of AMD, acting to delay progression of the disease.

molecular biology↗

bioRxiv2024

Photoprotective melanin is maintained within keratinocytes in a storage lysosome

In the skin, melanin is synthesized by melanocytes within melanosomes, and transferred to keratinocytes. After being phagocytosed by keratinocytes, melanin polarizes to supranuclear caps that protect against the genotoxic effects of ultraviolet radiation. We provide evidence that melanin-containing phagosomes undergo a canonical maturation process, with the sequential acquisition of early and late endosomal markers. Subsequently, these phagosomes fuse with active lysosomes, leading to the formation of a melanin-containing phagolysosome that we named melanokerasome. Melanokerasomes achieve juxtanuclear positioning via lysosomal trafficking regulators Rab7 and RILP. Mature melanokerasomes exhibit lysosomal markers, elude connections with the endo/phagocytic pathway, are weakly degradative, retain undigested cargo and are likely tethered to the nuclear membrane. We propose that they represent a lysosomal-derived storage compartment that has exited the lysosome cycle, akin to the formation of lipofuscin in aged cells and dysfunctional lysosomes in lysosomal storage and age-related diseases. This storage lysosome allows melanin to persist for long periods, where it can exert its photoprotective effect efficiently.

cell biology↗

bioRxiv2023

3D human retinal organoid model for the study of early diabetic retinopathy

Diabetic retinopathy (DR) is a significant complication of diabetes and a primary cause of visual impairment among working-age individuals. DR is a degenerative condition in which hyperglycaemia results in morphological and functional changes in certain retinal cells. Existing treatments mainly address the advanced stages of the disease, which involve vascular defects such as macular edema or neovascularization. However, it is now known that retinal neurodegeneration and inflammation precede these vascular changes. Therefore, there is a pressing need to identify new therapeutic approaches that target the early stages of DR and prevent its progression. In the last decade, the development of reliable in vitro models resembling the complexity of the retinal tissue has significantly improved. Namely, three-dimensional (3D) retinal organoids derived from human induced-pluripotent stem cells (hiPSCs) recapitulate the cellular organization and complexity of the human retina. Here, we used hiPSCs-derived retinal organoids to generate a model of early DR. In this model, we observe well-established molecular and cellular features of early DR: i) loss of retinal ganglion and amacrine cells; ii) glial reactivity and inflammation, with increased expression of the vascular endothelial-derived growth factor (VEGF) and interleukin-1{beta} (IL-1{beta}), and monocyte chemoattractant protein-1 (MCP-1) secretion; iii) increased levels of reactive oxygen species accompanied by activation of key enzymes involved in antioxidative stress response. The data provided highlight the utility of retinal organoid technology in modelling early-stage DR. This offers new avenues for the development of targeted therapeutic interventions on neurodegeneration and inflammation in the initial phase of DR, potentially slowing the diseases progression.

neuroscience↗

bioRxiv2022

Reconstructed human pigmented skin/epidermis models achieve epidermal pigmentation through melanocore transfer.

The skin acts as a barrier to environmental insults and provides many vital functions. One of these is to shield DNA from harmful UV radiation, which is achieved by skin pigmentation arising as melanin is produced and dispersed within the epidermal layer. This is a crucial defence against DNA damage, photo-ageing and skin cancer. The mechanisms and regulation of melanogenesis and melanin transfer involve extensive crosstalk between melanocytes and keratinocytes in the epidermis, as well as fibroblasts in the dermal layer. Although the predominant mechanism of melanin transfer continues to be debated and several plausible models have been proposed, we and others previously provided evidence for a coupled exo/phagocytosis of melanocore model. Herein, we performed histology and immunohistochemistry analyses and demonstrated that a newly developed full-thickness 3D reconstructed human pigmented skin model and an epidermis-only model exhibit dispersed pigment throughout keratinocytes in the epidermis. Transmission electron microscopy revealed melanocores between melanocytes and keratinocytes, suggesting that melanin is transferred through coupled exocytosis/phagocytosis of the melanosome core similar to our previous observations in human skin biopsies. We therefore present evidence that our in vitro models of pigmented human skin show epidermal pigmentation comparable to human skin. These findings have a high value for studies of skin pigmentation mechanisms and pigmentary disorders, whilst reducing the reliance on animal models and human skin biopsies. SignificanceCurrently, many approaches to study skin pigmentation and pigmentary disorders use 2D cultures or co-culture of melanocytes and/or keratinocytes, as well as skin biopsies obtained from consenting donors. However, challenges are faced when it is necessary to mimic the tissue microenvironment as there is a limited availability of skin biopsies, especially for the case of rare pigmentary disorders. Through this study, we show the potential of 3D in vitro reconstructed pigmented skin/epidermis models for both general and specific pigmentary research questions, which could reduce reliance on animal models and human skin biopsies.

cell biology↗

bioRxiv2021

Rab3a regulates melanin exocytosis induced by keratinocyte-conditioned medium

Skin pigmentation relies on melanin and is crucial for photoprotection against ultraviolet radiation-induced toxicity. Melanin is synthesized and stored in melanosomes, within melanocytes and then transferred to keratinocytes. While the molecular players involved in melanogenesis have been extensively studied, those underlying melanin transfer remain poorly characterized. Previously, our group proposed that coupled exo/phagocytosis is the predominant mechanism of melanin transfer in human skin and showed an essential role for Rab11b and the exocyst tethering complex in this process. Using a fluorescence-based assay, we show here that keratinocyte-conditioned medium (KCM) specifically induces melanin exocytosis from melanocytes. Moreover, we found that Rab3a, but not Rab11b, regulates melanin exocytosis upon KCM stimulation. In fact, melanosomes accumulate in melanocyte dendrites upon KCM stimulation, co-localizing with Rab3a mainly in the vicinity of the plasma membrane. Additionally, Rab3a silencing does not affect melanin transfer in melanocyte/keratinocyte co-cultures, in contrast with Rab11b depletion, indicating that Rab11b regulates non-KCM-stimulated melanin exocytosis. Thus, our results suggest the existence of at least two distinct routes of melanin exocytosis: one controlled by Rab11b and another Rab3a-dependent, stimulated by KCM. Furthermore, these results provide evidence that soluble factors secreted by keratinocytes can control skin pigmentation via induction of melanocyte signaling pathways that promote peripheral transport of melanosomes and a Rab3a-mediated exocytosis mechanism.

molecular biology↗

bioRxiv2021

Melanocore uptake by keratinocytes occurs through phagocytosis and involves Protease-activated receptor-2 activation

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred - either in a membrane-bound melanosome or as a melanosome core, i.e. melanocore. Here we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1 and CtBP1/BARS, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Furthermore, we confirmed that melanocore, but not melanosome uptake is dependent on the Protease-activated receptor-2 (PAR-2) and found that PAR-2 can be specifically activated by melanocores. As skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.

cell biology↗

bioRxiv2021

FORMATION OF LIPOFUSCIN-LIKE AUTOFLUORESCENT GRANULES IN THE RETINAL PIGMENT EPITHELIUM REQUIRES LYSOSOME DYSFUNCTION

PurposeWe aim to characterize the pathways required for autofluorescent granule (AFG) formation by retinal pigment epithelium (RPE) cells using cultured monolayers. MethodsWe fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24h the cells started accumulating AFGs similar to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking Cathepsin D by gene editing. ResultsAFGs appear to derive from incompletely digested POS-containing phagosomes and are surrounded after 72h by a single membrane containing lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation but that impairment of lysosomal pH or catalytic activity, particularly Cathepsin D activity, enhances AF accumulation. ConclusionsWe conclude that lysosomal dysfunction results in incomplete POS degradation and AFG accumulation.

cell biology↗

bioRxiv2019

Symmetric arrangement of mitochondria:plasma membrane contacts between adjacent photoreceptor cells regulated by Opa1

Mitochondria are known to play an essential role in photoreceptor function and wellbeing that enables normal healthy vision. Within photoreceptors they are elongated and extend most of the length inner segment, where they supply energy for protein synthesis and the phototransduction machinery in the outer segment as well as acting as a calcium store. Here we examined the arrangement of the mitochondria within the inner segment in detail using 3D electron microscopy techniques and show they are tethered to the plasma membrane in a highly specialised arrangement. This includes mitochondria running alongside each other in neighbouring inner segments, with evidence of alignment of the cristae openings. As the pathway by which photoreceptors meet their high energy demands is not fully understood, we propose this to be a mechanism to share metabolites and assist in maintaining homeostasis across the photoreceptor cell layer. In the extracellular space between photoreceptors, Muller glial processes were identified. Due to the often close proximity to the inner segment mitochondria, they may too play a role in the inner segment mitochondrial arrangement as well as metabolite shuttling. OPA1 is an important factor in mitochondrial homeostasis, including cristae remodelling; therefore, we examined the photoreceptors of a heterozygous Opa1 knock-out mouse model. The cristae structure in the Opa1+/- photoreceptors was not greatly affected, but there were morphological abnormalities and a reduction in mitochondria in contact with the inner segment plasma membrane. This indicates the importance of key regulators in maintaining this specialised photoreceptor mitochondrial arrangement.

cell biology↗