Search

The Amplified Fragment Length Polymorphism (AFLP) technique is employed for characterizing inbred breeds of animals and detecting strain-specific polymorphisms. The genetic differentiation of laboratory bred gerbils by means of AFLP markers are investigated.

Various approaches exist to quantify the aging process and estimate biological age on an individual level. Frailty indices based on an age-related accumulation of physical deficits have been developed for human use and translated into mouse models. However, declines observed in aging are not limited to physical functioning but also involve social capabilities. The concept of "social frailty" has been recently introduced into human literature, but no index of social frailty exists for laboratory mice yet. To fill this gap, we developed a mouse Social Frailty Index (mSFI) consisting of seven distinct assays designed to quantify social functioning which is relatively simple to execute and is minimally invasive. Application of the mSFI in group-housed male C57BL/6 mice demonstrated a progressively elevated levels of social frailty through the lifespan. Conversely, group-housed females C57BL/6 mice manifested social frailty only at a very old age. Female mice also showed significantly lower mSFI score from 10 months of age onward when compared to males. We also applied the mSFI in male C57BL/6 mice under chronic subordination stress and in chronic isolation, both of which induced larger increases in social frailty compared to age-matched group-housed males. Lastly, we show that the mSFI is enhanced in mouse models that show accelerated biological aging such as progeroid Ercc1 -/Δ and Xpg -/- mice of both sexes compared to age matched littermate wild types. In summary, the mSFI represents a novel index to quantify trajectories of biological aging in mice and may help elucidate links between impaired social behavior and the aging process., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the views of the funders, the Department of Veterans Affairs or the United States Government. Funders had no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript., (© 2024. The Author(s).)

Life stress can shorten lifespan and increase risk for aging-related diseases, but the biology underlying this phenomenon remains unclear. Here we assessed the effect of chronic stress on cellular senescence-a hallmark of aging. Exposure to restraint stress, a psychological non-social stress model, increased p21 Cip1 exclusively in the brains of male, but not female mice, and in a p16 Ink4a -independent manner. Conversely, exposure to chronic subordination stress (only males were tested) increased key senescent cell markers in peripheral blood mononuclear cells, adipose tissue and brain, in a p16 Ink4a -dependent manner. p16 Ink4a -positive cells in the brain of chronic subordination stress-exposed mice were primarily hippocampal and cortical neurons with evidence of DNA damage that could be reduced by p16 Ink4a cell clearance. Clearance of p16 Ink4a -positive cells was not sufficient to ameliorate the adverse effects of social stress on measured metrics of healthspan. Overall, our findings indicate that social stress induces an organ-specific and p16 Ink4a -dependent accumulation of senescent cells, illuminating a fundamental way by which the social environment can contribute to aging., Competing Interests: Competing interests: D.J.B. and J.M.v.D. have a potential financial interest related to this research. J.M.v.D. is a cofounder of Unity Biotechnology, D.J.B. and J.M.v.D. are co-inventors on patents held by Mayo Clinic and patent applications licensed to or filed by Unity Biotechnology, and D.J.B. and J.M.v.D. are Unity shareholders. Research in the Baker laboratory has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with Mayo Clinic Conflict of Interest policies. The other authors declare no conflicts of interest., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Brown adipocytes are defined based on a distinct morphology and genetic signature that includes, amongst others, the expression of the Purinergic 2 Receptor X5 (P2RX5). However, the role of P2RX5 in brown adipocyte and brown adipose tissue function is poorly characterized. In the present study, we conducted a metabolic characterization of P2RX5 knockout male mice; next, we characterized this purinergic pathway in a cell-autonomous context in brown adipocytes. We then tested the role of the P2RX5 receptor agonism in metabolic responses in vivo in conditions of minimal adaptive thermogenesis requirements. Our data show that loss of P2RX5 causes reduced brown adipocyte differentiation in vitro, and browning in vivo. Lastly, we unravel a previously unappreciated role for P2RX5 agonism to exert an anti-obesity effect in the presence of enhanced brown adipose tissue recruitment in male mice housed at thermoneutrality. Altogether, our data support a role for P2RX5 in mediating brown adipocyte differentiation and function that could be further targeted for benefits in the context of adipose tissue pathology and metabolic diseases.

For centuries, aging was considered inevitable and immutable. Geroscience provides the conceptual framework to shift this focus toward a new view that regards aging as an active biological process, and the biological age of an individual as a modifiable entity. Significant steps forward have been made toward the identification of biomarkers for and measures of biological age, yet knowledge gaps in geroscience are still numerous. Animal models of aging are the focus of this perspective, which discusses how experimental design can be optimized to inform and refine the development of translationally relevant measures and biomarkers of biological age. We provide recommendations to the field, including: the design of longitudinal studies in which subjects are deeply phenotyped via repeated multilevel behavioral/social/molecular assays; the need to consider sociobehavioral variables relevant for the species studied; and finally, the importance of assessing age of onset, severity of pathologies, and age-at-death. We highlight approaches to integrate biomarkers and measures of functional impairment using machine learning approaches designed to estimate biological age as well as to predict future health declines and mortality. We expect that advances in animal models of aging will be crucial for the future of translational geroscience but also for the next chapter of medicine., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Upper-limb spasticity, frequent after central nervous system lesions, is typically treated with botulinum neurotoxin type A (BoNT-A) injections to reduce muscle tone and increase range of motion. However, performing adjunct physical therapy post-BoNT-A can be challenging due to residual weakness or spasticity. This study evaluates the feasibility of hand therapy using a robotic hand orthosis (RELab tenoexo) with a mobile phone application as an adjunct to BoNT-A injections. Five chronic spastic patients participated in a two-session pilot study. Functional (Box and Block Test (BBT), Action Research Arm Test (ARAT)), and muscle tone (Modified Ashworth Scale (MAS)) assessments were conducted to assess functional abilities and impairment, along with usability evaluations. In the first session, subjects received BoNT-A injections, and then they performed a simulated unsupervised therapy session with the RELab tenoexo in a second session a month later. Results showed that BoNT-A reduced muscle tone (from 12.2 to 7.4 MAS points). The addition of RELab tenoexo therapy was safe, led to functional improvements in four subjects (two-cube increase in BBT as well as 2.8 points in grasp and 1.3 points in grip on ARAT). Usability results indicate that, with minor improvements, adjunct RELab tenoexo therapy could enhance therapy doses and, potentially, long-term outcomes.

Objective: Despite advances toward understanding the etiology of Alzheimer's disease (AD), it remains unclear which aspects of this disease are affected by environmental factors. Chronic life stress increases the risk of aging-related diseases including AD. The impact of stress on tauopathies remains understudied. We examined the effects of stress elicited by social (chronic subordination stress [CSS]) or psychological/physical (chronic restraint stress [CRS]) factors on the PS19 mouse model of tauopathy., Methods: Male PS19 mice (average age, 6.3 months) were randomized to receive CSS or CRS, or to remain as singly housed controls. Behavioral tests were used to assess anxiety-like behaviors and cognitive functions. Immunofluorescence staining and Western blotting analysis were used to measure levels of astrogliosis, microgliosis, and tau burden. Immunohistochemistry was used to assess glucocorticoid receptor expression., Results: PS19 mice exhibit neuroinflammation (glial fibrillary acidic protein, t tests: p = .0297; allograft inflammatory factor 1, t tests: p = .006) and tau hyperphosphorylation ( t test, p = .0446) in the hippocampus, reduced anxiety (post hoc, p = .046), and cognitive deficits, when compared with wild-type mice. Surprisingly, CRS reduced hippocampal levels of both total tau and phospho-tau S404 ( t test, p = .0116), and attenuated some aspects of both astrogliosis and microgliosis in PS19 mice ( t tests, p = .068-.0003); however, this was not associated with significant changes in neurodegeneration or cognitive function. Anxiety-like behaviors were increased by CRS (post hoc, p = .046). Conversely, CSS impaired spatial learning in Barnes maze without impacting tau phosphorylation or neurodegeneration and having a minimal impact on gliosis., Conclusions: Our results demonstrate that social or psychological stress can differentially impact anxiety-like behavior, select cognitive functions, and some aspects of tau-dependent pathology in PS19 male mice, providing entry points for the development of experimental approaches designed to slow AD progression., (Copyright © 2023 by the American Psychosomatic Society.)

Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle wasting disease caused by the loss of the structural protein dystrophin with secondary pathological manifestations including metabolic dysfunction, mood and behavioral disorders. In the mildly affected mdx mouse model of DMD, brief scruff stress causes inactivity, while more severe subordination stress results in lethality. Here, we investigated the kynurenine pathway of tryptophan degradation and the nicotinamide adenine dinucleotide (NAD+) metabolic pathway in mdx mice and their involvement as possible mediators of mdx stress-related pathology. We identified downregulation of the kynurenic acid shunt, a neuroprotective branch of the kynurenine pathway, in mdx skeletal muscle associated with attenuated peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) transcriptional regulatory activity. Restoring the kynurenic acid shunt by skeletal muscle-specific PGC-1α overexpression in mdx mice did not prevent scruff -induced inactivity, nor did abrogating extrahepatic kynurenine pathway activity by genetic deletion of the pathway rate-limiting enzyme, indoleamine oxygenase 1. We further show that reduced NAD+ production in mdx skeletal muscle after subordination stress exposure corresponded with elevated levels of NAD+ catabolites produced by ectoenzyme cluster of differentiation 38 (CD38) that have been implicated in lethal mdx response to pharmacological β-adrenergic receptor agonism. However, genetic CD38 ablation did not prevent mdx scruff-induced inactivity. Our data do not support a direct contribution by the kynurenine pathway or CD38 metabolic dysfunction to the exaggerated stress response of mdx mice., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The prevalence of hypertension increases with aging and is associated with increased arterial stiffness. Resistant hypertension is presented when drug treatments fail to regulate a sustained increased blood pressure. Given that the mechanisms between the sympathetic nervous system and the kidney play an important role in blood regulation, renal denervation (RDN) has emerged as a therapeutic potential in resistant hypertension. In this study, we investigated the effects of RDN on the biomechanical response and microstructure of elastic arteries. Common carotid arteries (CCA) excised from 3-month, 8-month, and 8-month denervated rats were subjected to biaxial extension-inflation test. Our results showed that hypertension developed in the 8-month-old rats. The sustained elevated blood pressure resulted in arterial remodeling which was manifested as a significant stress increase in both axial and circumferential directions after 8 months. RDN had a favorable impact on CCAs with a restoration of stresses in values similar to control arteries at 3 months. After biomechanical testing, arteries were imaged under a multi-photon microscope to identify microstructural changes in extracellular matrix (ECM). Quantification of multi-photon images showed no significant alterations of the main ECM components, elastic and collagen fibers, indicating that arteries remained intact after RDN. Regardless of the experimental group, our microstructural analysis of the multi-photon images revealed that reorientation of the collagen fibers might be the main microstructural mechanism taking place during pressurization with their straightening happening during axial stretching., (© 2024. The Author(s).)

G protein-coupled receptors (GPCRs) are leading druggable targets for several medicines, but many GPCRs are still untapped for their therapeutic potential due to poor understanding of specific signaling properties. The complement C3a receptor 1 (C3aR1) has been extensively studied for its physiological role in C3a-mediated anaphylaxis/inflammation, and in TLQP-21-mediated lipolysis, but direct evidence for the functional relevance of the C3a and TLQP-21 ligands and signal transduction mechanisms are still limited. In addition, C3aR1 G protein coupling specificity is still unclear, and whether endogenous ligands, or drug-like compounds, show ligand-mediated biased agonism is unknown. Here, we demonstrate that C3aR1 couples preferentially to Gi/o/z proteins and can recruit β-arrestins to cause internalization. Furthermore, we showed that in comparison to C3a 63-77 , TLQP-21 exhibits a preference toward Gi/o-mediated signaling compared to β-arrestin recruitment and internalization. We also show that the purported antagonist SB290157 is a very potent C3aR1 agonist, where antagonism of ligand-stimulated C3aR1 calcium flux is caused by potent β-arrestin-mediated internalization. Finally, ligand-mediated signaling bias impacted cell function as demonstrated by the regulation of calcium influx, lipolysis in adipocytes, phagocytosis in microglia, and degranulation in mast cells. Overall, we characterize C3aR1 as a Gi/o/z-coupled receptor and demonstrate the functional relevance of ligand-mediated signaling bias in key cellular models. Due to C3aR1 and its endogenous ligands being implicated in inflammatory and metabolic diseases, these results are of relevance toward future C3aR1 drug discovery., Competing Interests: Conflict of interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed., Methods: We developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R 21 →A)., Results: We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by an environmental temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue., Conclusions: The ΔTLQP-21 mouse line can be a valuable resource to conduct mechanistic studies on the necessary role of TLQP-21 in physiology and disease, while also serving as a platform to test the specificity of novel antibodies or immunoassays directed at TLQP-21. Our approach also has far-reaching implications by informing the development of knowledge-based genetic engineering approaches to generate selective loss of function of other peptides encoded by pro-hormones genes, leaving all other peptides within the pro-protein precursor intact and unmodified., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Chronic stress is a risk factor for numerous aging-related diseases and has been shown to shorten lifespan in humans and other social mammals. Yet how life stress causes such a vast range of diseases is still largely unclear. In recent years, the impact of stress on health and aging has been increasingly associated with the dysregulation of the so-called hallmarks of aging. These are basic biological mechanisms that influence intrinsic cellular functions and whose alteration can lead to accelerated aging. Here, we review correlational and experimental literature (primarily focusing on evidence from humans and murine models) on the contribution of life stress - particularly stress derived from adverse social environments - to trigger hallmarks of aging, including cellular senescence, sterile inflammation, telomere shortening, production of reactive oxygen species, DNA damage, and epigenetic changes. We also evaluate the validity of stress-induced senescence and accelerated aging as an etiopathological proposition. Finally, we highlight current gaps of knowledge and future directions for the field, and discuss perspectives for translational geroscience., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The prevalence of hypertension increases with aging and is associated with increased arterial stiffness. Resistant hypertension is presented when drug treatments fail to regulate a sustained increased blood pressure. Given that the mechanisms between the sympathetic nervous system and the kidney play an important role in blood regulation, renal denervation (RDN) has emerged as a therapeutic potential in resistant hypertension. In this study, we investigated the effects of RDN on the biomechanical response and microstructure of elastic arteries. Common carotid arteries (CCA) were excised from 3-, 8- and 8-month-old denervated rats, and subjected to biaxial extension-inflation test. Our results showed that hypertension developed in the 8-month-old rats. The sustained elevated blood pressure resulted in arterial remodeling which was manifested as a significant stress increase in both axial and circumferential directions after 8 months. RDN had a favorable impact on CCAs with a restoration of stresses in values similar to control arteries at 3 months. After biomechanical testing, arteries were imaged under a multi-photon microscope to identify microstructural changes in extracellular matrix (ECM). Quantification of multi-photon images showed no significant alterations of the main ECM components, elastic and collagen fibers, indicating that arteries remained intact after RDN. Regardless of the experimental group, our microstructural analysis of the multi-photon images revealed that reorientation of the collagen fibers might be the main microstructural mechanism taking place during pressurization with their straightening happening during axial stretching., Competing Interests: Competing interests The authors have no competing interests.

Objective . Transcutaneous electrical nerve stimulation (TENS) has been recently introduced in neurorehabilitation and neuroprosthetics as a promising, non-invasive sensory feedback restoration alternative to implantable neurostimulation. Yet, the adopted stimulation paradigms are typically based on single-parameter modulations (e.g. pulse amplitude (PA), pulse-width (PW) or pulse frequency (PF)). They elicit artificial sensations characterized by a low intensity resolution (e.g. few perceived levels), low naturalness and intuitiveness, hindering the acceptance of this technology. To address these issues, we designed novel multiparametric stimulation paradigms, featuring the simultaneous modulation of multiple parameters, and implemented them in real-time tests of performance when exploited as artificial sensory inputs. Approach . We initially investigated the contribution of PW and PF variations to the perceived sensation magnitude through discrimination tests. Then, we designed three multiparametric stimulation paradigms comparing them with a standard PW linear modulation in terms of evoked sensation naturalness and intensity. The most performant paradigms were then implemented in real-time in a Virtual Reality-TENS platform to assess their ability to provide intuitive somatosensory feedback in a functional task. Main results . Our study highlighted a strong negative correlation between perceived naturalness and intensity: less intense sensations are usually deemed as more similar to natural touch. In addition, we observed that PF and PW changes have a different weight on the perceived sensation intensity. As a result, we adapted the activation charge rate (ACR) equation, proposed for implantable neurostimulation to predict the perceived intensity while co-modulating the PF and charge per pulse, to TENS (ACR T ). ACR T allowed to design different multiparametric TENS paradigms with the same absolute perceived intensity. Although not reported as more natural, the multiparametric paradigm, based on sinusoidal PF modulation, resulted being more intuitive and subconsciously integrated than the standard linear one. This allowed subjects to achieve a faster and more accurate functional performance. Significance . Our findings suggest that TENS-based, multiparametric neurostimulation, despite not consciously perceived naturally, can provide integrated and more intuitive somatosensory information, as functionally proved. This could be exploited to design novel encoding strategies able to improve the performance of non-invasive sensory feedback technologies., (Creative Commons Attribution license.)

Sustained life stress and low socioeconomic status are among the major causes of aging-related diseases and decreased life expectancy. Experimental rodent models can help to identify the underlying mechanisms, yet very few studies address the long-term consequences of social stress on aging. We conducted a randomized study involving more than 300 male mice of commonly used laboratory strains (C57BL/6J, CD1, and Sv129Ev) chosen for the spontaneous aggression gradient and stress-vulnerability. Mice were exposed to a lifelong chronic psychosocial stress protocol to model social gradients in aging and disease vulnerability. Low social rank, inferred based on a discretized aggression index, was found to negatively impact lifespan in our study population. However, social rank interacted with genetic background in that low-ranking C57BL/6J, high-ranking Sv129Ev, and middle-ranking CD1 mice had lower survival, respectively, implying a cost of maintaining a given social rank that varies across strains. Machine learning linear discriminant analysis identified baseline fat-free mass as the most important predictor of mouse genetic background and social rank in the present dataset. Finally, strain and social rank differences were significantly associated with epigenetic changes, most significantly in Sv129Ev mice and in high-ranking compared to lower ranking subjects. Overall, we identified genetic background and social rank as critical contextual modifiers of aging and lifespan in an ethologically relevant rodent model of social stress, thereby providing a preclinical experimental paradigm to study the impact of social determinants of health disparities and accelerated aging.

Late onset, sporadic Alzheimer's disease (AD) accounts for the vast majority of cases. Unlike familial AD, the factors that drive the onset of sporadic AD are poorly understood, although aging and stress play a role. The early onset/severity of neuropathology observed in most genetic mouse models of AD hampers the study of the role of aging and environmental factors; thus alternate strategies are necessary to understand the contributions of these factors to sporadic AD. We demonstrate that mice acquiring a low social status (subordinate) in a lifelong chronic psychosocial stress (CPS) model, accrue widespread proteomic changes in the frontal/temporal cortex during aging. To better understand the significance of these stress-induced changes, we compared the differentially expressed proteins (DEPs) of subordinate mice to those of patients at varying stages of dementia. Sixteen and fifteen DEPs upregulated in subordinate mice were also upregulated in patients with mild cognitive impairment (MCI) and AD, respectively. Six of those upregulated proteins (CPE, ERC2, GRIN2B, SLC6A1, SYN1, WFS1) were shared by subordinate mice and patients with MCI or AD. Finally, comparison with a spatially detailed transcriptomic database revealed that the superior frontal gyrus and hippocampus had the greatest overlap between mice subjected to lifelong CPS and AD patients. Overall, most of the overlapping proteins were functionally associated with enhanced NMDA receptor mediated glutamatergic signaling, an excitotoxicity mechanism known to affect neurodegeneration. These findings support the association between stress and AD progression and provide valuable insight into potential early biomarkers and protein mediators of this relationship., (© 2021 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Childhood cancer survivors have a high risk for premature cardiovascular diseases, mainly due to cardiotoxic cancer treatments such as doxorubicin (DOX). Psychosocial stress is a significant cardiovascular risk factor and an enormous burden in childhood cancer survivors. Although observational studies suggest that psychosocial stress is associated with cardiovascular complications in cancer survivors, there is no translationally relevant animal model to study this interaction. We established a "two-hit" model in which juvenile mice were administered DOX (4 mg/kg/week for 3 weeks), paired to a validated model of chronic subordination stress (CSS) 5 weeks later upon reaching adulthood. Blood pressure, heart rate, and activity were monitored by radio-telemetry. At the end of CSS experiment, cardiac function was assessed by echocardiography. Cardiac fibrosis and inflammation were assessed by histopathologic analysis. Gene expressions of inflammatory and fibrotic markers were determined by PCR. Juvenile exposure to DOX followed by adult-onset CSS caused cardiac fibrosis and inflammation as evident by histopathologic findings and upregulated gene expression of multiple inflammatory and fibrotic markers. Intriguingly, juvenile exposure to DOX blunted CSS-induced hypertension but not CSS-induced tachycardia. There were no significant differences in cardiac function parameters among all groups, but juvenile exposure to DOX abrogated the hypertrophic response to CSS. In conclusion, we established a translationally relevant mouse model of juvenile DOX-induced cardiotoxicity that predisposes to adult-onset stress-induced adverse cardiac remodeling. Psychosocial stress should be taken into consideration in cardiovascular risk stratification of DOX-treated childhood cancer survivors.

Background: Both human and veterinary cancer chemotherapy are undergoing a paradigm shift from a "one size fits all" approach to more personalized, patient-oriented treatment strategies. Personalized chemotherapy is dependent on the identification and validation of biomarkers that can predict treatment outcome and/or risk of toxicity. Many cytotoxic chemotherapy agents, including doxorubicin, base their mechanism of action by interaction with DNA and disruption of normal cellular processes. We developed a high-resolution/accurate-mass liquid chromatography-mass spectrometry DNA screening approach for monitoring doxorubicin-induced DNA modifications (adducts) in vitro and in vivo. We used, for the first time, a new strategy involving the use of isotope-labeled DNA, which greatly facilitates adduct discovery. The overall goal of this work was to identify doxorubicin-DNA adducts to be used as biomarkers to predict drug efficacy for use in veterinary oncology., Results: We used our novel mass spectrometry approach to screen for adducts in purified DNA exposed to doxorubicin. This initial in vitro screening identified nine potential doxorubicin-DNA adduct masses, as well as an intense signal corresponding to DNA-intercalated doxorubicin. Two of the adduct masses, together with doxorubicin and its metabolite doxorubicinol, were subsequently detected in vivo in liver DNA extracted from mice exposed to doxorubicin. Finally, the presence of these adducts and analytes was explored in the DNA isolated from dogs undergoing treatment with doxorubicin. The previously identified nine DOX-DNA adducts were not detected in these preliminary three samples collected seven days post-treatment, however intercalated doxorubicin and doxorubicinol were detected., Conclusions: This work sets the stage for future evaluation of doxorubicin-DNA adducts and doxorubicin-related molecules as candidate biomarkers to personalize chemotherapy protocols for canine cancer patients. It demonstrates our ability to combine in one method the analysis of DNA adducts and DNA-intercalated doxorubicin and doxorubicinol. The last two analytes interestingly, were persistent in samples from canine patients undergoing doxorubicin chemotherapy seven days after treatment. The presence of doxorubicin in all samples suggests a role for it as a promising biomarker for use in veterinary chemotherapy. Future studies will involve the analysis of more samples from canine cancer patients to elucidate optimal timepoints for monitoring intercalated doxorubicin and doxorubicin-DNA adducts and the correlation of these markers with therapy outcome., (© 2021. The Author(s).)

The TLQP-21 neuropeptide has been implicated in functions as diverse as lipolysis, neurodegeneration and metabolism, thus suggesting an important role in several human diseases. Three binding targets have been proposed for TLQP-21: C3aR1, gC1qR and HSPA8. The aim of this review is to critically evaluate the molecular identity of the TLQP-21 receptor and the proposed multi-receptor mechanism of action. Several studies confirm a critical role for C3aR1 in TLQP-21 biological activity and a largely conserved mode of binding, receptor activation and signaling with C3a, its first-identified endogenous ligand. Conversely, data supporting a role of gC1qR and HSPA8 in TLQP-21 activity remain limited, with no signal transduction pathways being described. Overall, C3aR1 is the only receptor for which a necessary and sufficient role in TLQP-21 activity has been confirmed thus far. This conclusion calls into question the validity of a multi-receptor mechanism of action for TLQP-21 and should inform future studies., (© 2021. The Author(s).)

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of β-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3., Competing Interests: Declaration of interests O.S.J., Jakob Bondo Hansen, D.P.C., T.W.S., and Z.G.-H. work or have worked, in some capacity, for Embark Biotech ApS, a company developing therapeutics for the treatment of diabetes and obesity. All other authors declare no competing interests associated with this manuscript., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Psychosocial stressors can cause physical inactivity, cardiac damage, and hypotension-induced death in the mdx mouse model of Duchenne muscular dystrophy (DMD). Because repeated exposure to mild stress can lead to habituation in wild-type mice, we investigated the response of mdx mice to a mild, daily stress to determine whether habituation occurred. Male mdx mice were exposed to a 30-sec scruff restraint daily for 12 weeks. Scruff restraint induced immediate physical inactivity that persisted for at least 60 minutes, and this inactivity response was just as robust after 12 weeks as it was after one day. Physical inactivity in the mdx mice was not associated with acute skeletal muscle contractile dysfunction. However, skeletal muscle of mdx mice that were repeatedly stressed had slow-twitch and tetanic relaxation times and trended toward high passive stiffness, possibly due to a small but significant increase in muscle fibrosis. Elevated urinary corticosterone secretion, adrenal hypertrophy, and a larger adrenal cortex indicating chronic activation of the hypothalamic-pituitary-adrenal (HPA) axis were measured in 12-week stressed mdx mice relative to those unstressed. However, pharmacological inhibition of the HPA axis did not affect scruff-induced physical inactivity and acute corticosterone injection did not recapitulate the scruff-induced phenotype, suggesting the HPA axis is not the driver of physical inactivity. Our results indicate that the response of mdx mice to an acute mild stress is non-habituating and that when that stressor is repeated daily for weeks, it is sufficient to exacerbate some phenotypes associated with dystrophinopathy in mdx mice., (© 2021 Federation of American Societies for Experimental Biology.)

Background: Duchenne muscular dystrophy (DMD) is caused by the loss of dystrophin. Severe and ultimately lethal, DMD progresses relatively slowly in that patients become wheelchair bound only around age twelve with a survival expectancy reaching the third decade of life., Methods: The mildly-affected mdx mouse model of DMD, and transgenic DysΔMTB-mdx and Fiona-mdx mice expressing dystrophin or utrophin, respectively, were exposed to either mild (scruffing) or severe (subordination stress) stress paradigms and profiled for their behavioral and physiological responses. A subgroup of mdx mice exposed to subordination stress were pretreated with the beta-blocker metoprolol., Findings: Subordination stress caused lethality in ∼30% of mdx mice within 24 h and ∼70% lethality within 48 h, which was not rescued by metoprolol. Lethality was associated with heart damage, waddling gait and hypo-locomotion, as well as marked up-regulation of the hypothalamus-pituitary-adrenocortical axis. A novel cardiovascular phenotype emerged in mdx mice, in that scruffing caused a transient drop in arterial pressure, while subordination stress caused severe and sustained hypotension with concurrent tachycardia. Transgenic expression of dystrophin or utrophin in skeletal muscle protected mdx mice from scruffing and social stress-induced responses including mortality., Interpretation: We have identified a robust new stress phenotype in the otherwise mildly affected mdx mouse that suggests relatively benign handling may impact the outcome of behavioural experiments, but which should also expedite the knowledge-based therapy development for DMD., Funding: Greg Marzolf Jr. Foundation, Summer's Wish Fund, NIAMS, Muscular Dystrophy Association, University of Minnesota and John and Cheri Gunvalson Trust., Competing Interests: Declaration of Competing Interest The authors who have taken part in this study declare that they do not have anything to disclose regarding conflicts of interest concerning this manuscript., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The brown adipose tissue (BAT) is a thermogenic organ that plays a major role in energy balance, obesity, and diabetes due to the potent glucose and lipid clearance that fuels its thermogenesis, which is largely mediated via sympathetic nervous system activation. However, thus far there has been little experimental validation of the hypothesis that selective neuromodulation of the sympathetic nerves innervating the BAT is sufficient to elicit thermogenesis in mice. We generated mice expressing blue light-activated channelrhodopsin-2 (ChR2) in the sympathetic nerves innervating the BAT using two different strategies: injecting the BAT of C57Bl/6J mice with AAV6-hSyn-ChR2 (H134R)-EYFP; crossbreeding tyrosine hydroxylase-Cre mice with floxed-stop ChR2-EYFP mice. The nerves in the BAT expressing ChR2 were selectively stimulated with a blue LED light positioned underneath the fat pad of anesthetized mice, while the BAT and core temperatures were simultaneously recorded. Using immunohistochemistry we confirmed the selective expression of EYFP in TH positive nerves fibers. In addition, local optogenetic stimulation of the sympathetic nerves induced significant increase in the BAT temperature followed by an increase in core temperature in mice expressing ChR2, but not in the respective controls. The BAT activation was also paralleled by increased levels of pre-UCP1 transcript. Our results demonstrate that local optogenetic stimulation of the sympathetic nerves is sufficient to elicit BAT and core thermogenesis, thus suggesting that peripheral neuromodulation has the potential to be exploited as an alternative to pharmacotherapies to elicit organ activation and thus ameliorate type 2 diabetes and/or obesity., (© 2019 Federation of American Societies for Experimental Biology.)

Structural and functional diversity of peptides and GPCR result from long evolutionary processes. Even small changes in sequence can alter receptor activation, affecting therapeutic efficacy. We conducted a structure-function relationship study on the neuropeptide TLQP-21, a promising target for obesity, and its complement 3a receptor (C3aR1). After having characterized the TLQP-21/C3aR1 lipolytic mechanism, a homology modeling and molecular dynamics simulation identified the TLQP-21 binding motif and C3aR1 binding site for the human (h) and mouse (m) molecules. mTLQP-21 showed enhanced binding affinity and potency for hC3aR1 compared with hTLQP-21. Consistently, mTLQP-21, but not hTLQP-21, potentiates lipolysis in human adipocytes. These findings led us to uncover five mutations in the C3aR1 binding pocket of the rodent Murinae subfamily that are causal for enhanced calculated affinity and measured potency of TLQP-21. Identifying functionally relevant peptide/receptor co-evolution mechanisms can facilitate the development of innovative pharmacotherapies for obesity and other diseases implicating GPCRs., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

TLQP-21 is a multifunctional neuropeptide and a promising new medicinal target for cardiometabolic and neurological diseases. However, to date its clearance kinetics and plasma stability have not been studied. The presence of four arginine residues led us to hypothesize that its half-life is relatively short. Conversely, its biological activities led us to hypothesize that the peptide is still taken up by adipose tissues effectively. [ 125 I]TLQP-21 was i.v. administered in rats followed by chasing the plasma radioactivity and assessing tissue uptake. Plasma stability was measured using LC-MS. In vivo lipolysis was assessed by the palmitate rate of appearance., Results: A small single i.v. dose of [ 125 I]TLQP-21 had a terminal half-life of 110 min with a terminal clearance rate constant, k t , of 0.0063/min, and an initial half-life of 0.97 min with an initial clearance rate constant, k i , of 0.71/min. The total net uptake by adipose tissue accounts for 4.4% of the entire dose equivalent while the liver, pancreas and adrenal gland showed higher uptake. Uptake by the brain was negligible, suggesting that i.v.-injected peptide does not cross the blood-brain-barrier. TLQP-21 sustained isoproterenol-stimulated lipolysis in vivo. Finally, TLQP-21 was rapidly degraded producing several N-terminal and central sequence fragments after 10 and 60 min in plasma in vitro. This study investigated the clearance and stability of TLQP-21 peptide for the first time. While its pro-lipolytic effect supports and extends previous findings, its short half-life and sequential cleavage in the plasma suggest strategies for chemical modifications in order to enhance its stability and therapeutic efficacy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)