Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease (AD), mainly affecting the elderly population with an incidence of 1-3%. It is characterized by motor symptoms closely linked to dopaminergic cell death in the Substantia nigra pars compacta. From a molecular standpoint, the deposition of Lewy bodies, namely protein aggregates particularly enriched with misfolded α-synuclein, is a typical hallmark of the disease. In addition, recent findings have highlighted cholesterol dysmetabolism as another common peculiarity of PD profoundly contributing to changes in membrane composition and integrity. Notably, p75 neurotrophin receptor (p75NTR) was found to be upregulated in the brains of post-mortem PD patients and associated with a reduction in the expression of pivotal neuroprotective effectors, strongly suggesting a role for this receptor in PD. Given its ability to favor both pro-survival and pro-apoptotic cascades, the synthesis of small p75NTR-binding molecules (i.e. LM11A-31) promoting cell viability over death was of particular interest in research. Hence, the aim of this study was to evaluate whether the pharmacological modulation of p75NTR by LM11A-31 provides neuroprotection in a rotenone-induced cellular model of PD. Our data showed that LM11A-31 promoted cell viability and reduced rotenone-dependent neuromorphological aberrations, as well as α-synuclein downregulation. We also observed that p75NTR modulation counteracted rotenone-dependent cholesterol alterations: specifically, LM11A-31 normalized free cholesterol content, as well as the expression of proteins involved in cholesterol uptake and trafficking. Furthermore, LM11A-31 reduced oxidative stress damage upon macromolecules by both boosting the expression of transcriptional regulators of the antioxidant response and reducing the expression of NADPH-oxidase (NOX) modulatory subunits. Even though further studies are required to better dissect the molecular mechanisms linking p75NTR to PD physiopathology, our data point to p75NTR modulation as a promising therapeutic avenue in PD treatment.

Rett syndrome (RTT) is a neurological disorder with an early onset, primarily affecting females, and is characterized by severe cognitive and physical impairments. RTT is no longer regarded as an exclusively neurological disease; rather, it is now considered a multisystem syndrome that affects the brain and several other tissues/organs. Recent research suggests that disruptions in redox homeostasis and heightened inflammatory responses are of pivotal importance with regard to the disease’s clinical manifestations. Notably, emerging evidence points to the p75 neurotrophin receptor (p75NTR) as a regulator of oxidative stress (OS) and inflammation.

The objective of this study is to explore the impact of modulating p75NTR through the use of the small molecule LM11A-31 on fibroblasts derived from RTT patients. Fibroblasts were treated with 0.1 μM of LM11A-31 for 24 hours, and analyses were conducted through qPCR, immunofluorescence, ELISA, and Western blotting.

The results reveal that LM11A-31 significantly mitigates OS markers in RTT fibroblasts. Specifically, p75NTR modulation restored protein glutathionylation and reduced the expression of the pro-oxidant enzyme NADPH-oxidase 4 (NOX4). Moreover, LM11A-31 blunted the expression of pro-inflammatory mediators while simultaneously normalizing transcription factors involved in antioxidant response and inflammatory response.

These results suggest that targeting p75NTR with LM11A-31 may offer a potential therapeutic avenue for restoring redox balance and alleviating inflammation in RTT patients. The use of a p75NTR modulator, such as LM11A-31, could be more effective for treating RTT by targeting key processes, addressing multiple aspects of the disease simultaneously. Further research should be directed towards a more detailed investigation of the molecular mechanisms and the corroboration of these findings in in vivo models.

Introduction: Pequi (Caryocar brasiliense) is a fruit from Cerrado, Brazil, that has important therapeutic properties for the tissue repair of skin wounds. Methods: Balb/c mice were subjected to skin lesions and received daily topical treatment with the lipophilic fraction of pequi (Treated group) or saline solution (Control group). Animals from each group were sacrificed at 3, 7, and 14 days post injury (n=5). Macroscopic analyses were performed to assess wound contraction, and peripheral blood was collected for leukocyte quantification. Additionally, skin fragments were used for histopathological analysis (Hematoxylin–Eosin) and for quantification of the expression of the proteins Annexin A1 (AnxA1) and phosphorylated ERK (pERK) using the Western blot technique. CEUA-UFMT 23108.080623/2023-50. Results: At 3 days, the Treated animals exhibited a more robust scab on the lesion, providing greater physical and microbiological protection. At 7 days, wound contraction was significantly higher in the Treated group compared to the Control group (p ≤ 0.01), accelerating wound closure and preventing dehydration and infection risks. In peripheral blood, a higher number of neutrophils, eosinophils, and monocytes were identified in the Treated group compared to the Control group (p ≤ 0.0001; p ≤ 0.05; p ≤ 0.05, respectively), particularly during the early 3-day period. No difference was observed in AnxA1 protein expression between the experimental groups; however, the Treated group showed higher pERK expression at 7 days compared to the Control group (p ≤ 0.05). Conclusions: These data suggest that pequi oil acts as an effective modulator of the inflammatory phase, by inhibiting leukocyte transmigration, and of the proliferative phase, through the activation of the ERK pathway, preparing the tissue for regeneration without altering AnxA1 levels. Thus, it is evident that the lipophilic fraction of pequi accelerates tissue repair by modulating the inflammatory and proliferative phases, promoting more efficient healing. (Financial Support: Fapemat 000547-2023; CAPES 88887.976959/2024-00).

Introduction: The tissue repair process is regulated by various molecular and cellular elements that must be in balance to result in healthy tissue. Pequi oil (Caryocar brasiliense) possesses therapeutic properties that may contribute to this context. Thus, this study evaluated the action of this oil on skin lesions. Methods: Balb/c mice with induced skin lesions were divided into groups (n=5) receiving daily topical applications of saline solution (the Control group) or the lipophilic fraction of pequi (the Treated group). After 3, 7, and 14 days, the animals from each group were sacrificed, and fragments of their injured skin were analyzed using polarization microscopy to evaluate collagen; using Western blot to evaluate matrix metalloproteinase 2 (MMP2); and using a multiplex assay to evaluate tumor growth factor-beta 1 (TGF-β1). CEUA - UFMT 23108.080623/2023-50. Results: In the Treated group, a lower amount of type I collagen was observed on days 3 (p ≤ 0.05) and 14 (p ≤ 0.001) and of type III collagen on day 14 (p ≤ 0.001) compared to these levels in the Control group. Additionally, the Treated group exhibited slightly higher MMP2 expression (not significant) and lower TGF-β1 levels (p ≤ 0.05) on day 3 compared to the Control, indicating reduced fibrogenic stimulation. Conclusion: These results demonstrate that treatment with pequi oil promotes lower deposition of type I and III collagen in the late stages of skin regeneration, primarily by inhibiting TGF-β1 production in the early stage, suggesting the reduced activation of pathways that could lead to excessive fibrosis. Meanwhile, the stable levels of MMP2 reflect the necessary balance for tissue remodeling. These findings reveal that pequi oil regulates key factors in tissue remodeling, promoting less fibrosis and preserving extracellular matrix balance, highlighting its therapeutic potential for wound management (Financial Support: FAPEMAT 000547-2023).

Introduction

The cytoskeleton and trafficking pathway play a pivotal role in maintaining cellular homeostasis, particularly in retinal pigment epithelium (RPE), which is essential for photoreceptor functionality. Dysfunction in these pathways is increasingly recognized as a contributor to retinal degenerative diseases, such as Retinitis Pigmentosa (RP). This study explores transcriptomic alterations in RPE cells under oxidative stress conditions induced by oxidized low-density lipoproteins (oxLDLs).

Methods

Human RPE cells were exposed to oxLDL (100 µg/ml), and transcriptomic analyses were conducted across three time points (0h, 3h, and 6h). RNA-seq was utilized for differential gene expression profiling, and data were analyzed using Gene Set Enrichment Analysis (GSEA) to highlight significantly altered pathways. Focus was given to cytoskeleton-related genes and their association with vesicular trafficking and signal transduction.

Results

Significant changes were observed in genes regulating cytoskeleton integrity, vesicle trafficking, and motor protein function. The key findings include the 1) dysregulation of Retinitis Pigmentosa GTPase Regulator (RPGR), impairing ciliary transport; 2) alterations in actin filament organization and microtubule stabilization, disrupting cellular polarity and vesicular transport; 3) upregulation of genes like FAM161A and RP1, linked to microtubule binding and photoreceptor disk morphogenesis; and 4) progressive collapse of intermediate filaments and junctional complexes affecting RPE--photoreceptor interactions.

Conclusion

Cytoskeleton-related pathways undergo extensive remodeling under oxidative stress, contributing to the pathophysiology of RP. Disruption in vesicular trafficking and cytoskeletal organization underscores the mechanistic link between oxidative stress and retinal degeneration. This study identifies novel targets within the cytoskeleton pathway, providing a foundation for therapeutic exploration in retinal disorders

Background: Epigenetic modifications, particularly DNA methylation, play a critical role in regulating gene expression under stress conditions. This study investigates the methylation landscape of mitochondrial stress response genes in retinal pigment epithelium (RPE) cells exposed to oxidative stress, focusing on how these changes may drive pathophysiological alterations linked to retinal degenerative diseases.

Methods: Human RPE cells were treated with N-retinylidene-N-retinylethanolamine adduct (A2E) to induce oxidative stress. Whole-genome bisulfite sequencing (WGBS) was employed to profile methylation patterns. Differentially methylated regions (DMRs) were identified and analyzed for enrichment in mitochondrial stress-related pathways using bioinformatic tools, including Bismark, MethylKit, and ClueGO.

Results: Analysis revealed significant methylation changes in key mitochondrial stress pathway genes: 1) Hypomethylation of PGC1α and NRF1, genes essential for mitochondrial biogenesis and repair, indicating an adaptive response. 2) Hypermethylation of BCL2 and HSP70, associated with decreased anti-apoptotic signaling and impaired mitochondrial protein folding capacity. 3) Identification of novel DMRs in regulatory regions of genes involved in oxidative phosphorylation (e.g., ND5, COX2) and antioxidant defense mechanisms (SOD2, GPX1). Lastly, 4) clustering analysis of DMRs highlighted enrichment in pathways linked to mitochondrial unfolded protein response (UPRmt) and oxidative stress mitigation.

Conclusion: Oxidative stress induces significant alterations in the methylation profiles of mitochondrial stress pathway genes in RPE cells. These changes likely contribute to mitochondrial dysfunction and cellular apoptosis, underpinning the pathophysiology of retinal degenerative conditions. The identification of key methylation markers provides novel insights into the epigenetic regulation of mitochondrial function and potential therapeutic targets for restoring retinal allostasis.

Introduction: Lactate significantly influences the metabolism of melanoma by promoting tumor development and progression. However, the role of this metabolite and its main transporter MCT-1 in driving the propagation of melanoma cancer stem cells (CSCs) still needs to be elucidated.

Methods: Experiments were conducted on the A375 and WM115 melanoma cell lines. Cell quiescence was assessed by a proliferation and colony formation assay, cell-cycle and survival analysis (flow cytometry), and Western blotting. The spherogenic ability of tumor cells was evaluated through sphere formation and a propagation assay. The metabolic effects of lactate and AZ3965 treatment were analyzed by flow cytometry and Western blotting.

Results: We found that lactate-treated cells exhibited a quiescent state characterized by reduced proliferation, G1 cell-cycle phase arrest, and P27 upregulation; however, no changes in cell survival or clonogenic efficiency were found, suggesting that this quiescent state was not associated with cell death. More importantly, lactate treatment significantly increased melanoma cells' spherogenic ability and ABCG2 enrichment, two common CSC traits. Mechanistically, lactate was observed to activate PCG1-alpha, leading to enhanced OXPHOS protein expression and mitochondrial mass. Of note, treatment with AZ3965, a specific inhibitor of the lactate transporter MCT-1, significantly suppressed melanoma CSC expansion via PGC1-alpha downregulation.

Conclusions: Lactate plays a key role in sustaining melanoma CSC proliferation and survival. Targeting lactate metabolism via MCT1 inhibition holds promise for novel anti-cancer therapies.

Introduction: Melanoma is an aggressive cancer characterized by a rapid metastatic process. Thus, understanding the mechanisms underlying its progression is urgently needed to improve patient outcomes. In this regard, there is consistent evidence of a tumor-sustaining crosstalk between melanoma and subcutaneous adipose tissue; however, the role of EVs in this communication still needs to be clarified.

Methods: After isolation by SEC, EVs were characterized by NTA, TEM and Western blot analysis. The impact of these particles on melanoma cell growth was evaluated by cell proliferation and colony formation assays, and the effects on metastatic potential were assessed by transwell assay. Melanoma cell stem-like traits, including spherogenic ability, ABCG2 enrichment and vemurafenib response, were investigated by sphere formation assay, flow cytometry and cell viability assay. The metabolic consequences of EV treatment were analyzed by Western blot and cytofluorimetric assays.

Results: We demonstrated that the EVs derived from adipocytes did not alter melanoma cell proliferation, but significantly promoted tumor cell migration and invasion by determining an enrichment in mesenchymal markers, such as N-cadherin and vimentin. In particular, these changes were accompanied by the transition towards a stem-like phenotype, characterized by enhanced spherogenic ability and ABCG2 upregulation. Interestingly, this led to a reduced response to vemurafenib, with diminished apoptotic rates and decreased caspase 3 and PARP cleavage. Mechanistically, an increase in PGC-1α expression was found, resulting in higher mitochondrial mass and activity; of note, the treatment of melanoma cells with XCT790 and SR-18292, two specific inhibitors of mitochondrial biogenesis, successfully counteracted the above EV-related effects, suggesting that this process could be targeted to suppress the EV-mediated interactions between subcutaneous adipocytes and melanoma.

Conclusions: Taken together, these results highlight the crucial role played by EVs in the melanoma microenvironment, highlighting the ability of adipocyte-derived vesicles to sustain melanoma cell aggressiveness via PGC-1α activation.

BNCT (boron neutron capture therapy) is a cancer therapy combining neutron irradiation and the administration of boron carrier drug, such as ¹⁰B-boronophenylalanine (BPA). The clinical application of BNCT has recently been approved for oral and head-and-neck cancers in Japan. To evaluate the therapeutic efficacy and side effects of BNCT for various cancer stages, we evaluated factors related to early responses to BNCT in tumor cells. We showed that the expression of the long noncoding RNA SNHG12, a cancer-related molecule, is increased in oral cancer SAS cells after BPA-based BNCT. Here, the biological function of SNHG12 during BNCT and early cell responses was investigated. We showed that SNHG12 expression in SAS cells increased significantly after gamma irradiation and treatment with an alkylating agent, methylmethanesulfonate, within 24 hrs. SNHG12 overexpression triggered a decrease in the sub-G1 apoptotic fraction and increase in S-phase population. We thus hypothesized that SNHG12 may have a supportive role for cell survival in particular types of DNA damage responses. Furthermore, colony formation assay of SAS cells after BNCT irradiation showed that SNHG12 knockdown increased the sensitivity of SAS cells to BNCT. Knockdown of SNHG12 in SAS cells after gamma irradiation decreased the mRNA expression of IAP family protein within 24 hrs. The mechanisms by which SNHG12 is involved in the early cell response after BNCT will be discussed.

Necroptosis has been reported in post-mortem AD brains; however, the events that regulate this type of inflammatory cell death in AD are not fully understood. Autophagy has been shown to protect cells from necroptotic cell death and act as a negative regulator of necroptosis. Moreover, our previous studies indicate that intracellular cholesterol accumulation can disrupt autophagy function, a pathological hallmark in multiple age-related neurodegenerative diseases, including AD. This study aims to evaluate the relationship between high cholesterol levels, altered autophagy, and necroptosis in AD, using primary neurons isolated from APP-PSEN1 mice overexpressing the cholesterol-related transcription factor SREBF2 and dorsal root ganglion neurons (DRGs). Methods: Cholesterol enrichment was assessed by incubating the cells with a soluble cholesterol/methyl-cyclodextrin complex (50 mg/ml) for 1h, followed by 4h of recovery. The necroptotic pathway was induced through treatment with TZL (TNFα (60 ng/ml)) plus the SMAC mimetic (LCL-161, 10 mM) and the pan-caspase inhibitor zVAD (10 mM) for different durations. Results: Brains from APP-PSEN1-SREBF2 mice exhibited an up-regulated expression of necroptosis-related proteins, specifically RIPK1, RIPK3, and MLKL, which accumulated in the urea-soluble fraction, indicating necrosome assembly. Also, SREBF2 overexpression resulted in increased levels of active phosphorylated RIPK3 and MLKL. Similarly, phospho-RIPK3 and phospho-MLKL levels were significantly elevated in primary neurons derived from the APP-PSEN1-SREBF2 mice compared to wild-type (WT) neurons, even without necroptosis induction. In vitro cholesterol enrichment increased cell death following TZL exposure, which was mitigated by RIPK1 and RIPK3 inhibitors (Necrostatin and GSK’872, respectively). In DRG neurons, phosphorylated RIPK3 and MLKL were constitutively expressed in aged neurons with compromised autophagy. The induction of the necroptotic pathway was prevented by restoring the autophagy function in old neurons. Conclusions: Overall, these findings indicate that the activation of the necroptotic pathway and inflammatory cell death in AD may be favored by the increased intracellular cholesterol load and defective autophagy associated with aging.