Cell Death & Disease

The microRNAs miR-15a and miR-16 are key regulators of the anti-apoptotic oncogene BCL2, playing a significant role in tumorigenesis. These miRNAs function as tumor suppressors by directly targeting BCL2, whose overexpression contributes to cell survival and resistance to therapy in multiple malignancies, including chronic lymphocytic leukemia (CLL). The downregulation or deletion miR-15a/miR-16-1 cluster located on chromosome 13q occurs in about 50% of CLL patients and leads to the overexpression of the oncogenic BCL2, contributing to the survival and proliferation of cancer cells. In this confirmatory study, we provide additional evidence supporting the mechanism by which these miRNAs mediate the inhibition of BCL2 translation, leading to reduced levels of BCL2 protein with no significant effect on BCL2 mRNA degradation. This mechanism has been previously established as a critical pathway in the regulation of apoptosis, particularly in cancer cells where BCL2 overexpression is often associated with resistance to cell death. Our findings reinforce the notion that miRNAs, such as miR-15 and miR-16, bind to the 3-UTR of BCL2 messenger RNA (mRNA), specifically repressing its translation without inducing mRNA degradation. The results from our study align with previous research, confirming that the miRNA-mediated inhibition of BCL2 translation serves as a precise regulatory mechanism that targets protein synthesis rather than mRNA stability. These findings highlight the role of miRNAs in fine-tuning post-transcriptional gene regulation, offering a targeted approach to downregulate oncogenic proteins like BCL2 without disrupting the underlying mRNA, which could be leveraged for more refined therapeutic strategies.

Targeting mitochondrial Complex I (CI) is a currently emerging anti-cancer strategy, with several enzyme inhibitors entering clinical trials. Among others, aggressive high-grade serous tubo-ovarian cancer (HGSOC) may particularly benefit from this therapeutic approach due to the scarce response to first- and second-line treatments, with consequent high mortality, such as the anti-angiogenic bevacizumab. We here show that CI represents a vulnerability in HGSOC, which can be exploited for therapeutic intervention. Indeed, ablating CI function in OV-90 HGSOC cells led to significant in vivo tumor growth decrease, smaller masses, and lower KI-67 proliferative index. This was confirmed in a switch-off system in which CI deprivation was induced during tumor progression to mimic pharmacologic treatment, suggesting this result can be achieved in growing neoplasms. We also show that abolishing CI in HGSOC cells leads to failure in stabilizing the hypoxia inducible factor-1a and to respond to hypoxia through the transcriptional activation of its target genes, ultimately lowering vascular endothelial growth factor (VEGF) and generating an immature intratumor vascular system accompanied by a decreased blood flow. Last, we demonstrate that targeting CI sets the biological basis for increased sensitivity to anti-angiogenics, as CI-deprived tumors displayed growth arrest when bevacizumab was administered, unlike their CI-competent counterpart. Our findings point to CI inhibition as a booster for anti-VEGF therapies and pave the way for combined protocols in treatment of HGSOC.

Myocardial ischemia-reperfusion injury significantly exacerbates cardiac damage and worsens clinical outcomes, with oxidative stress in cardiomyocytes representing a central pathological mechanism. In this study, we reveal that APEX1, a key redox regulator, is markedly downregulated in cardiomyocytes under oxidative stress conditions. Functional analyses demonstrate that APEX1 knockdown intensifies oxidative stress-induced cardiomyocyte injury, whereas APEX1 overexpression confers robust protection against hypoxia reoxygenation mediated damage. Mechanistically, APEX1 exerts its cardioprotective effects by stabilizing the p53 protein and modulating its ubiquitination status. These findings establish APEX1 as a critical defender against oxidative injury in cardiomyocytes through direct regulation of p53 protein stability, highlighting its potential as a therapeutic target for ischemia-reperfusion related heart disease.

Infiltration of conventional immune cells has been ascribed as the fundamental drivers of innate immune signaling in the damaged myocardium. However, the emerging intrinsic immunoregulatory potential of cardiomyocytes still remains poorly understood. Interferon gamma (IFN{gamma}) is a pleiotropic cytokine with context-dependent detrimental as well protective role in regulating cardiac inflammatory circuits. The prevailing view of IFN{gamma} as a prime pro-inflammatory cytokine has been challenged due to its paradoxical actions both as an inducer as well as negative regulator of inflammation, but the players involved in these converse processes remains enigmatic. Here we show that cardiomyocytes exhibit a cell-autonomous immunocompetent response upregulating innate inflammatory signaling upon type I and type II IFN stimulus. Notably, hiPSC-derived cardiomyocytes display a robust increase in guanylate binding protein 5 (GBP5), one of the major IFN{gamma}-induced GTPase involved in inflammasome signaling, followed by upregulation of AIM2/CASP1 pathway whereas NLRP3 levels remain unaltered by IFN{gamma} stimulation. GBP5 knockdown and overexpression studies in hiPSC-derived cardiomyocytes identify GBP5/TGF{beta} axis as a non-canonical anti-inflammatory feedback regulation on the IFN{gamma}-induced inflammatory cascade.

Prostate cancer is the second most common malignancy among men, with androgen deprivation therapy (ADT) serving as the standard treatment due to the hormone sensitivity of prostate tumors. However, therapeutic resistance frequently develops, leading to castration-resistant prostate cancer (CRPC), an aggressive and lethal disease. A recently defined subtype, stem cell-like CRPC (CRPC-SCL), accounts for approximately 25% of CRPC cases and demonstrates poor responsiveness to ADT. CRPC-SCL is characterized by the expression of Cluster of Differentiation 44 (CD44), a glycoprotein that promotes hyaluronic acid binding and uptake. Within CRPC-SCL patient-derived xenograft (PDX) model, CD44 high (CD44hi) cells exhibit enhanced tumorigenicity and proliferative capacity. Importantly, iron metabolism emerges as a critical regulator of this population: CD44hi cells maintain elevated intracellular iron, which sustains CD44 expression and stem cell-like properties by modulating H3K9me2 modification. Leveraging this vulnerability, inhibition of the iron-regulatory factor NRF2 was shown to increase intracellular free iron and selectively induce ferroptosis in CD44hi cells. These findings highlight the therapeutic potential of targeting iron metabolism to induce ferroptosis as a novel treatment strategy for CRPC-SCL.

Ovarian cancer (OC) remains a lethal malignancy with limited therapeutic options, underscoring the need for the identification of novel agents. Natural products like clove (Syzygium aromaticum) have shown promising anti-cancer activity, but their mechanism in OC is poorly understood. This study investigates the anti-tumour effects and underlying mechanisms of a clove aqueous extract (CAE) on a panel of patient-derived OC cells. We found that CAE significantly inhibited cellular proliferation and induced cell death in a time-and dose-dependent manner. Mechanistically, CAE induced profound cellular stress, activating the transcription factor ATF-2. This was accompanied by a significantly increased lysosomal stress response, as evidenced by increased lysosomal mass/acidity, and a pathogenic hyperpolarisation of the mitochondrial membrane potential ({Delta}{Psi}m). The bioenergetic crisis induced as a consequence resulted in a sharp reduction in cellular oxygen consumption rate (OCR). Notably, the sensitivity to CAE-induced lysosomal and mitochondrial dysfunction varied across cell lines, revealing distinct phenotypic responses. Our results demonstrate that clove extract exerts its anti-tumour effects by orchestrating a multi-organellar stress response, positioning lysosomal disruption as a central event in its mechanism of action. This study provides a strong rationale for the further development of clove-based interventions for OC.

Nerve growth factor (NGF) exerts neuroprotective effects in the retina, and accumulating evidence indicates that microglia represent a key cellular target of NGF/TrkA signaling. However, evidence showing that the NGF/TrkA signaling in microglia is required for downstream neuroprotective actions remains unresolved. Here, we directly addressed this question by pharmacologically depleting microglia and assessing the impact on NGF pathway activity and retinal integrity. Adult C57BL/6J mice were treated with the CSF1R inhibitor PLX5622 for three weeks, resulting in a robust ([~]77%) depletion of retinal microglia. Microglial ablation induced marked structural and cellular alterations, including significant loss of retinal ganglion cells (RGCs) and thinning of retinal layers, in the absence of any other lesion or insult. Residual microglia exhibited layer-specific phenotypic changes, with a phagocytic profile in the ganglion cell layer and a more ramified morphology in the outer plexiform layer. Strikingly, microglial depletion led to a profound decrease of NGF signaling, with a strong reduction in total and phosphorylated TrkA, and decreased p75NTR levels, in retinal extracts. The amount of TrkA expression is strongly correlated with microglial levels, supporting a primary role of microglia in sustaining NGF signaling in the retina. Together, these findings demonstrate that microglia are required for NGF/TrkA signaling and identify these cells as essential mediators of NGF-dependent neuroprotection in the retina.

Recurrent hypoglycemia increases cognitive impairment in diabetes mellitus patients. Following cerebral neuron injury, endothelial cells provide morphological, metabolic and immune support to damaged neurons. We investigated the inflammatory mechanism involved in hippocampal neuron degeneration. Behavioral experiments, including the open field test (OFT) and the Morris water maze test, were performed to measure cognitive changes. Using a vascular ring experiment, we evaluated vasodilation of the carotid artery. ZBP1 expression was knocked down after transfection with small interfering RNA in a brain endothelial cell line (bEnd3). In this study, PANoptosis, a recently defined form of programmed cell death (PCD), was found to be increased by hypoglycemia in the hippocampus of type 2 diabetic mice in vivo and by low glucose in bEnd3 cells in vitro. ZBP1 knockdown decreased PANoptosis induced by low-glucose stimulation in high-glucose-cultivated bEnd3 cells. RNA transcriptomics sequencing revealed that AGE-RAGE signaling significantly changed after ZBP1 was knocked down in bEnd3 cells. Corresponding biochemical data confirmed that ZBP1 knockdown regulated the advanced glycation end products (AGEs)-Receptor for Advanced Glycation End Products (RAGE) axis in bEnd3 cells. We present the first evidence that hypoglycemia impaired cognition in mice with type 2 diabetes by activating brain endothelial ZBP1-mediated PANoptosis via the AGE-RAGE axis. ARTICLE HIGHLIGHTSO_LIPANoptosis, a newly defined form of programmed cell death, is induced in the hippocampus after recurrent hypoglycemia in male db/db mice. C_LIO_LIZBP1, a sensor of the PANoptosome, was activated in low glucose cultured brain endothelial cells. C_LIO_LIHypoglycemia impairs vasodilation and cognitive function in type 2 diabetic mice. C_LIO_LIOur study indicates that inhibiting ZBP1-PANoptosis and the AGE-RAGE axis may be a potential approach to prevent hypoglycemia-induced cognitive degeneration in individuals with type 2 diabetes. C_LI

Osteoarthritis (OA) is a leading cause of disability worldwide, with chronic pain representing its most debilitating symptom and frequently accompanied by affective and cognitive comorbidities. Increasing evidence implicates maladaptive supraspinal plasticity within cortical regions involved in pain affect, including the anterior cingulate cortex (ACC) and anterior insular cortex (AIC), however, the relationship between these behavioral impairments and neuronal alterations, as well as potential sex-specific vulnerability, remains poorly documented. Using a monosodium iodoacetate (MIA) model of knee OA in adult male and female mice, we examined the temporal progression of sensory, affective, and cognitive alterations at early (day 7) and advanced (day 28) stages of disease. Pain sensitivity, locomotor and gait changes, anxiety- and depression-like behaviors, and working-memory performance were assessed using established behavioral paradigms, followed by analyses of apoptosis-associated neuronal signaling in the ACC and AIC. MIA induced robust mechanical and thermal hypersensitivity and gait impairment in both sexes, while early emotional and cognitive alterations were not observed. In contrast, advanced OA was associated with pronounced anxiety- and depression-like behaviors and impaired working memory. Notably, analysis at day 28 post-MIA revealed a significant increase in apoptotic signaling and neuronal loss in both cortical regions, with females exhibiting greater vulnerability, particularly within the AIC, paralleling their more severe affective phenotypes. Together, these findings indicate that chronic OA pain is associated with progressive, sex-dependent neuronal loss within key cortical pain-affective circuits and highlight supraspinal remodeling as a potential substrate underlying the emotional and cognitive burden of OA pain.

Presenilin 1 (PS1), a key pathogenic factor in familial Alzheimers disease, is implicated in regulation of mitochondrial functions, yet its precise sub-mitochondrial localization and underlying mechanisms remain poorly understood. In this study, we generated PS1 knockout (PS1 KO) cell lines to investigate the role of PS1 in mitochondrial structure and function. Our results demonstrated that PS1 is directly localized to the mitochondrial inner membrane. PS1 deficiency led to reduced ATP production, impaired mitochondrial respiration capacity, decreased mitochondrial membrane potential, disrupted Ca2+ homeostasis, and elevated reactive oxygen species (ROS) accumulation. Moreover, loss of PS1 caused abnormal mitochondrial cristae structure. Further analysis revealed that PS1 interacts with mitochondrial inner membrane proteins. Its absence promotes ATAD3A oligomerization and disrupts its arrangement at mitochondrial cristae junctions, leading to expansion of the mitochondria-associated membrane (MAM) and instability of mitochondrial DNA (mtDNA). Our findings demonstrate that PS1 acts as a central regulator of mitochondrial cristae morphogenesis by modulating protein interaction networks at cristae junctions, thereby illuminating fundamental molecular mechanisms contributing to mitochondrial dysfunctions in Alzheimers disease.

Usher syndrome type 2A (USH2a), the most common form of hereditary deaf-blindness, is frequently accompanied by fatigue and poor sleep quality. As these sleep problems occur independently of visual decline, it is hypothesized that the USH2A-encoded protein usherin regulates sleep and circadian rhythmicity via an extra-retinal mechanism. Ush2a knockout zebrafish models were utilized to investigate this hypothesis. Immunohistochemical analysis demonstrated usherin localisation in pineal gland photoreceptor cells in wild-type larvae, alongside the USH2 complex proteins Adgrv1 and Whrna. Cross-species transcriptomic and proteomic analyses confirmed USH2A expression in all mammalian pineal gland tissues studied. Circadian clock gene expression was measured over 24 h and showed preserved oscillatory patterns in wild-type and mutant zebrafish. Ex vivo superfusion of pineal glands revealed sustained circadian melatonin release with comparable phase and period to controls, although potential differences in absolute melatonin levels could not be excluded. Despite intact clock gene expression and melatonin release in ush2a mutants, behavioural classification over 24-h recordings revealed altered sleep-wake behaviour: ush2a mutants displayed elevated daytime sleep and significantly prolonged and more variable sleep latency. The dissociation between intact molecular rhythms and abnormal sleep behaviour likely implicates that usherin plays a role in sleep-wake regulation independent of the circadian pacemaker and melatonin synthesis. These findings suggest a novel role of usherin in the pineal gland and establish a mechanistic link between usherin dysfunction and sleep disturbances, providing a biological basis for the fatigue and sleep problems reported in USH2a patients.

Altered iron homeostasis has long been implicated in Parkinson's Disease (PD), although the mechanisms have not been clear. Given the critical role of PD-related activating mutations in LRRK2 (leucine-rich repeat protein kinase 2) within membrane trafficking pathways we examined the impact of a homozygous mutant LRRK2G2019S on iron homeostasis within the RAW macrophage cell line with high iron capacity. Proteomics analysis revealed a dysregulation of iron-related proteins in steady state with highly elevated levels of ferritin light chain and a reduction of ferritin heavy chain. LRRK2G2019S mutant cells showed efficient ferritinophagy upon iron chelation, but upon iron overload there was a near complete block in the degradation of the ferritinophagy adaptor NCOA4. These conditions lead to an accumulation of phosphorylated Rab8 at the plasma membrane, which is selectively inhibited by LRRK type II kinase inhibitors. Iron overload then leads to increased oxidative stress and ferroptotic cell death. These data implicate LRRK2 as a key regulator of iron homeostasis and point to the need for an increased focus on the mechanisms of iron dysregulation in PD.

Ferroptosis is a form of regulated cell death that is characterized by iron-dependent lipid peroxidation. This process is regulated by specific metabolites, the lipid composition of the cells, redox-active iron, and antioxidant mechanisms. Although numerous regulators have been identified over the past decade, exploring other mechanisms, particularly from non-coding genomic regions, can build a thorough understanding of the multifaceted regulatory processes underlying ferroptosis. MicroRNAs (miRNAs) play a crucial role in gene regulation and cellular functions. Through a CRISPR KO screen, we identified miR-940 as a negative regulator of ferroptosis. Overexpression of miR-940 in several cell lines consistently suppressed ferroptosis induced by system xc- inhibition. Notably, multiple cancer patient cohorts with elevated miR-940 levels exhibit reduced survival. Integrated bioinformatic, transcriptomic, and proteomic analyses revealed that miR-940 decreases the expression of ACSL4, LPCAT3, DMT1, and NCOA4, and simultaneously increases levels of GPX4. Pharmacological inhibition of GPX4 attenuated the protective effect of miR-940, indicating that its primary anti-ferroptotic activity is mediated through GPX4. Overall, this gene rewiring is associated with reduced levels of redox-active iron and diminished lipid peroxidation, consistent with ferroptosis suppression. These findings suggest that miR-940 coordinates ferroptosis inhibition, which presents a novel regulatory layer for therapeutic exploration in susceptible cancers.

Parkinsons disease (PD) is characterized by progressive dopaminergic neurodegeneration driven by complex interactions among oxidative stress, impaired survival signaling, and protein homeostasis disruption. Emerging evidence suggests that endogenous retroelements, including human endogenous retrovirus K (HERV-K), may contribute to neurodegenerative processes; however, their role in PD remains poorly defined. Here, we investigated whether dopaminergic neurotoxic stress induces HERV-K activation and whether modulation of pro-survival signaling pathways influences this response in PD-relevant cellular models. Using undifferentiated SHSY5Y cells and neuron-like retinoic acid-differentiated SHSY5Y cells, we show that exposure to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) induces a rapid and robust transcriptional de-repression of HERV-K Env gene. HERV-K activation occurs early after toxin exposure, scales with the intensity of the insult, and is associated with alterations in oxidative stress defenses, survival signaling pathways, and protein homeostasis. Notably, 6-OHDA treatment promotes the accumulation and cytoplasmic mislocalization of phosphorylated TAR DNA-binding protein 43 (pTDP-43), a pathological feature linked to neurodegenerative proteinopathies. Pharmacological modulation of the Wnt/{beta}-catenin pathway by the natriuretic peptide atrial natriuretic peptide (ANP) significantly attenuates neurotoxin-induced HERV-K activation, restores oxidative stress-related and survival signaling markers, and limits pTDP-43 accumulation and mislocalization. These findings indicate that reinforcement of Wnt/{beta}-catenin dependent protective pathways constrains stress-driven HERV-K de-repression and associated molecular alterations. Overall, this study identifies HERV-K activation as an early stress-responsive feature in PD-like cellular models and supports the existence of a functional interplay between retroelement regulation, survival signaling, and protein homeostasis. Modulation of Wnt/{beta}-catenin signaling may represent a strategy to limit retroelement-associated pathological responses in PD.

The development of advanced therapies for stroke, spinal cord injury or neurodegenerative diseases -main causes of death, disability and dementia- requires a profound understanding of the complex interactions established among excitotoxic neuronal death, aberrant neurotrophic-signaling, glial reactivity, and neuroinflammation. However, the master proteins coordinating these mechanisms have not been yet defined. Different evidence suggests that the truncated form of the neurotrophin tyrosine kinase receptor, TrkB-T1, might play such a key role. The levels of this TrkB isoform increase in stroke while those of the full-length pro-survival isoform (TrkB-FL) are reduced. Additionally, ischemic stroke and, specifically, excitotoxicity induce TrkB-T1 regulated intramembrane proteolysis (RIP), a process releasing a receptor ectodomain able to bind the brain-derived neurotrophic factor (BDNF) and leading to decreased BDNF-signaling. We hypothesize that the second RIP product, TrkB-T1 intracellular domain (TrkB-T1-ICD), might similarly contribute to neurotoxicity but also reactive gliosis and neuroinflammation. Herein, we first demonstrate migration of the cytoplasmic TrkB-T1-ICD to the nuclei of neurons undergoing excitotoxicity, suggesting a possible role in the transcriptional control induced by injury. Then, taking advantage of cell-penetrating peptides (CPPs), we produce a TrkB-T1-ICD mock peptide (Bio-LTT1Ct) containing the short TrkB-T1 intracellular region (23 amino acids) and test it in vitro and in vivo. Notably, this peptide migrates to the nucleus of both neurons and astrocytes cultured in vitro and provokes cell death. Additionally, Bio-LTT1Ct induces early transcriptional changes in neurons resembling those triggered by excitotoxicity such as the inhibition of the promoter activity of pro-survival transcription factors CREB and MEF2, and altered mRNA levels of their regulated genes. In vivo, Bio-LTT1Ct is accessible to the brain cortex after intranasal delivery, being efficiently distributed into cortical neurons and astrocytes of both hemispheres. Moreover, peptide administration is sufficient to promote important pathological hallmarks of stroke such as the imbalance of the TrkB isoforms, and the reactivity of astrocyte and microglia, cells that acquire proinflammatory profiles. Altogether, these results establish TrkB-T1 RIP as a central mechanism of ischemic damage and demonstrate that the receptor intracellular region is sufficient to recapitulate stroke-like effects on neurotoxicity, glial reactivity and neuroinflammation.

This study describes the distribution of non-reactive brain-resident microglia densely populated along the borders of the lateral ventricles and choroid plexus in premature rabbit pups during early forebrain development. Following intraventricular hemorrhage (IVH), microglia become activated, proliferate, and migrate deeper into parenchymal regions. During this process, activated microglia exhibit a global expansion with a disproportionally elevated proinflammatory M1 nomenclature phenotype from 25% to 50% of the total; that shift was reduced by sulforaphane (SFN; Nrf2-antioxidant response element [ARE] activator of anti-inflammatory pathways) plus deferoxamine (DFN; iron chelator) treatment. Transcriptome analysis identified over expression of pro-inflammatory calcium-binding proteins S100A8 and S100A12 (intracellular damage signals), as well as chemokines CXCL8 and CXCL10 by neurons and microglia. The combination treatment of SFN-DFN mitigated M1 infiltration, suppressed the magnitude of inflammation and reduced ferroptosis after IVH in the developing postnatal brain. Moreover, SFN-DFN treatment reversed most dysregulated genes in inflammation and iron homeostasis networks, revealing potential molecular targets for additional pharmacologic interventions after IVH. We propose that reducing the toxic microcellular environment will attenuate both the injurious inflammatory responses and improve recovery of the trajectory toward normal brain development. Additionally, suppression of proinflammatory molecules and iron toxicity should promote better survival as well as salutary effects of "living stem cell therapy" as we have previously shown.

The TGF-{beta} signaling pathway has both tumor-suppressing and metastasis-promoting effects in cancer. However, the molecular determinants governing this switch remain unclear. Here, we explored the miR-16-1-3p/MDM2/p53 axis as a critical conductor of the TGF-{beta}-Smad pathway in osteosarcoma. Although miR-16-1-3p overexpression by itself markedly reduces proliferative and clonogenic potential of U2OS cells, when paired with TGF-{beta} treatment, it significantly increases arrest cells in G1 phase and nearly extinguishing the growth capability of these cells. MiR-16-1-3p overexpression inhibited TGF-induced actin remodeling and EMT featuring, significantly decreasing vimentin levels. TGF-{beta} enhances both 2D and 3D migration, but miR-16-1-3p overexpression, alone or with TGF-{beta}, strongly counteracts its pro-migratory effects. MiR-16-1-3p restored p53 stability by targeting MDM2, redirecting TGF-{beta}-Smad signaling toward p21 activation and proliferation inhibition while attenuating its EMT-promoting capacity. Administration of TGF-{beta} together with miR-16-1-3p dramatically increases the sensitivity of wild-type U2OS cells to cisplatin, exceeding that of TGF-{beta} therapy alone by more than an order of magnitude. Administering TGF-{beta} and miR-16-1-3p together significantly reduces the tumor nodule volume and Ki67 expression, while effectively eradicates metastases in the chicken chorioallantoic membrane (CAM) in vivo model. For the first time, our research demonstrates that miR-16-1-3p shifts TGF-{beta}1 signaling from a facilitator of metastasis to a promoter of anti-growth effects through MDM2 inhibition and p53 stabilization, effectively reducing the self-renewal and invasiveness of cancer stem cells in human osteosarcoma model. This process preserves TGF-{beta}s tumor-suppressive role while limiting its associated cancer risks.

As highly dynamic organelles, mitochondria play an essential role in neuronal survival and synaptic function. Excitotoxicity is as a critical factor that promotes mitochondrial dysfunction after traumatic brain injury (TBI). Intercellular mitochondrial transfer and exogenous mitochondrial transplantation are emerging concepts to understand mitochondrial trafficking in response to mitochondrial dysfunction; however, robust in vivo evidence remains limited on the extent of these processes in the central nervous system (CNS). There is a significant knowledge gap in our understanding of mitochondrial transfer mechanisms under both normal physiological conditions and after experimental TBI. Mouse lines expressing mitochondrial green-fluorescent dendra-2 (mtD2) and GFP (mtGFP) targeted to inner and outer mitochondrial membranes, respectively, were used to study astrocyte-specific (Aldh1l1-CreER; mtD2f/f - AmtD2 and Aldh1l1-CreER; mtGFPf/f - AmtGFP) and neuron-specific (CamK2aCre; mtD2f/f - NmtD2 and CamK2aCre; mtGFPf/f - NmtGFP) mitochondrial dynamics and bioenergetics in acute TBI and excitotoxicity. At 24 hrs following TBI, neurons in the NmtD2 mouse brain exhibited rapid and significant alterations in mitochondrial morphology, including changes in total mitochondrial volume, volume distribution, and sphericity. Synaptic neuronal (SN) mitochondria display robust deficits in mitochondrial bioenergetics and complex protein levels while non-synaptic neuronal (NSN) mitochondria show State III bioenergetics and complex proteins at control levels. These findings are accompanied by a marked increase in astrocyte-derived mitochondria (AmtGFP) transfer to neurons at 24 hrs post-injury, compared to control animals, but no increase in transfer to neuronal synapses. While TBI also altered astrocytic mitochondrial morphology in the cortex, astrocytic mitochondrial bioenergetics remained preserved. Single-cell RNA-seq analysis of astrocytes revealed significant transcriptional reprogramming following TBI, characterized by the upregulation of genes associated with mitochondrial homeostasis and the machinery for organelle trafficking. In vitro co-cultures of primary cortical astrocytes and neurons demonstrated that astrocytes can transfer mitochondria to neurons via direct contact and that NMDA-mediated excitotoxicity further enhanced this astrocyte-to-neuron mitochondrial transfer. Furthermore, astrocytic-derived extracellular vesicles containing mitochondria (EV-mito) deliver mitochondria to neurons and EV-mediated mitochondrial transfer significantly ameliorated NMDA-induced mitochondrial dysfunction in primary cortical neurons. Together, these findings show that astrocytes take on a TBI-related phenotype that facilitates dynamic changes in mitochondrial networks and mitochondrial trafficking to neurons. Astrocytic transfer of respiratory-competent mitochondria support is an intrinsic neuroprotective response to injury that supports mitochondrial function in neuronal soma, dendrites, and axons but not at the neuronal synapse. Finally, we show therapeutic potential of exogenous mitochondrial transfer, particularly via EV-mito, for treating neurological disorders associated with excitotoxicity, such as TBI.

Excessive iron accumulation is a pathological feature of several neurodegenerative diseases (NDDs) and a growing body of evidence suggests that ferroptosis, an iron-dependent form of regulated cell death (RCD) driven by lipid peroxidation, is implicated in their pathogenesis. Microglia, the brains resident immune cells, buffer iron overload but become susceptible to ferroptotic death, exacerbating neuroinflammation and neuronal loss. To uncover the molecular events leading to microglial ferroptosis, we established a human microglial ferroptosis model using the HMC3 cell line. This model recapitulates core features of ferroptosis, including increased reactive oxygen species (ROS) and peroxidation of lipids at the membrane, both rescued by Ferrostatin-1 (Fer-1). We used this model to perform integrated multi-omics profiling and identified significant dysregulation in lipid species, notably an accumulation of sterols, including oxysterols such as the 7-oxo-cholesterol, alongside the oxidation of polyunsaturated fatty acid (PUFA) characteristic of ferroptosis. Transcriptomic and proteomic analyses corroborated these findings, revealing the upregulation of genes and proteins involved in the mevalonate pathway and cholesterol metabolism. Importantly, the increased expression of some of these key metabolic genes was also reversed by Fer-1 treatment, indicating their role in a pre-ferroptotic signature. Our model provides a novel platform for investigating early molecular events in microglia ferroptosis. Integrating these findings into future investigations could uncover new protective mechanisms against microglia ferroptosis at the crossroad between ROS level mitigation and sterol metabolism.

Deregulation of amyloid precursor protein (APP) metabolism leads to the production of pathological proteoforms of A{beta} peptides, which ultimately form amyloid deposits, a primary pathological feature of Alzheimers disease (AD). The accumulation of misfolded proteins and alterations in protein degradation systems, such as the ubiquitin-proteasome system, endoplasmic reticulum-associated degradation, and autophagy-lysosomal pathways, also contribute to AD development. The Valosin-Containing Protein AAA-ATPase (p97/VCP) is a crucial regulator of proteostasis, facilitating the clearance of misfolded proteins by the proteasome and other protein degradation systems. Here, we investigated whether VCP influences APP processing. Reducing VCP expression or inhibiting its ATPase activity led to the accumulation of mature, full-length APP in cells, thereby decreasing APP trafficking to the cell surface. Downstream of APP-CTFs secretase processing, p97/VCP modulates APP-CTFs cleavage and degradation via autophagy and endolysosomal-dependent mechanisms. Our findings demonstrate that VCP is involved in APP metabolism at two levels: controlling APP trafficking within the cell secretory pathway and regulating autophagy-dependent degradation of APP-CTFs, suggesting a potential role for VCP in the APP deregulation observed in AD.