Cell Stress and Chaperones

Cells employ a bevy of transcriptional and post-translational stress responses to tolerate the burden of misfolded proteins induced by stress. In particular, the heat shock response facilitates the upregulation of molecular chaperones and protein remodeling factors that mediate proteostasis in response to accumulated misfolded proteins in the nucleus and cytosol. However, in response to stress neurons struggle to induce a canonical heat shock response, highlighting our poor understanding of how neurons maintain proteostasis. Specifically, the ability of post-mitotic respiring cells to regulate the heat shock response in comparison to their rapidly dividing, predominantly glycolytic counterparts has been under-studied. In this study, we employ yeast models that are easily manipulated to generate energy via glycolysis or mitochondrial respiration by changing the carbon source in the media. Using this model, we demonstrate that Hsf1 activity, the heat shock response and proteostasis are impaired in respiring cells. Interestingly, our data show that reduced Hsf1 activity regulates viability of respiring cells, with respiring cells poorly tolerating constitutively activated Hsf1. Finally, we describe alternative post-translational programming of the molecular chaperones Hsp70 and Hsp104 that plausibly enables respiring cells to mediate proteostasis despite a dampened heat shock response. Our findings offer new insights into possible proteostatic strategies employed by cells in different metabolic conditions.

Mice housed at room temperature (RT, 25{degrees}C) experience chronic mild cold stress compared with those housed at thermoneutrality (TN, 30{degrees}C). We hypothesized that cold stress suppresses circadian transcript expression in peripheral tissues. RNA-seq of hearts, livers, and diaphragms collected every 4 hours over 48 hours in constant darkness identified mRNA transcripts exhibiting {approx}24-hour rhythms (REGs). TN produced tissue-specific changes in REG number, identity, and phase without altering core circadian clock transcript levels. Cardiac REGs increased 4-fold, diaphragm REGs 1.5-fold, and hepatic REG identity shifted substantially. GO analysis revealed coordinated reorganization of rhythmic metabolic programs in the heart and liver. These data demonstrate that ambient housing temperature has tissue-specific effects on the number, identity, and temporal organization of rhythmically expressed transcripts in the heart, liver, and diaphragm.

ObjectiveN-acetylcysteine (NAC) is a clinically available antioxidant with potential applications in trauma-induced hypermetabolic states, including burn injury and crush syndrome. However, its effects on heat-stressed skeletal muscle cells remain incompletely characterized. This study conducted a secondary analysis of a publicly available dataset to quantify NACs protective effects against heat-stress-induced cellular damage. MethodsWe re-analyzed a publicly available dataset (Lu J, 2024, Mendeley Data, doi:10.17632/wffrtcgbnx.1) containing 21 observations across three conditions: Control (n=3), Heat Stress only (HS, n=3), and HS with NAC at five doses (0.5-8.0 mM, n=3 per dose). The primary outcome was the protective ratio [(HS+NAC - HS) / (Control - HS)], where 1.0 indicates complete protection. Statistical analyses included one-way ANOVA, post-hoc t-tests with Bonferroni correction, Cohens d effect sizes, and bootstrap confidence intervals. ResultsHeat stress significantly reduced cell viability by 56.3% (Control: 100.0 {+/-} 12.2 vs HS: 43.7 {+/-} 5.1; t(4)=7.37, p=0.002, Cohens d=6.02). NAC demonstrated a biphasic dose-response with maximal protection at 2.0 mM (66.7 {+/-} 14.4), yielding a protective ratio of 0.409 (95% CI: 0.146-0.675), representing 40.9% protection against heat stress damage. The comparison between HS and HS+NAC (2.0 mM) showed a large effect size (Cohens d = 2.12) but did not reach statistical significance (p = 0.060) due to the small sample size. One-way ANOVA confirmed overall group differences (F(2,18)=32.39, p<0.001, 2=0.783). ConclusionsNAC provides partial protection against heat stress-induced skeletal muscle cell damage at 2.0 mM, with a large effect size suggesting clinical relevance despite limited statistical power. These preliminary findings support further investigation of NAC as an adjunct therapy in trauma-induced hypermetabolic states. All analysis code is provided for reproducibility.

Background/ObjectivesNeuroinflammation-driven iron dysregulation and neurotoxic astrocyte polarization are increasingly recognized as interconnected pathological mechanisms in neurodegenerative diseases. Systemic inflammation triggered by strenuous exercise or infection can engage the central nervous system and astrocytic inflammatory responses and perturb iron homeostasis; however, targeted nutritional strategies to counteract these processes remain limited. Inflamate(R) is a multi-component botanical supplement comprising boswellic acids, astilbin, xanthohumol, and cinnamaldehyde, each with documented anti-inflammatory properties. However, whether this combined formulation can modulate the inflammatory-iron metabolic axis and astrocyte phenotypic polarization remains unexplored. This study aimed to investigate the effects of Inflamate(R) on LPS-induced pro-inflammatory gene expression, iron metabolism-related gene regulation, and A1/A2 astrocyte phenotypic polarization in mouse astrocytes. MethodsMouse astrocytes (AWT) were pre-treated with Inflamate(R) (0.0375 g/mL) or DMSO vehicle for 24 h, followed by lipopolysaccharide (LPS; 1 g/mL) stimulation for an additional 24 h. The non-cytotoxic working concentration was determined by morphological assessment, CCK-8 cell viability, and LDH cytotoxicity assays. Expression of 14 target genes spanning pro-inflammatory mediators (NOS2, IL6, C3, COX2, PLA2g15, SOCS3), iron metabolism regulators (FTH1, Hepcidin, TFRC, SLC40A1, RGMa, RGMb), and astrocyte polarization markers (S100A10, GFAP) was quantified by qRT-PCR. ResultsUnder normal culture conditions, Inflamate(R) did not significantly alter the expression of any target gene except S100A10, confirming the absence of baseline cytotoxicity or transcriptional homeostatic perturbation. Upon LPS stimulation, Inflamate(R) selectively suppressed NOS2 (approximately 64% reduction, p < 0.0001), IL6 (approximately 37% reduction, p < 0.0001), and C3 (approximately 47% reduction, p < 0.0001), while COX2, PLA2g15, and SOCS3 remained unaffected. Concurrently, Inflamate(R) significantly reduced LPS-induced Hepcidin expression to approximately 17% of the control level (p < 0.05) and attenuated FTH1 upregulation (p < 0.01), without altering the expression of iron transporters (TFRC, SLC40A1) or BMP-SMAD pathway components (RGMa, RGMb). Furthermore, Inflamate(R) upregulated the neuroprotective A2 marker S100A10 under both basal (p < 0.05) and LPS-stimulated conditions (p < 0.01), while the general reactivity marker GFAP remained unchanged. ConclusionsInflamate(R) exerts a selective, multi-target modulatory effect at the transcriptional level in LPS-stimulated astrocytes, encompassing suppression of the iNOS-NO and IL-6 signaling axes, attenuation of inflammation-driven hepcidin-ferritin iron dysregulation via the IL-6-STAT3 pathway, and promotion of a phenotypic shift from neurotoxic A1 toward neuroprotective A2 astrocyte polarization. Given that the IL-6-JAK-STAT3-hepcidin axis is also activated during exercise-induced systemic inflammation, these findings suggest that Inflamate(R) may represent a targeted nutritional strategy for preserving CNS iron homeostasis and supporting neuroprotective astrocyte function in both neurodegenerative and exercise-related neuroinflammatory contexts. Further validation in in vivo neurodegenerative and exercise models, including protein-level analyses, is warranted to confirm these transcriptional findings.

Many marine invertebrates are characterized by broad and highly plastic thermal limits, though the dynamic molecular mechanisms that enable extended thermal acclimation remain poorly understood. A classic example is the green crab (Carcinus maenas), which is a prolific and damaging non-indigenous species. Using a 22-day thermal exposure to cold (5{degrees}C), ambient (13{degrees}C), or warm (30{degrees}C) temperatures, we characterized plastic shifts in C. maenas performance using respirometry and time-to-right. We then used untargeted metabolomics and lipidomics analysis of heart tissues from days 4 and 22 to identify the molecular mechanisms underpinning plastic responses over time. Crabs at 30{degrees}C exhibited higher oxygen consumption rates than counterparts at 5{degrees}C. Interestingly, oxygen consumption rate increased over time at both temperatures, indicating thermal plasticity of aerobic respiration. Temperature-dependent metabolic reprogramming was employed by crabs to sustain aerobic respiration across temperature. Catabolism of branched-chain amino acids was important for energy production at elevated temperatures, while catabolism of arginine may have sustained the minimal energy needs of crabs exhibiting metabolic depression at cold temperatures. Righting response was positively correlated with temperature, and did not exhibit any changes over time. Lipidome remodeling consistent with homeoviscous adaptation could have enabled motor activity across temperature. Higher abundances of saturated and monounsaturated lipids likely provided structural integrity to cell membranes at 30{degrees}C, while lower abundances of these compounds may have enabled membrane fluidity at 5{degrees}C. Our work demonstrates the importance of ongoing molecular reprogramming in long-term acclimation, even when whole-animal physiology remains relatively stable. Summary StatementThis study demonstrates how the highly invasive green crab regulates metabolite and lipid pathways over time to maintain physiological performance across different temperatures.

An expectation of a hypothesis that proposes cell-to-cell signalling pathways are redundant due to the redundancy of pathway terminal transcription factors (TFs) was tested by screening 35 signalling ligands (SLs) for rescue of a decapentaplegic (dpp) hypomorphic wing growth phenotype. The screen identified three examples of partial rescue: Hedgehog (HH), Semphorin 1a (SEMA1A) and Wnt ortholog 2 (WNT2). HH overexpression with dppGAL4 may increase the expression of DPP activity from the hypomorphic dpp alleles. However, SEMA1A and WNT2 did not phenocopy ectopic expression of HH or DPP and neither SEMA1A nor WNT2 were required for wing growth suggesting substitution of DPP for partial restoration of wing growth. The WNT2 rescue was dependent on the Frizzled 4 (FZ4) WNT receptor excluding the possibility that WNT2 weakly binds the DPP receptor. Although examples of phenotypic nonspecificity of SL function were identified, this is an expectation, and not direct proof, of the hypothesis of TF redundancy. Screen Report SummaryAn expectation of a hypothesis proposing that cell-to-cell signalling pathways are redundant due to the redundancy of the pathway terminal transcription factors was tested by screening for replacement of one signalling ligand (DPP; SLa) with another SLb for wing growth. Three non-DPP SLs were identified in the screen of 35SLs: HH, SEMA1A and WNT2. Genetic analysis of Sema1a and Wnt2 suggests functional complementation of dpp for wing growth suggesting that SEMA1A and WNT2 partially replace DPP for wing growth. Therefore, an expectation of the hypothesis is met.

BackgroundTrained immunity is a durable functional reprogramming of innate immune cells characterized by enhanced responsiveness upon secondary challenge. While metabolic rewiring and epigenetic remodeling are well-established features of this process, the contribution of ubiquitin-mediated post-translational regulation remains poorly defined. MethodsWe performed an integrative analysis of publicly available human transcriptomic datasets derived from monocytes, macrophages, and PBMCs exposed to established training stimuli ({beta}-glucan, Bacillus Calmette-Guerin [BCG], and hemin-{beta}-glucan) followed by secondary stimulation. A curated panel of deubiquitinating enzymes (DUBs) and E3 ubiquitin ligases with established immune functions was analyzed for differential expression. Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted to evaluate higher-order convergence across independent datasets. ResultsAcross multiple trained immunity models, we identified reproducible transcriptional remodeling of ubiquitin-modifying enzymes. USP25, OTUB1, and TRIM25 were consistently upregulated following restimulation, whereas several chromatin- and cytokine-regulatory DUBs--including USP3, USP4, USP7, USP16, MYSM1, and USP38--were downregulated. Normalization to RPMI-restimulated controls reduced many activation-associated signals; however, USP25 remained persistently elevated, suggesting a stable training-associated signature. Pathway enrichment analysis independently demonstrated significant engagement of ubiquitin-related functional categories across datasets, supporting coordinated reorganization of ubiquitin regulatory networks. ConclusionThese findings identify selective transcriptional remodeling of the ubiquitin- proteasome system as a recurring feature of trained immunity. Integrating ubiquitin signaling into the established metabolic-epigenetic framework expands the conceptual model of innate immune memory and suggests that ubiquitin-modifying enzymes function as modulatory rheostats shaping immune amplitude and stability. Future functional and proteomic studies are required to determine whether these transcriptional signatures directly mediate trained immunity phenotypes.

Adaptation to local environments enables species to thrive in diverse and challenging habitats. Steep elevational gradients provide a compelling natural adaptation laboratory, because abiotic conditions change progressively over short geographical differences. Given that elevation can strongly reshape physiology and behavior of insects, neuromodulatory systems offer a promising lens through which to examine elevation-specific adaptation. We challenged the hypothesis that adaptation to elevation involves octopaminergic signaling in honey bees (Apis mellifera), an important pollinator species occupying different elevations along East African mountains. We collected foragers from two distinct elevations at Mount Kenya (1,150 m and 1,900 m above sea level) and analyzed elevation-dependent changes in octopaminergic signaling. Tissue-specific analysis revealed a striking upregulation of all three octopamine {beta} receptor genes in the thoracic flight muscles and elevated octopamine brain concentrations at high elevation. Expression differences in the brain and fat body were rather modest. We subjected CRISPR/Cas9-mediated octopamine {beta}2 receptor knockouts to cold stress to study the function of octopaminergic signaling in thermoregulation. Loss of AmOAR{beta}2 reduced both the slope and amplitude of heating phases, indicating altered thermogenic dynamics. Together, these results identify the octopaminergic system as a central neuromodulatory regulator of thermogenic performance across elevations in honey bees. More broadly, our study highlights how modulation of conserved aminergic signaling pathways can shape physiological resilience to environmental gradients, pointing to a general mechanism by which insects adapt to changing thermal landscapes. Highlights- Bees from high and low elevation differ in expression of octopamine {beta} receptor genes and octopamine brain concentrations - CRISPR/Cas9-mediated octopamine receptor knockout alters thermogenic behavior - Octopaminergic signaling emerges as a key neuromodulator in thermal adaptation to elevation in honey bees Significance statementAnimals living along mountain gradients must cope with rapidly changing temperatures, yet the mechanisms enabling this adaptation remain poorly understood. We show that honey bees from higher elevations have increased brain octopamine levels and enhanced expression of octopamine receptors in heat-producing flight muscles. Using gene editing, we demonstrate that disrupting one key receptor alters how bees generate heat under cold stress. These findings identify octopamine signaling as a central regulator of thermogenesis and reveal a mechanism by which insects adjust to colder environments. More broadly, our results highlight how conserved neuromodulatory systems can fine-tune physiological performance, offering insight into how insects may respond to changing climates and expanding environmental extremes.

Birds provide a unique model for ageing research, as they exhibit higher mass-adjusted metabolic rates and blood glucose levels than other vertebrate groups, yet demonstrate greater longevity and slower senescence compared to mammals of similar body size. This challenges the "pace of life syndrome" hypothesis, which predicts that high metabolic rates and elevated glucose should correlate with shorter lifespans. While the effects of glucose, glycation, and advanced glycation end-products (AGEs) on ageing are well-documented in humans and the conventional models used in biomedical research, their impact on avian physiology and ageing remains poorly understood. Some evidence suggests that birds possess adaptations mitigating the potential detrimental effects of glucose levels, which are much higher than those of all other vertebrate groups. However, previous studies indicate that elevated glucose predicts reduced lifespan, and protein glycation--varying with age--can influence survival and some fitness-related traits. This implies that glycation or AGE accumulation may have relevant effects on avian longevity. In this study, we experimentally investigated how one year of dietary supplementation with glucose or methylglyoxal affects survival and ageing markers (metabolic rate, flying performance, and beak coloration) in captive zebra finches (Taeniopygia guttata). Our results reveal a significant increase in mortality exclusively in glucose-supplemented birds. Although glucose treatment elevated albumin glycation rate and AGE formation--the latter also observed with methylglyoxal supplementation--these variables did not directly explain the increased mortality in glucose-treated birds, which was absent in methylglyoxal-treated individuals despite similar AGE accumulation. Additionally, we observed some effects on the assessed senescence markers, with an age-related constraint on seasonal metabolic adjustment, and a treatment-influenced age decline in secondary sexual traits expression. These findings support the use of these markers as proxies for senescence in zebra finches. We also discuss alternative mechanisms, independent of the glycation cascade, which may contribute to mortality. A seasonal decline in flight performance, particularly during peak mortality periods, suggests a broader deterioration of health. Thus, although we demonstrate glucose supplementation to be more deleterious than methylglyoxal, the underlying mechanisms for the observed increase in mortality induced by the treatment remain unresolved.

Small heat-shock proteins (sHSPs) are an ancient and diverse class of molecular chaperones, acting as a first line of defense against proteotoxic stresses. While the canonical sHSPs prevent uncontrollable aggregation of a broad range of non-native substrates, a subset of sHSPs do not exhibit this broad activity in vitro, and their functions in vivo are poorly understood. Interestingly, several such sHSPs are selectively expressed in muscle tissues, including by myogenic programs, indicating likely functional roles. We examined in vivo function of C. elegans HSP-12.6, which possesses no chaperone activity in vitro but regulates lifespan, and is developmentally induced in the muscles of long-lived dauer animals. We found that HSP-12.6 exhibits exceptional selectivity in protecting the muscle function against folding or assembly mutations in thick filament proteins, but not in thin filament or non-filament proteins. This reflected its exclusive chaperone-like binding to the healthy myosin-containing thick filaments, and to their aggregates. HSP-12.6 did not bind other muscle structures or aggregates, including those of thin filaments, and retained its selectivity to either healthy thick filaments or their aggregates when challenged with a toxic aggregation-prone polyQ protein. Our data establish HSP-12.6 as a highly-selective myoprotective chaperone, with client spectrum distinct from other sHSPs.

To elucidate the early mechanisms underlying the long-term neuroprotective effect of FLASH-RT in the normal brain, spatial transcriptomics (Nanostring) were performed after whole-brain irradiation of C57BL/6J mice with either 1 or 3 fractions of 10 Gy at 5.6x106 Gy/s (1 pulse-FLASH) or at conventional dose-rate 0.1 Gy/s. FLASH -RT induced a distinct transcriptomic signature in the CA3 and DG neurons, with upregulation of genes encoding glutamate receptors, involved in calcium signaling, long-term potentiation and mitochondrial OXPHOS. Early transcriptional upregulation of Gria gene translated into increased AMPAR protein levels at 48h in the DG and CA3 region and sustained higher AMPAR expression at 2 and 4 weeks post-FLASH. These findings support a durable activation of AMPAR. We propose a mechanism to explain FLASH-induced neuroprotection initiated by early calcium influx and subsequent sustained expression of glutamate receptor AMPAR in neurons and/or neural progenitors of the CA3, potentially contributing to long-term cognitive sparing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/725423v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1ae125forg.highwire.dtl.DTLVardef@138357aorg.highwire.dtl.DTLVardef@13f128dorg.highwire.dtl.DTLVardef@1db1cf6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFLASH-RT induces a stronger transcriptional response in the hippocampus than the cortex. C_LIO_LIFLASH-RT induces calcium signaling, LTP and mitochondrial OXPHOS genes. C_LIO_LIEarly AMPAR upregulation leads to sustained protein expression. C_LIO_LIFLASH-RT induces a AMPAR-dependent signaling program in CA3 neurons. C_LI

Circadian regulation of proteostasis, a key determinant of muscle health, remains poorly understood. Here, we identified DNAJB6, an Hsp40 (DnaJ) co-chaperone, as a substrate of the circadian E3 ligase FBXL21. FBXL21 mediated the ubiquitination-dependent proteasomal degradation of both DNAJB6 and its client proteins including Desmin; causative mutations of DNAJB6 in myopathies, however, rendered resistance to FBXL21-directed degradation. Fbxl21 KO C2C12 cells displayed aberrant accumulation of Desmin, and showed aggravated cytoplasmic accumulation of TDP-43, another DNAJB6 client protein, in heat shock response. Under timed exercise as a physiological stressor, WT mice displayed robust diurnal rhythms in the levels of stress granule markers (G3BP1 and FUS) and TDP-43 as a function of exercise timing. In contrast, the Fbxl21 hypomorph Psttm mutant mice showed elevated expression of these proteins without exercise, which was exacerbated under exercise-induced stress conditions; importantly, these abnormalities were rescued by skeletal muscle-specific FBXL21 expression. Our study elucidates a novel diurnal regulatory mechanism of skeletal muscle proteostasis via FBXL21 as a chaperone-linked E3 ligase, highlighting the FBXL21-DNAJB6 axis as a potential therapeutic target for myopathies.

Adult females of the two-spotted spider mite, Tetranychus urticae Koch, enter a photoperiodically induced diapause to overwinter. Diapause in T. urticae is accompanied by reproductive arrest and the orange body coloration that arises from the accumulation of astaxanthin esters. How these two traits are coordinated at the molecular level remains poorly understood. Here, we compared the proteomes of adult females reared under diapause-inducing (long-night) and non-diapause-inducing (short-night) photoperiods using liquid chromatography-tandem mass spectrometry, followed by RNA interference (RNAi) of candidate genes. The carotenoid biosynthesis enzymes phytoene desaturase (TuPDS) and lycopene cyclase/phytoene synthase (TuLCPS), both encoded by genes horizontally transferred from fungi, were more abundant in diapausing females than in non-diapausing females. RNAi of the genes encoding TuPDS and TuLCPS markedly reduced orange pigmentation as well as {beta}-carotene and astaxanthin contents, demonstrating that these enzymes are required for diapause-associated pigmentation. Our proteomic analysis further identified a single PLAT (Polycystin-1, Lipoxygenase, Alpha-toxin) domain protein, TuPLAT10, as one of the most strongly upregulated proteins in diapausing females. The PLAT domain is a lipid-binding module, suggesting a role for TuPLAT10 in lipid metabolism. In addition to the suppression of orange pigmentation, RNAi of the TuPLAT10 gene restored reproduction even under diapause-inducing conditions and selectively reduced TuPDS and TuLCPS protein levels, despite the absence of sequence similarity to their genes. We propose that TuPLAT10 acts as a lipid-allocation switch that, in response to photoperiodic information, partitions fatty acids between astaxanthin esterification and yolk lipid supply, thereby coupling reproductive arrest and carotenoid pigmentation during diapause in T. urticae.

Autophagy is a hallmark of aging, but autophagy-related proteins have not been exclusively targeted to attenuate the progressive decline in physical function associated with aging. Here, we combined Tat-Beclin1, an autophagy agonist, and endurance training to determine whether Tat-Beclin1 enhances exercise adaptation in old male mice. Tat-Beclin1 was administered intraperitoneally (TB group, 15 mg/kg, 2x/week) as a standalone therapy, or in combination with endurance training (TB+Exe group, 70% of maximal running speed 3x/week) for 1 month in 23-month-old male C57BL/6J mice. Control groups were age-matched cage controls and exercise-only groups. Animals were assessed for grip strength, endurance capacity on a treadmill, and balance and coordination on a rotarod. Gastrocnemius/plantaris (G/P) and tibialis anterior muscles were harvested for western blotting, myofiber typing, and proteomic profiling (G/P only). TB+Exe led to significant increases in grip strength, endurance capacity, and balance and coordination performance beyond those observed in the TB and Exe groups alone. Autophagy markers, including Beclin1, the LC3B-II/I ratio, and p62, did not differ among groups. A proteomic analysis of the G/P muscle revealed that TB upregulated biological processes involved in muscle contraction and adaptation, whereas TB+Exe increased mitochondrial bioenergetic processes and, surprisingly, upregulated acute inflammatory responses, including proteins such as haptoglobin and orosomucoid-1. We conclude that combining Tat-Beclin1 and endurance training may represent a new approach to attenuate aging-related decline in physical function. New & NoteworthyWe show evidence that combining Tat-Beclin1 and endurance training (TB+Exe) resulted in greater improvements in physical function in 24-month-old male mice than either standalone therapy. We also show that TB+Exe upregulates traditional exercise-like biological processes and unexpectedly upregulates acute-inflammatory proteins (e.g., orosomucoid-1), which are thought to improve physical function in preclinical studies. Our study suggests that TB may be a new drug enhancing physical function, especially when combined with endurance training in old male mice.

Adeno-associated viral (AAV) vectors are foundational tools for dissecting brain structure-function relationships, but AAV serotype tropism varies across brain regions and species, requiring empirical validation to inform experimental design. This need is especially important in non-model organisms, where molecular neuroscience tools remain underdeveloped and access to research subjects is often limited. The Arctic ground squirrel (AGS, Urocitellus parryii) is a valuable model for studying extreme physiology, including metabolic suppression during hibernation and resistance to cerebral ischemia/reperfusion, yet no studies have evaluated AAV performance in the AGS brain. Here, we investigated the ability of AAV serotypes 1, 8, 9, and DJ to transduce the AGS hypothalamus using the human synapsin (hSyn) promoter and directly compared cellular transduction rates in a region implicated in thermoregulation and hibernation. To maximize data collection from a limited experimental population, we used a within-animal, contralateral stereotaxic injection design. Recombinant AAV vectors expressing enhanced green fluorescent protein or mCherry were delivered bilaterally, and reporter expression was analyzed four weeks later. All tested serotypes produced clear and reproducible reporter expression, establishing AAV as a viable molecular tool in the AGS hypothalamus. AAV1 produced significantly greater cellular transduction rates than AAV-DJ (17.2% {+/-} 3.5% vs 8.4% {+/-} 2.9%, paired t-test, p = 0.032). AAV8 and AAV9 showed transduction rates of 22.8% {+/-} 0.6% and 20.1% {+/-} 1.5%, respectively; however, with only two biological replicates per serotype, formal statistical comparison was not performed. These findings provide the first direct characterization of AAV-mediated gene delivery in the AGS brain and establish a foundation for future molecular interrogation of hypothalamic circuits in this extreme mammalian hibernator.

The adult skeletal muscle regenerates robustly upon injury, but this regenerative capacity rapidly declines with age. In this study, we identify the lanthionine synthetase C-Like (LanCL) proteins, mammalian homologs of the bacterial peptide cyclase LanC, as positive regulators of muscle regeneration in middle-aged mice. In a barium chloride-induced injury model, we found the protein levels of LanCL1 and LanCL2 to increase during an early phase of regeneration in middle-aged (12-month-old) but not young adult (4-month-old) mice. Utilizing a mouse line lacking all three LanCL proteins (LanCL triple KO or LTKO), we examined a potential role of LanCL in injury-induced muscle regeneration. Consistent with an age-dependent function of LanCL, we observed a delayed regeneration of the tibialis anterior (TA) muscle after injury, as reflected by reduced sizes of regenerating myofibers in middle-aged (but not young) LTKO compared to age-matched WT mice. Although the pool size of quiescent satellite cells (Pax7+) was comparable between 12-month-old LTKO and WT muscles without injury, the number of Pax7+ cells was significantly higher in regenerating LTKO muscles at day 5 after injury, accompanied by drastically decreased numbers of MyoD+ and MyoG+ cells, as well as increased numbers of proliferating cells. In addition, we detected elevated expression of pro-inflammatory cytokines in regenerating LTKO muscles, while the number of macrophages was similar comparing LTKO and WT muscles. Taken together, our observations suggest that in aging muscles LanCLs are important for proper timing of inflammation resolution and regeneration upon injury. New & NoteworthyPhysiological roles of the mammalian homologs of bacterial LanC, LanCLs, are poorly understood. Our work uncovers a function of LanCLs in post-injury regeneration of aging skeletal muscles. Middle-aged LanCL triple KO mice displayed a delay in satellite cell differentiation and regenerative myofiber formation, as well as persistent inflammatory cytokine expression, suggesting that LanCLs may have an age-dependent role in modulating inflammation in the injured muscles to facilitate regeneration.

Circadian disruption (CD) is increasingly recognized as a sex-specific risk factor for Alzheimers disease (AD). However, the mechanisms linking CD to AD, and the role of biological sex in this interaction, are unclear. Immunometabolic regulation is extensively circadianly timed, has sex-specific phenotypes, and plays a role in AD. Therefore, we hypothesized that CD affects the timing of immunometabolism, contributing to the sex-specific effects of CD on AD. To demonstrate this, we subjected male and female APP/PS1 mice to chronic disruptive lighting to model circadian disruption, finding CD induced a female-specific reduction in amyloid plaque burden but an increase in the infiltration of peripheral macrophages into the brain. Concomitantly, we found macrophages exhibited CD-associated immune reprogramming, which in females led to altered immunometabolic timing, an increase of macrophages in the activated state, and elevated levels of reactive oxygen species (ROS), supporting a role for immunometabolism in the sex-specific effects of CD in AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/721994v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@b9a59forg.highwire.dtl.DTLVardef@289219org.highwire.dtl.DTLVardef@18fe804org.highwire.dtl.DTLVardef@c96bb8_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICircadian disruption reduces A{beta} plaque load specifically in female mice C_LIO_LIPeripheral immune infiltration correlates with reduced A{beta} plaques in females C_LIO_LICircadian disruption coordinates phasing of circadian immunometabolic proteins C_LIO_LIIn females, circadian phase advancement correlates with increased ROS C_LI

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.

Bdelloid rotifers are known to survive desiccation and high doses of ionizing radiation. This extreme resistance is notably due to their capacity to cope with numerous DNA double-strand breaks (DSBs). Genes encoding key components of the non-homologous end joining (NHEJ) DNA repair pathway are strongly upregulated in the bdelloid rotifer Adineta vaga following exposure to ionizing radiation. Considering the notably high doses tolerated by these organisms, their capacity to efficiently restore genome integrity is particularly striking. Although NHEJ is generally regarded as less accurate than homologous recombination (HR), the absence of major genomic rearrangements in the descendants of irradiated rotifers suggests that DNA repair occurs with high fidelity. Terwagne et al. recently reported a delayed repair in germline nuclei, occurring during oocyte development when homologous chromosomes pair, thereby enabling template-based repair through HR. In this study, we established an in situ hybridization approach on A. vaga cryosections to investigate the spatial and temporal expression of key actors involved in NHEJ, HR, and Base excision repair (BER) pathways in somatic and germline tissues. We show that NHEJ (KU80) and BER-related genes (PARPs) as well as A. vaga Ligase E (putatively involved in DNA repair) are expressed early after radiation exposure in the somatic syncytium. In contrast, HR-related genes (Rad51: two paralogs, Rad54), as well as PCNA (involved in DNA replication, NER, BER, HR) are expressed later in maturing oocytes, indicating the activation of a delayed homologous recombination repair pathway in germline nuclei. Nurse cells, which express genes associated with both HR and NHEJ pathways, may rely on both mechanisms for their own DNA repair while also supplying mRNAs to the maturing oocyte. Our results provide new evidence for a differential regulation of DNA DSB repair pathways between soma and germline in bdelloids, with NHEJ predominating in somatic tissues and HR in the germline of A. vaga. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722046v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3b1f3borg.highwire.dtl.DTLVardef@17f5eb5org.highwire.dtl.DTLVardef@122ef14org.highwire.dtl.DTLVardef@7e4413_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure:C_FLOATNO Summary of in situ hybridization results: genes coding for actors of NHEJ are expressed in the somatic nuclei and in the nurse nuclei of Adineta vaga individuals 2.5 hours post X-rays radiation, while genes coding for HR actors and PCNA (involved in multiple pathways including DNA replication and DNA repair: NER, BER, MR, HR) are expressed in the nurse nuclei 2.5 hours post radiation, and later in the maturing oocyte during oogenesis and in the laid eggs. Genes coding for actors highly expressed post-radiation, involved in the BER pathway appear to be only expressed in the somatic syncytium 2.5 hours post radiation, as well as the gene coding for the Ligase E, likely involved in DNA repair. C_FIG

BackgroundNeonatal sepsis is a major cause of infant morbidity and mortality worldwide, particularly in preterm and very low birthweight babies. Fundamental differences between neonates and adults warrant clinically relevant models of neonatal sepsis. Here, we describe a preclinical fecal-slurry (FS)-induced peritonitis model of polymicrobial sepsis in neonatal rats, along with a novel neonatal rat sepsis score (nRSS) to monitor illness severity. MethodsPeritonitis was induced in 3-day-old Sprague Dawley rats by intraperitoneal injection of various doses (0.3-1.5mg/g body weight) of fecal slurry (FS); control pups received equivalent doses of vehicle. All pups received analgesics (buprenorphine), antibiotics (ampicillin and gentamicin), and fluids (saline) to model clinical standards of sepsis treatment. Time-dependent changes in circulating cytokines (IL-6, IL-1{beta}) and biomarkers of sepsis pathology (hemoglobin, glucose, alanine transaminase [ALT] levels) were assessed and correlated with nRSS scores. ResultsFS administration caused a dose-dependent increase in severity of sepsis over time, as indicated by increases in mortality rates (based on predefined criteria for euthanasia), nRSS scores, as well as time-dependent changes in circulating glucose, hemoglobin, IL-6, IL-1{beta}, and ALT activity levels. nRSS scores correlated with all quantitative measures of sepsis pathology. Notably, females showed higher mortality and higher early NRSS scores than males at moderate to high FS doses, yet biochemical markers and time of death did not differ between sexes, suggesting that the apparent female vulnerability may reflect more conspicuous behavioral manifestations of illness rather than greater underlying physiological severity. ConclusionInduction of peritonitis in rats at postnatal day 3 produced a consistent and reproducible model of polymicrobial neonatal sepsis. Illness severity was monitored using a newly developed nRSS. By minimizing distress and incorporating standards of care, this model and scoring system may serve as a platform for future investigations into the underlying mechanisms and potential therapeutic interventions for neonatal sepsis. ImpactO_LIA clinically relevant rat model of neonatal polymicrobial sepsis was developed, incorporating standards of care (analgesics, antibiotics, and fluid resuscitation) to better reflect the clinical context in which preclinical findings must ultimately translate. C_LIO_LIA novel neonatal rat sepsis scoring system (nRSS) was developed and validated, providing a sensitive, non-invasive measure of disease severity that correlates with biochemical markers and predicts mortality. C_LIO_LIFemale pups showed higher mortality and earlier behavioral signs of illness than males despite equivalent biochemistry, highlighting that clinical scores may capture sex-dependent vulnerability not apparent in standard biochemical measures. C_LIO_LITogether, this model and scoring system offer a refined platform for mechanistic and therapeutic studies of neonatal sepsis while advancing the welfare-conscious 3Rs principles essential to rigorous preclinical research C_LI