Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.

Inactivating negative regulators of cortical branched actin enhances persistence of single cell migration.

The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knockdown (KD) or knockout (KO), we combine the inactivation of the Arp2/3 inhibitory protein arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit CYFIP2, all of which enhance the polymerization of cortical branched actin. Inactivation of the three negative regulators of cortical branched actin increases migration persistence of human breast MCF10A cells and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO cells, but not in triple KD cells, the 'super-migrator' phenotype was associated with a heterogenous downregulation of vimentin (VIM) expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells largely restored normal persistence of single cell migration, suggesting that vimentin downregulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression.

Cell-intrinsic regulation of phagocyte function by interferon lambda during pulmonary viral, bacterial super-infection.

Influenza infections result in a significant number of severe illnesses annually, many of which are complicated by secondary bacterial super-infection. Primary influenza infection has been shown to increase susceptibility to secondary methicillin-resistant Staphylococcus aureus (MRSA) infection by altering the host immune response, leading to significant immunopathology. Type III interferons (IFNs), or IFNλs, have gained traction as potential antiviral therapeutics due to their restriction of viral replication without damaging inflammation. The role of IFNλ in regulating epithelial biology in super-infection has recently been established; however, the impact of IFNλ on immune cells is less defined. In this study, we infected wild-type and IFNLR1-/- mice with influenza A/PR/8/34 followed by S. aureus USA300. We demonstrated that global IFNLR1-/- mice have enhanced bacterial clearance through increased uptake by phagocytes, which was shown to be cell-intrinsic specifically in myeloid cells in mixed bone marrow chimeras. We also showed that depletion of IFNLR1 on CX3CR1 expressing myeloid immune cells, but not neutrophils, was sufficient to significantly reduce bacterial burden compared to mice with intact IFNLR1. These findings provide insight into how IFNλ in an influenza-infected lung impedes bacterial clearance during super-infection and show a direct cell intrinsic role for IFNλ signaling on myeloid cells.

Elucidation of the molecular mechanism of the breakage-fusion-bridge (BFB) cycle using a CRISPR-dCas9 cellular model.

Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanism for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrate that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we show that targeting dCas9 to telomeres using sgTelo impedes DNA replication at telomeres and induces a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigate the genome-wide features of telomeres in the dCas9/sgTelo cells and observe a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS-. Consistently, we also observe an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncover many interesting molecular and structural features of the ITCB and demonstrate that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance.

BCG vaccination induces cross-protective immunity against pathogenic microorganisms.

Bacillus Calmette-Guérin (BCG) is an attenuated Mycobacterium bovis strain used as a vaccine to prevent Mycobacterium tuberculosis (M. tb) infection. Its ability to potentiate the immune response induced by other vaccines and to promote nonspecific immunomodulatory effects has been described. These effects can be triggered by epigenetic reprogramming and metabolic shifts on innate immune cells, a phenomenon known as trained immunity. The induction of trained immunity may contribute to explain why BCG vaccination effectively decreases disease symptoms caused by pathogens different from M. tb. This article explains the importance of BCG immunization and the possible mechanisms associated with the induction of trained immunity, which might be used as a strategy for rapid activation of the immune system against unrelated pathogens.

The Arp2/3 inhibitory protein Arpin inhibits homology-directed DNA repair.

BACKGROUND INFORMATION: Arpin, an Arp2/3 inhibitory protein, inhibits lamellipodial protrusions and cell migration. Arpin expression is lost in tumor cells of several cancer types. RESULTS: Here we analyzed expression levels of Arpin and various markers using Reverse Phase Protein Array (RPPA) in human mammary carcinomas. We found that Arpin protein levels were correlated with those of several DNA damage response markers. Arpin-null cells display enhanced clustering of double stand breaks (DSBs) when cells are treated with a DNA damaging agent, in line with a previously described role of the Arp2/3 complex in promoting DSB clustering for homologous DNA repair (HDR) in the nucleus. Using a specific HDR assay, we further showed that Arpin depletion increased HDR efficiency two-fold through its ability to inactivate the Arp2/3 complex. CONCLUSIONS: Arpin regulates both cell migration in the cytosol and HDR in the nucleus. SIGNIFICANCE: Loss of Arpin expression coordinates enhanced cell migration with up-regulated DNA repair, which is required when DNA damage is induced by active cell migration.

Large influence of soil moisture on long-term terrestrial carbon uptake.

Although the terrestrial biosphere absorbs about 25 per cent of anthropogenic carbon dioxide (CO2) emissions, the rate of land carbon uptake remains highly uncertain, leading to uncertainties in climate projections1,2. Understanding the factors that limit or drive land carbon storage is therefore important for improving climate predictions. One potential limiting factor for land carbon uptake is soil moisture, which can reduce gross primary production through ecosystem water stress3,4, cause vegetation mortality5 and further exacerbate climate extremes due to land-atmosphere feedbacks6. Previous work has explored the impact of soil-moisture availability on past carbon-flux variability3,7,8. However, the influence of soil-moisture variability and trends on the long-term carbon sink and the mechanisms responsible for associated carbon losses remain uncertain. Here we use the data output from four Earth system models9 from a series of experiments to analyse the responses of terrestrial net biome productivity to soil-moisture changes, and find that soil-moisture variability and trends induce large CO2 fluxes (about two to three gigatons of carbon per year; comparable with the land carbon sink itself1) throughout the twenty-first century. Subseasonal and interannual soil-moisture variability generate CO2 as a result of the nonlinear response of photosynthesis and net ecosystem exchange to soil-water availability and of the increased temperature and vapour pressure deficit caused by land-atmosphere interactions. Soil-moisture variability reduces the present land carbon sink, and its increase and drying trends in several regions are expected to reduce it further. Our results emphasize that the capacity of continents to act as a future carbon sink critically depends on the nonlinear response of carbon fluxes to soil moisture and on land-atmosphere interactions. This suggests that the increasing trend in carbon uptake rate may not be sustained past the middle of the century and could result in accelerated atmospheric CO2 growth.

Lipoprotein-associated phospholipase A2: A paradigm for allosteric regulation by membranes.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein's phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA2 binding to a phospholipid surface. This allosteric regulation of an enzyme's activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA2's specificity toward oxidized phospholipids.

IL-2 family cytokines IL-9 and IL-21 differentially regulate innate and adaptive type 2 immunity in asthma.

BACKGROUND: Asthma is often accompanied by type 2 immunity rich in IL-4, IL-5, and IL-13 cytokines produced by TH2 lymphocytes or type 2 innate lymphoid cells (ILC2s). IL-2 family cytokines play a key role in the differentiation, homeostasis, and effector function of innate and adaptive lymphocytes. OBJECTIVE: IL-9 and IL-21 boost activation and proliferation of TH2 and ILC2s, but the relative importance and potential synergism between these γ common chain cytokines are currently unknown. METHODS: Using newly generated antibodies, we inhibited IL-9 and IL-21 alone or in combination in various murine models of asthma. In a translational approach using segmental allergen challenge, we recently described elevated IL-9 levels in human subjects with allergic asthma compared with nonasthmatic controls. Here, we also measured IL-21 in both groups. RESULTS: IL-9 played a central role in controlling innate IL-33-induced lung inflammation by promoting proliferation and activation of ILC2s in an IL-21-independent manner. Conversely, chronic house dust mite-induced airway inflammation, mainly driven by adaptive immunity, was solely dependent on IL-21, which controlled TH2 activation, eosinophilia, total serum IgE, and formation of tertiary lymphoid structures. In a model of innate on adaptive immunity driven by papain allergen, a clear synergy was found between both pathways, as combined anti-IL-9 or anti-IL-21 blockade was superior in reducing key asthma features. In human bronchoalveolar lavage samples we measured elevated IL-21 protein within the allergic asthmatic group compared with the allergic control group. We also found increased IL21R transcripts and predicted IL-21 ligand activity in various disease-associated cell subsets. CONCLUSIONS: IL-9 and IL-21 play important and nonredundant roles in allergic asthma by boosting ILC2s and TH2 cells, revealing a dual IL-9 and IL-21 targeting strategy as a new and testable approach.

Augmentation of Endothelial S1PR1 Attenuates Postviral Pulmonary Fibrosis.

Respiratory viral infections are frequent causes of acute respiratory distress syndrome (ARDS), a disabling condition with a mortality of up to 46%. The pulmonary endothelium plays an important role in the development of ARDS as well as the pathogenesis of pulmonary fibrosis; however, the therapeutic potential to modulate endothelium-dependent signaling to prevent deleterious consequences has not been well explored. Here, we used a clinically relevant influenza A virus infection model, endothelial cell-specific transgenic gain-of-function and loss-of-function mice as well as pharmacologic approaches and in vitro modeling, to define the mechanism by which S1PR1 expression is dampened during influenza virus infection and determine whether therapeutic augmentation of S1PR1 has the potential to reduce long-term postviral fibrotic complications. We found that the influenza virus-induced inflammatory milieu promoted internalization of S1PR1, which was pharmacologically inhibited with paroxetine, an inhibitor of GRK2. Moreover, genetic overexpression or administration of paroxetine days after influenza virus infection was sufficient to reduce postviral pulmonary fibrosis. Taken together, our data suggest that endothelial S1PR1 signaling provides critical protection against long-term fibrotic complications after pulmonary viral infection. These findings support the development of antifibrotic strategies that augment S1PR1 expression in virus-induced ARDS to improve long-term patient outcomes.

BCL-2 Modulates IRE1α Activation to Attenuate Endoplasmic Reticulum Stress and Pulmonary Fibrosis.

BCL-2 family members are known to be implicated in survival in numerous biological settings. Here, we provide evidence that in injury and repair processes in lungs, BCL-2 mainly acts to attenuate endoplasmic reticulum (ER) stress and limit extracellular matrix accumulation. Days after an intratracheal bleomycin challenge, mice lose a fraction of their alveolar type II epithelium from terminal ER stress driven by activation of the critical ER sensor and stress effector IRE1α. This fraction is dramatically increased by BCL-2 inhibition, because IRE1α activation is dependent on its physical association with the BCL-2-proapoptotic family member BAX, and we found BCL-2 to disrupt this association in vitro. In vivo, navitoclax (a BCL-2/BCL-xL inhibitor) given 15-21 days after bleomycin challenge evoked strong activation of IRE-1α in mesenchymal cells and markers of ER stress, but not apoptosis. Remarkably, after BCL-2 inhibition, bleomycin-exposed mice demonstrated persistent collagen accumulation at Day 42, compared with resolution in controls. Enhanced fibrosis proved to be due to the RNAase activity of IRE1α downregulating MRC2 mRNA and protein, a mediator of collagen turnover. The critical role of MRC2 was confirmed in precision-cut lung slice cultures of Day-42 lungs from bleomycin-exposed wild-type and MRC2 null mice. Soluble and tissue collagen accumulated in precision-cut lung slice cultures from navitoclax-treated, bleomycin-challenged mice compared with controls, in a manner nearly identical to that of challenged but untreated MRC2 null mice. Thus, apart from mitochondrial-based antiapoptosis, BCL-2 functions to attenuate ER stress responses, fostering tissue homeostasis and injury repair.

Advancement in the development of mRNA-based vaccines for respiratory viruses.

Acute respiratory infections are the leading cause of death and illness in children under 5 years old and represent a significant burden in older adults. Primarily caused by viruses infecting the lower respiratory tract, symptoms include cough, congestion, and low-grade fever, potentially leading to bronchiolitis and pneumonia. Messenger ribonucleic acid (mRNA)-based vaccines are biopharmaceutical formulations that employ mRNA molecules to induce specific immune responses, facilitating the expression of viral or bacterial antigens and promoting immunization against infectious diseases. Notably, this technology had significant relevance during the COVID-19 pandemic, as these formulations helped to limit SARS-CoV-2 virus infections, hospitalizations, and deaths. Importantly, mRNA vaccines promise to be implemented as new alternatives for fighting other respiratory viruses, such as influenza, human respiratory syncytial virus, and human metapneumovirus. This review article analyzes mRNA-based vaccines' main contributions, perspectives, challenges, and implications against respiratory viruses.

Delayed Diagnosis of Locally Acquired Lyme Disease, Central North Carolina, USA.

Healthcare providers in North Carolina, USA, have limited experience diagnosing and managing Lyme disease because few cases occur annually statewide. We outline the prolonged diagnostic course for a patient with locally acquired Lyme disease in North Carolina. This case highlights the need for greater awareness and professional education.

Rapid Increase in Seroprevalence of Borrelia burgdorferi Antibodies among Dogs, Northwestern North Carolina, USA, 2017-20211.

We evaluated spatial-temporal risk for Lyme disease in northwestern North Carolina, USA, by using individual-level canine Borrelia burgdorferi seroprevalence data collected during 2017-2021 at routine veterinary screenings for tickborne diseases. Seroprevalence in dogs increased from 2.2% (47/2,130) in 2017 to 11.2% (339/3,033) in 2021. The percentage of incident seropositivity increased from 2.1% (45/2,130) in 2017 to 7.6% (231/3,033) in 2021. Exploratory geographic analyses found canine seroprevalence shifted from clustered (2017, Moran's I = 0.30) to dispersed (2021, Moran's I = -0.20). Elevation, slope, aspect, and forest land cover density were associated with canine seroprevalence within various household buffer regions in 2017. Slope was associated with seroprevalence at the household level in 2021. Results support the use of individual-level canine seroprevalence data for monitoring human risk for Lyme disease. Establishing sentinel veterinary clinics within Lyme disease-emergent communities might promote prevention and control efforts and provide opportunities for educational and behavioral interventions.

22q11.2 Deletion-Associated Blood-Brain Barrier Permeability Potentiates Systemic Capillary Leak Syndrome Neurologic Features.

We present a case study of a young male with a history of 22q11.2 deletion syndrome (22qDS), diagnosed with systemic capillary leak syndrome (SCLS) who presented with acute onset of diffuse anasarca and sub-comatose obtundation. We hypothesized that his co-presentation of neurological sequelae might be due to blood-brain barrier (BBB) susceptibility conferred by the 22q11.2 deletion, a phenotype that we have previously identified in 22qDS. Using pre- and post-intravenous immunoglobulins (IVIG) patient serum, we studied circulating biomarkers of inflammation and assessed the potential susceptibility of the 22qDS BBB. We employed in vitro cultures of differentiated BBB-like endothelial cells derived from a 22qDS patient and a healthy control. We found evidence of peripheral inflammation and increased serum lipopolysaccharide (LPS) alongside endothelial cells in circulation. We report that the patient's serum significantly impairs barrier function of the 22qDS BBB compared to control. Only two other cases of pediatric SCLS with neurologic symptoms have been reported, and genetic risk factors have been suggested in both instances. As the third case to be reported, our findings are consistent with the hypothesis that genetic susceptibility of the BBB conferred by genes such as claudin-5 deleted in the 22q11.2 region promoted neurologic involvement during SCLS in this patient.

The innate immune response in tauopathies.

Tauopathies, which include frontotemporal dementia, Alzheimer's disease, and chronic traumatic encephalopathy, are a class of neurological disorders resulting from pathogenic tau aggregates. These aggregates disrupt neuronal health and function leading to the cognitive and physical decline of tauopathy patients. Genome-wide association studies and clinical evidence have brought to light the large role of the immune system in inducing and driving tau-mediated pathology. More specifically, innate immune genes are found to harbor tauopathy risk alleles, and innate immune pathways are upregulated throughout the course of disease. Experimental evidence has expanded on these findings by describing pivotal roles for the innate immune system in the regulation of tau kinases and tau aggregates. In this review, we summarize the literature implicating innate immune pathways as drivers of tauopathy.

Immune status of the murine 22q11.2 deletion syndrome model.

Mice modeling the hemizygous deletion of chromosome 22q11.2 (22qMc) have been utilized to address various clinical phenotypes associated with the disease, including cardiac malformations, altered neural circuitry, and behavioral deficits. Yet, the status of the T cell compartment, an important clinical concern among 22q11.2 deletion syndrome (22qDS) patients, has not been addressed. While infancy and early childhood in 22qDS are associated with deficient T cell numbers and thymic hypoplasia, which can be severe in a small subset of patients, studies suggest normalization of the T cell counts by adulthood. We found that adult 22qMc do not exhibit thymic hypoplasia or altered thymic T cell development. Our findings that immune cell counts and inflammatory T cell activation are unaffected in 22qMc lend support to the hypothesis that human 22qDS immunodeficiencies are secondary to thymic hypoplasia, rather than intrinsic effects due to the deletion. Furthermore, the 22q11.2 deletion does not impact the differentiation capacity of T cells, nor their activity and response during inflammatory activation. Thus, 22qMc reflects the T cell compartment in adult 22qDS patients, and our findings suggest that 22qMc may serve as a novel model to address experimental and translational aspects of immunity in 22qDS.

Pooled analysis of multiple sclerosis findings on multisite 7 Tesla MRI: Protocol and initial observations.

Although 7 T MRI research has contributed much to our understanding of multiple sclerosis (MS) pathology, most prior data has come from small, single-center studies with varying methods. In order to truly know if such findings have widespread applicability, multicenter methods and studies are needed. To address this, members of the North American Imaging in MS (NAIMS) Cooperative worked together to create a multicenter collaborative study of 7 T MRI in MS. In this manuscript, we describe the methods we have developed for the purpose of pooling together a large, retrospective dataset of 7 T MRIs acquired in multiple MS studies at five institutions. To date, this group has contributed five-hundred and twenty-eight 7 T MRI scans from 350 individuals with MS to a common data repository, with plans to continue to increase this sample size in the coming years. We have developed unified methods for image processing for data harmonization and lesion identification/segmentation. We report here our initial observations on intersite differences in acquisition, which includes site/device differences in brain coverage and image quality. We also report on the development of our methods and training of image evaluators, which resulted in median Dice Similarity Coefficients for trained raters' annotation of cortical and deep gray matter lesions, paramagnetic rim lesions, and meningeal enhancement between 0.73 and 0.82 compared to final consensus masks. We expect this publication to act as a resource for other investigators aiming to combine multicenter 7 T MRI datasets for the study of MS, in addition to providing a methodological reference for all future analysis projects to stem from the development of this dataset.

Coupled social and ecological change drove the historical extinction of the California grizzly bear (Ursus arctos californicus).

Large carnivores (order Carnivora) are among the world's most threatened mammals due to a confluence of ecological and social forces that have unfolded over centuries. Combining specimens from natural history collections with documents from archival records, we reconstructed the factors surrounding the extinction of the California grizzly bear (Ursus arctos californicus), a once-abundant brown bear subspecies last seen in 1924. Historical documents portrayed California grizzlies as massive hypercarnivores that endangered public safety. Yet, morphological measurements on skulls and teeth generate smaller body size estimates in alignment with extant North American grizzly populations (approx. 200 kg). Stable isotope analysis (δ13C, δ15N) of pelts and bones (n = 57) revealed that grizzlies derived less than 10% of their nutrition from terrestrial animal sources and were therefore largely herbivorous for millennia prior to the first European arrival in this region in 1542. Later colonial land uses, beginning in 1769 with the Mission era, led grizzlies to moderately increase animal protein consumption (up to 26% of diet), but grizzlies still consumed far less livestock than otherwise claimed by contemporary accounts. We show how human activities can provoke short-term behavioural shifts, such as heightened levels of carnivory, that in turn can lead to exaggerated predation narratives and incentivize persecution, triggering rapid loss of an otherwise widespread and ecologically flexible animal.

Seasonal fluctuations in BDNF regulate hibernation and torpor in golden-mantled ground squirrels.

Aphagic hibernators such as the golden-mantled ground squirrel (GMGS; Callospermophilus lateralis) can fast for months and exhibit profound seasonal fluctuations in body weight, food intake, and behavior. Brain-derived neurotrophic factor (BDNF) regulates cellular and systemic metabolism via mechanisms that are conserved across mammalian species. In this study, we characterized regional changes in BDNF with hibernation, hypothermia, and seasonal cycle in GMGS. Analysis of BDNF protein concentrations by ELISA revealed overlapping seasonal patterns in the hippocampus and hypothalamus, where BDNF levels were highest in summer and lowest in winter. BDNF is the primary ligand for receptor tyrosine kinase B (TrkB), and BDNF/TrkB signaling in the brain potently regulates energy expenditure. To examine the functional relevance of seasonal variation in BDNF, hibernating animals were injected with the small molecule TrkB agonist 7,8-dihydroxyflavone (DHF) daily for 2 wk. When compared with vehicle, DHF-treated animals exhibited fewer torpor bouts and shorter bout durations. These results suggest that activating BDNF/TrkB disrupts hibernation and raise intriguing questions related to the role of BDNF as a potential regulatory mechanism or downstream response to seasonal changes in body temperature and environment.NEW & NOTEWORTHY Golden-mantled ground squirrels exhibit dramatic seasonal fluctuations in metabolism and can fast for months while hibernating. Brain-derived neurotrophic factor is an essential determinant of cellular and systemic metabolism, and in this study, we characterized seasonal fluctuations in BDNF expression and then administered the small molecule BDNF mimetic 7,8-dihydroxyflavone (DHF) in hibernating squirrels. The results indicate that activating BDNF/TrkB signaling disrupts hibernation, with implications for synaptic homeostasis in prolonged hypometabolic states.