Traumatic Brain Injury (TBI) is a major global health concern and a leading cause of death and disability, especially in young adults. It triggers complex secondary injury cascades, e.g., calcium dysregulation, mitochondrial dysfunction and protease activation, that extend well beyond the initial mechanical insult to drive ongoing neurodegeneration. The calcium-dependent protease calpain has emerged as a central mediator of TBI cellular pathology. Calpain cleaves a broad range of cytoskeletal and regulatory proteins across neuronal compartments, disrupting axonal integrity, synaptic function and calcium homeostasis. Despite decades of research, calpain remains an elusive therapeutic target. In this review, we examine the spatial and temporal patterns of calpain activation in the traumatically injured brain, categorize key calpain substrates by structure and location, and assess their mechanistic roles in TBI pathology. We also review recent advances in next-generation calpain-2 selective inhibitors with enhanced specificity and preclinical efficacy and discuss the emerging use of calpain-cleaved protein fragments such as SBDP145 and SNTF as candidate biomarkers for TBI diagnosis and progression. Drawing on molecular, preclinical, and clinical data, we argue that calpain warrants renewed attention as both a therapeutic target and mechanistic biomarker in TBI. It may be time for Cinderella to leave the basement.

Physical constraints like compression influence cancer cell invasion and transcriptional dynamics in various tumors. Liver cancer is characterized by the rapid proliferation of tumor cells within a densely packed tissue matrix, subjecting the cancer cells to crowding and compression. The highly dysregulated mechanical environment highlights the need to elucidate the broader impact of compression on liver cancer development and evolution. In this study, we investigated and described a unique adaptive response of liver cells to prolonged compression. Liver cells presented significant transcriptional changes due to compression, including the loss of liver-specific markers and enrichment of epithelial-to-mesenchymal transition genes. Compression elevated Rac1 activity, which promoted cellular protrusions and YAP nuclear translocation and maintained cell viability under mechanical stress. Furthermore, compression disrupted intracellular calcium signaling, leading to resistance to apoptosis. Counteracting the effects of compression by inhibiting Rac1 or manipulating intracellular calcium facilitated death of compression-adapted cells. This study highlights compression as a critical biophysical signal in the tissue microenvironment that can induce cell state transitions and disease-driving phenotypes in the liver. SIGNIFICANCE:Compression in liver cancer affects cell states, signaling, and survival, which can be counteracted by inhibiting Rac1 or targeting intracellular calcium as potential avenues to eradicate compression-induced aggressive cancer cells.

Background/Objectives: Repeat low-level blast exposure has emerged as a significant concern for military populations exposed to explosive events. Blast-Related Traumatic Brain Injury (bTBI) is a unique form of brain trauma with poorly understood molecular mechanisms. Loss of calcium homeostasis has emerged as a mediator of early neuronal dysfunction after blast injury. This review aims to examine the role of calcium signaling in bTBI, focusing on the dual function of calcium channels as mediators and modulators of injury, and to explore therapeutic strategies targeting calcium homeostasis. Methods: We conducted a review of peer-reviewed articles published between 2000 and 2024, using the databases PubMed, Scopus, and EBSCO. Search terms included “blast traumatic brain injury”, “calcium channels”, and “calcium”. Studies investigating intracellular calcium dynamics after bTBI were included. Exclusion criteria included studies lacking evaluation of calcium signaling, biomarker studies, and studies on extracellular calcium. Results: We identified 13 relevant studies, primarily using preclinical models. Dysregulated calcium signaling was consistently linked to cellular dysfunction, including plasma membrane abnormalities, cytoskeletal destabilization, mitochondrial dysfunction, and proteolytic enzyme activation. Studies highlighted spatially compartmentalized vulnerabilities across neurons and astrocytes, suggesting that targeting specific cellular regions, such as the neuronal soma or axons, could enhance the therapeutic outcome. Therapeutic strategies included pharmacological inhibitors, plasma membrane stabilizers, and modulators of secondary injury. Conclusions: Calcium signaling is implicated in the pathophysiology of bTBI. Standardized experimental approaches would reduce variability in findings and improve the understanding of the relationship between calcium channel dynamics and bTBI and help guide the development of neuroprotective interventions that mitigate injury and promote recovery.

Loss of calcium homeostasis, a shared feature of Alzheimer’s Disease (AD) and Traumatic Brain Injury (TBI), activates enzyme-dependent cascades that promote protein misfolding, degrade synaptic architecture, impair axonal transport, and lead to neuronal death. Epidemiological studies identify TBI as a major risk factor for AD, yet the mechanistic basis for this association remains incompletely understood. Evidence from human and experimental studies implicate calcium dysregulation as a central link, triggering interconnected kinase, phosphatase, and protease networks that drive AD hallmark pathology, including amyloid-β (Aβ) accumulation and tau hyperphosphorylation. The calcium-dependent protease calpain is a key node in this network, regulating downstream enzyme activity, and cleaving essential scaffolding and signaling proteins. Selective vulnerability of the hippocampus and white matter to calcium-mediated damage may underlie cognitive deficits common to both conditions. In preclinical TBI and AD models, pharmacological inhibition of calcium-dependent enzymes confers neuroprotection. Recognizing disrupted calcium signaling as an upstream driver of post-traumatic neurodegeneration may enable early interventions to reduce AD risk among TBI survivors.

Antibody-drug conjugates (ADCs) are a promising class of targeted cancer therapies that deliver cytotoxic agents to cancer cells. Although designed to reduce side effects, ADCs utilizing a microtubule-targeting agent, exemplified by monomethyl auristatin E (MMAE), as the drug cargo, frequently induce chemotherapy-induced peripheral neuropathy (CIPN) at rates comparable to taxanes like paclitaxel (PTX). Despite their clinical success, ADC-associated neurotoxicity remains a significant challenge, necessitating a deeper mechanistic understanding and potential neuroprotective strategies. This study examines the neurotoxic effects of MMAE and PTX and evaluates lithium as a protective agent. Zebrafish models demonstrated that MMAE exhibits greater neurotoxicity than PTX, yet lithium co-treatment significantly improved survival. In human cell lines PTX treatment transiently increased intracellular calcium levels before reducing calcium signaling, whereas MMAE induced an immediate and sustained reduction in calcium signaling. Despite these distinct effects, lithium mitigates functional changes in both cases. The calcium binding protein neuronal calcium sensor 1 (NCS-1) is an essential component of the PTX pathological pathway; however, docking and immunoprecipitation assays showed that MMAE and PTX bind to NCS-1 at different sites, leading to differential impacts on calcium homeostasis. PTX enhances NCS-1 interaction with the inositol trisphosphate receptor (ITPR), whereas MMAE does not, underscoring mechanistic differences in their neurotoxicity. Lithium's ability to prevent neuronal dysfunction suggests it as a prophylactic agent against CIPN. By elucidating how these drugs disrupt calcium homeostasis, this study provides critical insights into ADC-induced neurotoxicity and highlights lithium as an intervention to enhance chemotherapy tolerability and patient quality of life.

The increasing number of cancer survivors, thanks to improved cancer treatments, has escalated the prevalence of adverse effects, especially chemotherapy-induced peripheral neuropathy (CIPN) and chemotherapy-induced cognitive impairment (CICI). New drug classes, including antibody-drug conjugates (ADCs), are being developed to target cancer cells and avoid noxious effects. Despite the efforts, ADCs present a high prevalence of neuropathy. A drug often employed in approved ADCs is Monomethyl Auristatin E (MMAE), a microtubule-based agent. The aim of this study was to investigate the sensory and cognitive effects of MMAE in a mouse model and test the potential use of lithium to alleviate MMAE-induced neuropathy. We developed a model of MMAE-induced CIPN and CICI and used behavior and sensory tests to analyze these conditions. We also evaluated calcium signaling and protein levels in neuropathic tissues and tumor progression upon treatments with lithium and MMAE. MMAE administration leads to loss of peripheral sensitivity and cognitive impairment and lithium prevents both central and peripheral neuropathies induced by chemotherapy, without affecting the antitumor activity of MMAE. This study shows that strategies including lithium pretreatment can prevent both central and peripheral neuropathies induced by chemotherapy to improve quality of life of cancer survivors.

Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, nondestructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here, we report a completely different mechanism by which neutrophils act nondestructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of Serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy individuals. This nondestructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.

Compressive stress can arise as cancer cells rapidly expand in the dense solid tumor environment. The mechanisms by which mechanical signals modulate cancer evolution and treatment outcomes in the liver are not well understood. In this study, we first identify and characterize mechanically compressed subdomains in liver tumor tissues. Next, we subject a liver cell line (HepG2) and primary hepatocytes to compressive stresses using two in vitro approaches: (i) hyperosmotic pressure and (ii) mechanical force.

The physical and functional interaction between transient receptor potential channel ankyrin 1 (TRPA1) and neuronal calcium sensor 1 (NCS-1) was assessed. NCS-1 is a calcium (Ca2+) sensor found in many tissues, primarily neurons, and TRPA1 is a Ca2+ channel involved not only in thermal and pain sensation but also in conditions such as cancer and chemotherapy-induced peripheral neuropathy, in which NCS-1 is also a regulatory component.We explored the interactions between these two proteins by employing western blot, qRT-PCR, co-immunoprecipitation, Ca2+ transient monitoring with Fura-2 spectrophotometry, and electrophysiology assays in breast cancer cells (MDA-MB-231) with different levels of NCS-1 expression and neuroblastoma cells (SH-SY5Y).Our findings showed that the expression of TRPA1 was directly correlated with NCS-1 levels at both the protein and mRNA levels. Additionally, we found a physical and functional association between these two proteins. Physically, the NCS-1 and TRPA1 co-immunoprecipitate. Functionally, NCS-1 enhanced TRPA1-dependent Ca2+ influx, current density, open probability, and conductance, where the functional effects depended on PI3K. Conclusion: NCS-1 appears to act not only as a Ca2+ sensor but also modulates TRPA1 protein expression and channel function in a direct fashion through the PI3K pathway. These results contribute to understanding how Ca2+ homeostasis is regulated and provides a mechanism underlying conditions where Ca2+ dynamics are compromised, including breast cancer. With a cellular pathway identified, targeted treatments can be developed for breast cancer and neuropathy, among other related diseases.

Alterations in calcium (Ca2+) signaling is a major mechanism in the development of chemotherapy-induced peripheral neuropathy (CIPN), a side effect caused by multiple chemotherapy regimens. CIPN is associated with numbness and incessant tingling in hands and feet which diminishes quality of life during treatment. In up to 50% of survivors, CIPN is essentially irreversible. There are no approved, disease-modifying treatments for CIPN. The only recourse for oncologists is to modify the chemotherapy dose, a situation that can compromise optimal chemotherapy and impact patient outcomes. Here we focus on taxanes and other chemotherapeutic agents that work by altering microtubule assemblies to kill cancer cells, but also have off-target toxicities. There have been many molecular mechanisms proposed to explain the effects of microtubule-disrupting drugs. In neurons, an initiating step in the off-target effects of treatment by taxane is binding to neuronal calcium sensor 1 (NCS1), a sensitive Ca2+ sensor protein that maintains the resting Ca2+ concentration and dynamically enhances responses to cellular stimuli. The taxane/NCS1 interaction causes a Ca2+ surge that starts a pathophysiological cascade of consequences. This same mechanism contributes to other conditions including chemotherapy-induced cognitive impairment. Strategies to prevent the Ca2+ surge are the foundation of current work.

Liver cancer involves tumor cells rapidly growing within a packed tissue environment. Patient tumor tissues reveal densely packed and deformed cells, especially at tumor boundaries, indicative of physical crowding and compression. It is not well understood how these physical signals modulate tumor evolution and therapeutic susceptibility. Here we investigate the impact of volumetric compression on liver cancer (HepG2) behavior. We find that conditioning cells under a highly compressed state leads to major transcriptional reprogramming, notably the loss of hepatic markers, the epithelial-to-mesenchymal transition (EMT)-like changes, and altered calcium signaling-related gene expression, over the course of several days. Biophysically, compressed cells exhibit increased Rac1-mediated cell spreading and cell-extracellular matrix interactions, cytoskeletal reorganization, increased YAP and β-catenin nuclear translocation, and dysfunction in cytoplasmic and mitochondrial calcium signaling. Furthermore, compressed cells are resistant to chemotherapeutics and desensitized to apoptosis signaling. Apoptosis sensitivity can be rescued by stimulated calcium signaling. Our study demonstrates that volumetric compression is a key microenvironmental factor that drives tumor evolution in multiple pathological directions and highlights potential countermeasures to re-sensitize therapy-resistant cells. Significance statement Compression can arise as cancer cells grow and navigate within the dense solid tumor microenvironment. It is unclear how compression mediates critical programs that drive tumor progression and therapeutic complications. Here, we take an integrative approach in investigating the impact of compression on liver cancer. We identify and characterize compressed subdomains within patient tumor tissues. Furthermore, using in vitro systems, we induce volumetric compression (primarily via osmotic pressure but also via mechanical force) on liver cancer cells and demonstrate significant molecular and biophysical changes in cell states, including in function, cytoskeletal signaling, proliferation, invasion, and chemoresistance. Importantly, our results show that compressed cells have impaired calcium signaling and acquire resistance to apoptosis, which can be countered via calcium mobilization.

Figure 1: NCS-1 and its Known Binding Partners. Figure 2: Assessment of the effect of NCS-1 on Motility. Figure 3: NCS-1 May Promote Tumor Aggressiveness by Altering Invasion and Chemotaxis. Figure 4: NCS-1 May Promote Tumor Aggressiveness by Altering Adhesion. Figure 5: Assessment of NCS-1 in Normal Breast Tissue. Figure 6: Validation of Prognostic Value of NCS-1 mRNA using SurvExpress, a biomarker validation tool with publicly available cancer datasets.

Figure 1: NCS-1 and its Known Binding Partners: This web diagram illustrates the binding partners of NCS-1. Figure 2: Assessment of the effect of NCS-1 on Motility: Representative photomicrographs from scratch motility assays for A. Figure 3: NCS-1 May Promote Tumor Aggressiveness by Altering Invasion and Chemotaxis: Representative photomicrographs from Transwell invasion assays show that overexpression of NCS-1 increases invasion in both A. MCF-7 and B. MB-231 cell lines. Figure 4: NCS-1 May Promote Tumor Aggressiveness by Altering Adhesion: Representative images for Collagen IV cell-adhesion assay for A. MCF-7 and B. MB-231 cell lines. Figure 5: Assessment of NCS-1 in Normal Breast Tissue. A. AQUA scores for NCS-1 in the tumor mask (y-axis) for each individual patient (x-axis). B. Normal distribution of NCS-1 AQUA scores for collective normal breast tissue cohort.

The destructive role of neutrophils in inflammation is well known 1 but they also have less damaging effects such as tissue remodeling and modulation of metabolism 2, 3 . Usually, neutrophils in tissues release toxic or digestive compounds into the extracellular region 4–8 . Here we report that neutrophils can inject their granule contents directly into hepatocytes. Neutrophil elastase within the hepatocytes selectively degrades the inositol trisphosphate receptor (ITPR), especially the type 2 isoform which is the predominant intracellular calcium release channel in these cells 9 . This action reduces calcium signals and cell proliferation without cellular damage. In response, the hepatocytes increase expression of serpins E2 and A3, which block the effect of elastase. This phenomenon is also observed in liver biopsies from patients with alcoholic hepatitis, a condition characterized by infiltration of neutrophils 10, 11 . This non-destructive and reversible effect on hepatocytes defines a previously unappreciated role of neutrophils in transiently regulating signaling mechanisms in epithelia.

Wolfram syndrome is a rare genetic disorder characterized by endocrine dysfunction and progressive neurodegeneration. By targeting intracellular calcium dysregulations, a sigma-1 receptor agonist rescued neurological deficits in preclinical models of Wolfram syndrome.

Calcium’s importance in signaling has been known since Ringer showed that it was needed for contraction of isolated hearts in 1883. Calcium remained a curiosity until the mid 1940s when Heilbrunn showed that intracellular calcium is the trigger for muscle contraction (1). Since then, calcium has been implicated in so many cellular processes, some have wondered how one ion can do so many things. The large difference in calcium concentration between the cytoplasm (∼0.1 μM), the intracellular organelles (400 μM), and the extracellular fluid (1.5 mM) allows small fluxes of calcium to have a big impact. But there are several features of the intracellular calcium signaling network that make it possible for this one ion to invoke specific responses within the cell. Two of these properties are the location of the calcium release into the cytoplasm and the location at which calcium itself binds and regulates components of the signaling complex. In PNAS, Arige et al. (2) identify a critical calcium binding site on the inositol trisphosphate receptor (IP3R), a major component of calcium signaling that addresses both aspects of location. The mammalian IP3R has three isoforms, and, although remarkably similar, each isoform has unique cellular locations and properties. Even though most mammalian cells express all three isoforms, the primary IP3R isoform expressed differs among cell types. For example, cerebellar Purkinje cells express primarily InsP3R type 1 (IP3R1), and hepatocytes express primarily InsP3R type 2 (IP3R2) (3–5). Additionally, isoforms are found in distinct subcellular locations. In pancreatic acinar cells, IP3R2 is found near the apical end of the cell, providing a barrier to calcium transients invading the secretory end of the cell (6), and IP3R3 and IP3R1 are localized to regions where the mitochondria and endoplasmic reticulum (ER) meet (mitochondrial-associated membrane; e.g., refs. 7–9) to enhance calcium flow between these two organelles. These subcellular locations and the type of IP3Rs at specific sites can be changed by disease (7, 10, 11).

Drug treatment against liver cancer has limited efficacy due to heterogeneous response among liver cancer subtypes. In addition, the functional biophysical phenotypes which arise from this heterogeneity and contribute to aggressive invasive behavior remain poorly understood. This study interrogated how heterogeneity in liver cancer subtypes contributes to differences in invasive phenotypes and drug response. Utilizing histological analysis, quantitative 2D invasion metrics, reconstituted 3D hydrogels, and bioinformatics, our study linked cytoskeletal dynamics to differential invasion profiles and drug resistance in liver cancer subtypes. We investigated cytoskeletal regulation in 2D and 3D culture environments using two liver cancer cell lines, SNU-475 and HepG2, chosen for their distinct cytoskeletal features and invasion profiles. For SNU-475 cells, a model for aggressive liver cancer, many cytoskeletal inhibitors abrogated 2D migration but only some suppressed 3D migration. For HepG2 cells, cytoskeletal inhibition did not significantly affect 3D migration but did affect proliferative capabilities and spheroid core growth. This study highlights cytoskeleton driven phenotypic variation, their consequences and coexistence within the same tumor, as well as efficacy of targeting biophysical phenotypes that may be masked in traditional screens against tumor growth.