Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution.

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability.

BMP signaling: at the gate between activated melanocyte stem cells and differentiation.

Through recurrent bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate into pigment-producing melanocytes. The signaling factors orchestrating these events remain incompletely understood. Here, we use single-cell RNA sequencing with comparative gene expression analysis to elucidate the transcriptional dynamics of McSCs through quiescence, activation, and melanocyte maturation. Unearthing converging signs of increased WNT and BMP signaling along this progression, we endeavored to understand how these pathways are integrated. Employing conditional lineage-specific genetic ablation studies in mice, we found that loss of BMP signaling in the lineage leads to hair graying due to a block in melanocyte maturation. We show that interestingly, BMP signaling functions downstream from activated McSCs and maintains WNT effector, transcription factor LEF1. Employing pseudotime analysis, genetics, and chromatin landscaping, we show that following WNT-mediated activation of McSCs, BMP and WNT pathways collaborate to trigger the commitment of proliferative progeny by fueling LEF1- and MITF-dependent differentiation. Our findings shed light upon the signaling interplay and timing of cues that orchestrate melanocyte lineage progression in the hair follicle and underscore a key role for BMP signaling in driving complete differentiation.

A gene regulatory network combining Pax3/7, Sox10 and Mitf generates diverse pigment cell types in medaka and zebrafish.

Neural crest cells generate numerous derivatives, including pigment cells, and are a model for studying how fate specification from multipotent progenitors is controlled. In mammals, the core gene regulatory network for melanocytes (their only pigment cell type) contains three transcription factors, Sox10, Pax3 and Mitf, with the latter considered a master regulator of melanocyte development. In teleosts, which have three to four pigment cell types (melanophores, iridophores and xanthophores, plus leucophores e.g. in medaka), gene regulatory networks governing fate specification are poorly understood, although Mitf function is considered conserved. Here, we show that the regulatory relationships between Sox10, Pax3 and Mitf are conserved in zebrafish, but the role for Mitf is more complex than previously emphasized, affecting xanthophore development too. Similarly, medaka Mitf is necessary for melanophore, xanthophore and leucophore formation. Furthermore, expression patterns and mutant phenotypes of pax3 and pax7 suggest that Pax3 and Pax7 act sequentially, activating mitf expression. Pax7 modulates Mitf function, driving co-expressing cells to differentiate as xanthophores and leucophores rather than melanophores. We propose that pigment cell fate specification should be considered to result from the combinatorial activity of Mitf with other transcription factors.

Endothelin signaling in development.

Since the discovery of endothelin 1 (EDN1) in 1988, the role of endothelin ligands and their receptors in the regulation of blood pressure in normal and disease states has been extensively studied. However, endothelin signaling also plays crucial roles in the development of neural crest cell-derived tissues. Mechanisms of endothelin action during neural crest cell maturation have been deciphered using a variety of in vivo and in vitro approaches, with these studies elucidating the basis of human syndromes involving developmental differences resulting from altered endothelin signaling. In this Review, we describe the endothelin pathway and its functions during the development of neural crest-derived tissues. We also summarize how dysregulated endothelin signaling causes developmental differences and how this knowledge may lead to potential treatments for individuals with gene variants in the endothelin pathway.

α-Melanocyte-stimulating hormone alleviates pathological cardiac remodeling via melanocortin 5 receptor.

α-Melanocyte-stimulating hormone (α-MSH) regulates diverse physiological functions by activating melanocortin receptors (MC-R). However, the role of α-MSH and its possible target receptors in the heart remain completely unknown. Here we investigate whether α-MSH could be involved in pathological cardiac remodeling. We found that α-MSH was highly expressed in the mouse heart with reduced ventricular levels after transverse aortic constriction (TAC). Administration of a stable α-MSH analog protected mice against TAC-induced cardiac hypertrophy and systolic dysfunction. In vitro experiments revealed that MC5-R in cardiomyocytes mediates the anti-hypertrophic signaling of α-MSH. Silencing of MC5-R in cardiomyocytes induced hypertrophy and fibrosis markers in vitro and aggravated TAC-induced cardiac hypertrophy and fibrosis in vivo. Conversely, pharmacological activation of MC5-R improved systolic function and reduced cardiac fibrosis in TAC-operated mice. In conclusion, α-MSH is expressed in the heart and protects against pathological cardiac remodeling by activating MC5-R in cardiomyocytes. These results suggest that analogs of naturally occurring α-MSH, that have been recently approved for clinical use and have agonistic activity at MC5-R, may be of benefit in treating heart failure.

Re-examining the evidence that ivermectin induces a melanoma-like state in Xenopus embryos.

Modeling metastasis in animal systems has been an important focus for developing cancer therapeutics. Xenopus laevis is a well-established model, known for its use in identifying genetic mechanisms underlying diseases and disorders in humans. Prior literature has suggested that the drug, ivermectin, can be used in Xenopus to induce melanocytes to convert into a metastatic melanoma-like state, and thus could be ideal for testing possible melanoma therapies in vivo. However, there are notable inconsistencies between ivermectin studies in Xenopus and the application of ivermectin in mammalian systems, that are relevant to cancer and melanoma research. In this review, we examine the ivermectin-induced phenotypes in Xenopus, and we explore the current uses of ivermectin in human research. We conclude that while ivermectin may be a useful drug for many biomedical purposes, it is not ideal to induce a metastatic melanocyte phenotype in Xenopus for testing the effects of potential melanoma therapeutics.

Aldh2 is a lineage-specific metabolic gatekeeper in melanocyte stem cells.

Melanocyte stem cells (McSCs) in zebrafish serve as an on-demand source of melanocytes during growth and regeneration, but metabolic programs associated with their activation and regenerative processes are not well known. Here, using live imaging coupled with scRNA-sequencing, we discovered that, during regeneration, quiescent McSCs activate a dormant embryonic neural crest transcriptional program followed by an aldehyde dehydrogenase (Aldh) 2 metabolic switch to generate progeny. Unexpectedly, although ALDH2 is well known for its aldehyde-clearing mechanisms, we find that, in regenerating McSCs, Aldh2 activity is required to generate formate - the one-carbon (1C) building block for nucleotide biosynthesis - through formaldehyde metabolism. Consequently, we find that disrupting the 1C cycle with low doses of methotrexate causes melanocyte regeneration defects. In the absence of Aldh2, we find that purines are the metabolic end product sufficient for activated McSCs to generate progeny. Together, our work reveals McSCs undergo a two-step cell state transition during regeneration, and that the reaction products of Aldh2 enzymes have tissue-specific stem cell functions that meet metabolic demands in regeneration.

Lrig1- and Wnt-dependent niches dictate segregation of resident immune cells and melanocytes in murine tail epidermis.

The barrier-forming, self-renewing mammalian epidermis comprises keratinocytes, pigment-producing melanocytes and resident immune cells as first-line host defense. In murine tail skin, interfollicular epidermis patterns into pigmented 'scale' and hypopigmented 'interscale' epidermis. Why and how mature melanocytes accumulate in scale epidermis is unresolved. Here, we delineate a cellular hierarchy among epidermal cell types that determines skin patterning. Already during postnatal development, melanocytes co-segregate with newly forming scale compartments. Intriguingly, this process coincides with partitioning of both Langerhans cells and dendritic epidermal T cells to interscale epidermis, suggesting functional segregation of pigmentation and immune surveillance. Analysis of non-pigmented mice and of mice lacking melanocytes or resident immune cells revealed that immunocyte patterning is melanocyte and melanin independent and, vice versa, immune cells do not control melanocyte localization. Instead, genetically enforced progressive scale fusion upon Lrig1 deletion showed that melanocytes and immune cells dynamically follow epithelial scale:interscale patterns. Importantly, disrupting Wnt-Lef1 function in keratinocytes caused melanocyte mislocalization to interscale epidermis, implicating canonical Wnt signaling in organizing the pigmentation pattern. Together, this work uncovers cellular and molecular principles underlying the compartmentalization of tissue functions in skin.

mTORC1 activity negatively regulates human hair follicle growth and pigmentation.

Dysregulation of the activity of the mechanistic target of rapamycin complex 1 (mTORC1) is commonly linked to aging, cancer, and genetic disorders such as tuberous sclerosis (TS), a rare neurodevelopmental multisystemic disease characterized by benign tumors, seizures, and intellectual disability. Although patches of white hair on the scalp (poliosis) are considered as early signs of TS, the underlying molecular mechanisms and potential involvement of mTORC1 in hair depigmentation remain unclear. Here, we have used healthy, organ-cultured human scalp hair follicles (HFs) to interrogate the role of mTORC1 in a prototypic human (mini-)organ. Gray/white HFs exhibit high mTORC1 activity, while mTORC1 inhibition by rapamycin stimulated HF growth and pigmentation, even in gray/white HFs that still contained some surviving melanocytes. Mechanistically, this occurred via increased intrafollicular production of the melanotropic hormone, α-MSH. In contrast, knockdown of intrafollicular TSC2, a negative regulator of mTORC1, significantly reduced HF pigmentation. Our findings introduce mTORC1 activity as an important negative regulator of human HF growth and pigmentation and suggest that pharmacological mTORC1 inhibition could become a novel strategy in the management of hair loss and depigmentation disorders.

Disorganisation of basement membrane zone architecture impairs melanocyte residence in vitiligo.

The basement membrane zone is the interface between the epidermis and dermis, and it is disrupted in several skin conditions. Here, we report the results of a comprehensive investigation into the structural and molecular factors of the basement membrane zone in vitiligo, a dermatological disorder characterised by depigmented patches on the skin. Using electron microscopy and immunofluorescence staining, we confirmed abnormal basement membrane zone morphology and disrupted basement membrane zone architecture in human vitiliginous skin. Furthermore, we identified elevated expression of matrix metalloproteinase 2 (MMP2) in human dermal fibroblasts as a key factor responsible for basement membrane zone matrix degradation. In our in vitro and ex vivo models, overexpression of MMP2 in fibroblasts led to basement membrane zone disruption and melanocyte disappearance. Importantly, we reveal that the loss of melanocytes in vitiligo is primarily linked to their weakened adhesion to the basement membrane, mediated by binding between integrin ß1 and laminin and discoidin domain receptor 1 and collagen IV. Finally, inhibition of matrix metalloproteinase 2 expression reversed depigmentation in a mouse model of vitiligo. In conclusion, our research shows the importance of basement membrane zone integrity in melanocyte residence and offers new avenues for therapeutic interventions to address this challenging skin condition. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cell-type-specific meQTLs extend melanoma GWAS annotation beyond eQTLs and inform melanocyte gene-regulatory mechanisms.

Although expression quantitative trait loci (eQTLs) have been powerful in identifying susceptibility genes from genome-wide association study (GWAS) findings, most trait-associated loci are not explained by eQTLs alone. Alternative QTLs, including DNA methylation QTLs (meQTLs), are emerging, but cell-type-specific meQTLs using cells of disease origin have been lacking. Here, we established an meQTL dataset by using primary melanocytes from 106 individuals and identified 1,497,502 significant cis-meQTLs. Multi-QTL colocalization with meQTLs, eQTLs, and mRNA splice-junction QTLs from the same individuals together with imputed methylome-wide and transcriptome-wide association studies identified candidate susceptibility genes at 63% of melanoma GWAS loci. Among the three molecular QTLs, meQTLs were the single largest contributor. To compare melanocyte meQTLs with those from malignant melanomas, we performed meQTL analysis on skin cutaneous melanomas from The Cancer Genome Atlas (n = 444). A substantial proportion of meQTL probes (45.9%) in primary melanocytes is preserved in melanomas, while a smaller fraction of eQTL genes is preserved (12.7%). Integration of melanocyte multi-QTLs and melanoma meQTLs identified candidate susceptibility genes at 72% of melanoma GWAS loci. Beyond GWAS annotation, meQTL-eQTL colocalization in melanocytes suggested that 841 unique genes potentially share a causal variant with a nearby methylation probe in melanocytes. Finally, melanocyte trans-meQTLs identified a hotspot for rs12203592, a cis-eQTL of a transcription factor, IRF4, with 131 candidate target CpGs. Motif enrichment and IRF4 ChIP-seq analysis demonstrated that these target CpGs are enriched in IRF4 binding sites, suggesting an IRF4-mediated regulatory network. Our study highlights the utility of cell-type-specific meQTLs.

A UVB-responsive common variant at chromosome band 7p21.1 confers tanning response and melanoma risk via regulation of the aryl hydrocarbon receptor, AHR.

Genome-wide association studies (GWASs) have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). Because ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWASs identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. Because AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 introduced via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression and altering melanocyte growth phenotypes upon exposure.

Cyclical fate restriction: a new view of neural crest cell fate specification.

Neural crest cells are crucial in development, not least because of their remarkable multipotency. Early findings stimulated two hypotheses for how fate specification and commitment from fully multipotent neural crest cells might occur, progressive fate restriction (PFR) and direct fate restriction, differing in whether partially restricted intermediates were involved. Initially hotly debated, they remain unreconciled, although PFR has become favoured. However, testing of a PFR hypothesis of zebrafish pigment cell development refutes this view. We propose a novel 'cyclical fate restriction' hypothesis, based upon a more dynamic view of transcriptional states, reconciling the experimental evidence underpinning the traditional hypotheses.

Effect of Ibuprofen as an Albumin Binder on Melanoma-Targeting Properties of 177Lu-Labeled Ibuprofen-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides.

The purpose of this study was to examine how the introduction of ibuprofen (IBU) affected tumor-targeting and biodistribution properties of 177Lu-labeled IBU-conjugated alpha-melanocyte-stimulating hormone peptides. The IBU was used as an albumin binder and conjugated to the DOTA-Lys moiety without or with a linker to yield DOTA-Lys(IBU)-GG-Nle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(IBU)-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2}, DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex, DOTA-Lys(Asn-IBU)-GGNle-CycMSHhex, and DOTA-Lys(Dab-IBU)-GGNle-CycMSHhex peptides. Their melanocortin-receptor 1 (MC1R) binding affinities were determined on B16/F10 melanoma cells first. Then the biodistribution of 177Lu-labeled peptides was determined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to choose the lead peptide for further examination. The full biodistribution and melanoma imaging properties of 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex were further evaluated using B16/F10 melanoma-bearing C57 mice. DOTA-Lys(IBU)-GG-Nle-CycMSHhex, DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex, DOTA-Lys(Asn-IBU)-GGNle-CycMSHhex, and DOTA-Lys(Dab-IBU)-GGNle-CycMSHhex displayed the IC50 values of 1.41 ± 0.37, 1.52 ± 0.08, 0.03 ± 0.01, and 0.58 ± 0.06 nM on B16/F10 melanoma cells, respectively. 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex exhibited the lowest liver and kidney uptake among all four designed 177Lu peptides. Therefore, 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex was further evaluated for its full biodistribution and melanoma imaging properties. The B16/F10 melanoma uptake of 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex was 19.5 ± 3.12, 24.12 ± 3.35, 23.85 ± 2.08, and 10.80 ± 2.89% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. Moreover, 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex could clearly visualize the B16/F10 melanoma lesions at 2 h postinjection. The conjugation of IBU with or without a linker to GGNle-CycMSHhex affected the MC1R binding affinities of the designed peptides. The charge of the linker played a key role in the liver and kidney uptake of 177Lu-Asp-IBU, 177Lu-Asn-IBU, and 177Lu-Dab-IBU. 177Lu-Asp-IBU exhibited higher tumor/liver and tumor/kidney uptake ratios than those of 177Lu-Asn-IBU and 177Lu-Dab-IBU, underscoring its potential evaluation for melanoma therapy in the future.

Long-Term Dynamics of Serum α-MSH and α-MSH-Binding Immunoglobulins with a Link to Gut Microbiota Composition in Patients with Anorexia Nervosa.

INTRODUCTION: Immunoglobulins (Ig) reactive with α-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, are present in humans and were previously associated with eating disorders. In this longitudinal study involving patients with anorexia nervosa (AN), we determined whether α-MSH in serum is bound to IgG and analyzed long-term dynamics of both α-MSH peptide and α-MSH-reactive Ig in relation to changes in BMI and gut microbiota composition. METHODS: The study included 64 adolescents with a restrictive form of AN, whose serum samples were collected at hospital admission, discharge, and during a 1-year follow-up visit and 41 healthy controls, all females. RESULTS: We found that in both study groups, approximately 40% of serum α-MSH was reversibly bound to IgG and that levels of α-MSH-reactive IgG but not of α-MSH peptide in patients with AN were low at hospital admission but recovered 1 year later. Total IgG levels were also low at admission. Moreover, BMI-standard deviation score correlated positively with α-MSH IgG in both groups studied but negatively with α-MSH peptide only in controls. Significant correlations between the abundance of specific bacterial taxa in the gut microbiota and α-MSH peptide and IgG levels were found in both study groups, but they were more frequent in controls. CONCLUSION: We conclude that IgG in the blood plays a role as an α-MSH-binding protein, whose characteristics are associated with BMI in both patients with AN and controls. Furthermore, the study suggests that low production of α-MSH-reactive IgG during the starvation phase in patients with AN may be related to altered gut microbiota composition.

Role of melanocortin system in the locomotor activity rhythms and melatonin secretion as revealed by agouti-signalling protein (asip1) overexpression in zebrafish.

Temporal signals such as light and temperature cycles profoundly modulate animal physiology and behaviour. Via endogenous timing mechanisms which are regulated by these signals, organisms can anticipate cyclic environmental changes and thereby enhance their fitness. The pineal gland in fish, through the secretion of melatonin, appears to play a critical role in the circadian system, most likely acting as an element of the circadian clock system. An important output of this circadian clock is the locomotor activity circadian rhythm which is adapted to the photoperiod and thus determines whether animals are diurnal or nocturnal. By using a genetically modified zebrafish strain known as Tg (Xla.Eef1a1:Cau.asip1)iim04, which expresses a higher level of the agouti signalling protein 1 (Asip1), an endogenous antagonist of the melanocortin system, we observed a complete disruption of locomotor activity patterns, which correlates with the ablation of the melatonin daily rhythm. Consistent with this, in vitro experiments also demonstrated that Asip1 inhibits melatonin secretion from the zebrafish pineal gland, most likely through the melanocortin receptors expressed in this gland. Asip1 overexpression also disrupted the expression of core clock genes, including per1a and clock1a, thus blunting circadian oscillation. Collectively, these results implicate the melanocortin system as playing an important role in modulating pineal physiology and, therefore, circadian organisation in zebrafish.

MIF inhibition alleviates vitiligo progression by suppressing CD8+ T cell activation and proliferation.

In vitiligo, autoreactive CD8+ T cells have been established as the main culprit considering its pathogenic role in mediating epidermal melanocyte-specific destruction. Macrophage migration inhibitory factor (MIF) is a pleiotropic molecule that plays a central role in various immune processes including the activation and proliferation of T cells; but whether MIF is intertwined in vitiligo development and progression and its involvement in aberrantly activated CD8+ T cells remains ill-defined. In this study, we found that MIF was overabundant in vitiligo patients and a mouse model for human vitiligo. Additionally, inhibiting MIF ameliorated the disease progression in vitiligo mice, which manifested as less infiltration of CD8+ T cells and more retention of epidermal melanocytes in the tail skin. More importantly, in vitro experiments indicated that MIF-inhibition suppressed the activation and proliferation of CD8+ T cells from the lymph nodes of vitiligo mice, and the effect extended to CD8+ T cells in peripheral blood mononuclear cells of vitiligo patients. Finally, CD8+ T cells derived from MIF-inhibited vitiligo mice also exhibited an impaired capacity for activation and proliferation. Taken together, our results show that MIF might be clinically targetable in vitiligo treatment, and its inhibition might ameliorate vitiligo progression by suppressing autoreactive CD8+ T cell activation and proliferation. © 2023 The Pathological Society of Great Britain and Ireland.

Bridging Molecular Mechanism and Clinical Practice in Vitiligo Treatment: An Updated Review.

BACKGROUND: Treatment of vitiligo seeks to achieve three goals: cessation of disease progression, regeneration of pigmentation, and prevention of recurrence. SUMMARY: Number of nonsurgical interventions are available that suppress the autoimmune response and regenerate the melanocytes from the reservoir: phototherapy including psoralen and ultraviolet A, narrowband ultraviolet B, and 308-nm excimer and 311-nm Titanium:Sapphire lasers; topical agents including topical calcineurin inhibitors, topical corticosteroids, and topical 5-fluorouracil; and systemic agents including corticosteorids, mycophenolate mofetil, cyclosporine, methotrexate, minocycline, afamelanotide, and antioxidants. In recent years, a great advance has been made in the understanding of pathogenesis of vitiligo, and JAK inhibitors are being investigated as a new treatment. Minimally invasive procedures such as fractional lasers or microneedling can help achieve the optimal treatment outcome when used properly. KEY MESSAGES: Our review describes various treatment modalities for vitiligo based on their molecular mechanism of action. Bridging the gap between molecular mechanisms and therapeutic options would be a valuable reference for physicians in clinical practice.

Transient receptor potential mucolipin 1 circumvents oxidative stress in primary human melanocytes.

BACKGROUND: Transient Receptor Potential Mucolipin 1 (TRPML1) serves as a pivotal reactive oxygen species (ROS) sensor in cells, which is implicated in the regulation of autophagy. However, its function in melanocyte autophagy under oxidative stress remains elusive. METHODS: The expression and ion channel function of TRPML1 were investigated using immunofluorescence and calcium imaging in primary human melanocytes (MCs). After activating TRPML1 with MLSA1 (TRPML1 agonist), autophagy-related molecules were investigated via western blot. ROS level, apoptosis- and autophagy-related molecules were investigated after pretreatment with MLSA1. After interference with TRPML1 expression, mitochondrial structures were visualized by electron microscopy with hydrogen peroxide (H2O2）treatment. RESULTS: TRPML1 was expressed and functionally active in primary human MCs, and its activation promotes elevated expression of LC3-II and reduced apoptosis and ROS levels under oxidative stress. TRPML1 downregulation caused mitochondrial swelling and disruption of cristae structures under oxidative stress in primary human MCs. CONCLUSIONS: TRPML1 might mediate lysosomal autophagy in primary human MCs under oxidative stress, participating in mechanisms that maintain the oxidative and antioxidant systems in balance.

Clinicopathologic profiles of canine ocular melanosis: A comparative study between cairn terriers and non-cairn terriers.

OBJECTIVES: To identify canine breeds at risk for ocular melanosis and to compare the clinical and histologic features between affected Cairn Terriers (CTs) and non-Cairn Terriers (NCTs). DESIGN: Relative risk (RR) analysis and retrospective cohort study of dogs histologically diagnosed with ocular melanosis. PROCEDURES: The COPLOW archive was searched for globe submissions diagnosed with ocular melanosis. Six hundred fifty globes were included, and RR analysis was performed to identify at-risk NCT breeds. A cohort of 360 CT and NCT globes diagnosed from 2013 to 2023 were included in the retrospective cohort study. Clinical data were collected from submission forms, medical records, and follow-up surveys. One hundred fifty-seven submissions underwent masked histologic review. Immunohistochemical staining for CD204 was performed to determine the predominance of melanophages in affected uvea from five NCTs. RESULTS: At-risk NCT breeds included the Boxer, Labrador Retriever, and French Bulldog. Glaucoma was the reported reason for enucleation in 79.4% of submissions. At enucleation, clinical features less prevalent in NCTs than CTs included pigmentary abnormalities in the contralateral eye (33.7% vs. 63.1%, p = .0008) and abnormal episcleral/scleral pigmentation in the enucleated globe (25.4% vs. 53.6%, p = .0008). Histologic involvement of the episclera was also less frequent in NCTs than in CTs (39.7% vs. 76.9%, p = .008). Concurrent melanocytic neoplasms arising in melanosis were more common in NCTs (24.4%) than CTs (3.9%). Melanophages were not predominant in any samples evaluated immunohistochemically. CONCLUSIONS: Several popular NCT breeds carry risk for ocular melanosis, and some clinicopathologic disease features may differ from those described in CTs.