Dysregulation of the fluid homeostasis system by aging.

Chronic dehydration is a leading cause of morbidity for the elderly, but how aging alters the fluid homeostasis system is not well understood. Here, we used a combination of physiologic, behavioral and circuit analyses to characterize how fluid balance is affected by aging in mice. We found that old mice have a primary defect in sensing and producing the anti-diuretic hormone vasopressin, which results in chronic dehydration. Recordings and manipulations of the thirst circuitry revealed that old mice retain the ability to sense systemic cues of dehydration but are impaired in detecting presystemic, likely oropharyngeal, cues generated during eating and drinking, resulting in disorganized drinking behavior on short timescales. Surprisingly, old mice had increased drinking and motivation after 24-hour water deprivation, indicating that aging does not result in a general impairment in the thirst circuit. These findings reveal how a homeostatic system undergoes coordinated changes during aging.

Neuropeptide inactivation regulates egg-laying behavior to influence reproductive health in Caenorhabditis elegans.

Neural communication requires both fast-acting neurotransmitters and neuromodulators that function on slower timescales to communicate. Endogenous bioactive peptides, often called "neuropeptides," comprise the largest and most diverse class of neuromodulators that mediate crosstalk between the brain and peripheral tissues to regulate physiology and behaviors conserved across the animal kingdom. Neuropeptide signaling can be terminated through receptor binding and internalization or degradation by extracellular enzymes called neuropeptidases. Inactivation by neuropeptidases can shape the dynamics of signaling in vivo by specifying both the duration of signaling and the anatomic path neuropeptides can travel before they are degraded. For most neuropeptides, the identity of the relevant inactivating peptidase(s) is unknown. Here, we established a screening platform in C. elegans utilizing mass spectrometry-based peptidomics to discover neuropeptidases and simultaneously profile the in vivo specificity of these enzymes against each of more than 250 endogenous peptides. We identified NEP-2, a worm ortholog of the mammalian peptidase neprilysin-2, and demonstrated that it regulates specific neuropeptides, including those in the egg-laying circuit. We found that NEP-2 is required in muscle cells to regulate signals from neurons to modulate both behavior and health in the reproductive system. Taken together, our results demonstrate that peptidases, which are an important node of regulation in neuropeptide signaling, affect the dynamics of signaling to impact behavior, physiology, and aging.

Temperature-dependent differences in mouse gut motility are mediated by stress.

Researchers have advocated elevating mouse housing temperatures from the conventional ~22 °C to the mouse thermoneutral point of 30 °C to enhance translational research. However, the impact of environmental temperature on mouse gastrointestinal physiology remains largely unexplored. Here we show that mice raised at 22 °C exhibit whole gut transit speed nearly twice as fast as those raised at 30 °C, primarily driven by a threefold increase in colon transit speed. Furthermore, gut microbiota composition differs between the two temperatures but does not dictate temperature-dependent differences in gut motility. Notably, increased stress signals from the hypothalamic-pituitary-adrenal axis at 22 °C have a pivotal role in mediating temperature-dependent differences in gut motility. Pharmacological and genetic depletion of the stress hormone corticotropin-releasing hormone slows gut motility in stressed 22 °C mice but has no comparable effect in relatively unstressed 30 °C mice. In conclusion, our findings highlight that colder mouse facility temperatures significantly increase gut motility through hormonal stress pathways.

The association of race, ethnicity, and socioeconomic status on the severity of menopause symptoms: a study of 68,864 women.

OBJECTIVE: This study aimed to evaluate if and how race, ethnicity, and socioeconomic status (SES) are associated with the severity of menopause symptoms in a large, diverse sample of women. METHODS: For this cross-sectional study conducted between March 24, 2019, and January 13, 2023, a total of 68,864 women were enrolled from the Evernow online telehealth platform. Participants underwent a clinical intake survey, which encompassed demographic information, detailed medical questionnaires, and a modified Menopause Rating Scale. The modified scale was adapted for ease of use online and is available in the supplementary material along with the full intake. Symptom severity was evaluated using a multivariate binomial generalized linear model, accounting for factors such as race, ethnicity, age, body mass index, smoking status, bilateral oophorectomy status, and SES. Odds ratios (OR) and CIs were calculated based on the linear regression coefficients. RESULTS: Of the participants, 67,867 (98.6%) were included in the analysis after excluding outliers and those with unknown oophorectomy status. The majority of respondents identified as White (77.4%), followed by Hispanic (9.0%), Black (6.7%), two or more races/ethnicities (4.4%), Asian (1.2%), Indigenous/First Nations (0.8%), Middle Eastern (0.3%), and South Asian (0.2%). Notably, individuals identifying as Black (hot flashes OR, 1.91; 97.5% CI, 1.75-2.09; P < 0.001), Hispanic (skin/hair changes OR, 1.58; 97.5% CI, 1.45-1.71; P < 0.001), Indigenous/First Nations (painful sex OR, 1.39; 97.5% CI, 1.19-2.75; P = 0.007), Middle Eastern (weight changes OR, 2.22; 97.5% CI, 1.25-4.37; P = 0.01), or with two or more races/ethnicities (skin/hair changes OR, 1.41; 97.5% CI, 1.26-1.58; P < 0.001) reported higher levels of symptom severity compared with their White counterparts. Conversely, Asian and South Asian participants reported lower symptom severity. Even after incorporating SES into the linear model, racial and ethnic groups with lower SES (Black, Hispanic, Indigenous, and multiple ethnicities) exhibited slight shifts in OR while maintaining high statistical significance (Black [hot flashes OR, 1.87; 97.5% CI, 1.72-2.04; P < 0.001], Hispanic [skin/hair changes OR, 1.54; 97.5% CI, 1.42-1.68; P < 0.001], Indigenous/First Nations [painful sex OR, 1.74; 97.5% CI, 1.17-2.70; P = 0.009], multiple ethnicities [skin/hair changes OR, 1.41; 97.5% CI, 1.26-1.58; P < 0.001]). CONCLUSIONS: Our study suggests that the relationship between race and ethnicity and the severity of menopause symptoms is not solely explained by differences in SES but is itself an independent factor. Understanding and addressing social, cultural, and economic factors are crucial to reduce disparities in menopausal symptoms.

A role for worm cutl-24 in background- and parent-of-origin-dependent ER stress resistance.

BACKGROUND: Organisms in the wild can acquire disease- and stress-resistance traits that outstrip the programs endogenous to humans. Finding the molecular basis of such natural resistance characters is a key goal of evolutionary genetics. Standard statistical-genetic methods toward this end can perform poorly in organismal systems that lack high rates of meiotic recombination, like Caenorhabditis worms. RESULTS: Here we discovered unique ER stress resistance in a wild Kenyan C. elegans isolate, which in inter-strain crosses was passed by hermaphrodite mothers to hybrid offspring. We developed an unbiased version of the reciprocal hemizygosity test, RH-seq, to explore the genetics of this parent-of-origin-dependent phenotype. Among top-scoring gene candidates from a partial-coverage RH-seq screen, we focused on the neuronally-expressed, cuticlin-like gene cutl-24 for validation. In gene-disruption and controlled crossing experiments, we found that cutl-24 was required in Kenyan hermaphrodite mothers for ER stress tolerance in their inter-strain hybrid offspring; cutl-24 was also a contributor to the trait in purebred backgrounds. CONCLUSIONS: These data establish the Kenyan strain allele of cutl-24 as a determinant of a natural stress-resistant state, and they set a precedent for the dissection of natural trait diversity in invertebrate animals without the need for a panel of meiotic recombinants.

Cold Survival and Its Molecular Mechanisms in a Locally Adapted Nematode Population.

Since Darwin, evolutionary biologists have sought to understand the drivers and mechanisms of natural trait diversity. The field advances toward this goal with the discovery of phenotypes that vary in the wild, their relationship to ecology, and their underlying genes. Here, we established resistance to extreme low temperature in the free-living nematode Caenorhabditis briggsae as an ecological and evolutionary model system. We found that C. briggsae strains of temperate origin were strikingly more cold-resistant than those isolated from tropical localities. Transcriptional profiling revealed expression patterns unique to the resistant temperate ecotype, including dozens of genes expressed at high levels even after multiple days of cold-induced physiological slowdown. Mutational analysis validated a role in cold resistance for seven such genes. These findings highlight a candidate case of robust, genetically complex adaptation in an emerging model nematode, and shed light on the mechanisms at play.

An oxytocin/vasopressin-related neuropeptide modulates social foraging behavior in the clonal raider ant.

Oxytocin/vasopressin-related neuropeptides are highly conserved and play major roles in regulating social behavior across vertebrates. However, whether their insect orthologue, inotocin, regulates the behavior of social groups remains unknown. Here, we show that in the clonal raider ant Ooceraea biroi, individuals that perform tasks outside the nest have higher levels of inotocin in their brains than individuals of the same age that remain inside the nest. We also show that older ants, which spend more time outside the nest, have higher inotocin levels than younger ants. Inotocin thus correlates with the propensity to perform tasks outside the nest. Additionally, increasing inotocin pharmacologically increases the tendency of ants to leave the nest. However, this effect is contingent on age and social context. Pharmacologically treated older ants have a higher propensity to leave the nest only in the presence of larvae, whereas younger ants seem to do so only in the presence of pupae. Our results suggest that inotocin signaling plays an important role in modulating behaviors that correlate with age, such as social foraging, possibly by modulating behavioral response thresholds to specific social cues. Inotocin signaling thereby likely contributes to behavioral individuality and division of labor in ant societies.

Soma-Targeted Imaging of Neural Circuits by Ribosome Tethering.

Neuroscience relies on techniques for imaging the structure and dynamics of neural circuits, but the cell bodies of individual neurons are often obscured by overlapping fluorescence from axons and dendrites in surrounding neuropil. Here, we describe two strategies for using the ribosome to restrict the expression of fluorescent proteins to the neuronal soma. We show first that a ribosome-tethered nanobody can be used to trap GFP in the cell body, thereby enabling direct visualization of previously undetectable GFP fluorescence. We then design a ribosome-tethered GCaMP for imaging calcium dynamics. We show that this reporter faithfully tracks somatic calcium dynamics in the mouse brain while eliminating cross-talk between neurons caused by contaminating neuropil. In worms, this reporter enables whole-brain imaging with faster kinetics and brighter fluorescence than commonly used nuclear GCaMPs. These two approaches provide a general way to enhance the specificity of imaging in neurobiology.

Oxytocin/vasopressin-related peptides have an ancient role in reproductive behavior.

Many biological functions are conserved, but the extent to which conservation applies to integrative behaviors is unknown. Vasopressin and oxytocin neuropeptides are strongly implicated in mammalian reproductive and social behaviors, yet rodent loss-of-function mutants have relatively subtle behavioral defects. Here we identify an oxytocin/vasopressin-like signaling system in Caenorhabditis elegans, consisting of a peptide and two receptors that are expressed in sexually dimorphic patterns. Males lacking the peptide or its receptors perform poorly in reproductive behaviors, including mate search, mate recognition, and mating, but other sensorimotor behaviors are intact. Quantitative analysis indicates that mating motor patterns are fragmented and inefficient in mutants, suggesting that oxytocin/vasopressin peptides increase the coherence of mating behaviors. These results indicate that conserved molecules coordinate diverse behavioral motifs in reproductive behavior.

Molecular profiling of activated neurons by phosphorylated ribosome capture.

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue.

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation.

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120-160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway.

Secretory protein profiling reveals TNF-α inactivation by selective and promiscuous Sec61 modulators.

Cotransins are cyclic heptadepsipeptides that bind the Sec61 translocon to inhibit cotranslational translocation of a subset of secreted and type I transmembrane proteins. The few known cotransin-sensitive substrates are all targeted to the translocon by a cleavable signal sequence, previously shown to be a critical determinant of cotransin sensitivity. By profiling two cotransin variants against a panel of secreted and transmembrane proteins, we demonstrate that cotransin side-chain differences profoundly affect substrate selectivity. Among the most sensitive substrates we identified is the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Like all type II transmembrane proteins, TNF-α is targeted to the translocon by its membrane-spanning domain, indicating that a cleavable signal sequence is not strictly required for cotransin sensitivity. Our results thus reveal an unanticipated breadth of translocon substrates whose expression is inhibited by Sec61 modulators.

Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes.

Evolution can follow predictable genetic trajectories, indicating that discrete environmental shifts can select for reproducible genetic changes. Conspecific individuals are an important feature of an animal's environment, and a potential source of selective pressures. Here we show that adaptation of two Caenorhabditis species to growth at high density, a feature common to domestic environments, occurs by reproducible genetic changes to pheromone receptor genes. Chemical communication through pheromones that accumulate during high-density growth causes young nematode larvae to enter the long-lived but non-reproductive dauer stage. Two strains of Caenorhabditis elegans grown at high density have independently acquired multigenic resistance to pheromone-induced dauer formation. In each strain, resistance to the pheromone ascaroside C3 results from a deletion that disrupts the adjacent chemoreceptor genes serpentine receptor class g (srg)-36 and -37. Through misexpression experiments, we show that these genes encode redundant G-protein-coupled receptors for ascaroside C3. Multigenic resistance to dauer formation has also arisen in high-density cultures of a different nematode species, Caenorhabditis briggsae, resulting in part from deletion of an srg gene paralogous to srg-36 and srg-37. These results demonstrate rapid remodelling of the chemoreceptor repertoire as an adaptation to specific environments, and indicate that parallel changes to a common genetic substrate can affect life-history traits across species.

Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels.

In traditional folk medicine, Xanthoxylum plants are referred to as 'toothache trees' because their anesthetic or counter-irritant properties render them useful in the treatment of pain. Psychophysical studies have identified hydroxy-alpha-sanshool as the compound most responsible for the unique tingling and buzzing sensations produced by Szechuan peppercorns or other Xanthoxylum preparations. Although it is generally agreed that sanshool elicits its effects by activating somatosensory neurons, the underlying cellular and molecular mechanisms remain a matter of debate. Here we show that hydroxy-alpha-sanshool excites two types of sensory neurons, including small-diameter unmyelinated cells that respond to capsaicin (but not mustard oil) as well as large-diameter myelinated neurons that express the neurotrophin receptor TrkC. We found that hydroxy-alpha-sanshool excites neurons through a unique mechanism involving inhibition of pH- and anesthetic-sensitive two-pore potassium channels (KCNK3, KCNK9 and KCNK18), providing a framework for understanding the unique and complex psychophysical sensations associated with the Szechuan pepper experience.

Photo-leucine incorporation reveals the target of a cyclodepsipeptide inhibitor of cotranslational translocation.

Photoaffinity labeling is a powerful tool to identify protein targets of biologically active small molecules, yet is often limited by the size, chemical properties, and availability of photoreactive groups. We report an improved synthesis of photo-leucine, a diazirine-based photoreactive analogue of leucine, and demonstrate its incorporation into a cyclodepsipeptide inhibitor of cotranslational translocation. Photoaffinity labeling in a crude membrane fraction, followed by "click chemistry" with a rhodamine-azide reporter, enabled the identification of Sec61alpha, the structural core of the Sec61 translocation channel, as the inhibitor's target.

The role of p58IPK in protecting the stressed endoplasmic reticulum.

The preemptive quality control (pQC) pathway protects cells from acute endoplasmic reticulum (ER) stress by attenuating translocation of nascent proteins despite their targeting to translocons at the ER membrane. Here, we investigate the hypothesis that the DnaJ protein p58(IPK) plays an essential role in this process via HSP70 recruitment to the cytosolic face of translocons for extraction of translocationally attenuated nascent chains. Our analyses revealed that the heightened stress sensitivity of p58-/- cells was not due to an impairment of the pQC pathway or elevated ER substrate burden during acute stress. Instead, the lesion was in the protein processing capacity of the ER lumen, where p58(IPK) was found to normally reside in association with BiP. ER lumenal p58(IPK) could be coimmunoprecipitated with a newly synthesized secretory protein in vitro and stimulated protein maturation upon overexpression in cells. These results identify a previously unanticipated location for p58(IPK) in the ER lumen where its putative function as a cochaperone explains the stress-sensitivity phenotype of knockout cells and mice.

Substrate-specific translocational attenuation during ER stress defines a pre-emptive quality control pathway.

Eukaryotic proteins entering the secretory pathway are translocated into the ER by signal sequences that vary widely in primary structure. We now provide a functional rationale for this long-observed sequence diversity by demonstrating that differences among signals facilitate substrate-selective modulation of protein translocation. We find that during acute ER stress, translocation of secretory and membrane proteins is rapidly and transiently attenuated in a signal sequence-selective manner. Their cotranslational rerouting to the cytosol for degradation reduces the burden of misfolded substrates entering the ER and represents a pathway for pre-emptive quality control (pQC). Bypassing the pQC pathway for the prion protein increases its rate of aggregation in the ER lumen during prolonged stress and renders cells less capable of viable recovery. Conversely, pharmacologically augmenting pQC during ER stress proved protective. Thus, protein translocation is a physiologically regulated process that is utilized for pQC as part of the ER stress response.

Cotranslocational degradation protects the stressed endoplasmic reticulum from protein overload.

The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58(IPK)/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58(IPK) recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58(IPK-/-) mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58(IPK) is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.