Objective: To develop a workflow that transforms electronic health record data into machine learning-ready features for molecular endotype assignment and to evaluate whether clinician-informed feature engineering improves model performance and interpretability. Materials and Methods: We developed parallel clinician-informed and clinician-agnostic feature engineering pipelines to prepare raw EHR data from mechanically ventilated patients with respiratory failure. Molecular endotype labels derived from paired deep lung and blood profiling of subjects with acute lung injury were used to train candidate machine learning classifiers. Champion models from each pipeline were compared on predefined performance metrics. Results: Bayesian network classifiers were the top-performing models in both pipelines. The clinician-informed pipeline generated fewer features than the clinician-agnostic pipeline (645 vs 1,127) and produced a lower misclassification rate in the final Bayesian network model (0.047 vs 0.14). In an independent cohort of subjects with acute lung injury, the clinician-informed model better distinguished corticosteroid-responsive from non-responsive subgroups. Discussion: Clinical context improved feature engineering efficiency, model interpretability, and classification performance. These findings support the integration of domain expertise into machine learning workflows intended for critical care implementation. Conclusions: Clinician-informed feature engineering can simplify machine learning models while improving performance and preserving clinical relevance. AI tools developed for healthcare should incorporate subject matter expertise early in the feature engineering and analytic workflow. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project was supported by the North Carolina Collaboratory at The University of North Carolina at Chapel Hill with funding appropriated by the North Carolina General Assembly (V.V. and M.C.W) and by the Rapidly Emerging Antiviral Drug Development Initiative at the University of North Carolina at Chapel Hill with funding from the North Carolina Coronavirus State and Local Fiscal Recovery Funds program, appropriated by the North Carolina General Assembly (J.C.S., R.S.H., and M.C.W.) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the institutional review board at the University of North Carolina at Chapel Hill with a waiver of informed consent (IRB 22-3196, January 11, 2023). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

The carboxyl terminus of Hsp70-interacting protein (CHIP, encoded by STUB1) combines co-chaperone and E3 ubiquitin ligase activities to regulate protein quality control. Heterozygous mutations in STUB1 cause spinocerebellar ataxia type 48 (SCA48), a progressive cerebellar ataxia with variable extrapyramidal and cognitive features. To understand the molecular basis of this variability, we systematically analyzed 13 SCA48-associated variants spanning the TPR and U-box domains through recombinant protein biochemistry and cellular models. TPR variants retained intrinsic ligase activity but showed significantly reduced HSP70 binding, impaired substrate ubiquitination, and decreased stability. Conversely, U-box variants abolished ligase function, promoted the formation of high-molecular-weight oligomers, and often increased CHIP levels while only partially impairing co-chaperone activity. Many mutants displayed temperature-sensitive defects and defective stress-induced nuclear translocation. Principal component analysis revealed distinct biochemical clustering specific to each domain. RNA-seq following STUB1 knockdown modeled CHIP insufficiency and showed preserved HSF1-dependent transactivation, but loss of CHIP’s capacity to amplify ubiquitination, chaperone function, and stress-related transcriptional programs. Meta-analysis of 87 SCA48 patients linked TPR-like biochemical signatures to upper motor neuron involvement and U-box-like profiles to prominent dysarthria. Overall, these data indicate that SCA48 results from domain-specific disruption of CHIP’s dual functions, producing varying degrees of CHIP insufficiency and/or gain-of-toxic effects that together contribute to the phenotypic diversity observed across patients. This work refines the mechanistic framework for SCA48 pathogenesis and highlights strategies for therapeutic modulation of residual CHIP activity.

Future deep-space missions will expose astronauts to microgravity and ionizing radiation, which induce molecular changes that mirror biological aging, including inflammation, cellular senescence, and fibrotic remodeling. However, the regulatory mechanisms linking spaceflight stressors to aging-associated molecular responses in metabolically important organs such as the liver remain incompletely understood. In this study, we investigated the effects of simulated microgravity and deep-space radiation analogs on microRNA (miRNA) and mRNA networks associated with the transforming growth factor-β (TGF-β) signaling pathway in mouse liver. We further evaluated an antagomir cocktail (miR-16-5p, miR-125b-5p, and let-7a-5p) as a potential countermeasure. Notably, while antagomir treatment influenced genes associated with senescence and inflammatory responses, it increased the expression of fibrosis-associated transcripts. To explore translational relevance, we compared these findings with transcriptomic data from the NASA Twins Study and the Inspiration4 mission. Despite lower radiation exposures, similar regulatory patterns affecting TGF-β-associated genes and miRNAs were observed in human circulating blood cells. Together, these findings suggest that spaceflight-associated stressors perturb hepatic miRNA-TGF-β regulatory networks linked to senescence, inflammation, and fibrotic signaling. Our results support that radiation and microgravity exposures may act as drivers of aging-like molecular responses and highlight miRNA regulatory networks as potential targets for countermeasures during long-duration spaceflight.

Summary Spaceflight is associated with systemic effects, including central nervous system syndromes. To mitigate the effects from spaceflight syndromes, mouse spinal and ocular tissue were exposed to simulated galactic cosmic radiation and microgravity, and also treated with antagomirs for let-7a-5p, miR-125b-5p, and miR-16-5p. In spinal tissue, there were increases in DNA damage repair responses, mitochondrial genes, and neurodegenerative genes in antagomir-treated groups. In ocular tissue, there were no changes in DNA damage repair, but decreased antioxidant enzyme and inflammatory gene expression in antagomir-treated groups. There were differential effects of how antagomirs modulated gene expression based on spaceflight stressor and tissue.

Trametinib (Trm) is a highly selective mitogen-activated protein kinase kinase (MEK) inhibitor that potently and persistently abrogates extracellular signal-regulated kinase 1/2 activation. Trm initially was used to treat BRAF Val600→Glu (V600E)-mutated melanoma, but its Food and Drug Administration-approved indications are expanding rapidly. Trm generally is well tolerated, but it can cause dose-limiting cardiomyopathy and heart failure. Here, we characterize a mouse model of Trm cardiotoxicity using complementary in vitro approaches to show that Trm induces mitochondrial dysfunction in cardiomyocytes and some cancer cell types. In vivo, Trm caused contractile dysfunction within 3 days and heart failure within 2 weeks. High-resolution respirometry using isolated cardiac mitochondria revealed that Trm compromises oxidative metabolism, in part, through blunted activity of electron transport system complexes. Trm-mediated mitochondrial injury led to the release of mitochondrial damage-associated molecular patterns including mitochondrial DNA in both mice and humans, triggering activation of canonical innate immune pathways including cGAS-STING. In multiple rodent and human cardiomyocyte platforms, Trm diminished mitochondrial respiratory capacity at nanomolar concentrations, but this lesion was reversed by expression of a phosphomimetic signal transducer and activator of transcription 3-S727 construct. We also found that Trm induced mitochondrial dysfunction in some but not all cancer cell lines, identifying a previously unrecognized effect that could contribute to Trm's anticancer efficacy.

Heat shock protein 70 (HSP70) and its E3 ligase co-chaperone CHIP (STUB1) form a critical quality-control complex that directs client proteins toward folding or degradation. Phosphorylation of HSP70 at a conserved threonine in the C-terminal tail influences the fate of clients during cellular stress, yet the structural basis for this regulation remains unclear. Here, we present crystal structures of the CHIP tetratricopeptide repeat (TPR) domain bound to unphosphorylated and phosphorylated HSP70 C-terminal peptides at 1.6-1.9 Å resolution. Phosphate occupancy at Thr636 (HSPA1A numbering) causes steric clashes and electrostatic repulsion within the TPR-binding groove, decreasing affinity by more than 10-fold, as shown by biolayer interferometry and fluorescence polarization. Molecular dynamics simulations confirm destabilization of key hydrogen bonds. A structure-guided G132N substitution in CHIP introduces new hydrogen bonds to the phosphate group, restoring affinity for phosphorylated peptides in isolated TPR domains without losing native ubiquitination activity. However, in full-length CHIP, interface modifications do not restore phosphorylation-impaired stable binding but yield only partial recovery of transient interactions in cells, indicating additional context-dependent constraints on HSP70-CHIP regulation. These findings reveal the atomic mechanism by which phosphorylation impairs HSP70-CHIP interaction during stress and demonstrate that targeted interface engineering can compensate for post-translational changes in isolated domains. Overall, the results explain how cells switch chaperone-mediated triage pathways and offer a framework for understanding how proteostasis becomes dysregulated in neurodegenerative diseases and cancer.

Organotypic slice culture is a sophisticated technique historically used in cellular and developmental neurobiology to investigate three-dimensional architecture and cellular interactions. Maintaining thin tissue slices preserves organotypic structure and more accurately reflects the in vivo microenvironment than traditional monolayer cell cultures. These models are crucial in neurobiology, especially for studying age-dependent neurodegenerative disorders. However, preserving tissue viability in organotypic slices from adult brain tissue remains challenging. This protocol focuses on culturing organotypic slices from adult cerebellar tissue to address the critical need for discovery within this brain region. The cerebellum's unique cellular architecture and specialized circuitry have made culturing organotypic slices challenging. This protocol presents a novel finding: careful manipulation of culture conditions reduces early neuroinflammatory responses, enhances cerebellar tissue viability, and promotes the long-term maintenance of well-preserved cerebellar slices. This innovative system enables detailed analyses of neuronal morphology and functional connectivity at both cellular and circuit levels, incorporating advanced imaging and electrophysiological techniques to fulfill a critical need in neurobiology.

Microgravity and space radiation experienced during spaceflight have adverse effects on musculoskeletal health, yet their impact on articular cartilage has not been fully understood. In this study, we demonstrated that simulated spaceflight on Earth leads to cartilage degradation in the knees of mice. Similar changes were also observed in mice exposed to actual spaceflight. To investigate mechanisms underlying spaceflight-associated cartilage loss, human chondrocytes were encapsulated in a hydrogel scaffold and subjected to rotary culture to simulate microgravity-induced alterations. Simulated microgravity increased the expression of biomarkers related to inflammation and cellular senescence. Additionally, rotary culture decreased mitochondrial respiration and increased reactive oxygen species production. Through RNA sequencing and bioenergetic profiling, we identified NADPH oxidase 4 (NOX4) as a crucial factor driving these changes. Moreover, kaempferol, a naturally occurring flavonoid that directly binds to NOX4, was found to partially reverse the harmful effects of microgravity on chondrocytes. Finally, systemic administration of kaempferol reduced cartilage degradation in mice subjected to simulated spaceflight on the ground. These findings establish that NOX4-mediated mitochondrial dysfunction is a key mechanism underlying spaceflight-induced cartilage degradation and highlight kaempferol as a potential protective measure for joint health in space.

Background:Corticosteroids reduce mortality in severe COVID-19 requiring oxygen or invasive mechanical ventilation, yet emerging data suggest that SARS-CoV-2-associated acute lung injury is biologically heterogeneous and that treatment response may vary across molecularly defined disease states. Lung-derived molecular endotypes of severe COVID-19-associated acute lung injury have been described, but direct molecular profiling is not routinely available at the bedside. We evaluated whether a clinical predictor of previously defined lung molecular endotype identifies heterogeneity in corticosteroid treatment effect among mechanically ventilated patients with COVID-19. Methods:We utilized a single-center cohort of 5,000 patients with COVID-19 treated at the University of North Carolina Hospital between January 1, 2020, and December 31, 2022, to emulate a target trial assessing the effect of corticosteroid receipt on mortality, length of stay, and incident organ support. Confounding was addressed through inverse probability of treatment weighting (IPTW). Outcomes for severely ill patients requiring mechanical ventilation were compared to the RECOVERY trial results, with subsequent moderation analysis and stratified analysis by clinically predicted lung molecular endotype and vaccination status. The primary outcome was 28-day mortality. Secondary Outcomes were time to discharge alive and progression to additional organ support. Results:This emulated target trial showed a directionally favorable but non-statistically significant association between corticosteroid treatment and reduced 28-day mortality in patients requiring mechanical ventilation for SARS-CoV-2 infection. A clinical predictor of lung molecular endotype moderated the effect of corticosteroids on 28-day mortality (p-value for interaction 0.038) and identified distinct predicted endotype-specific treatment effect. Corticosteroid treatment was associated with lower 28-day mortality in the predicted Hyper-Inflammatory endotype (OR 0.62, 95% CI 0.39, 0.99) but not in the predicted Metabolic Dysregulation endotype (OR 1.15, 95% CI 0.82, 1.61). We did not detect significant effect modification by vaccination status (p-value for interaction 0.65), although inference was limited by the small, vaccinated subgroup (28-mortality OR 0.78, 95% CI 0.37, 1.65 in vaccinated vs 0.94, 95% CI 0.70, 1.26 in unvaccinated). Conclusions:In this target trial emulation of mechanically ventilated patients with severe COVID-19, corticosteroid treatment showed a directionally favorable but non-statistically significant association with reduced 28-day mortality in the overall cohort. However, a clinical predictor of lung molecular endotype identified significant heterogeneity in treatment effect, with benefit concentrated in the predicted Hyper-Inflammatory endotype and no apparent benefit in the predicted Metabolic Dysregulation endotype. These findings support prospective validation of clinically deployable endotype-guided corticosteroid treatment strategies in acute lung injury and ARDS.

The carboxyl terminus of HSC70-interacting protein (CHIP, encoded by STUB1 ) integrates co-chaperone and E3 ubiquitin ligase activities to maintain proteostasis. Heterozygous STUB1 mutations cause the dominant cerebellar ataxia SCA48 through incompletely understood mechanisms. We characterized 13 SCA48-associated variants in the TPR and U-box domains using recombinant protein and cellular assays. TPR mutations retained intrinsic ligase activity but lost HSC70 binding, substrate ubiquitination efficiency, and protein stability. Conversely, U-box mutations abolished ligase function, induced aberrant high-molecular-weight oligomerization, and frequently elevated steady-state CHIP levels while only partially impairing co-chaperone activity. Many variants exhibited temperature-sensitive defects and stress-induced nuclear mislocalization. Principal component analysis revealed robust domain-specific biochemical clustering. RNA-seq following STUB1 knockdown demonstrated preserved HSF1-dependent transactivation but a loss of CHIP-dependent amplification of ubiquitination, chaperone, and transcriptional pathways under heat stress. Meta-analysis of 87 SCA48 patients linked TPR-like biochemical profiles to upper motor neuron involvement and U-box profiles to prominent dysarthria. Collectively, SCA48 mutations decouple CHIP’s dual functions in a domain-dependent manner, exerting dominant-negative or gain-of-toxic effects that drive the observed clinical heterogeneity. These findings establish a direct biochemical–clinical correlation in SCA48 and provide a framework for domain-targeted therapeutic strategies exploiting residual ligase or chaperone activity. ### Competing Interest Statement The Brown laboratory receives research funding from Amgen.

Protein quality control is crucial for cellular homeostasis, involving the heat shock response, the ubiquitin-proteasome system, and the autophagy-lysosome pathway. Central to these systems are the chaperone homologs heat shock protein 70 (HSP70) and heat shock cognate 70 (HSC70), which manage protein folding and degradation. This study investigated the impact of the C-terminal phosphorylation of HSP70 on its interaction with the co-chaperone CHIP (C-terminus of HSC70 interacting protein), an E3 ligase that ubiquitinates protein substrates for degradation. Using both cell-free and cell-based approaches, including X-ray crystallography, biolayer interferometry, and live cell biocomplementation assays, we demonstrate that phosphorylation at HSP70 T636 reduces CHIP's binding affinity, shifting the preference toward other co-chaperones like HOP. Structural analysis reveals that phosphorylation disrupts key hydrogen bonds, altering binding dynamics. We engineered a CHIP variant (CHIP-G132N) to restore binding affinity to phosphorylated HSP70. While CHIP-G132N effectively restored binding without additional functional domains, its effectiveness was diminished in full-length phosphomimetic constructs in cell-free and in-cell assays, suggesting that additional interactions may influence binding. Functional assays indicate that phosphorylation of HSP70 affects its stability and degradation, with implications for diseases such as cancer and neurodegeneration. Our findings highlight the complexity of chaperone-co-chaperone interactions and underscore the importance of post-translational modifications in regulating protein quality control mechanisms. By elucidating the molecular details of HSP70 and CHIP interactions, our study provides a foundation for developing therapeutic interventions for diseases characterized by proteostasis imbalance.

The natively unfolded tau (MAPT) protein is extremely soluble, which poses challenges when modeling neurofibrillary tangle (NFT) pathology in Alzheimer’s disease (AD). To overcome this hurdle, we combined P301L and S320F mutations (PL-SF) to generate a rapid and reliable tau pathology platform to expedite the discovery of factors that modify tau aggregation. Using this model, we evaluated heat-shock proteins (Hsp), which have been linked to tau pathology, but whose role in AD remains enigmatic and controversial. In primary neurons, expression of Hsp70, but not Hsc70 or Hsp90, exacerbated tau aggregation. Conversely, lowering Hsp70 or employing a chaperone-deficient tau mutant (PL-SF-4Δ) reduced tau phosphorylation and abrogated tau aggregation, highlighting Hsp70 as a key driver of tau aggregation. Hsp70 foci clustered within and surrounding neuritic plaques and NFTs in post-mortem AD brain. Functionally, mature aggregate-bearing neurons showed deficits in neuronal firing and network communication, which were restored by chaperone-binding deficient tau variants that abrogated tau pathology. This study provides a powerful cell-intrinsic model for accelerated tau aggregation, which can be harnessed to identify potent regulators of tau aggregation as promising therapeutic targets.

The carboxyl terminus of Hsc70-interacting protein (CHIP) is pivotal for managing misfolded and aggregated proteins via chaperone networks and degradation pathways. In a preclinical rodent model of CHIP-related ataxia, we observed that CHIP mutations lead to increased levels of phosphodiesterase 9A (PDE9A), whose role in this context remains poorly understood. Here, we investigated the molecular mechanisms underlying the role of PDE9A in CHIP-related ataxia and demonstrated that CHIP binds to PDE9A, facilitating its polyubiquitination and autophagic degradation. Conversely, dysfunctional CHIP disrupts this process, resulting in PDE9A accumulation, increased cGMP hydrolysis, and impaired PKG phosphorylation of CHIP at serine 19. This cascade further amplifies PDE9A accumulation, ultimately disrupting mitophagy and triggering neuronal apoptosis. Elevated PKA levels inhibit PDE9A degradation, further exacerbating this neuronal dysfunction. Notably, pharmacological inhibition of PDE9A via Bay 73-6691 or virus-mediated CHIP expression restored the balance of cGMP/cAMP signalling. These interventions protect against cerebellar neuropathologies, particularly Purkinje neuron mitophagy dysfunction. Thus, PDE9A upregulation considerably exacerbates ataxia associated with CHIP mutations, and targeting the interaction between PDE9A and CHIP is an innovative therapeutic strategy for CHIP-related ataxia.

Trametinib (Trm) is a highly selective MEK inhibitor that potently and persistently abrogates ERK1/2 activation. Trm initially was used to treat BRAF V600E-mutated melanoma but its FDA-approved indications are expanding rapidly. Trm generally is well tolerated but it can cause dose-limiting cardiomyopathy and heart failure. Here we characterize a mouse model of Trm cardiotoxicity using complementary in vitro approaches to show that Trm induces mitochondrial dysfunction in cardiomyocytes and some cancer cell types. In vivo , Trm caused contractile dysfunction within 3 days and heart failure within 2 weeks. High resolution respirometry using isolated cardiac mitochondria revealed that Trm compromises oxidative metabolism, in part through blunted activity of Electron Transport System Complexes. Trm-mediated mitochondrial injury led to the release of mitochondrial Damage-Associated Molecular Patterns including mitochondrial DNA in both mice and humans, triggering activation of canonical innate immune pathways including cGAS-STING. In multiple rodent and human cardiomyocyte platforms, Trm diminished mitochondrial respiratory capacity at nanomolar concentrations but this lesion was reversed by expression of a phosphomimetic STAT3-S727 construct. We also found that Trm induced mitochondrial dysfunction in some but not all cancer cell lines, identifying a previously unrecognized effect that could contribute to Trm’s anti-cancer efficacy.

The thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT) plays an important role in sodium reabsorption, blood pressure and potassium homeostasis. Dietary potassium (K + ) intake modulates NCC abundance and activity, with high K + intake promoting NCC degradation and reducing BP. We hypothesized in our previous study that ubiquitin-dependent NCC degradation involves the ubiquitin E3 ligase CHIP (C-terminus of Hsc70-interacting protein)(1). In this study, our aim was to elucidate CHIP's role in NCC degradation and its impact on BP under varying dietary K + conditions. Methods: To explore CHIP’s role in regulating NCC and BP we used complementary in vivo and ex vivo experimental approaches. For in vivo studies, CHIP knockout (KO) and wildtype (WT) control mice were fed diets with varying K + content; low (0%), normal (1%), or high (5%) for five days. BP was monitored using telemetry (24 h recordings) or via tail-cuff plethysmography (early evening). To evaluate the contribution of NCC to BP regulation, mice were treated with the NCC inhibitor hydrochlorothiazide (HCTZ, 37.5 mg/kg body weight). For ex vivo studies, renal tubule suspensions isolated from WT mice were treated with a CHIP-specific inhibitor (10 µM) to evaluate its impact on NCC protein abundance. Additionally, tubules from WT and CHIP KO mice were incubated for various time points with cycloheximide and actinomycin to assess NCC half-life using immunoblotting. Results: Pharmacological CHIP inhibition in renal tubules significantly increased NCC protein levels, demonstrating a role for CHIP in NCC biogenesis. In CHIP KO mice on a normal K + diet, NCC protein abundance was significantly higher compared to WT, with no differences in NCC mRNA, suggesting the effects of CHIP on NCC are post-translational. CHIP KO mice had elevated BP that normalized with HCTZ, highlighting a role for CHIP in BP control. CHIP KO mice demonstrated an attenuated reduction in NCC abundance in response to a high K + diet. Ex vivo, NCC half-life was prolonged in tubules isolated from CHIP KO compared to WT, further confirming CHIP’s role in NCC degradation. Conclusion: Our findings highlight CHIP as a key regulator of NCC degradation, linking CHIP activity to BP regulation. Preliminary studies suggest a role for CHIP in mediating the effects of higher dietary potassium intake on NCC. Understanding the mechanisms by which CHIP regulates NCC, especially under varying dietary K + conditions, offers valuable insights into hypertension pathophysiology and may inform future therapeutic strategies targeting BP control. (1) Kortenoeven, MLA. et al., J Biol Chem 297, (2):100915 (2021). M.A. is supported by a Danish Diabetes and Endocrine Academy postdoctoral fellowship (NNF22SA0079901) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

The pro-inflammatory form of cellular death, necroptosis, is critical to age-related pathologies. Necroptosis primarily functions as an antipathogenic and antitumor biological mechanism by triggering inflammatory pathways within rogue cell bodies, resulting in cell death. Several neurodegenerative conditions have hallmarks of necroptosis, suggesting a potential role for this cell death pathway in the pathogenesis of neuroinflammation and neuronal cell death, likely through the release of pro-inflammatory cytokines that perpetuate inflammatory signaling and neurodegeneration. The receptor-interacting protein kinases 1 and 3 (RIPK1/3) signaling cascade is critical to necroptosis regulation; however, the complete mechanism behind necroptotic activation, regulation, and resolution remains incomplete. In cases where necroptosis is disadvantageous, such as neurodegenerative diseases, we lack effective pharmacological suppressors of necroptosis that could mitigate disease progression. Targeting regulatory proteins within the necroptotic signaling pathway has shown promise; however, the need for specific inhibitors limits therapeutic opportunities. This review focuses on necroptosis and its role in neuroinflammation and neurodegeneration in age-dependent disorders. We comprehensively detail the known necroptotic signaling pathways and potential signaling partners and discuss the ongoing therapeutic efforts in targeting and preventing active necroptotic signaling and their relevance to neuroprotection.