Patrick G. Sullivan

Mitochondria are dynamic organelles essential for neuronal survival and synaptic function, and their dysfunction is a key consequence of excitotoxicity following traumatic brain injury (TBI). While intercellular mitochondrial transfer and exogenous mitochondrial transplantation have emerged as mechanisms to restore cellular bioenergetics, its in vivo relevance in the central nervous system remains incompletely understood. Here, we used astrocyte and neuron-specific mitochondrial reporters (GFP or Dendra2) in mice to assess cell-type-specific mitochondrial morphology, bioenergetics, and transfer 24 h after TBI. Neurons exhibited marked mitochondrial dysfunction, including altered morphology and reduced bioenergetic capacity across somatic, synaptic, and non-neuronal fractions. In contrast, astrocytic mitochondria showed morphological changes but preserved bioenergetic function. Concomitantly, astrocyte-to-neuron mitochondrial transfer was significantly increased following injury, although transfer to synapses remained limited. Single-cell RNA sequencing of astrocytes revealed upregulation of genes involved in extracellular vesicle (EV) biogenesis and mitochondrial translation following injury compared to controls. In vitro co-culture studies confirmed that astrocytes transfer mitochondria to neurons via EVs containing mitochondria (EV-mito). Isolated EV-mito from astrocyte-conditioned media improves neuronal mitochondrial function under NMDA (N-methyl-D-aspartate) induced excitotoxic conditions. Together, these findings demonstrate that TBI drives astrocyte-mediated mitochondrial transfer as an adaptive response, occurring alongside neuronal mitochondrial dysfunction. This process preserves neuronal bioenergetics in the soma and neurites but not at synapses, highlighting both its therapeutic potential and spatial limitations.

Spinal cord injury (SCI) induces a robust inflammatory response involving resident microglia and infiltrating macrophages. Macrophage activation states are closely associated with cellular metabolism, with pro-inflammatory macrophages relying primarily on glycolysis and reparative macrophages favoring mitochondrial oxidative phosphorylation (OXPHOS). Aging is a major determinant of functional recovery post-SCI and is associated with enhanced pro-inflammatory macrophage activation; however, the metabolic mechanisms underlying these age-dependent responses remain poorly understood. We investigated whether aging alters macrophage metabolism following SCI. Female C57BL/6 mice aged 4 months (young) or 14 months (aged) underwent T9 contusion SCI. At 7 days post-injury, CD11b⁺ intraspinal macrophages/microglia were isolated by magnetic-activated cell sorting and assessed for their OXPHOS and glycolytic activity. In parallel, an age-comparison subset of a published single-nucleus RNA-sequencing atlas was analyzed to identify age-associated changes in myeloid cell composition and metabolic transcriptional programs. Ex vivo analysis demonstrated that aging significantly reduced basal, ATP-linked, maximal respiration, and spare respiratory capacity, in CD11b⁺ cells isolated from injured spinal cord tissue, whereas glycolytic activity remained largely unchanged. Single-nucleus analysis revealed a relative reduction in resident microglia and expansion of inflammatory and chemotaxis-associated macrophages in 14-month-old mice. Metabolic gene programs were broadly altered across myeloid populations, with subtype-specific reductions in mitochondrial oxidative and pyruvate metabolism pathways in chemotaxis-associated macrophages. Together, these findings indicate that aging impairs mitochondrial respiratory capacity while reshaping myeloid composition and metabolic programs after SCI, suggesting reduced metabolic flexibility within the inflammatory lesion environment. Targeting macrophage immunometabolism may improve SCI outcomes, particularly in aging populations.

Pulmonary fibrosis (PF) is a progressive lung condition characterized by irreversible scarring and high mortality, with limited effective treatments. Mitochondrial dysfunction has emerged as a critical factor in fibroblast activation in PF, although approaches to restore mitochondrial function remain underexplored. The present study investigated whether mitochondrial transfer from alveolar type II epithelial cells (A549) to patient-derived fibroblasts could restore mitochondrial function and bioenergetics. Histological analysis of fibrotic lungs reveals increased collagen deposition and elevated profibrotic markers, accompanied by reduced expression of mitochondrial biogenesis and respiratory proteins compared to non-fibrotic controls, indicating mitochondrial impairment. Freshly isolated donor mitochondria were functionally validated before mitoception using Seahorse analysis and patient-derived fibroblasts were confirmed by qRT-PCR using fibroblast-specific markers. In vitro transfer of mitochondria to diseased patient-derived fibroblasts exhibited a modest, dose and time-dependent increase in mitochondrial membrane potential compared to normal fibroblasts. Gene expression analysis revealed decreased fibrosis-associated markers and increased expression of mitochondrial and antioxidant genes following mitoception. Seahorse analysis after mitoception revealed enhanced ATP-linked respiration and improved selected mitochondrial bioenergetic parameters, whereas maximal respiration and spare respiratory capacity demonstrated variable responses. In contrast, normal fibroblasts displayed minimal changes. Collectively, these findings indicate that mitochondrial transfer modulates fibroblast bioenergetics and profibrotic signaling, supporting its potential as a therapeutic strategy for pulmonary fibrosis.

Mitochondria are dynamic organelles essential for neuronal survival and synaptic function, and their dysfunction is a key consequence of excitotoxicity following traumatic brain injury (TBI). While intercellular mitochondrial transfer and exogenous mitochondrial transplantation have emerged as mechanisms to restore cellular bioenergetics, its in vivo relevance in the central nervous system remains incompletely understood. Here, we used astrocyte and neuron-specific mitochondrial reporters (GFP or Dendra2) in mice to assess cell-type-specific mitochondrial morphology, bioenergetics, and transfer 24hrs after TBI. Neurons exhibited marked mitochondrial dysfunction, including altered morphology and reduced bioenergetic capacity across somatic, synaptic, and non-neuronal fractions. In contrast, astrocytic mitochondria showed morphological changes but preserved bioenergetic function. Concomitantly, astrocyte-to-neuron mitochondrial transfer was significantly increased following injury, although transfer to synapses remained limited. Single-cell RNA sequencing of astrocytes revealed upregulation of genes involved in extracellular vesicle (EV) biogenesis and mitochondrial translation following injury compared to controls. In vitro co-culture studies confirmed that astrocytes transfer mitochondria to neurons via EVs containing mitochondria (EV-mito). Isolated EV-mito from astrocyte-conditioned media improves neuronal mitochondrial function under NMDA (N-methyl-D-aspartate) induced excitotoxic conditions. Together, these findings demonstrate that neuronal mitochondrial dysfunction drives astrocyte-mediated mitochondrial transfer as an adaptive neuroprotective response after TBI. This process preserves neuronal bioenergetics in the soma and neurites but not at synapses, highlighting both its therapeutic potential and spatial limitations.

Traumatic brain injury (TBI) triggers complex and evolving secondary cascades that disrupt mitochondrial homeostasis and contribute to progressive neurodegeneration. Although mitochondrial impairment is a well-recognized driver of post-traumatic pathology, the spatial and temporal progression of mitochondrial dysfunction, particularly in regions distal to the injury site, remains poorly defined, and potential sex-specific responses remain understudied. Here, we performed a comprehensive mitochondrial-focused analysis in a mouse model of controlled cortical impact (CCI), quantifying mtDNA copy number (mtDNA-CN), mitochondrial gene expression, and protein markers regulating biogenesis, transcription, electron transport chain integrity, and mitophagy. Mitochondrial profiles were assessed across four brain regions (cortex at 2, 4, and 6 mm from the injury epicenter, and hippocampus) at four time points (6h, 12h, 24h, and 48h) in both female and male C57BL/6J mice. While mtDNA content exhibited only modest and region-restricted reduction, particularly near the injury core, transcriptional and protein-level changes were far more pronounced and sex-divergent. Females displayed extensive early cortical gene activation followed by widespread hippocampal suppression at 48 h across mitochondrial dynamics, OXPHOS, transcriptional regulation, and biogenesis pathways, accompanied by 48h in PGC-1α, TFAM, and NDUFS1. In contrast, males showed minimal transcriptional disruption but demonstrated delayed compensatory increases in TFAM, NDUFS1, and p62 protein levels, suggesting activation of mitochondrial maintenance and recovery programs. These spatially and temporally distinct responses reveal fundamental sex-specific vulnerabilities in mitochondrial regulation after TBI. Together, our findings provide a direction to an integrated mitochondrial landscape of early post-injury events and identifies critical windows and pathways that may support sex-specific therapeutic targeting to restore mitochondrial function after TBI.

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; mtD2 ^f/f – AmtD2 and Aldh1l1-CreER; mtGFP ^f/f – AmtGFP) and neuron-specific (CamK2aCre; mtD2 ^f/f – NmtD2 and CamK2aCre; mtGFP ^f/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.

Although metabolism was originally studied across an array of mammals, contemporary metabolic studies moved towards human and mouse cells. A newfound interest in how metabolism regulates cell state during homeostasis, tissue repair and disease has uncovered key roles for energy flux inside and outside of mitochondria. Fibroblasts are key mediators of wound healing outcomes and prior work uncovered that cells from highly regenerative mammals (spiny mice and rabbits) exhibit enhanced resistance to oxidative stress compared to those from non-regenerating laboratory mice and rats. Using a battery of cellular tests in primary ear pinna fibroblasts from spiny mice, rabbits, laboratory mice and rats, we show that cells from spiny mice and rabbits exhibit a baseline preference for glycolysis supporting lower ROS-production. Mitochondria from spiny mouse fibroblasts were generally low respiring and depolarized while exhibiting a large, spherical morphology. We observed this large, spherical phenotype consistently across lifespan in ear pinna fibroblasts from fetal, young and old spiny mice and cells from all ages were highly resistant to oxidative stress. While rabbit, mouse and rat fibroblasts had polarized tubular mitochondrial networks typical of adult mammalian fibroblasts, isolated rabbit and spiny mice fibroblasts shared lower oxygen consumption efficiency even in the absence of a potential gradient. Taken together, our results support that a shared metabolic signature exists in stromal cells from highly regenerative mammals, although possibly driven by different mechanisms, to converge on a ROS-resistant phenotype that increases cellular resilience.

Cerebral hypometabolism occurs in both traumatic brain injury (TBI) and Alzheimer’s disease (AD), but whether these conditions act through distinct or overlapping mechanisms is unclear. TBI disrupts cerebral metabolism via blood–brain barrier damage, altered glucose transporter expression, calcium buffering abnormalities, and oxidative damage to metabolic enzymes. AD-related hypometabolism is linked to amyloid-β (Aβ) effects on mitochondria, including impaired respiration, oxidative stress, and altered mitophagy, fusion, and fission. We tested whether TBI-induced mitochondrial dysfunction exacerbates Aβ-mediated impairment using a closed-head injury (CHI) model in APP/PS1 knock-in (KI) mice. Injuries were delivered at 4-5 months of age, before plaque formation and mitochondrial deficits in KI mice. Bioenergetics were measured at 1, 4, and 8 months post-injury in hippocampus and cortex using Seahorse assays on isolated mitochondria. At 1 month, genotype-by-injury interactions revealed greater dysfunction in KI mice than either condition alone, with males more vulnerable than females. At 4-8 months, amyloid-mediated effects predominated, while TBI-specific changes were no longer apparent, suggesting recovery or convergence onto shared mechanisms. These results indicate that TBI can temporarily worsen mitochondrial dysfunction in the context of early amyloidosis, with sex influencing vulnerability. Findings provide insight into the temporal relationship between TBI and amyloid-induced mitochondrial deficits and support the importance of sex as a biological variable in neurodegenerative disease progression. Highlights: TBI and APP/PS1 genotype interact to worsen early mitochondrial dysfunction. Hippocampus exhibits greater susceptibility to combined TBI and amyloid pathology. Sex-specific effects and temporal patterns underscore TBI’s role in AD risk. Male mice show heightened vulnerability to TBI-induced mitochondrial dysfunction. GRAPHICAL