TOPIC:

This study is focused on the expression levels of AQP4 during neurodevelopment and aging in mice are influenced and potentially regulated by the antisense transcripts GM50048-201 and GM50048-202 (collectively referred to as AQP4-AS).

THEORETICAL FOCUS

AQP4 is expressed as two isoforms, M1 and M23, which assemble into orthogonal arrays of particles (OAPs) (Nicchia et al., 2008; Rossi et al., 2010). A novel AQP4 isoform, AQP4ex, results from translational readthrough, extending the C-terminus by 29 amino acids (De Bellis et al., 2017) is crucial for anchoring AQP4 at astrocytic endfeet near blood vessels, and its absence in AQP4ex-KO mice disrupts perivascular localization (Palazzo et al., 2019) and impairs glymphatic activity (Abbrescia et al., 2024). Emerging evidence highlights long non-coding RNAs (lncRNAs) as regulators of aging and senescence, acting at various molecular levels (Abraham et al., 2017; Ghafouri-Fard et al., 2022). For AQP4, the antisense lncRNA AQP4-AS1 (GM50048 in mice) includes two main transcripts: GM50048-201 (long variant) and GM50048-202 (short variant). In this study we analyzed AQP4 mRNA, AQP4-AS transcripts, and AQP4 protein levels across different stages of mouse brain development, from the prenatal period through postnatal development and adulthood, up to advanced aging

METHODS

Experiments were carried out with C57BL/6 AQP4WT. We quantified the RNA expression of AQP4, GM50048-201, and GM50048-202 across mouse brain development stages (E18 to 32 months) using qPCR. We then investigated regional differences in adult brains—specifically in the hippocampus, cortex, and cerebellum—at 3, 13, and 32 months of age, and WB analysis to assess AQP4 expression along mice brain developmental stages. Gene expression levels were normalized using beta Actin (ACTB) and relative gene expression was calculated by the 2−ΔΔCt method, WB were performed using specific antibodies: rabbit polyclonal anti-AQP4; rabbit polyclonal anti-mouse and a custom rabbit polyclonal anti-mouse p-AQP4ex that specific binds phosphorylated serine residues.

RESULTS

During embryonic and early postnatal stages (E18–P0), AQP4 mRNA and AQP4-AS transcript levels were both low and comparable, and AQP4 protein was undetectable. AQP4 protein expression began at postnatal day 7 (P7), increased progressively, peaked between P14 and P21, and remained stable in adult mice (1–3 months).When AQP4 protein expression emerged, both AQP4 mRNA and AQP4-AS transcript levels increased, although AQP4-AS levels were significantly higher than those of AQP4 mRNA, suggesting a possible role in fine-tuning and stabilizing AQP4 transcript levels to ensure efficient protein expression. In aged brains (13–32 months), AQP4 protein levels significantly declined, particularly in 32-month-old mice. This reduction was paralleled by a decrease in both AQP4-AS and AQP4 mRNA levels, reaching values similar to those observed in early developmental stages. Our findings reveal a time-dependent interplay between AQP4 and AS, suggesting that AQP4-AS may act as a cis-regulatory element that sustains AQP4 transcript levels to support appropriate protein expression.

EXPECTING FINDINGS

Future studies employing siRNA-mediated silencing or overexpression of AS may help uncover their potential as therapeutic targets in age-related brain disorders.

Despite receiving antiretroviral therapy (ART), an increasing number of adolescents and young adults with perinatally acquired HIV (PHIVAYA) are at risk of developing premature senescence and aging-associated illnesses, including cancer. Given this concern, it is crucial to assess aging biomarkers and their correlation with the HIV reservoir in order to comprehensively characterize and monitor these individuals. Fifty-five PHIVAYA (median age: 23, interquartile range [IQR]: 20–27 years, and 21 [18–23] years on ART at the time of study sampling) were studied along with 23 age-matched healthy controls. The PHIVAYA exhibited significantly higher percentages of activated, senescent, exhausted CD4 and CD8 T cells, shorter telomeres, reduced thymic output, and higher levels of circulating inflammatory markers (PAMPs, DAMPs, and pro-inflammatory cytokines IL-6, IL-8, and TNFα) as well as denervation biomarkers (neural cell adhesion molecule 1 [NCAM1] and C-terminal Agrin fragment [CAF]), compared to controls. HIV-DNA levels positively correlated with activated, senescent, exhausted CD4 and CD8 T cells, circulating biomarkers levels, and inversely with regulatory T and B cells and telomere length. According to their viremia over time, PHIVAYA were subgrouped into 14 Not Suppressed (NS)-PHIVAYA and 41 Suppressed (S)- PHIVAYA, of whom 6 who initiated ART within one year of age and maintained sustained viral suppression overtime were defined as Early Suppressed (ES)-PHIVAYA and the other 35 as Late Suppressed (LS)-PHIVAYA. ES-PHIVAYA exhibited significantly lower HIV-DNA reservoir, decreased percentages of senescent and exhausted CD4 and CD8 T cells, reduced levels of circulating inflammatory and denervation biomarkers, but longer telomere compared to LS- and NS-PHIVAYA. They differed significantly from healthy controls only in a few markers, including higher percentages of regulatory T and B cells, and higher levels of DAMPs. Overall, these results underscore the importance of initiating ART early and maintaining viral suppression to limit the establishment of the viral reservoir and to counteract immune and cellular premature aging. These findings also suggest new approaches for minimally invasive monitoring of individuals at high risk of developing premature aging and age-related illnesses.

https://doi.org/10.1371/journal.ppat.1012547

Skeletal muscle regeneration is a complex process requiring the precise coordination between multiples cell types, such as inflammatory cells, muscle stem cells and endothelial cells. Progressive impairment of the interplay between the inflammatory cells, mainly the infiltrated macrophages, and the different muscle cellular components is a key event in switching regeneration from compensatory to pathogenic in disease and aging. Macrophages exhibit substantial plasticity transitioning between pro-inflammatory (M1) and anti-inflammatory (M2) states and can quickly change their phenotype in response to different stimuli and with aging. Here, we investigate the role of the membrane protein Cripto in this complex scenario, exploring its role in regulating macrophage identity/plasticity and their metabolic adaptation during skeletal muscle regeneration. To this end, we combined both in vitro and in vivo approaches. Specifically, we performed a time course single-cell RNA sequencing analysis of injured skeletal muscle from myeloid-specific Cripto loss-of-function mice (LOF), at day 3 and 5 after injury. Preliminary results revealed a distinct M2-like macrophage cluster in Cripto LOF mice with significant differential gene expression. Gene Ontology analysis of these deregulated genes pointed to prominent alterations in cellular respiration, mitochondrial function, and protein synthesis pathways. These results were corroborated by preliminary in vitro studies suggesting that Cripto is required for proper pro- and anti-inflammatory macrophage polarization, and its absence leads to defective macrophage metabolism. Specifically, seahorse analysis shows that Cripto KO macrophages exhibit an aberrantly active metabolic profile compared to control cells. Moreover, while control M2 macrophages exhibit high oxidative phosphorylation, Cripto KO M2 macrophages appear to become metabolically quiescent. Overall, these preliminary results highlight a previously unidentified key role of Cripto in macrophage polarization and associated metabolic reprogramming, providing novel insights into its impact on the cellular landscape during muscle regeneration.

Chronic kidney disease (CKD) is a progressive condition classified into five stages, affecting about 13% of adults worldwide. Its silent onset often delays diagnosis until advanced stages. CKD is associated with dysregulation of the tryptophan/kynurenine (Trp/Kyn) pathway, characterized by reduced Trp and elevated kynurenine and quinolinic acid (QA), which may contribute to neurocognitive decline through neurotoxic metabolite accumulation.

While histopathology remains the diagnostic gold standard, it is invasive and costly, and conventional biomarkers such as eGFR and albuminuria have limitations. This has driven interest in non-invasive approaches, including the analysis of volatile organic compounds (VOCs) in biofluids as potential indicators of renal function.

This pilot study examined VOC profiles in blood and urine from 26 subjects: healthy controls (n=6), CKD patients at stages G2–G3 (n=10), and G4–G5 (n=10). Due to sampling challenges in dialysis patients, urine comparisons were limited to healthy and intermediate
groups. VOCs were analyzed using SPME-GC/MS and semi-quantified with internal standards. Multivariate analyses (PLS, VIP scores, Random Forest) identified distinct VOC patterns. Thirty-eight VOCs in blood and ten in urine were significant (p<0.05). Random Forest classification achieved 84.6% accuracy for blood and 93.8% for urine, confirming strong discriminatory power.

Serum Trp, Kyn, and QA were measured via ELISA, and Spearman’s correlation revealed significant associations between VOCs and neurocognitive biomarkers. These findings suggest VOC profiles not only reflect metabolic dysregulation but may serve as early, non-invasive predictors of CKD and related neurological impairment.